Developing Sustainable Food with Hermetia illuscens &

Macroalgae by Coupling Material Balances to

Physicochemical Properties

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University

By

Jeffrey Caminiti
Graduate Program in Food Science and Technology

The Ohio State University

2023

Dissertation Committee
Dennis Heldman, Advisor
Macdonald Wick, Advisor
Christopher Simons
Yael Vodovotz
 Copyrighted by

Jeffrey T. Caminiti

2023

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 Abstract
Underutilized biomasses hold promise as sustainable food, feed, and fuel. As food, novel

materials must be nutritious, safe, and desirable to consumers while causing minimal

environmental damage. This dissertation hopes to encourage future cultivations of

macroalgae and an insect, Hermetia illuscens (the Black Soldier Fly larvae, BSFL).

Macroalgae and BSFL are associated with environmental benefits including low land,

water, and energy demands. They are both nutritious but are not readily accepted by

modern consumers as food. To facilitate their adoption, novel materials must take the

shape of familiar foods. This requires processing into novel food ingredients.

Environmental impacts increase as processing and materials are employed during the

creation of novel ingredients. While the desirability of the final product may benefit, the

process must be scrutinized to reduce resource use and ensure sustainability. Extraction

and separation operations aim to concentrate a specific component, such as protein, in a

single fraction. Depending on the composition, the remaining materials will also have

value. Optimal co-product development requires that the influence of each process

parameter be well understood. This is advantageous to processors since each additional

product shares the costs and environmental impacts associated with production. The

research aimed to evaluate hypotheses concerning the influence of processing parameters

on material separation and physicochemical properties after biomass fractionation. The

following specific objectives were addressed during this research 1) understand the

nutritional quality of macroalgae and BSFL, 2) evaluate aqueous extracts from BSFL and
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macroalgae, 3) develop an experimental fractionation system to monitor component

separation using experimental data and mass balance relationships, and 4) evaluate the

effects of process parameters on the outputs of the fractionation.

Macroalgae and insects were found to be promising future foods after a review of their

environmental impact, nutritional quality, and historic use as food. A broad analysis of

macroalgae amino acid profiles found less variation within and between phyla than

previously reported. Investigation into the proteolytic activity of commercial BSFL

demonstrates the capacity for autohydrolysis. Experimental analysis of protein extraction

from BSFL and macroalgae (brown kelp) demonstrates that NaOH is the most effective

solution for extracting proteins.

Improved experimental procedures were paired with explicit mass balance relationships

and accompanying assumptions used to define process metrics. These metrics were used

to evaluate the moisture, solids, and protein balances after separations. The experimental

fractionation system was found to be repeatable and versatile after experimental runs with

BSFL, two macroalgae genera, and soy flour. The evaluation showed variables such as

homogenization, temperature (20-37°C), and batch-to-batch variation did not have a

significant influence on the process metrics. Changes to NaOH concentration (0-2.5M)

and exposure time (0-24h) were found to influence the extraction of biomass solids

including proteins, the water-holding capabilities of the remainder, and the protein profile

of BSFL extracts.

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The experimental fractionation system developed herein has identified critical process

parameters and provides a systematic and repeatable approach to fractionation evaluation.

Sustainability is an ongoing process but experimental fractionation systems that couple

material balances with output properties are needed to facilitate positive development.

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 Dedication
I dedicate this work to Angelo, Ron, and Katie, for your inspiration to lead a good life.

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 Acknowledgments
A special thanks to my parents and brothers, your continued support throughout my

education has been everything. My years of school have been full of unexpected

challenges; I would not have made it this far without your love and guidance. An Extra

Special thanks to my partner Cori Overs, your love and support during this process has

been instrumental.

Thank you to The Ohio State University Department of Food, Science, and Technology

as well as the department of Animal Sciences. The education I have received through my

time at this University has been incredibly valuable. The facilities and opportunities

available are greatly appreciated.

I want to thank my committee: Dr. Dennis Heldman, Dr. Macdonald Wick, Dr.

Christopher Simons, and Yael Vodovotz. Your support and guidance before and during

my Ph.D. work have been invaluable. You have all helped me hone my interest in

scientific pursuits.

I am especially grateful for the generosity of Dale A. Seiberling to the Food Engineering

Research Laboratory at The Ohio State University for providing a home to my research

project. To Dr. David Phinney, thank you for being a friend and mentor. A special thanks

to Dr. Brian Roe for his instrumental role in acquiring my initial funding to study BSFL. I

am still waiting to deploy your slogan for the BSFL: “Locally thrown to locally grown.”

To Dr. Michael Huesemann, thank you for taking a chance and committing to algal

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protein. I hope to find success with you in the future. Thank you, Damen Reynolds and

Scott Edmundson, for your collaboration and acceptance. Thank you, Matt Bernier, for

your work in conducting amino acid profiles.

To all the students I have shared time with at OSU, thank you for making the experience

enjoyable. A special thanks to Dr. Aishwarya Badiger, Dr. Katie Williamson, and Dr.

Holly Huellemeier your support and knowledge helped define my research. Finally,

Thank you Hayden Riley for all the help in the lab, especially after my arm injury.

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 Vita
Vita

2011................................................................Elder High School

2014, 2015......................................................Internship, Perfetti van Melle

2016................................................................B.S. Food Science, Ohio State University

2018................................................................M.S. Food Science, Ohio State University

2016 to present ...............................................Graduate Research Associate, Department of Food,

Science, & Technology, The Ohio State University

Publications

Reynolds, D., Caminiti, J., Edmundson, S., Gao, S., Wick, M., & Huesemann, M. (2022).

Seaweed proteins are nutritionally valuable components in the human diet Running head:

Nutritional value of seaweed proteins. American Society for Nutrition.

https://doi.org/10.1093/ajcn/nqac190/6640420

Fields of Study

Major Field: Food, Science, & Technology,

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 Table of Contents
Abstract ................................................................................................................................ i
Dedication .......................................................................................................................... iv
Acknowledgments................................................................................................................v
Vita .................................................................................................................................... vii
Table of Contents ............................................................................................................. viii
List of Tables ..................................................................................................................... xi
List of Figures .................................................................................................................. xiii
Chapter 1 Introduction .........................................................................................................1
1.1 References ..................................................................................................................8
Chapter 2 Review of Literature: “Understanding New Foods: Alternative Protein
Sources” from Sustainable Food Innovation .....................................................................11
Abstract ..........................................................................................................................11
2.1 Introduction ..............................................................................................................13
2.2 Algae ........................................................................................................................15
2.3 Fermentation-based ingredients ...............................................................................22
2.3.1 Mycoprotein ......................................................................................................22
2.3.2 Precision fermentation ......................................................................................24
2.4 Insects ......................................................................................................................25
2.5 Conclusions ..............................................................................................................28
2.6 References ................................................................................................................30
Chapter 3 Evaluations of commercial Black Soldier Fly larvae (BSFL) as a novel source
of nutrients, aqueous protein extraction, and protease activity ..........................................38
Abstract ..........................................................................................................................38
3.1 Introduction ..............................................................................................................40
3.1.1 BSFL as a nutritious part of a sustainable food system. ...................................41
3.1.2 Potential markets for fractionated BSFL components and the associated
processing ..................................................................................................................47
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 3.1.3 Proteolytic activity of the BSFL .......................................................................54
3.1.4 Protein quantification ........................................................................................56
3.1.5 Rationale and objectives: ..................................................................................59
3.2 Materials and Methods .............................................................................................60
3.2.1 BSFL preparation and extractions ....................................................................60
3.2.2 Analytical methods ...........................................................................................60
3.3 Results and Discussion ............................................................................................62
3.3.1 Nitrogen extraction versus time from BSFL using NaOH and deionized water
....................................................................................................................................62
3.3.3 BSFL protein extraction using NaOH washes ..................................................65
3.3.4 Identifying BSFL proteolytic activity with casein plates .................................68
3.3.5 Characterization of BSFL proteases with SDS-PAGE zymogram ...................69
3.3.6 Protein characterization of ground and soaked BSFL to monitor autohydrolysis
....................................................................................................................................70
3.4 Conclusions ..............................................................................................................73
3.5 References ................................................................................................................74
Chapter 4 Macroalgae amino acid analysis and extraction screening for protein yield
using enzyme-assisted extraction (EAE) for two species of Brown Kelp .........................87
Abstract ..........................................................................................................................87
4.1 Introduction ..............................................................................................................89
4.1.1 Rationale and objectives ...................................................................................92
4.2 Methods....................................................................................................................93
4.2.1 Extraction Materials and methods ....................................................................93
4.2.2 Amino acid profile methods: ............................................................................98
4.3 Results and discussion ...........................................................................................103
4.3.1 Extraction analysis ..........................................................................................103
4.3.2 Amino acid analysis ........................................................................................108
4.4 Conclusions ............................................................................................................120
4.5 References: .............................................................................................................121

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Chapter 5 Experimental fractionation system including a process model, experimental
procedure, and associated mass balances used for novel food evaluation .......................124
Abstract ........................................................................................................................124
5.1 Introduction ............................................................................................................126
5.1.1 Emerging raw materials for sustainable food development ............................127
5.1.2 Sustainability-driven food development .........................................................131
5.1.3 Generic and systematic fractionation system for food ingredient development.
..................................................................................................................................134
5.1.4 Rationale and objectives .................................................................................144
5.2 Materials and Methods ...........................................................................................146
5.2.1 Fractionation process analysis ........................................................................146
5.2.2 Experimental designs and statistical analysis .................................................166
5.3 Results and Discussion: .........................................................................................172
5.3.1 Analysis 1 – Retrospective process analysis of the experimental fractionation
system ......................................................................................................................172
5.3.2 Analysis 2 – Deionized water fractionation among four biomasses (BSFL,
Alaria, Ulva, and soy) ..............................................................................................175
5.3.3 Analysis 3 – Retrospective sensitivity analysis for BSFL process decisions .180
5.3.4 Experiment 1 – Ulva fractionation with different solution conditions ...........183
5.3.5 Experiment 2 – BSFL fractionation with different solutions and versus NaOH
exposure time ...........................................................................................................186
5.4 Conclusions ............................................................................................................196
5.5 References ..............................................................................................................199
Chapter 6 Conclusions .....................................................................................................214
Chapter 7 Future works ....................................................................................................218
References ........................................................................................................................219
Appendix A: Images from preliminary BSFL experiments .............................................249
Appendix B: Fractionation system data management and analysis code ........................251
Appendix C: Author permission, Chapter 2 ....................................................................255

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 List of Tables
Table 4.1: Validation table for GC/MS identification of amino acids ........................... 101
Table 4.2: Proximate analysis for the Alaria and Saccharina samples used in the protein
extraction experiments (see Figure 4.1) compared to a report by Schiener et al., (2014)
on seasonal variation of brown kelp species. .................................................................. 103
Table 4.3: List of amino acid references used for comparison and validation of the
macroalgae samples and purified protein controls.......................................................... 113
Table 4.4: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Laminaria and stock
protein samples ............................................................................................................... 114
Table 4.5: Amino acid profile (AA mg/Σ(AA mg)) * 100, Alaria, Macrcytis, Eularia,
and Nereocytis species .................................................................................................... 115
Table 4.6: Amino acid profile (AA mg/Σ(AA mg)) * 100, Saccharina species ............ 116
Table 4.7: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Palmaria and Porphyra
species ............................................................................................................................. 117
Table 4.8: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Ulva species ................. 118
Table 4.9: Average amino acid content (AA mg/ Σ (AA mg)) * 100 for blue evolution
and commercial samples separated by phyla and species where more than two samples
were measured ................................................................................................................ 119
Table 5.1: Summary of terms used for fractionation analysis........................................ 149
Table 5.2: Decision points corresponding to “D” labels in Figure 5.2. Each decision
point represents one or more explanatory variables that may be manipulated during
experiments. The variables and variable types are included in the table. ....................... 153
Table 5.3: Process metrics with their explicit definition referring to Figure 5.2 and the
rationale for why each metric was included in the research. .......................................... 166
Table 5.4: Summary statistics for retrospective analysis of mass collection after
separation and drying, N=88. Difference metrics (Sec. 5.2.2.1) were presented as
normalized percentages (fractionsC-slurry) C/slurryC)*100). Where C refers to component.
......................................................................................................................................... 174

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Table 5.5: Preliminary sensitivity analysis of BSFL explanatory variables (N=28); table
body contains ANOVA F-ratio values representing a relative effect size, “n.s” means
non-significant effect (α=0.05). ...................................................................................... 180

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 List of Figures
Figure 3.1: Amino acid profile of BSFL compared to whey protein isolate and soy
protein isolate amino acid profile (% of amino acids; Kalman, 2014; Müller et al., 2017;
Spranghers et al., 2017). *indicates essential amino acid ..................................................46
Figure 3.2: Extraction of nitrogen versus time (0-24 h) for BSFL protein using deionized
water and alkali solvent conditions; mg protein/ml was converted from total N analysis of
liquid samples. ...................................................................................................................63
Figure 3.3: SDS-PAGE gel showing the effect of solvent on the protein extracted from
BSFL flour. Lanes include: 1) diH2O (0.1x dilution), 2) PBS (0.5x dilution), 3) NaOH
(0.5x dilution), 4) standard. Protein ladder (right) shows standard lane molecular weight.
Protein concentrations are not normalized. ........................................................................65
Figure 3.4: Rinsing experiment, (A) shows a picture of BSFL in NaOH after shaking but
before centrifugation, (B) shows a plot of protein in the supernatant after repeated washes
with 1 M NaOH .................................................................................................................67
Figure 3.5: Casein plates showing proteolytic activity of diH2O-soluble BSFL extract
(left) and the same extract dialyzed against PBS (right). ...................................................68
Figure 3.6: Zymogram of BSFL and BSFL extracts. Left panel shows two concentrations
of whole BSFL (25, 50 mg sample/ml) suspended in non-reducing dissolution buffer. The
right panel shows BSFL extracts from diH2O and PBS. Protein ladders show standard
lane molecular weight. .......................................................................................................70
Figure 3.7: Soaking experiment showing whole larvae, larvae flour, and flour soaked in
diH2O for one hour on a 12.5% acrylamide gel, loadings were standardized to BSFL
concentration. Protein ladder (right) shows standard lane molecular weight. ...................72
Figure 4.1: Top: Experimental design diagram showing the 16 extraction treatment
conditions. Bottom: graphical overview of experimental procedure showing initial
treatment, adjustment, neutralization, centrifugation, and separation. ..............................97
Figure 4.2: Pellet dry matter collected for all conditions tested showing pH7 or “neutral”
samples on the left and 2.5 N NaOH or “alkali” samples on the right for Saccharina and

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Alaria. Initial mass was approximately 10 grams for all samples. Capital letters indicate a
difference between sample groups (α<0.05). Each bar represents 1 run. ........................105
Figure 4.3: Supernatant % solids for all conditions determined via drying at 105 °C for
17 hours showing pH7 or “neutral” samples on the left and 2.5 N NaOH or “alkali”
samples on the right for Saccharina and Alaria. Initial mass was approximately 10 grams
for all samples. Capital letters indicate a difference between sample groups (α<0.05).
Each bar represents 1 run. ................................................................................................106
Figure 4.4: Extracted protein collected for all conditions tested showing pH7 or “neutral”
samples on the left and 2.5 N NaOH or “alkali” samples on the right for Saccharina and
Alaria. Initial mass is approximately 10 grams for all samples. Values are the product of
the BCA protein content times the supernatant volume. Capital letters indicate a
difference between sample groups (α<0.05). Each bar represents 1 run. ........................108
Figure 5.1: Theoretical dry-fractionation process shows the transformation of a biomass
into n generic fractions after a separation process. Each fraction is labeled with an Fi as
unique identifier, where i is a count of fractions. Inputs (I) and outs (O) are identified and
make up the overall process mass balance. ......................................................................150
Figure 5.2: Experimental fractionation scheme featuring twelve possible unique fractions
created from five operations with parameters dependent on five process decision points
(D1-5). Each fraction is labeled with an Fi as a unique identifier. Inputs (I) and outs (O)
transverse the process boundary, are identified, and make up the overall process mass
balance. All operations and fractions not identified as input or output are within the
process boundary. Intermediate groups (G1-5) identify groups of fractions that are
outputs of the same operation. Dotted lines indicate operations and associated fractions
that are included as explanatory variables. ......................................................................153
Figure 5.3: Data collection flow chart for the extraction, desalting, and drying operations.
Masses and volumes are identified and numbered with W/ V in boxes outlined in black,
processes are numbered corresponding to Figure 5.2 in dark gray boxes, unmeasured
intermediates are named in light gray boxes, and additional analytics are identified in
black boxes nearest the intermediate fraction the analysis is conducted on. Analytical

xiv
measurements are identified for moisture content (MC), protein content (via BCA), and
SDS-PAGE. .....................................................................................................................158
Figure 5.4: Retrospective analysis of mass collection after separation and drying (N=88),
Box plot of differences metrics (Sec. 5.2.2.1) for “total wet weight” and components
presented as normalized percentages (fractionsC-slurryC) /slurryC)*100). Where C refers
to component. ...................................................................................................................175
Figure 5.5: Retrospective analysis of mass collection for deionized water extractions
after separation and drying (N=22), Box plot of differences metrics (Sec. 5.2.2.1) for
“total wet weight” and components presented as normalized percentages (fractionsC-
slurryC) /slurryC)*100). Where C refers to component. ...................................................176
Figure 5.6: Process metric and measurements for distilled water fractionation of four
different biomass (Alaria, n=4; Ulva, n=4; BSFL, n= 12; Soy, n=2); Tukey HSD
pairwise comparisons, α=0.05, different letters indicate difference within metric category
..........................................................................................................................................179
Figure 5.7: The relationship between, protein yield, protein purity; non-protein, non-
additives pellet yield, and WHC% as a function of NaOH concentration in molarity used
during extraction for 1 hour at 20 °C. Error bars for all metrics on 0 M (diH2O, n=4), and
0.1 M NaOH (n=3), remaining concentrations had 1 replication. ...................................183
Figure 5.8: Process metrics describing Ulva fractionation experiment in three plots: Plot
I contain supernatant protein metrics, II contains moisture and solids related metrics, III
contains % WHC of the pellet. Error bars show standard error. Tukey HSD pairwise
comparisons, α=0.05, different letters indicate difference within metric category .........185
Figure 5.9: Process metrics describing BSFL fractionation experiment in three plots: Plot
I illustrates supernatant protein metrics, II contains moisture- and solids-related metrics,
and III contains WHC. All NaOH samples have undergone defatting as described in Sec.
5.2.1. Error bars show standard error and statistical differences among groups were
determined with Tukey HSD pairwise comparisons, α=0.05, different letters indicate
difference within metric category ....................................................................................191
Figure 5.10: NaOH versus time process metrics. top) protein-based process metrics,
bottom) moisture and solids process metrics. ..................................................................192

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Figure 5.11: SDS-PAGE gel (12.5% acrylamide, 0.75 mm) with deionized water, and
NaOH samples (0, 1, 6, 12, and 24 h) between two protein ladders. Loadings were not
normalized by protein concentration. ..............................................................................195
Figure 5.12: SDS-PAGE gel (12.5% acrylamide, 0.75 mm) with deionized water,
dialyzed 3M NaCl and dialyzed NaOH samples (0, 2, 6, 12, and 24 h) with protein
ladders. Loadings were not normalized by protein concentration. ..................................195
Figure A.1: Processing of raw BSFL from collection to dried flour. BSFL are gown in
large bins (A) at waterman farms. There were transported to Dr. Heldman’s lab (B).
Thermal blanching was then conducted in a makeshift hot water bath (C). Photo D shows
the enzymatic activity associated with BSFL and the results of grinding without heat
treatment. The cleaned, inactivated and frozen product is shown in photo E. The final
image (F) shows ground BSFL during the sieving process to obtain a uniform flour prior
to protein extraction. ........................................................................................................249
Figure A.2: Ground BSFL flour suspend in aqueous solvents after different treatment.
Image A ground flour was mixed vigorously by hand with 20x its mass in water then left
to settle at 4 °C for 24 hours. Image B demonstrates the extraction process for different
solvents: Alkali (1M NaOH), di water, and salt (0.5 M NaCl) water after shaking in a
room temperature water bath for 24 hours. ......................................................................250
Figure A.3: “Soldier Croutons.” A product created by a student group using BSFL flour
for the American Society of Baking product development competition. ........................250

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 1. Chapter 1 Introduction
The research presented in this dissertation encourages the future development of

sustainable food through the evaluation of novel biomasses and their fractionation. Many

underutilized biomasses have been identified for their potential to increase sustainability

through conversion to bioproducts in the food, feed, fuel, plastic, or pharmaceutical

industry. Food has been recognized as the most profitable and impactful application for

most biomasses (Parodi et al., 2018). Thus, a food-first approach to biomass fractionation

became the focus of the experimental portion of the research. In general, a fractionation

or biorefinery (where biofuel is a co-product) approach is designed to separate valuable

components for utilization in various industries. With food as the main product, co-

products in one of the other industrial sectors mentioned may also be viable. For this

reason, efforts to systematically determine the composition of all fractions produced from

a fractionation were taken. In the case of food, extracted protein is expected to undergo

additional processing as it is transformed from a raw extract to a high-protein ingredient

such as a protein concentrate or isolate. Protein’s role in foods is often dynamic as it

provides physical form dictated by certain physicochemical properties as well as nutrition

(Foegeding et al., 2017). The research aimed to evaluate hypotheses concerning the

influence of processing parameters on material separation and physicochemical

properties after biomass fractionation. By coupling a material-balance based process

analysis to physicochemical evaluation, considerations for sustainability are possible as

processes for novel ingredients are identified. The adoption of novel biomasses and

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attempts to sustainably process and valorize them is a response to the environmental

damages associated with agricultural industries and a need for resiliency in the face of

climate change.

Climate change, combined with a large and growing global population, has renewed

concerns about how humanity will feed itself in the coming century. Humans have altered

global climates through excess greenhouse gas emissions (GHGE), and it is changing the

way we live, eat, and interact with the natural world. Numerous scientific investigations

have been conducted to study the greenhouse effect with the first occurring in 1856 by

Eunice Foote at the end of the industrial revolution. Eunice speculated that increases in

atmospheric CO2 concentrations due to our use of fossil fuels will lead to significant

warming (Sorenson, 2011). Climate science has advanced a great deal in the last 150

years, but Eunice’s theory has yet to be disproven. In a report from the Intergovernmental

Panel on Climate Change (IPCC, United Nations), they state “It is unequivocal that

human influence has warmed the atmosphere, ocean, and land” (Arias et al., 2021). The

industrialization of global economies has increased global energy demand, resulting in

the increased emission of greenhouse gases. Agriculture as an industrial sector, while

essential to the well-being of humanity, is a leading emitter of greenhouse gasses

contributing up to 20% globally (Bezner Kerr et al., 2022). Along with its energy

demands, agriculture also requires large amounts of land and water which may be

insufficient to feed a growing population (Molotoks et al., 2021). Due to agriculture's role

in and vulnerability to climate change, novel food sources became a focus of this

dissertation.

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According to the US Environmental Protection Agency (EPA) sustainability is based on

the principle that:

“Everything that we need for our survival and well-being depends, either directly

or indirectly, on our natural environment. To pursue sustainability is to create and

maintain the conditions under which humans and nature can exist in productive

harmony to support present and future generations.” (EPA, 2021)

The EPA states that sustainability is a guideline for their organization’s work

(https://www.epa.gov/sustainability). Sustainable pursuits take vastly different forms

depending on the context but often require cooperation from governments, businesses,

and private individuals (Bennett et al., 2019). With an ultimate goal of zero hunger,

sustainability efforts must consider a broad range of disciplines from the environmental

impacts of global warming and habitat loss to the logistical realities of poor food access

(Foegeding et al., 2017; O’Hara & Toussaint, 2021). A detailed discussion of the political

and economic underpinnings of sustainability at this level is beyond the scope of this

dissertation but has been briefly discussed in Chapter 2 during a detailed discussion of

insects and macroalgae as novel foods.

Sustainability is a multifaceted concept that requires holistic evaluations; the best tool

available to researchers today is Life cycle Assessment methodologies (LCA). LCA is

used to answer critical sustainability questions about GHGE, land, and water use through

the comprehensive life of a product. When studying global warming potential (gwp),

LCAs typically make comparisons based on mass, however, with food, mass alone may

be insufficient. Berardy et al., (2019) were able to create an algorithm to include

3
nutritional protein quality in their LCA assessment of protein sources. When comparisons

were based on mass the top ten protein sources studied were all animal-derived.

However, when a weighted protein quality factor is included, other products such as rice,

bread, pasta, and tofu enter the top ten GHGE emitters. Their analysis is limited by the

consumption habits of these products since it is not recommended that consumers rely on

rice or bread as a major source of protein. Regardless, the most culpable food item in

both versions of the analysis was beef which produces double the GHGE of the next

closest animal product regardless of the metric used. Aside from GHGE, bovine meat

production is a major driver of deforestation in South America (Recanati et al., 2015).

The inefficiencies of beef have been described by independent reports sponsored by

Beyond Burger (Heller & Keoleian, 2018), Impossible Foods (Khan et al., 2019), and in

additional peer-reviewed literature (Oonincx & de Boer, 2012; Shafiullah et al., 2021; zur

Strassen et al., 2020). Reducing the demand for beef and other animal products is one

way to reduce GHGE in the modern food system.

The transition from a novel raw biomass to a competitive meat mimetic or other product

replacement requires significant research and development beyond the scope of this

dissertation. The main ingredient in novel meat mimetics is typically a protein

concentrate or isolate that is used to add texture and bind other ingredients together (Joshi

& Kumar, 2015). Physicochemical properties associated with the protein ingredients

allow for the necessary textures to produce desirable meat and other replacements

(Foegeding et al., 2017). Developing these types of ingredients starts with mixing and

separation operations to extract and concentrate proteins. These operations were a focus

of the research as fundamental steps for protein ingredient development. Mixing and
4
separation also dictate co-product potential in feed or fuel applications. For all three

applications composition is critical thus this dissertation presents a significant focus on

evaluating component mass distribution after separation. During the development of

procedures to achieve this evaluation, considerations for the assessment of

physicochemical properties were also taken. These attempts to couple material balances

with physicochemical properties hope to enhance the development of novel biomasses

into ingredients that are acceptable for meat mimicry and other food applications.

This dissertation has focused on two biomass types: macroalgae (or seaweed) and insects

through the Black Soldier Fly larvae (Hermetia illucens; BSFL). An overwhelming body

of literature describing the historic uses, nutritional quality, and environmental

considerations has been presented in the literature. This background is captured in

Chapter 2 which provides context for both classes of organisms and background for their

development into novel ingredients, addressing Objective 1 below. Gaps in the research

were identified specific to each class of organism that if filled would be beneficial to

assessing their sustainability upon production or informative for consumers hoping to

pick the best food for their situation. Nutritionally, both sources have been well

characterized, but in the case of macroalgae, knowledge of protein quality was

insufficient. Chapter 4 presents the most comprehensive review and experimental

investigation of the macroalgae amino acid profile, also addressing Objective 1 below.

Research is available about protein extraction and biomass fractionation efficiencies and

the physicochemical properties of both organism types. However, past research rarely

presents physicochemical analysis across multiple processing parameters with associated

component distributions. Similarly, evaluations rarely involve two disparate biomasses

5
using the same protocols. These gaps were identified as a lack of continuity in our

knowledge of biomass fractionation that could provide unbiased and robust assessments

of a range of highly diverse and numerous future food sources. The concept of a

generalized experimental fractionation system is presented and evaluated in Chapter 5 to

address these deficiencies in the literature and to address Objective 3 & 4 below.

Chapters 3 & 4 provide early extraction investigations that led to the development of the

experimental fractionation system. Chapter 3, while focusing on the BSFL, also discusses

the role of endogenous enzymatic activity and a selection of investigations used for its

study. Chapters 3 & 4 address Objective 2 below for BSFL and macroalgae, respectively.

As research progresses with novel foods, the systematic fractionation procedure

developed herein provides an approach to study the physicochemical and bioactive

properties of novel biomasses while ensuring sustainability, via the monitoring of

component masses, is put in context among traditional food sources.

The main objective of this research was to investigate two disparate biomasses (BSFL

and macroalgae) as sustainable future foods. This was done through the evaluation of

their proteins and their response to various fractionation conditions. The goal for the

analysis was to establish an experimental fractionation system with capabilities to relate

material balance outcomes to the physicochemical properties of fractions. The specific

objectives of this dissertation were to:

1. Evaluate Hermetia illuscens’ (BSFL) & macroalgal nutritional quality and

environmental impacts when used as novel food sources.

6
2. Create aqueous soluble extracts from BSFL and macroalgae and assess the

changes different chemical and biological processing aids have on their properties

3. Develop a repeatable bench-scale experimental fractionation system to measure,

calculate, or estimate biomass components during a generalized wet fractionation

of organic flours from various sources.

4. Identify processing parameters that affect process metrics associated with water,

solids, and protein separation

7
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Berardy, A., Johnston, C. S., Plukis, A., Vizcaino, M., & Wharton, C. (2019). Integrating
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Bezner Kerr, R., Hasegawa, T., Lasco, R., Bhatt, I., Deryng, D., Farrell, A., Gurney-
Smith, H., Ju, H., Lluch-Cota, S., Meza, F., Nelson, G., Neufeldt, H., & Thornton,
P. (2022). Ch 5. Food, fibre, and other ecosysem products. In IPCC WGII Sixth
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EPA. (2021, December 21). Learn About Sustainability.
Https://Www.Epa.Gov/Sustainability/Learn-about-Sustainability.

Foegeding, E. A., Stieger, M., & van de Velde, F. (2017). Moving from molecules, to
structure, to texture perception. Food Hydrocolloids, 68, 31–42.
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Heller, M. C., & Keoleian, G. A. (2018). Beyond Meat’s Beyond Burger Life Cycle
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environmental LCA of the Impossible Burger with conventional ground beef
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19. Ecological Economics, 180. https://doi.org/10.1016/j.ecolecon.2020.106859

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van Zanten, H. H. E. (2018). The potential of future foods for sustainable and
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Recanati, F., Allievi, F., Scaccabarozzi, G., Espinosa, T., Dotelli, G., & Saini, M. (2015).
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10
 2. Chapter 2 Review of Literature: “Understanding New
Foods: Alternative Protein Sources” from Sustainable
Food Innovation
Jeff Caminitia, Aishwarya Badigera, Omega Amoafob, Luca Serventib

a The Ohio State University Department of Food Science and Technology, The Ohio State

University, Parker Food Science and Technology, 2015 Fyffe Road, Columbus, OH

43210

bLincoln University. Department of Wine, Food and Molecular Biosciences, Faculty of

Agriculture and Life Sciences, RFH Building, PO Box 85054, Lincoln University, Lincoln,

7647, Christchurch, New Zealand.

*Corresponding Author: Jeff Caminiti, Parker Food Science and Technology, 2015

Fyffe Road, Columbus, OH 43210. Email: [email protected]

Abstract

This chapter discusses alternative protein sources and their potential to make novel and

sustainable foods. A constantly increasing human population, global warming, and

reduced availability of arable land for agriculture has been the cause of concerns for

scientists and governments in recent years. The exploration of food and protein sources

that require less land and water while emitting fewer greenhouse gasses during

cultivation is important are of future development. Algae, Insects, fungi, and other

11
fermented food sources were identified and discussed for their sustainable production

potential. Algae, including seaweed and microalgae, are photosynthetic organisms with a

high diversity of nutritional benefits. Seaweeds have additional benefits of

bioremediation when grown in the open ocean. Insects are high in protein and require less

land and water needed to produce staple crops such as soy. Algae and insects’ main

challenges are consumer acceptance requiring transformation into familiar forms for a

better appeal. Fungi can produce fibers that allow for meat mimicry increasing consumer

acceptance. Fermentation technologies, including microalgae, single-celled protein, and

lab-grown meat promise sustainable production of highly specific and functional proteins

for use in meat mimicry or other products. Agriculture is a fundamental aspect of life that

is also responsible for a considerable amount of emissions and damage to global

ecosystems. Developments of novel technologies and foods are needed to combat these

issues. More work is needed to develop palatable foods from algae, insects, and fungi.

These alternative protein sources are one answer to more sustainable systems that reduce

emissions and increase overall resilience.

Keywords:

Algae, insects, fungi, alternative protein, sustainability

12
2.1 Introduction

It should not come to a surprise that, in the recent years, consumers have increasingly

sought alternative sources of protein. Terms such “flexitarian”, “vegan” and “plant

based” have appeared on food labels. Flexitarians and plant-based consumers are those

who reduce their meat intake, while vegans abstain entirely from any kind of animal

derived food (Dagevos, 2021). Consumers’ main drivers were ethics, health, and

environment (Aschemann-Witzel et al., 2021; Dagevos, 2021; Esterl et al., 2021; Michel

et al., 2021). Online surveys carried in Australia and Germany depicted as high as 20%

flexitarian, 2/3 of which being women (Estel et al., 2021; Michel et al., 2021). This

nutritional shift is a growing phenomenon in western countries of America, Europe and

Oceania, whereas it does not appear to be popular in Africa and Asia. This was possibly

associated to the socio-economic status of emerging economies which typically is

associated to increase meat intake (Dagevos, 2021). What does it entail? Soy, pea, wheat

isolates, but also microalgae, insects, mycroprotein and cultured meat (Onwezen et al,

2021). Consumers’ main drivers were ethics, health, and environment. Convenience and

taste are not optimal yet, (Aschemann-Witzel et al., 2021). In addition, some products

sold as alternative proteins tend to be highly processed and perceived as less natural by

consumers (Varela et al., 2022). Thus, improvements are needed for such products.

Veggie-burgers have had a niche role in the American food system for the last half

century, but recently Impossible FoodsTM hit mainstream headlines when it began

producing a plant-based burger that bleeds. The founder, Pat Brown, set out to make a

better veggie burger and save the planet from the massive emissions associated with

13
factory farming during a sabbatical from Stanford in 2009. For many consumers the

Impossible is their first experience with “veggie burgers,” while the company is leading

an emerging billion-dollar industry of plant-based burgers and other alternative meat

products. The major companies in this emerging U.S. alternative protein industry are

Impossible Foods and Beyond Meat, both of whom were founded for the sole purpose of

displacing future growth of the meat industry. Increasingly, food technology companies

are making use of combinations of protein sources and novel ingredients, which in the

past have either been consumed separately or hadn’t existed until a few years back. There

is an interplay of different processes in the alternative protein space to generate the

desired final product convincing enough for consumers as meat replacements. For

example, the Impossible burger uses soy protein as its primary ingredient which has been

used as a key ingredient in many cultures for centuries, however, their product is set apart

by the inclusion of “heme” derived from a highly scaled precision fermentation process

(Impossiblefoods.com, accessed May 2022).

In this chapter, novel sources of foods and emerging food ingredients were discussed

which may be implemented in sustainable food systems of the future. This chapter hopes

to move past the regional novelty of certain food sources and focus on novelties that were

created or promoted for a singular purpose – sustainability. With many environmentally

favorable novel food-based ingredients, such as insect, microalgae, seaweed, or fungus,

commercial products are still under development so the food source will be the major

focus.

14
2.2 Algae

Algae is a collective term for photosynthetic Eukaryotic organisms not classified as

plants, most of which do not share a common ancestor. While prokaryotic, certain

cyanobacteria, such as Spirulina, are referred to as blue-green algae and are typically

included in discussions of algae (Torres-Tiji et al., 2020). This diverse group of

organisms are divided into two broad groups based primarily on size including the

microscopic (microalgae) and the macroscopic (macroalgae). Algae should be considered

a promising food of the future, but maybe not a novel source as they have been a source

of nutrients to humans for much of our history with the oldest known records have

identified its use 14,00 years ago (Torres-Tiji et al., 2020). The diversity in the biology of

these organisms provides both promise and challenge for the development of novel food

products. While attempts to use algae as a source of nutrients for a growing world

population were attempted in the last century, technological advancements and shifts in

consumer priorities have increased interest from investors in recent years. Broadly

speaking, algae has been championed as food of the future, a nutrient source to meet the

needs of our growing population, for their: low land use, and sustainable production

(Baghel et al., 2015), diverse functionality, and robust nutritional properties (Wells et al.,

2017).

Macroalgae have been a consistent staple of global cuisines. A great deal of seaweed’s

rich history has been published previously (Kaori O & Connor, 2017). The most

recognizable seaweed-derived food item is Nori, a product made from Porphyra and

widely used in sushi. Nori, Wakame (Undaria pinnatifida), Kombu (Laminaria

15
japonica), Dulse (Palmaria palmata or Rhodymenia sp) are three of the most

commercially successful varieties of seaweed (Griffiths et al., 2016). Microalgae have

followed a different path as governments in the 1940’s and ’50s viewed microalgae,

specifically Chlorella pyrenoidosa, as a high-tech solution to the rising population and

hunger in the world (Belasco, 1997). Much of these same hopes are shared within the

food industry today with the added motivation of developing sustainable crops to feed the

world in the face of climate change and for ecological repair. While research and

development in transforming microalgae into food were significant after WWII, it is

obvious to many readers that these efforts did not produce the desired outcome. Both

technical and economic factors led to microalgae being a poor vehicle to combat hunger

but instead, a few select varieties become moderately popular health supplements

(Belasco, 1997). Arhrospira, commercially known as Spirulina, was reintroduced into

western minds after a Belgian botanist observed it in African markets (Matufi &

Choopani, 2020), which reignited interest in microalgae’s superior physical and

nutritional properties in the late 1960’s (Belasco, 1997). Unfortunately, these, and other,

single-celled proteins were not able to deliver as cheap protein sources through the latter

part of the 20th century but instead found niche markets for health conscience consumers

able to pay a premium (Belasco, 1997; Matufi & Choopani, 2020).

Though their utilization is low compared to many traditional crops, micro- and

macroalgae provide diverse solutions to enhance the sustainability of the food system. To

better understand this potential, a brief description of their cultivation is helpful. First, all

algae photosynthesize making them primary sources of nutrients and providing a carbon

sink as they use atmospheric CO2 to create energy. This does not mean algae foods will
16
be carbon negative, but it helps reduce their final carbon footprint after processing,

storage, transportation, and other necessary steps in food production. Microalgae are

rarely collected from the wild for commercial purposes. Instead, a single strain is

identified and cultivated in large ponds or raceways with controlled substrates, agitation,

and other parameters to maximize growth (Suparmaniam et al., 2019). While control and

yield are maximized if this process is conducted indoors with advanced light management

systems, being able to forgo artificial light and the associated energy costs often make

outdoor cultivation the preferred method from a sustainability perspective. However, with

the rise of renewable energy, increased process controls, and more flexibility in the site of

the farm, indoor cultivation remains a viable option. The cultivation of microalgae is

another example of fermentation technology. Microalgae have also been the subject of

selective breeding and genetic modification to enhance specific traits such as growth rate

or a particular biomolecule (Torres-Tiji et al., 2020). The high levels of control and ease

genetic manipulations make microalgae an exciting source of novel and sustainable

proteins.

Certain seaweeds are also grown indoors, but this is a much smaller portion compared to

microalgae. The benefits of seaweed cultivation are wide-ranging. Like microalgae,

seaweed is a primary source of nutrients in that it photosynthesizes to sequester

atmospheric carbon and create new material. Macroalgae’s sustainability attributes are

wide-ranging aside from the carbon sequestration, land, and water footprints (Baghel et

al., 2015). Macroalgae are typically farmed in the ocean; when placed strategically, they

sequester excess nitrogen from the water benefiting the ecosystem, if harvested (Murphy

et al., 2015). A small-scale push to create 3-D farms that include a variety of sea crops is
17
expected to increase fish populations and biodiversity while providing a stable year-

round harvest (Bren Smith, 2019). The specifics of the impact will depend on many

factors and need to be studied for unintended impacts. If grown in polluted waters

bioaccumulation may be problematic so producers should always maintain quality control

for clean and potentially hazardous harvests of algae.

A biorefinery approach for maximizing utilization is an important way to enhance

sustainability by maximizing the utility of all algal components regardless of quality.

This process aims to mimic oil refinery in producing multiple saleable goods, but, in the

case of biorefinery, food and feed are often potential products that can help valorize the

original biomass if food safety is maintained (Chew et al., 2017). This process has been

studied extensively and may be the ideal choice when creating extracts or isolates from

both micro- and macroalgae biomass (Baghel et al., 2015; Greene et al., 2020a; Subhadra

& Grinson-George, 2011).

Whole seaweeds contain unique and diverse flavors and textures when eaten whole, but

their extracts and microalgae are most likely to be consumed as an ingredient in a more

complex food product. The local seaweed and the regional cuisines of whole macroalgae

have been discussed thoroughly in association with the Chinese (Xia & Abbott, n.d.)and

Alaskan (Garza & Alaska Sea Grant College Program., 2005) regions. Due to logistics

and consumer perception, whole algae is expected to have only a limited impact. Whole

microalgae powders are sold as supplements and are often mixed into food or drink

(Griffiths et al., 2016). Powders along with various extracts of either type of algae are

sold as emulsifiers, thickeners, gelling agents, and colorants (Gouveia et al., 2008). In

18
efforts to curtail the use of unstainable animal-based agricultural products, algae have

been studied as an ingredient in meat mimetic products (Grahl et al., 2018). Perhaps the

most important aspect of algal sustainability is its ability to provide sufficient protein to

displace the need for future animal agriculture investment. The topic of replacing meat

with algae has been studied along with other alternative proteins and it was found that

nutrition or wellness benefits associated with algae were a highly influential factor in

increasing the novel, algae-containing, products’ willingness-to-eat in consumers

(Onwezen et al., 2021).

The nutritional profiles are possibly the most compelling reason for the continued

adoption of algae foods. The best word to describe algal nutrition is diversity. As with

any large class of organism, the specific nutritional benefits of one variety are not

guaranteed to be present in others. This natural diversity is amplified in the algae due to

the loose qualifications for what makes an organism an alga. Furthermore, the way

macroalgae grow naturally, freely in an open body of water, makes them susceptible to

changes in the composition, temperature, and light available in that water (Renaud &

Luong-Van, 2006). Diversity in nutrition does pose a challenge to food processors, but it

should also be celebrated as an opportunity to develop diets capable of providing

complete and wholesome nutrition for the globe.

Only a small fraction of the micro- and macro- algae have been used in food and within

that, a smaller fraction has been approved by a governmental agency for sale as food

(Barros de Medeiros et al., 2021). More than 6,500 species of macroalgae are known

worldwide categorized into three groups: brown (Phaeophyta; 1,500), red (Rhodophyta;

19
4,000), or green (Chlorophyta; 1,000) (Garza & Alaska Sea Grant College Program.,

2005). Brown seaweed often referred to as kelp, is typically the largest of the seaweeds.

High carbohydrate contents containing alginates and fucoidans are often characteristics of

kelp. While low in protein, these seaweeds are the group most commonly used by the

food industry due to the diverse and useful properties of their carbohydrates (Afonso et

al., 2019). Red seaweed is commonly used in the production of agar and carrageenan as

stabilizers, emulsifiers, and homogenizers. They are typically lower in fat but contain a

diverse array of micronutrients and pigments while being as high as 47% protein making

them a desirable source of novel protein (Cotas et al., 2020). Green seaweed has been

called “Green Caviar” for its “delicate flavor and crisp texture” and like other seaweeds is

packed with macro- and micro- nutrients (Magdugo et al., 2020). The fatty acid, amino

acid, and mineral profiles of a given seaweed will vary depending on the phyla and

species as well as on harvest location and season (Renaud & Luong-Van, 2006).

Seaweeds have great potential to enhance human nutrition around the world while

providing unique physical and bioactive functionality for broad incorporation into many

diets and cuisines.

The number of microalgae species on the planet is not known and due to the nature of

microorganisms, is constantly changing. However, with our current understanding, the

number of microalgal species dwarfs that of macroalgae by orders of magnitude with

estimates coming in from 200,000 to over a million (Koyande et al., 2019; Matos, 2019).

Nonetheless, the diversity issues discussed with macroalgae are not as prevalent in

microalgae food production, primarily due to higher levels of cultivation control. In

microalgae production, the challenges come with isolating and mass-cultivating a GRAS
20
(generally recognized as safe) strain with a sufficiently high growth rate. Only a few

microalgae have been granted GRAS status by the US FDA Arthrospira

platensis,Chlamydomonas reinhardtii, Auxenochlorella protothecoides,

Chlorellavulgaris, Dunaliella bardawil, and Euglena gracilis; while many other species

are likely safe to eat obtaining GRAS status can be costly. (Torres-Tiji et al., 2020)

provides an in-depth review of these microalgae which is beyond the scope of this

chapter. Spirulina, and Chlorella are the most used microalgae in human nutrition,

animal nutrition, and cosmetics (Barros de Medeiros et al., 2021; Torres-Tiji et al., 2020).

Dunaliella is the next most cultivated for human nutrition and is of great interest for its

beta-carotene content (Koyande et al., 2019; Torres-Tiji et al., 2020). The remaining

GRAS strains are produced at lower quantifies for human nutrition products and oil

production(Torres-Tiji et al., 2020). In general microalgae are higher in protein than their

macroalgal cousins and often express favorable amino acid profiles (Matos, 2019; Y.

Wang et al., 2021). As a human food and as a biofuel feedstock the lipid fraction of

microalgae is highly sought after (Talebi et al., 2013). While microalgae are often higher

in “good fats,” those high in polyunsaturated fatty acids (PUFA’s), it is difficult to predict

their fatty acid profile as they are dependent on environment and species (Lang et al.,

2011; Teoh et al., 2004). Microalgae provide B vitamins, an essential nutrient; often

deficient in vegan diets (Koyande et al., 2019). A great deal of algae research, outside

what is cited in this chapter, has been conducted on a multitude of different species,

strains and with differing conditions. This research shows that microalgae can be a

reliable source of nutrients to meet the rapidly changing demands of our global food

system.
21
2.3 Fermentation-based ingredients

In the context of the alternative protein industry, fermentation-based innovations can be

categorized into traditional fermentation, biomass fermentation and precision

fermentation. Traditional fermentation has been done for centuries and can be commonly

seen in products such as bread or beer to modify functional properties of the product such

as flavor, nutrition, and physical structure. Biomass fermentation uses the ability of

micro-organisms to rapidly multiply and break down substrates to generate high protein

end products efficiently. Precision fermentation uses microorganisms to produce certain

key ingredients that can improve the sensory properties of alternative protein products.

2.3.1 Mycoprotein
One of the reasons the mycoprotein industry started growing rapidly was the fact that

most edible fungi exhibit complete amino acid profiles, grow rapidly, and are able to use

waste biomass as a substrate, thus vastly reducing costs associated with production. Fungi

are natural decomposers due to their ability to break down complex biomass mainly

through the production of enzymes. This can also be categorized under the upcycling

(adding value to food waste) industry which according to ReFED’s (Rethink Food Waste

through Economics and Date) Insights Engine has an estimated impact of 4.85M Metric

Tons of carbon dioxide emissions per year. Coupled with the scalability of fermentation

technologies, mycoprotein has been disrupting the market ever since the discovery and

regulatory approval of the QuornTM fungus.

Although the consumption of edible fungi dates back centuries, it wasn’t until the late

twentieth century that their full potential was explored due to developments in industrial

22
microbiology (Wiebe, 2002). Fusarium venenatum or the QuornTM fungus was first

studied as a mycoprotein source in the 1960s. It took 12 years of safety testing to ensure

it wasn’t a potential plant pathogen or mycotoxin producing before it was approved for

use as a food in 1984. For a long time, this was the primary source of mushroom protein

with regulatory approval on the market. In fact, a lot of literature on fungi protein uses

the term mycoprotein interchangeably with protein derived from Fusarium venenatum.

However, in this chapter, we consider mycoprotein as a broader class encompassing

protein derived from any edible fungi.

There have been considerable advances and newer innovations in the mycoprotein space.

In addition to a mass of research (Ahmad et al., 2022) investigating newer strains,

nutritional characteristics, more efficient forms of fermentation, the mycoprotein space

has seen a few commercial successes as well. Fusarium flavolapis is the primary fungus

strain trademarked by Nature’s Fynd and also known as FyTM. The strain was discovered

in 2009 in soil samples from the Yellowstone National Park in the United States.

Nature’s Fynd recently launched their first food products in the U.S. in 2021, a breakfast

patty and cream cheese made from Fy. Mycelium fermentation has also been used to

create one of the first whole cut meat replacements by U.S. based company, Meati Foods.

They use a proprietary strain and utilize the fibrous network formed by the fungi’s

hyphae to mimic the familiar fibrous texture characteristic to most meat products.

While mycoprotein has been extremely promising in the search for alternative protein

sources with considerably lower environmental impact and resource demands, one of the

major concerns has been consumer acceptance due to concerns about mycotoxin

23
production. While extensive toxicological work has been performed on the Quorn fungus,

newer strains of fungi being used need to be evaluated for the same, which is time

consuming.

2.3.2 Precision fermentation

Ingredients produced using precision fermentation typically use an organism modified to

efficiently produce specific compounds of interest at scale and in a cost-effective way

(Teng et al., 2021). For example, leghemoglobin is a molecule derived from legume

nodules and has properties similar to that of hemoglobin responsible for the meaty flavor

and color of animal meat products. However, extracting leghemoglobin from legume

nodules is costly and would require impractical amounts of starting material. To solve

this problem, Impossible foods capitalized on the desirable properties of leghemoglobin

by genetically engineering a yeast, Pichia pastoris, to efficiently produce leghemoglobin

using precision fermentation. Such fermentation-based innovations are rapidly occupying

a space in the food industry. Perfect Day is another company using fungal precision

fermentation to make milk proteins (casein and whey), whereas the EVERY company

(formerly known as Clara Foods) harnesses precision fermentation by modified yeasts to

create animal protein replacements such as egg white protein. Precision fermentation

offers a lot of flexibility in producing ingredients that are not only more cost-effective but

also can be designed to have sensory appeal for alternative protein products. Finally,

mycoprotein and precision fermentation-based products, unlike most other most novel

innovations in the food space, face fewer challenges with consumer acceptance primarily

due to consumers’ familiarity with mushrooms as compared to protein derived from

insects or algae.
24
2.4 Insects

Insects have been identified as future food and a sustainable source of protein (Parodi et

al., 2018). They are included in this chapter for their potential as a sustainable food

despite their not being plants. Insects are a highly diverse class of organisms leading to

diversity in cuisine (Melgar-lalanne, 2019). They provide a highly efficient link for the

development of circular food economies by transforming organic byproducts or waste

into homogenous and stable biomass (Cadinu et al., 2020; Ojha et al., 2020). Insects have

become a popular feedstock to supplement animal feed making the process more

environmentally and economically efficient. The Black Soldier Fly alone has been

studied as feed for swine (Veldkamp & Bosch, 2015), poultry (Cullere, et al., 2017), and

fish (Belghit et al., 2019; Irungu et al., 2018c) with many promising results. (Sánchez-

Muros et al., 2016) previously summarized global utilization of insects as feed, showing

increased adoption of these sustainable protein sources. While contributing to the

sustainability of animal agriculture is an important goal, to maximize the sustainability of

the entire system, direct consumption by humans is more efficient. This route will likely

provide the greatest economic benefit to producers since human food typically sells at a

higher price than animal feeds. Increasing demand of insects as human food and the

displacement of less sustainable food sources is a goal shared by many in industry and

research.

The western edible insect market is currently dominated by cricket-based products while

a wide variety of whole insects are consumed globally. The global edible insect market is

growing approximately 28% a year and is expected to reach 1.18 billion dollars globally

25
in 2023 (Statista 2019). Industrialization of insect foods is still developing which

accounts for the rapid growth, along with a growing demand for protein-rich foods

(Drewnowski, 2010). Traditionally, insects such as true bugs, caterpillars, beetles, ants,

grasshoppers, and many others were harvested from the wild (van Huis & Oonincx,

2017). However, wild caught insects have drawbacks as a mechanism for increasing

sustainability due to multiple factors including destruction of habitats, food safety, and

scalability. Traditional insect cuisine is also associated with eating insects whole often

after being cooked. Seeing whole insects on their plate is major turn off for many

westerns as well as younger generations not accustomed to the traditional cuisine of their

region. Forward thinking food developers and researchers have learned that the “ick”

factor might be avoided by creating foods which do not resemble insects (Lammers et

al., 2019; Mishyna et al., 2020; Sogari et al., 2018). This phenomenon might explain the

early success of cricket powder, but other insects are emerging as well.

Edible insects are thought to be a mechanism for environmental sustainability in the food

system. For this to occur, consumers must be willing to eat a variety of critters. While

learning about insects’ environmental benefits does increase consumers’ willingness-to-

eat (Kauppi et al., 2019; Verneau et al., 2016; Wendin & Nyberg, 2021), the nutritional

quality of many insects will help establish a robust long-term market. When considering

insect nutrition, it is difficult to comprehend the scope and diversity present within this

emerging class of food. Multiple estimates suggest there are between 1,700 and 2,100

different species of edible insects in the world (Govorushko, 2019). This diversity is

amplified further after considering life stage as some insects are edible throughout their

life cycle. Different species and different life stages bring about a variety of nutritional
26
profiles as well as different textural and flavor profiles. Generalizing is difficult and this

topic has been reviewed in great detail over the years (Baiano, 2020; da Silva Lucas et

al., 2020; Kouřimská & Adámková, 2016; Rumpold & Schlüter, 2013; van Huis, 2020).

Insect protein, fat, carbohydrate, and micronutrient contents content can vary greatly

among species and life stages while also being affected by cultivation conditions and diet.

Many insects such as crickets, grasshoppers, and mealworms are actively being pursued

for their high protein content. The salt content of many insects is one potential drawback

in developed nations where salt intake has historically been beyond the needs of the

population (He et al., 2019).

Aside from the “ick” factor and the potentially high salt content, other safety related

concerns will be encountered. Fortunately for the promotion of entomophagy, there are

no novel microbiological concerns. No insect-specific human pathogens have been

identified in insects (Rumpold et al., 2017; Wade & Hoelle, 2019). This simple fact

should provide comfort to food processors as they will not need to prepare for any

additional pathogens during processing. (Sandrock et al., 2018) have shown known food-

borne pathogens such as Salmonella and Bacillus make up a portion of the BSFL’s gut

microbiota. These hazards should be treated the same as any other plant, fungi, or animal

food sources. Bioaccumulation is another concern that has not shown to be a great risk.

(Erickson et al., 2004) and (Schlüter et al., 2017) suggest the BSFL, regardless of

feedstock would not pose a serious risk due to minimal bioretention. Quality control

techniques at rearing facilities have been implemented to reduce toxins or heavy metals

entering animal feeds and are readily translated to human food operations (Barragan-

Fonseca et al., 2017a; van der Spiegel & Noordam, 2013).

27
Insects are a promising avenue to increase the global food systems sustainability.

Traditional food sources including animals and staple crops have been identified for their

high land, water, and carbon emissions (Smetana et al., 2021). Insects achieve greater

efficiency by consuming organic materials that would otherwise be wasted; diverting

them from the landfills and preventing the associated methane production in much

smaller areas(Ojha et al., 2020). With their desirable nutritional profiles, low additional

safety risk, and growing support; insects are primed to be an essential element of a global

sustainable food industry.

2.5 Conclusions

The world is constantly changing; shifts in climate and geography naturally produce

evolution of the life on Earth. Our current knowledge regarding climate change describes

how human activity is accelerating changes in climate that are resulting in changes to

regional geography and weather. These human-driven changes are occurring more

rapidly than the natural ebbs and flows, putting many species and many humans in

danger. The industrialization of food shares responsibility for this situation while having

the tools to build more sustainable systems. There are two main areas of action that will

be needed to produce a healthy planet and provide food security: 1) emission-reducing

and 2) resilience-building actions. All changes to the food system will result in dietary

changes on the consumer level. Encouraging and exploiting the most sustainable nutrient

sources and ensuring consumer acceptance is essential for new systems. This chapter

focused on algae, insects, and fungi which can serve as both emission reducers and

resilience builders. Small land footprints are common among these emerging food

28
sources which reduce emissions thorough allowing carbon sequestering forests to be

grown instead. Reduced demand for land is also important for resilience as it can serve

the growing population even as land becomes more and more scarce. These emerging

sources are also high in quality protein which can help displace animal agriculture and

potentially provide emissions reduction. Animal agriculture also lacks resiliency due to

the high rates of disease and incredibly high demands for land and water. Transitioning

from animal-based agriculture does provide challenges, especially from a consumer

standpoint as many consumers have become accustomed to animal products. Research

shows that education can help get consumers to try new things while appealing forms and

flavors can increase liking. These alternative foods will not be adopted overnight. It is

important that researchers continually develop novel products while considering the

consumer and the sustainability within the system.

29
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Wells, M. L., Potin, P., Craigie, J. S., Raven, J. A., Merchant, S. S., Helliwell, K. E.,
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37
 3. Chapter 3 Evaluations of commercial Black Soldier Fly
larvae (BSFL) as a novel source of nutrients, aqueous
protein extraction, and protease activity
Abstract

Black Soldier Fly larvae (Hermetia illuscens; BSFL) and insects have the potential to

increase the sustainability of the food system. Consumer disgust is a major hurdle to the

utilization of foods derived from insect sources, and research suggests creating familiar

foods from unfamiliar materials will increase acceptance. The objective of this research

was to explore various ways of creating sustainable food ingredients from the BSFL. To

increase protein yield, sodium hydroxide (NaOH) was investigated as an extraction aid.

NaOH was used to extract protein over time (0-24 h) resulting in increasing nitrogen

solubility over time while it remained constant when using deionized water (diH2O). It

was found that three washes with NaOH removed all detectable protein from the initial

BSFL flour. Enzymatic activity was observed and confirmed while characterizing the

physicochemical and bioactive properties of water-soluble BSFL extract. Autohydrolysis

of BSFL enzymes resulted in the deterioration of high and low molecular weight proteins.

The presence of enzyme activity in commercial BSFL indicate that a blanching step was

omitted during the harvesting processes. BSFL produce a wide range of enzymes so

understanding their activity during processing is important to achieve desirable product

properties. The enzymes may be exploited to create hydrolysates but if left unchecked

could result in reduced protein content. This chapter highlights the importance of

processing decisions by demonstrating the potential to extract maximal protein with

38
NaOH or to target an enzymatically active ingredient but with a lower protein yield.

Future research may investigate sequential extractions to optimize for both enzyme

activity and protein yield.

39
3.1 Introduction

Direct use of the Black Soldier Fly larvae (Hermetia illuscens; BSFL) proteins for human

food attempts to accomplish efficient and sustainable capture and conversion of organic

waste into human nutrition. Given the rising global demand for human protein and a

shrinking land base, such solutions have the potential to sustainably address global food

security (Goldstein et al., 2017). The BSFL was popularized as an organic waste

management tool as a non-vector, non-invasive insect with high rates of conversion for a

variety of organic wastes into a homogenous and safe larval biomass (Rehman et al.,

2022). This larval biomass has been the focus of a growing body of literature to optimize

its cultivation and maximize its potential in applications ranging from fuel to feed to food

as well in other niche sectors. This interest is in part due to the BSFL’s potential for food

waste disposal followed by conversion to high-quality food ingredients producing an

environmentally friendly circular economy (Cappellozza et al., 2019; Ojha et al., 2020).

To maximize the environmental benefits of BSFL-based circular economies, human food

markets must accept insect-based foods. Unfortunately, whole insects are considered

undesirable or disgusting by many western consumers and increasingly by the young

generations of traditionally entemophagic cultures (Müller, 2019). Approaches to finding

solutions for these problems are discussed in the next section but first an overview of use

in other applications provides important background knowledge.

First, BSFL biomass has been investigated and is considered a viable stock for anaerobic

digestion and biofuel production in proposed biorefinery pathways (Win et al., 2018).

However, from the context of maximizing a biomasses’ economic and environmental

40
potential, fuel production is considered a lower priority until food needs are met (Muscat

et al., 2020). Under this paradigm, feed applications are typically an improvement over

fuel applications. BSFL have become a popular source of livestock feed; studied as feed

for swine (Veldkamp & Bosch, 2015), poultry (Cullere, et al., 2017), and fish (Belghit et

al., 2019; Irungu et al., 2018) with many promising results. While the BSFL provides an

environmental improvement over many current animal feedstocks (Salomone et al.,

2017), using animals as an intermediate to human nutrition will always be less

environmentally friendly than direct consumption by humans.

BSFL and ingredients derived from BSFL must be adopted with high demand by

consumers. Understanding and overcoming reservations to insects and insect-derived

ingredients among even a sub-population of consumers should provide benefits to food

security and resiliency in the food system (Parodi et al., 2018). For these reasons,

persistent research into the development of insects and the BSFL ingredients for human

consumption should be maintained. This chapter will first cover literature associated with

BSFL sustainability, nutrition, evaluations, and applications for its protein fraction then it

will describe experimental work conducted to advance the understanding of BSFL and

insect extraction and biochemical properties.

3.1.1 BSFL as a nutritious part of a sustainable food system.

Entomophagy or the practice of consuming insects is observed across the globe, with an

estimated 2.5 billion people supplementing their diet with these highly nutritious raw

materials. Insects are a highly diverse class of organisms leading to diversity in cuisine

(Melgar-lalanne, 2019). While disgust is a common reaction to insect foods for most

consumers in westernized cultures (Chan, 2019), an understanding of consumer

41
preferences can help food developers avoid the worst reactions. These preferences are

likely driven by culture and social norms, which suggests consumer preferences are

learned and may change over time for a given population (Jensen & Lieberoth, 2019). In

a study of 97 young (mean: 27 y/o) adults who were asked to eat meat, plant-based, and

insect-based burgers; comparable liking was found between the plant- and insect-based

samples (Schou et al., 2016). Possibly more important, this author found that by revealing

the protein source to the panelists, the perceived liking of insect-based samples increased.

The results of this study may be attributed to youthful excitement to try new things.

Consumers are significantly more willing to consume insect-based foods if the form is

familiar (Lammers et al., 2019; Mishyna et al., 2020; Sogari et al., 2018). Early attempts

of using ground whole BSFL flour in a summer sausage product (Bessa et al., 2019) and

into bread (González et al., 2019) have occurred. González et al., (2019), demonstrated

the value of removing fat from the BSFL flour before bread-making which resulted in a

loaf that was a superior color and volume. While both authors published positive

conclusions, it was clear there is still work needed to produce high-value products from

BSFL. Even with these modest results, sufficient justification for BSFL as future food

exists, justifying further investigation into protein extraction and ingredient development.

Efforts toward developing BSFL-based foods is based on a strong and growing body of

literature detailing the sustainable production of this highly nutritious biomass. As the use

and demand for BSFL protein grows it would see the most impact if it were able to

displace less sustainable food sources such as meat. Meat production accounts for 56% of

the greenhouse gas emissions (GHGE’s) produced by the food system (Rust et al., 2020),

while agriculture is a larger producer of GHGE than any other industrial sector
42
(Rockström et al., 2020). Livestock and the feed needed for their growth are major

drivers of deforestation due to their high land and water demands (da Silva et al., 2022;

Hecht, 1993). Soy is often a component of animal feed; along with its high land demands

It is grown as a monoculture which leads to reduced biodiversity affecting bee

populations (de Groot et al., 2021). As previously mentioned, animal consumption is a

major driver of our diet’s environmental impacts, but BSFL adoption may provide

improvement. For instance, one acre of production space dedicated to BSFL production

can yield approximately eleven times the protein from the same land dedicated to cattle

production (Do et al., 2020). Furthermore, BSFL are likely more environmentally

friendly than leading plant-based protein sources for food or feed; rearing requires a

significant energy input, similar to soy production, but requires 8.65 m2 less land per kg

of protein produced (Salomone et al., 2017). As a potential mechanism for food waste

disposal, BSFL are even more efficient than composting and especially landfilling. BSFL

converted 41% of the feed to body mass in 7 days; a process that took 45 days for

bacteria digestion which resulted in a 20% higher release of carbon-based greenhouse

gases (Perednia et al., 2017). BSFL exhibits environmental benefits and a waste reduction

mechanism that few other food sources provide while being more environmentally

friendly than our current food sources. These benefits require that the BSFL be desirable

as food – from a nutritional perspective, there is a strong case.

The environmental benefits discussed are from a raw biomass very high in fat (ca. 40%

dw.) and high-quality protein (ca. 40% dw.; Enterra, 2019) because all essential amino

acids are produced (Kalman, 2014; Müller et al., 2017; Spranghers et al., 2017). The

environmental and basic nutritional benefits of the BSFL could make it a desirable raw
43
material for novel food ingredients. As a case is built for BSFL to be adopted as an

approved human food item, work from animal feed research can provide a basis for the

nutritional quality of the larvae (see Figure 3.1). The academic literature provides robust

research on BSFL’s use as swine, poultry, and/ or fish feed (Barragan-Fonseca et al.,

2017; Belghit et al., 2019; Cullere et al., 2016; Irungu et al., 2018; Schiavone et al., 2017;

Veldkamp & Bosch, 2015). Similar to most livestock, the proportions of lipids to protein

and the fatty acid profile are dependent on the larval diet (Barragan-Fonseca et al., 2017;

Belghit et al., 2019; Cullere et al., 2016; Ewald et al., 2020; Irungu et al., 2018; Leong et

al., 2016; Schiavone et al., 2017; Spranghers et al., 2017; St-Hilaire et al., 2007;

Veldkamp & Bosch, 2015). These effects of diet are most significant in the fatty acid

profile. For instance, BSFL reared on fish offal, scraps of fish muscle, were found to be

high in omega-3 fatty acids (St-Hilaire et al., 2007). Regardless of their feed, the

predominant fatty acids in BSFL are C12:0, C16:0, and C18:1n9 (Leong et al., 2016); due

to a high trilaurin content, the lipids have a high melting point (Bogevik et al., 2022). The

high levels of C12:0, lauric acid, may make the lipid fraction attractive in the fragrance

and cosmetic industry (Kalustian, 1985). Due to the high saturated fat BSFL lipids have

not been targeted as a potential human nutrition source, but the high energy density has

made it appealing as a chicken feed supplement (Kim et al., 2020). An additional

environmentally friendly outlet for the lipids, once human and animal feed markets have

been exhausted, is as feedstock for biofuel production. The biofuel application of BSFL

lipids is promising but will not be covered in depth; the topic has been studied and

reviewed elsewhere (Feng et al., 2018; Leong et al., 2016)

44
Unlike the fatty acid profile, the amino acid profile is minimally affected by feed

differences (Müller et al., 2017; Spranghers et al., 2017). The amino acid profiles of

BSFL compared to whey and soy protein isolates are presented in Figure 3.1. BSFL is

comparable to whey protein in branched-chain amino acids (leucine, iso-leucine, and

valine) (Blomstrand et al., 2006), which have been shown to aid in muscle recovery

(Norton & Layman, 2006). The amino acid profile of BSFL is promising however, by

itself is not necessarily a reliable gauge for nutritional quality for humans. The digestible

indispensable amino acid scores (DIAAS) are recognized as the standard for protein

nutritional quality (Mathai et al., 2017). A study of the DIAAS for BSFL in humans has

not been conducted and is important next to further understand BSFL’s protein quality,

but if BSFL is consumed. A few studies have used a cecectomized rooster assay to infer

the DIAAS in cats and dogs for the pet food industry (Do et al., 2020). While these

studies showed high absorption in their model systems true human digestibility is

dependent on the physical form of the food product.

45
 25%

BSFL (Sprangher, 2016) BSFL (Muller, 2017) Whey (WPI) Soy (SPI)

20%
% of amino acids

15%

10%

5%

0%

Figure 3.1: Amino acid profile of BSFL compared to whey protein isolate and soy
protein isolate amino acid profile (% of amino acids; Kalman, 2014; Müller et al., 2017;
Spranghers et al., 2017). *indicates essential amino acid

The protein and energy dense BSFL also contain micronutrients. Commercial BSFL are

reported as containing calcium (10,000 ppm) and phosphorus (7,300 ppm) (Enterra,

2019). Calcium in BSFL has been reported in the literature as the most abundant

micronutrient, with BSFL containing about 5% more calcium than other insects by dry

weight (Wang & Shelomi, 2017). Potassium, magnesium, B vitamins, and choline were

present in substantial amounts as well (Finke, 2013).

The microbiological risk factors are an additional and important consideration for the

BSFL to undergo mass production as food. Sandrock et al., (2018) have shown known
46
food-borne pathogens such as Salmonella and Bacillus make up a portion of the BSFL’s

gut microbiota. However, to date, no insect-specific human pathogens have been

identified (Rumpold et al., 2017). However, the production of ingredients from BSFL still

needs typical safeguards against common microbiological hazards. Erickson et al., (2004)

and Schlüter et al., (2017) suggest BSFL, regardless of feedstock, insects would not pose

a serious risk due to minimal bioretention; however, the authors note that, like most

livestock, prions could be a potential hazard.

3.1.2 Potential markets for fractionated BSFL components and the associated
processing
Protein extraction and refinement coupled with component fractionation is a potential

approach to valorize BSFL and other insects. The lipid fraction of BSFL is valuable as an

individual component due to its high lauric acid content which is used in the fragrance

industry (Kalustian, 1985). While the lipids are typically high in this and other saturated

fats, making them less desirable for human consumption, they will likely find a place in

the animal feed market or as a biofuel stock (Su et al., 2019) before becoming waste.

Chitin is typically the third most abundant carbohydrate in BSFL accounting for 3% to

14% dw which is highly dependent on the life stage (Caligiani et al., 2018; Spranghers et

al., 2017 H. Wang et al., 2020) . The pre-metamorphosis stage, puparium, is

characteristic of a harder darker exoskeleton formed. Wang et al., (2020) provides the

first characterization of BSFL chitin fraction and found similar properties among all life

stages. The group analyzed the structure, crystallinity, and heat stability for four life

stages with only minor differences reported. Chitin has many applications with some

overlap in the food industry. Chitin or its N-deacetylated derivative chitosan has been

47
used or tried as an edible film, antimicrobial agent dye-binding agent, emulsifying agent,

gelling agent, antioxidant, to increase water holding and many more (Agulló et al., 2003).

Deacetylation and depolymerization are two processes that often result in concerns about

economic feasibility but are often essential to their functionality in downstream

applications (Jeon et al., 2000). Utilizing the chitin fraction may provide economic

support to a BSFL fractionation, but the protein and fat are more desirable targets for

early-stage valorization of the biomass. The mineral content detailed above is another

potential value stream that will need to be the subject of post-industrialization

optimizations. Beneficial minerals may serve to enrich unrefined ingredients or protein

extracts.

The protein fraction has the potential for a broad array of applications which is highly

dependent on the physicochemical properties of an extract or ingredient. The intrinsic

properties of a protein system may allow for desirable food attributes such as the

formation of gels, emulsions, and foams (Day, 2013). The alternative meat protein market

represents the greatest potential for valorization with sales in 2019 at $5 billion, up 29%

from 2017 (GFI, 2020). The largest sector in plant-based foods (40%) are milk analogs and

beverage products (soy/nut milk, juices/smoothies, coffee cream alternatives, etc.), which

exceeded $2 billion in sales in 2019 gfi.org/marketresearch. Plant-based meats are also

growing at an average rate of 17% during this same time frame with sales just shy of $1

billion (cbinsights.com). Recently, Beyond Meat™ revenues went up by 140% months after

COVID-19 outbreaks affected meat processing facilities (FastCompany.com); Grothaus,

2020). These numbers are projected to continue growing and are estimated to be $35.54

billion in 2024 (marketsandmarkets.com, 2020).

48
To enter these protein markets, a substantial amount of processing is needed after the

cultivation of BSFL. The first steps require that the larvae are euthanized, cleaned, and

dried. The BSFL express high levels of enzymes making them a robust composter, but

this also produces challenges during the early processing steps. Leni et al., (2019) found

that slow killing through freezing allows for enzymatic pathways to continue compared to

blanching which destroys the enzymatic activity more rapidly resulting in less

blackening. When active, enzymes lead to black discoloration of the larvae, the color

change is the result of an iron-polyphenol complex that results from their decomposition

(Janssen et al., 2019). Results from Caligiani et al., (2019) show less lipase activity in

heat-treated samples. Preliminary experiments using homegrown larvae were conducted

and a blanching system was used which is shown in Appendix A. This system proved to

be effective, but the use of homegrown larvae was reconsidered, instead commercial

animal feed grade larvae were purchased. It is interesting to note that the larvae

purchased from commercial suppliers were not noticeably discolored after undergoing

only drying with no blanching step. This was discovered after observing proteolytic

activity in the larvae which will be discussed later in this chapter (Sec. 3.3.3). The drying

step has been investigated and modeled by previous researchers as well (da Silva Lucas et

al., 2020). Huang et al., (2019) have demonstrated that convective air drying at 60 °C

provided superior digestibility than microwave dried BSFL. Once dried, the larvae must

be ground into a flour before successful fractionation can be achieved.

3.1.2.1 Review BSFL fractionation operations

Lipid extraction and purification is often the first of the major macromolecular

components to be separated during a fractionation. This step is often referred to as

49
“defatting” and can involve chemical or physical methods to separate lipids from the

remaining biomass. Chemical methods of identifying lipids have been studied for decades

with two major contributions reported in the late 1950’s (Bligh & Dyer, 1959; Folch et

al., 1956). Ravi et al., (2019) optimized the solvent extraction of lipids from BSFL and that

2-methyloxolane provided optimal extraction. In this dissertation, hexane was used for all

lipid extractions due to cost. Partial defatting can be achieved with water and centrifugation

which was found to be improved by the addition of organic acids (Soetemans et al., 2019).

Fats can be removed mechanically through a pressing operation (Kierończyk et al., 2022).

Pressing is of interest for future work due to organic solvent’s effect on protein structure and

protease activity (Demiralp et al., 2000; Purohit et al., 2022). However, organic solvents have

provided increases in food functionality performance, such as foaming capacity (Gravel et

al., 2021; Queiroz et al., 2021)

Removing the fat from the BSFL has been used to produce a flour that is more concentrated

in protein but is also enriched in salt and other minerals. Since the fat is often highly saturate,

a defatted BSFL flour might have a more appealing nutritional profile to consumers with

minimal processing required. Researchers have shown that defatted BSFL flour will contain

about 10% more protein, up to 45%, but the effect of defatting on functional properties such

as water/oil binding and protein solubility were not greatly affected. (Bußler et al., 2016).

González et al., (2019) showed that defatted BSFL flour resulted in softer and more visually

appealing bread loafs. However, it should be mentioned that BSFL performed the worst

(smallest volume, densest, and darkest) among three insect flours incorporated into bread,

even when defatted. These humble results from defatting are an indication that mineral or

carbohydrate fractions could be interfering with the protein’s ability to provide physical food

50
functionality. Aqueous extraction and downstream processing are an approach for separating

these components.

Aqueous extraction is a common method used to solubilize and separate proteins from a

biomass. Pure water is not used industrially but may be used in benchtop experiments as a

control. Salts, buffers, and bases are employed to increase the solubility of different types of

protein. A classical approach to fractionation is to use water, followed by a salt (0.5 M NaCl)

solution, followed by 70% ethanol, and finally dilute NaOH to obtain a albumin, globulin,

prolamin, and glutelin fraction, respectively (Caligiani et al., 2018; Osborne, 1907). While

this approach was first used on wheat, characterizing the proteins of the BSFL based on their

solubility provides valuable information and expectations for industrial protein extraction.

Caligiani et al., (2018) performed an “Osborne fractionation” measuring protein using

nitrogen determination and found that the BSFL were 23% albumin, 15% globulin, 9%

prolamin, and 31% glutelin, with the remaining 23% of the nitrogen in the pellet.

Comparable results were found by (Leni et al., 2019) after performing a similar Osborne

fractionation on BSFL. Low ionic strength solutions are a desirable extraction aid due to their

relatively low cost and safety. Miron et al., (2019) conducted an array of experiments with

various concentrations of phosphate buffer saline (PBS) at different basic pHs. Their

experimental approach was preliminary, but they found that increased buffer concentrations

resulted in greater protein recovery up to 17.16%.

Sodium hydroxide is a strong base that requires safety precautions when handling and an

environmental plan for disposal. Its use as a processing aid to extract protein should be

reduced if possible. However, NaOH and other strong bases have a unique ability to

solubilize proteins that weak bases cannot provide. Further complications arise after

51
separation of the supernatant as the protein must be then separated from NaOH or the

NaOH neutralized. This is accomplished through membrane filtration or precipitation of

proteins using acid. Comparisons of the two methods showed filtration to be slightly

more environmentally friendly than precipitation while also resulting in a superior protein

product from a functionality standpoint (Alonso-Miravalles et al., 2019). Since filtration

is more energy intensive than precipitation it stands to improve its environmental

footprint as green energy is adopted. Dilute NaOH and acid were used by (Huang et al.,

2019) to produce small batches of BSFL protein isolate then conducting amino acid

profile and digestibility studies. Leni et al., (2019) used 1 M NaOH resulting in a fraction

containing 73% of the larvae’s proteins and recovered 63% of this protein using

trichloroacetic acid precipitation (TCA). NaOH and precipitation has been shown to be `

for recovering protein from the BSFL (Caligiani et al., 2018) .

Once recovered with sufficient yield, it is important for the protein ingredients to express

functionalities i.e., physicochemical properties relating to an application to improve the

quality. For fuel and feed, functionality generally is defined as the chemical composition

as this can predict most of the value for these applications. Human foods are often

complex. Meeting the dynamic expectations of the consumer is necessary for success. For

beverage products and any application that requires solubilized proteins, solubility and

electrochemical stability is important (Laclair & Etzel, 2009). Meat mimicry requires a

dynamic protein group to mimic the highly functional animal proteins. Insects are of

kingdom Animalia, like livestock animals, and contain similar proteins, but this has not

yet led to a successful insect-based meat mimetic. One of the most important properties

for meat mimicry is gelation or the ability for proteins to unfold and aggregate with each
52
other under heat (Foegeding et al., 2017). Since meat products often contain fat,

emulsification, oil holding, and water-holding are also critical. Properties concerning the

stability of fat are also important for breads and other confections (Lam & Nickerson,

2013). In a similar manner, foaming capacity also plays a role in confectionery

applications. Foaming is the ability for a protein to hold air allowing breads to rise and

providing aesthetic benefits in an array of products (Amagliani et al., 2021). Queiroz et

al., (2021) studied the colloidal properties and foamability of defatted, 0.25 M NaOH

extracted, acid precipitated BSFL protein extract. Their results showed a nearly 100%

overrun with extended foam stability indicating the BSFL protein extract has potential to

be used as a foaming agent. The processing of this extract likely played a role and

requires further investigation to achieve functionality while limiting the environmental

impact of the processing.

This dissertation did not focus on these physicochemical functionalities. While they are

important and are the next step in developing food ingredients from the BSFL, organic

and NaOH extractions require further analysis to ensure a minimal environmental

footprint is created and were the focus of this dissertation. Investigations using NaOH are

briefly described in this chapter and thoroughly investigated in Chapter 5. While

physicochemical functionality was not investigated, enzymatic functionality was.

Preliminary experiments using water to extract protein from BSFL showed a large

amount of small molecular weight peptides in the protein profile. Water extraction was

reasoned to be the most sustainable extraction method from a material utilization

standpoint so enzymatic activity became a more prominent focus. Leading to Objectives

2 and 3 of this chapter.

53
3.1.3 Proteolytic activity of the BSFL
A detailed investigation into commercially produced BSFL protease activity has not been

reported. Protease activity has economic importance since it will influence processing

decisions and result in chemical and physical changes to the biomass and all downstream

products (Asaithambi et al., 2022). As a waste management system, the BSFL’s ability to

digest organic material and the bacteria and enzymes necessary to do so have been

studied thoroughly. Researchers have proposed a 2-reactor system to better understand

the digestive ability of the BSFL and its variable microbiota. The first reactor includes

the enzymes excreted into the local environment where digestion begins before materials

enter the second reactor, or the internal digestive system of the larvae. There is a distinct

difference between the environment and reactions occurring outside the body of the

larvae where a diverse group of microorganisms lives and where the BSFL excrete

certain enzymes. After larval mastication, the pre-digested organic materials enter the

larvae’ digestive tract where a different set of microorganisms exist. Once inside the

midgut, the material encounters enzymes that complete the digestion of macromolecules

and organic polymers into smaller molecules that can be absorbed. Undigested material is

then excreted which can be collected and sold as fertilizer, also known as frass (Gold et

al., 2018).

In the context of food and feed the components, enzymes, and microbiota of the exterior

bioreactor will not be considered since BSFL will be thoroughly washed, removing

enzymes and microbes associated with the first stage of the bioreactor system. Thus, the

focus of the remaining discussion will be on the enzymes secreted within the digestive

tract. The enzymes within the BSFL are not constant and fluctuate based on multiple
54
factors; many of which BSFL farmers will have some control over. While studying the

digestion of an insect, Aedes aegypti, of the same order as BSFL, Noriega & Wells,

(1999) discussed two types of trypsin being released at different times after a meal. These

variations in enzyme excretion are also based on the composition of the diet (Gold et al.,

2018). The non-uniformity of enzymes within the individual BSFL has implications for

the results reported in this research and for future works. The result presented herein

should be qualified such that any enzymatic activity observed depends on the feed and

feeding time prior to euthanasia, the effectiveness of the rinse, as well as the drying and

processing completed before the analysis took place. The larvae were purchased

commercially so cultivation conditions are unknown. Each of these steps represents an

area of research that should be investigated and optimized as applications for the

enzymes are identified.

BSFL produce a broad range of enzymes including proteases, lipases, amalyses,

chitinases, and cellulases (Rabani et al., 2019). Proteases became the focus of this

dissertation after observations were made while characterizing the protein profile of

BSFL extracts. Proteolytic activity has a well-established application in animal feed

markets (Eugenio et al., 2022; Kwasek et al., 2021). Hydrolyzed proteins are also valued

in human food markets such as infant formula, protein beverages, and can also augment

physical functionality (Asaithambi et al., 2022). Targeted studies of freshly grown BSFL

have identified trypsin-like and chymotrypsin-like proteases (Rabani et al., 2019). Since

the identification had already been investigated, the purpose of the research was to

confirm that the initial observation of enzymatic activity was accurate, then to observe

autohydrolytic effects of the BSFL enzymes on BSFL protein. Past researchers showed
55
that bromelain successfully created protein hydrolysates with antioxidant properties from

defatted BSFL (Firmansyah & Yusuf, 2019). Future production of BSFL hydrolysates

may find success with autohydrolysis of non- or mechanically- defatted BSFL.

The present work utilized multiple methodologies to confirm enzymatic activity after

initial observations suggested the presence of active proteolytic enzymes in commercial

larvae. While studying electrophoretic mobilities of water-soluble BSFL proteins, the

lack of high molecular weight (mw) proteins visible in gels presented by Leni et al.,

(2019); Miron et al., (2019) and the simultaneous appearance of low mw proteins led to

the hypothesis that proteolysis was occurring. A casein plate and zymogram (García-

Cano et al., 2019, 2020) were used to confirm the presence of and characterize the mw

range of the proteolytic enzymes. Following, an experiment was conducted using sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) whereby the larvae

were ground and “soaked” in water then dehydrated before mixing with the sample buffer

which would inhibit the enzyme activity. This chapter covers proteolytic activity of the

BSFL including the casein plates, zymograms, and “soaked” larvae experiment along

with a NaOH extraction experiments to be described.

3.1.4 Protein quantification

Trends such as the increasing popularity of protein supplementation and alternatives to

animal-based agriculture have amplified the need for quality food protein research. For

novel sources of protein, especially sustainable biomasses, fundamental knowledge is

needed to provide accurate and rapid protein quantification to properly evaluate products

and processes. An introduction to proteins followed by the three categories of protein

quantification is discussed.
56
At the molecular level, proteins are polymers or chains of a specific class of molecules

called amino acids (AA). There are 21 biologically relevant amino acids with diverse

chemical properties some are considered essential for human nutrition while all amino

acids play an important role in protein synthesis (Hou et al., 2015). For the purposes of

understanding protein quantification three facts are critical (1) each AA has a specific and

known mass, (2) all amino acids contain nitrogen, and (3) polypeptides can bind to dyes

and absorb light.

The most accurate, but expensive technique is called “Amino Acid Analysis” which

directly measures the quantity of each amino acid in a sample. To perform this, a highly

trained technician will use chromatographic techniques to separate the amino acids after

protein hydrolysis has digested the proteins. Modern methodology utilizes mass

spectrometry to quantify each amino acid (Kambhampati et al., 2019). The sum of the

masses of all the amino acids makes up the most accurate and reliable estimate of a

product’s protein content. An amino acid analysis is not perfect and may report errors of

10-20% however it is still a superior option to other protein quantification methods since

interference by other nitrogen-containing molecules and matrix effects are not as

prevalent (Mariotti et al., 2008).

The next type of method, nitrogen analysis, is less expensive and more user-friendly if

the appropriate equipment is available. There are two common techniques that can be

followed to quantify nitrogen – Kjeldahl and Dumas (Jung et al., 2003). These techniques

rely on the digestion of protein and AA so that the nitrogen in the sample is liberated and

quantified. For protein determination, the technique must be paired with Amino Acid

57
Analysis to develop a relationship between N and AA (nitrogen conversion factor). With

a reliable conversion factor, this method can be completed routinely and cheaply but is

sample specific.

The next and most ubiquitous methods to quantify protein are spectrophotometric assays.

The first color reaction with proteins was reported in 1833 and again in 1857,

independently, which later became known as the biuret method. This method uses alkali

conditions and copper sulfate reacts with peptide bonds, a principle that has been

modified and improved in the decades following (Lee, 1914). Lowry et al., (1951) found

improved sensitivity and fewer interference effects by adding Folin-Ciocaltaeu reagent

adding while Bradford, (1976) improved on both Lowery and the biuret by incorporating

brilliant blue dye, a pigment that changes color after binding to protein. The method

followed for all colorimetric protein quantification in this dissertation is the bicinchoninic

acid (BCA) method. The BCA reagent also reacts under alkali conditions to form a deep

purple color with copper (I) when it is complexed with proteins. BCA analysis is the least

susceptible to interference, the most sensitive, and is highly consistent among different

proteins compared to previous colorimetric assays (Smith et al., 1985). These methods

are all based on the relationship between a molecule of interest (proteins) and interactions

with light (absorbance). This relationship was first reported by August Beer, who

determined that the concentration of an analyte varied linearly with the absorbance of

light measured (Beer, 1857; Berberan-Santos, 1990; Beer, 1857 is the original German

publication). This relationship is still used for direct protein quantification for purified

systems of a known protein using OD280 measurements. In these special cases, dye-

binding colorimetric assays are not needed. The spectophotometric assays are the
58
workhorse of a food protein lab and can be very robust when paired with N and AA

analyses. Colorimetric assays are especially useful in situations where relative

concentrations can be used. Method selection requires evaluating a laboratory’s

capabilities, the current knowledge of the protein source, and the goal of the evaluation.

3.1.5 Rationale and objectives:

The previous discussion addresses the first specific objective in this dissertation by

providing background on BSFL nutritional and environmental qualities while informing

the experiments presented in this chapter. This chapter aims to evaluate hypotheses

concerning the influence of processing parameters on material separation and to test the

hypothesis that endogenous BSFL proteases are active against their own proteins. The

major extraction parameter under investigation in this chapter is sodium hydroxide

(NaOH). NaOH is a strong base commonly used for protein extraction during the creation

of protein concentrates. The experiments presented herein explore the use of NaOH as an

extraction aid for BSFL processing. Deionized water (diH2O) and phosphate buffer saline

(PBS) are also used as extraction conditions. Enzymes are regularly used industrially as

processing aids to disrupt cellular matrices or to produce protein hydrolysates.

Investigating the endogenous proteolytic activity of the BSFL was pursued to better

understand their potential as an endogenous extraction aide. The following specific

objectives were developed to investigate NaOH extraction and proteolytic activity:

1. Evaluate NaOH extraction as a function of time and after sequential NaOH

washes

2. Confirm the presence of active proteolytic enzymes in commercial Black Soldier

Fly larvae
59
 3. Determine the effect of autohydrolysis on the Black Soldier Fly larvae proteins

3.2 Materials and Methods

3.2.1 BSFL preparation and extractions

Dry whole BSFL were purchased from a North American animal feed supplier and stored

at -30 °C upon reception. Larvae were ground in a food processor (Brevill, Sydney, AUS)

after being submerged in liquid nitrogen. The ground larvae were immediately placed in a

vacuum chamber to remove moisture while the ground larvae were equilibrated to

ambient temperature overnight. This was followed by sieving through a 0.75 mm screen.

The flour was combined with diH2O and 1M NaOH at a fixed 1:10 ratio in 50-ml

centrifuge tubes, mixed, then placed in a lateral shaker. Different tubes were pulled at

prescribed time points then centrifuged (2,000 x g, 1 h, 4 °C). The supernatant was tested

for protein concentration using Dumas, described below. The flour was also used to study

the effect of washing with NaOH on protein extraction. In an Erlenmeyer flask, the same

concentration and ratio were used during mixing. The contents of the flasks were then

transferred to 525 ml centrifuge bottles and centrifuged (2,000 x g, 1 h, 4 °C). The

supernatant was stored for BCA protein analysis. The pellet was added back to the flask

with additional NaOH. This process was repeated six times

3.2.2 Analytical methods

5.2.2.1 Moisture, Fat, and Ash content of materials

Moisture content was determined using an alteration of a method described by Belluco et

al., (1983) in which samples (2.5-3.5 g) were measured on to pans of known weight and

60
recorded. The pans were then placed in a moisture drying oven set to 110 °C for 17 h.

After drying, the pans were weighed.

Fat content was determined according to (ISO, 1973) using a Soxhlet apparatus. Dried

samples from the moisture content analysis were used.

5.2.2.2 Protein concentration of liquid samples

The protein concentration of liquid samples was measured via the Bicinchoninic Acid

(BCA) protein assay according to (Smith et al., 1985) with modification. Standard

procedures using BSA to create a standard curve and absorption measurements a 562 nm

were conducted. Dried products were reconstituted in phosphate-buffered saline (PBS,

137 mM NaCl, 2.7 mM KCl 10 mmol Na2HPO4, 1.8 mmol KH2PO4, pH 7.4) and then

diluted across the linear dynamic range, n=4). All measurements used in the above

analyses were the average of, at minimum, three dilutions within the linear dynamic

range.

5.3.2.2 % Nitrogen via Dumas

Total nitrogen was measured using the Dumas method on a Rapid N Exceed Nitrogen

Analyzer (Elementar, Ronkonkoma, NY.) using the Dumas method (Barka et al., 2018;

Spranghers et al., 2017)

5.2.2.4 SDS-PAGE

Standard electrophoretic analysis of the samples was conducted with sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) with modifications of the method described

by Updike et al., (2006). Samples were dissolved in dissociation buffer (8 M urea, 2M

thiourea, 60 mM Tris, pH 6.8, containing 2% SDS, 15% glycerol, 350 mM DTT, and 0.1%

61
bromophenol blue) overnight a 25 °C with agitation then diluted with dissociation buffer as

needed. Approximately 15 μl of sample dilutions were loaded onto each lane of a 12.5%

denaturing resolving gel (30:0.8, acrylamide: N, N’- bis-methylene acrylamide) with a 3%

stacking gel containing 1 % SDS. The proteins were resolved at 100 V cm-1 until the dye front

reached the bottom of the gel. Gels were stained with Coomassie Brilliant Blue G-250

overnight and then destained overnight with 10% acetic acid. Gels were imaged using near-

infrared with automatic exposure with an Azure Biosystems imager (Dublin, CA).

Zymogram SDS-PAGE followed the same procedure as above with modifications to the

dissolution buffer and resolving gel. The dissolution buffer (250 mM Tris-HCl, pH 6.8, 4%

SDS, 40% (w/v) glycerol, 0.02% bromophenol blue) for zymogram was mixed with

samples then homogenized and held overnight at 25 °C. The resolving gel was 10%

acrylamide containing 0.1% gelatin. Before staining zymogram gels were held at 37 °C in a

renaturation buffer (Tris-HCl 100 mM, pH 8.0, Triton X-167 100 1.0 %) for 18 h. Staining,

destaining, and imaging followed as previously described.

3.3 Results and Discussion

3.3.1 Nitrogen extraction versus time from BSFL using NaOH and deionized water
Defining the changes in protein extraction over time is of interest to conserve resources

while maximizing the extracted protein. Figure 3.2 shows nitrogen extracted from BSFL

with NaOH as an added solute compared to deionized water (diH2O) over time (0-24 h).

The % nitrogen refers to the nitrogen in the extracted solution, so the protein extracted is

not an absolute measure. When using diH2O no difference over time was observed

indicating that time and energy can be saved by shortening water-based extractions in the

future. When using NaOH during extraction N continues to increase over the 24-hour
62
period. Since BSFL contains chitin, a nitrogen-containing molecule, and chitin is known

to have partial solubility in alkali conditions (Einbu et al., 2004), it is uncertain whether

the increases in nitrogen are equally associated with extracted protein throughout the

entirety of the time series. Proteins are also known to exist in complexes with chitin in

the structural components of insects (Finke, 2007; Pillai et al., 2009), so the increase in

nitrogen might be due to an increase in both chitin and protein as chitin solubilizes,

liberating proteins. This concept was explored further and discussed in Ch. 5.3.5.

0.8
water
0.7
NaOH
0.6

0.5
%N (wet wt.)

0.4

0.3

0.2

0.1

0
0 5 10 15 20 25
time (hour)

Figure 3.2: Extraction of nitrogen versus time (0-24 h) for BSFL protein using deionized
water and alkali solvent conditions; mg protein/ml was converted from total N analysis of
liquid samples.
63
The above experiment also includes single time point extractions with PBS, not shown in

Figure 3.2, which was studied on SDS-PAGE with diH2O and NaOH extracts. The

addition of PBS and NaOH resulted in more proteins being resolved on the gel. Miron et

al., (2019) similarly published an SDS-PAGE gel containing low-ionic strength extracted

proteins. The results presented here showed more distinct banding than Miron et al.,

(2019) as well as the presence of the heavy band at 75 kDa. These differences are

attributable to the addition of buffers and may suggest protein degradation occurred in the

samples presented in Figure 3.3.

64
Figure 3.3: SDS-PAGE gel showing the effect of solvent on the protein extracted from

BSFL flour. Lanes include: 1) diH2O (0.1x dilution), 2) PBS (0.5x dilution), 3) NaOH

(0.5x dilution), 4) standard. Protein ladder (right) shows standard lane molecular weight.

Protein concentrations are not normalized.

3.3.3 BSFL protein extraction using NaOH washes

Extended extraction times have many practical drawbacks including long process times

and the potential for protein denaturation and degradation, which may be undesirable

(Ursu et al., 2014). Repeated short-duration washes in NaOH may provide a high protein

65
yield, but at the cost of greater material (NaOH) usage. Achieving full protein removal is

also needed for the isolation of a pure chitin fraction as protein removal is a necessary

step prior to mineral removal with acid (Smets et al., 2020). In another experiment,

presented in Figure 3.4B three washes with 1M NaOH were found to be sufficient to

remove all detectable protein (BCA). Figure 3.4A shows the BSFL slurry after mixing,

prior to centrifugation and protein analysis. Queiroz et al., (2021) performed two NaOH

washes with 0.25 M NaOH before analyzing the extracted proteins for their colloidal and

foaming properties.

66
 A

20
B
mg/ml (relative, BSA)

15

10

0
0 2 4 6
1 M NaOH washes

Figure 3.4: Rinsing experiment, (A) shows a picture of BSFL in NaOH after shaking but
before centrifugation, (B) shows a plot of protein in the supernatant after repeated washes
with 1 M NaOH

67
3.3.4 Identifying BSFL proteolytic activity with casein plates
Casein pour plates are a rapid and repeatable way to observe the proteolytic activity of a

complex mixture. Figure 3.5 shows a diH2O-soluble extract and a dialyzed diH2O-

soluble extract from BSFL exhibiting proteolytic activity. The diameter of the circles

surrounding each letter is related to the amount of proteolytic activity, with more

proteolysis being associated with a greater diameter.

Plate 1: a. 250 mg BSFL/ml; b. 125 mg BSFL Plate 2: a. 100% Dialyzed, b. 50% Dialyzed,

/ml; c. 50 mg BSFL /ml; d. 5 mg BSFL /ml, e. c. 25% Dialyzed, d. 10% Dialyzed, e. 1%

0.5mg BSFL /ml, f. 0.05mg trypsin/ml, g. Dialyzed f. 0.05mg trypsin/ml

0.5mg trypsin /ml

Figure 3.5: Casein plates showing proteolytic activity of diH2O-soluble BSFL extract (left)
and the same extract dialyzed against PBS (right).

68
3.3.5 Characterization of BSFL proteases with SDS-PAGE zymogram
Zymograms incorporate substrate (gelatin) as part of the resolving gel during SDS-PAGE

(García-Cano et al., 2019, 2020). Figure 3.6 presents a zymogram confirming the

proteolytic activity previously observed on SDS-PAGE and with the casein plate

discussed above while also showing multiple active enzymes. Zones of inhibition (dark

bands) were observed at approximately 35 and 55 kDa in the whole larvae samples while

the extracts showed inhibition in the 55 kDa region, 30-40 range, and in the 20-25 kDa

range (protein ladder lost visibility). A brief literature search turned up proteases

generated from bacteria identified within the ranges (Wandersman, 1989).

69
Figure 3.6: Zymogram of BSFL and BSFL extracts. Left panel shows two concentrations
of whole BSFL (25, 50 mg sample/ml) suspended in non-reducing dissolution buffer. The
right panel shows BSFL extracts from diH2O and PBS. Protein ladders show standard
lane molecular weight.

3.3.6 Protein characterization of ground and soaked BSFL to monitor autohydrolysis

While preliminary identification of the proteases can provide utility for future

optimizations, the effectiveness of the enzymes at auto hydrolysis has implications for

food and feed applications as described previously. Figure 3.7 shows the SDS-PAGE

70
results from a simple experiment where three BSFL samples were tested during the

extraction process. The first sample was “whole” BSFL homogenized in the SDS-PAGE

sample buffer to create a control lane on the left of the gel presented in Figure 3.7. Next,

BSFL were ground under liquid nitrogen, re-dried under vacuum resulting in “flour”,

then homogenized in SDS-PAGE sample buffer. The flour was also added to diH2O,

“soaked” for an hour, vacuum dried, then mixed with SDS-PAGE sample buffer. This

experiment was designed to control for all variables except grinding, and the reactions

occurring while suspended in water. The whole sample provides the best representation

of the proteins in the commercial BSFL; there is evidence that some hydrolysis has

occurred since larval deactivation due to the numerous faint bands throughout the middle

of lane one. SDS-PAGE protein profiles of whole larvae have not been published

previously. However, PAGE protein profiles of flour have been presented by Queiroz et

al., (2021); and Rabani et al., (2019). The “whole” BSFL presented in Figure 3.7 was

able to resolve more discrete bands above 100 kDa and below 3 kDa than the previous

reports. There is an observable loss of a 120 kDa band, and three bands in the 35 kDa

region in the “flour” after grinding which is an indication of proteolysis. The “soaked”

condition has not been presented in the literature. The lane representing the “soaked”

condition lost almost all banding above the heavy 60 kDa band as well as much of the 18

kDa band. The soaked condition also appears to have resulted in the general loss of intact

protein due to less intensity in the overall staining pattern of the lane. This may indicate

that the proteolysis reacted to completion, creating ammonia, other by-products, or small

peptides below the detectable range of this analytical technique (Parodi et al., 2020).

71
Figure 3.7: Soaking experiment showing whole larvae, larvae flour, and flour soaked in
diH2O for one hour on a 12.5% acrylamide gel, loadings were standardized to BSFL
concentration. Protein ladder (right) shows standard lane molecular weight.

72
3.4 Conclusions

The review of BSFL and edible insects suggests that BSFL has great potential to increase

the sustainability of food systems if the challenge of consumer acceptance can be

overcome. This might be overcome by the production of protein extracts of BSFL.

Therefore, protein extraction for the development of food ingredients was investigated.

The effect of time on protein extraction using diH2O and NaOH measured with nitrogen

analysis showed that prolonged diH2O exposure provides no protein yield increase. When

NaOH is used, nitrogen solubility continually increases over a 24 h period. Multiple

washes with NaOH are more effective at extracting all protein, but this requires more

resources as each wash used a fresh NaOH solution. High protein yield was possible with

NaOH extraction, but this comes with tradeoffs such as the costs associated with

neutralizing NaOH and the need for downstream processing to recover the protein.

Enzymatic activity was confirmed in commercial BSFL and extracts produced using

diH2O and PBS by zymography. Autohydrolysis observed after soaking BSFL in diH2O

showed degradation of both large and small proteins while suggesting the full hydrolysis

of some proteins. The presence of active proteases has the potential for increased

valorization but may result in protein loss. This chapter highlighted the importance of

processing decisions. NaOH was employed to achieve maximal protein extraction while

diH2O produced lower protein yields but resulted in an extract that was enzymatically

active. Future research may investigate sequential extractions to optimize for both

enzyme activity and protein yield.

73
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86
 4. Chapter 4 Macroalgae amino acid analysis and
extraction screening for protein yield using enzyme-
assisted extraction (EAE) for two species of Brown Kelp
Abstract

The goal of this research was to evaluate the protein extraction from two species of

brown kelp (Saccharina and Alaria) and to characterize amino acid profiles from 29

commercial macroalgae. The protein extracted into the liquid phase has the potential to

be a food ingredient while retention of non-protein materials in the insoluble fraction may

become a biofuel feedstock. Objectives included: 1) compare protein and mass yield

using water, alkali, and enzymatic treatments; 2) compare soluble protein yields after

long and short enzymatic treatments; 3) identify differences in extraction yields between

Alaria and Saccharina sources; 4) characterize the amino acid profiles of 29 commercial

macroalgae samples.

Twenty-nine commercially sourced macroalgae samples covering three phyla and nine

genera were studied for the amino acid profiles. Harvest month and source were also

studied. Only minor trends were observed among the different factors. The profiles were

compared to the literature which showed greater phyla-phyla variability than the present

study.

The insoluble fraction masses (dw.) for the macroalgae incubated in dilute NaOH were

larger (p<0.001) than those incubated in concentrated NaOH. The use of 2.5 N NaOH

resulted in the dissolution of cellular components more than the enzymatic treatment.

87
The protein content of the soluble fraction was also significantly (p<0.001) influenced by

the presence of NaOH. Extraction of protein with 2.5 N NaOH was 10 times greater than

with diH2O. The 1-hour enzyme treatment extracted more protein than the diH2O only

treatments. Among samples treated with dilute NaOH, additional enzymatic exposure for

18 hours resulted in the most protein extracted. Enzymatic treatment was effective in

increasing protein extraction while minimizing the extraction of non-protein components.

However, compared to enzymatic treatment, NaOH alone was 5 times more effective at

extracting proteins, yet the mass of the remaining solids fraction was reduced.

Maximizing protein extraction is important, but it is desirable to avoid strong

concentrations of NaOH for environmental and safety reasons. To maximize the

valorization of a bio-refinement pathway, it is important to understand the full mass

balance of extraction and the associated production costs.

88
4.5 Introduction

Macroalgae have gained interest for applications as both food and fuel. During the

biorefinery process, multiple processing steps are needed to convert kelp biomass into

biofuel. Biofuel production from algae is not yet carbon neutral, nor cost-effective (Cruce

& Quinn, 2019). Co-products from the underutilized protein fraction provide an avenue

for the increased valorization of macroalgae biomass. The human food market is a good

match, considering market size and the value associated with the plant-based beverage

and protein markets. The goal of this research was to evaluate the amino acid profiles and

protein extraction from macroalgae. The extraction study focused on the technical and

economic aspects of producing food and fuel from two species of brown kelp, Saccharina

and Alaria. While the amino acid study included a broad selection of macroalgae genera

from the three major phyla

Twenty-nine unique commercially sourced macroalgae samples, including samples from

9 different species, were the subject of the analysis. Harvest information for two species

(Saccharina, Alaria) was provided which included the biological component (blade or

stipe) and collection months (April, May, or June). Twenty-nine samples were analyzed

for amino acid composition. Amino acid analysis (AA) provides a definitive measure of

the quantity and nutritional quality of protein. By pairing the AA with total nitrogen

analysis, an estimation for the protein-nitrogen conversion factor can be made.

Blue Evolution provided multiple different samples of Alaria and Saccharina

macroalgae, thus these species became the focus of the extraction component of the

analysis. Commercially produced Alaria and Saccharina were used to screen for enzyme-
89
assisted extraction (EAE) efficiency and to evaluate the mass and protein balance

associated with the initial extraction and separation procedures. Details of the

experimental design are provided in Figure 4.1 below.

The separation operation is the major determinant in the protein yield from the extraction

and the value of each new fraction. It is desirable to solubilize the maximum amount of

protein from the biomass and retain it in the supernatant. At the same time, fat and

carbohydrate must be retained in the pellet fraction. Carbohydrates and fats contaminate

the protein whose price is linked to purity, so the more efficient the initial separation, the

lower the downstream processing costs will be. Poor separation efficiency also reduces

the value of the insoluble material. A smaller insoluble mass would suggest the

carbohydrates destined for biofuel conversion were solubilized (likely via alkali or

enzymatic hydrolysis) and incorporated into the supernatant. Further, sub-maximal

protein extraction could result in a pellet that does not meet biofuel conversion efficiency

targets due to its protein content.

Enzymatic treatment employed a commercially available enzyme mixture which targets

carbohydrates: Viscozyme (Novozymes Corp) containing hemicellulose, cellulase, β-

glucanase, hemicellulose, and xylanase. The mixtures were originally developed for the

brewing industry and have been used on brown seaweed to liberate phenolics

(Habeebullah et al., 2020; Sánchez-Camargo et al., 2016), and Sancez, 2016). Hammed et

al., (2013) identified 7 reports of Viscozyme’s use on macroalgae for the extraction of

antioxidants.

90
Previous attempts to extract and increase protein yield in macroalgae have explored the

effects of enzymes, various extraction times, substrate: enzyme ratios, and temperature.

While studying an enzyme blend (Cellic cTec3: cellulases, ß-glucosidases, and

hemicellulose), Vásquez et al., (2019) measured protein content as part of a response

surface model showing the substrate: enzyme ratio has a greater effect on protein yield

compared to enzyme exposure time (6-18 h)

The major carbohydrate in Alaria and Saccharina is alginate containing 37% and 28% of

the carbohydrates, respectively. Cellulose was found to be the second most abundant

polysaccharide with approximately 10-15% of the carbohydrates (Schiener et al., 2014).

The enzyme mixtures selected were expected to hydrolyze the cellulosic materials

degrading the structure and releaseing protein. Specific digestion of alginate has been

explored by Nguyen et al., (2020), using alginate lyase treatment to extract fucoidan.

Future studies could investigate this process for protein extraction applications from

brown kelp.

Minimal protein content in a biodiesel feedstock, through extraction or low initial

concentrations, is desirable to keep nitrogen content at a minimum for more efficient

biofuel conversion (Biller & Ross, 2011; Costanzo et al., 2015; Jones et al., 2014; Zhu et

al., 2020). However, the production of high-quality co-products from a protein fraction

could be a desirable revenue stream for producers if extractions are selective and

efficient. This could be achieved by using the protein as ingredients in novel foods.

Extraction of biomolecules such as phenolics, antioxidants, and carbohydrates from

macroalgae has been studied using wet fractionation and enzymatic-assisted-extractions

91
(EAE; Habeebullah et al., 2020; Sánchez-Camargo et al., 2016; and Sancez, 2016).

Protein has also been a target of extraction investigations (Fleurence et al., 1995), but to a

lesser extent than lipids or carbohydrates. As interest in alternative protein sources

continue to rise, amino acid profiling is an essential first step to understand the potential

value of a given macroalgae biomass.

Significant consumer demand exists for high-quality, non-animal derived protein sources.

Amino acid profiles are an important aspect of a food’s nutritional profile. However,

nutrient digestion and absorption are critical in evaluating a material’s nutritional value.

The present study reports only chemical compositions without digestibility analysis.

Previous research has reported in vitro analyses of macroalgae digestibility, but more

work will be needed in this area.

4.1.1 Rationale and objectives

The experiments conducted in this chapter address the dissertation’s specific Objectives 1

and 2. This chapter aims to evaluate hypotheses concerning the influence of processing

parameters on material separation and to test the hypothesis that macroalgae of different

phyla and genus exhibit different amino acid profiles. Aqueous extractions with chemical

and enzymatic aids were studied to better understand the limitations of removing protein

from macroalgae biomass. To evaluate the nutritional quality of macroalgae a broad

analysis of macroalgae amino acid analysis was conducted across 9 genera. This research

was driven by the collaborative goal with the Pacific Northwest National Laboratory to

remove nitrogen from the macroalgae biomass prior to biofuel conversion. This focus on

protein removal with a need to understand the remaining biomass was influential in the

92
development of the dissertation’s specific Objectives 3 and 4 as well as the material

presented in Ch 5. The following specific objectives were addressed:

1. Compare protein and mass extraction from Alaria and Sacharina yield after

water, sodium hydroxide, and enzymatic treatments

2. Evaluate soluble protein yield as a function of extraction time.

3. Identify major differences in protein and mass extraction between Alaria and

Saccharina sources

4. Identify differences in amino acid profiles among macroalgae from different

phyla, genus, location, and harvest date.

5. Compare the measured amino acid profiles to available profiles of macroalgae

found in the literature

4.2 Methods

4.2.1 Extraction Materials and methods

4.2.1.1 Materials

Macroalga samples were sourced commercially. Viscozyme enzymatic cocktail

(hemicellulose, cellulase, β-glucanase, hemicellulose, and xylanase; Sigma Aldrich) and

bicinchoninic acid BCA kits were purchased from Sigma Aldrich.

4.2.1.2 Particle size reduction

Alaria (AL-HME) was ball milled and passed easily through a 300 μm sieve. Whole,

freeze-dried Saccharina (SA-HME) was ground frozen in a food processor for 1 min and

sieved, passing through 1.18 mm screen. The dry ground powders were stored in airtight

containers at -30 °C until use.

93
4.2.1.3 Proximate analysis of ground samples

The moisture content (MC) was determined gravimetrically comparing the mass change

after 17 h of drying at 105 °C (Minor et al., 1984). Total fat (FC) was determined using

hexane in a Soxhlet apparatus, recirculating for 2 hours. Total nitrogen was measured

using the Dumas method on a Rapid N Exceed Nitrogen Analyzer (Elementar,

Ronkonkoma, NY.)

4.2.1.4 Calculation

Moisture content (MC) was calculated using:

𝑚𝑖 −𝑚𝑑
MC = %𝑀𝐶 = ∗ 100 (4.1)
𝑚𝑖

Where, mi was the initial sample mass, and md is the mass after 17 hours at 105 °C.

Fat content (FC) was calculated using:

𝑚𝑑−𝑚𝑛
FC = %𝐹𝐶 = *100 (4.2 )
𝑚𝑖

Where, md are the same initial and dried masses from equation 4.1, and mn is the non-fat

dried solids mass. Non-fat dried solids are obtained by recirculating hexane over the dried

biomass for 2 hours in a Soxhlet apparatus.

Protein content (PC) via Dumas is determined from the percent nitrogen (%N) according

to:

PC= %PC = %N*F (4.3)

Where F is the proportionality factor that relates % N to protein content (PC) for specific

organisms. The value can be determined from amino acid composition and is reported in

94
the literature to be 5.17 for brown macroalgae (Biancarosa et al., 2017). Carbohydrates

and ash were not measured directly.

4.2.1.5 Extraction screening experiment

I) All samples were extracted in 250-ml tubes with lateral shaking of 120 rpm at 53

°C. The procedure was to: Combine the samples (10 ±0.5 g) with the specified

solvent solution (100 ml) at a 1:10 ratio (w/v) and begin shaking.

II) Shake for 1 or 18 hours to provide agitation and test the effect of time on enzyme

exposure.

III) Heat treat to deactivate enzymes (submerged in boiling water for 10 min).

IV) Adjust the pH of buffered samples using 0.1 N NaOH or 5 N NaOH (100ml);

proteins are more soluble at neutral and high pH than under the acidic

conditions the enzyme requires.

V) Shake for an additional 1 h to reach equilibration. For samples exposed to a final

concentration of 2.5 N NaOH, neutralization with HCl and volume adjustment

to 400 ml was conducted prior to separation.

VII) Centrifuge (27,000 x g at 4 °C for 30 min) and separation into the liquid

supernatant and solid fraction pellet.

VIII) Filter the supernatants to remove excess particulates using a 2 μm filter.

IX) Freeze dry both fractions for preservation.

Independent extraction variables included: macroalgae species, solvent solution,

treatment time, and post treatment adjustment. The species include the ground SA-HME

95
and AL-HME powders, the solvent solution included diH2O, 0.1 M acetate buffer pH 4.5,

and 2.5 N NaOH, the adjustments included a dilute (0.1 N) or concentrated 5 N NaOH

solution. All samples treated with NaOH were neutralized with HCl, and diH2O was

added to achieve a 400 ml volume addition.

96
Figure 4.1: Top: Experimental design diagram showing the 16 extraction treatment
conditions. Bottom: graphical overview of experimental procedure showing initial
treatment, adjustment, neutralization, centrifugation, and separation.

97
4.2.1.6 Post-extraction analysis

After centrifugation, the mass of the liquid fraction (supernatant) and the solid fraction

(pellet) were collected. The MC of each was determined. Prior to freeze drying, the

supernatant was analyzed for protein concentration using Bicinchoninic acid (BCA)

method.

Component mass yields were calculated with the following formulae:

𝑚
% supernatant = 𝑚𝑠 Equation 4.4
𝑡

𝑚𝑝
% pellet = Equation 4.5
𝑚𝑡

Where mt is the is the total mass added to the extraction jars, ms is the mass of the

supernatant or liquid fraction, and mp is the mass of the pellet or solid fraction.

Extracted protein was calculated using:

Extracted protein (mg) = 𝑉𝑠 ∗ 𝐶𝑠

Where Vs equals the volume (ml) of the supernatant collected and Cs is the protein

concentration (mg/ml), as determined by BCA.

Data analysis was performed using JMP® Statistical software’s fit model functionality.

4.2.2 Amino acid profile methods:

4.2.2.1 Sample preparation:

For amino acid quantification, samples were first hydrolyzed by combining 50 µL of 2

mg/mL of solutions of solid macroalgae and protein standards with 200 µL of a

hydrolysis solution of 4 M methane-sulfonic acid with 0.2% tryptamine containing 10

µg/mL of a C13-labeled amino acid mixture (Cambridge Isotope Laboratories;

98
Tewksbury, MA). Standard solutions of amino acid mixture were also hydrolyzed the

same way at concentrations of 0.0, 0.025, 0.25, 2.5, 25, and 250 µM using blank

hydrolyzed solution with 50 µL of each solution mixed with 200 µL of the hydrolysis

solution. Both the hydrolyzed samples and calibration solution were then sealed in glass

vials with nitrogen gas and incubated for 22 hours at 100 °C. Following hydrolysis,

samples were adjusted to pH 8 with a 10% ammonium hydroxide solution, and

completely dried down and reconstituted at an equivalent volume in 1:1, H2O: ACN

mixture, sonicated and centrifuged before being placed in LC vials for quantification.

4.2.2.2 LC-MS Amino acid analysis methods:

The samples were quantified using a heated electrospray ionization (HESI) source

on a Thermo Scientific (Waltham, MA) Q-Exactive™ Plus Orbitrap mass spectrometer

and separation was achieved using a Thermo Scientific UltiMate 3000 HPLC on an

Agilent (Santa Clara, CA) Poroshell 120 HILIC-Z (2.1 x 100 mm, 2.7 µm particle size)

column kept at 40 ⁰C. Samples were injected at 5 µL and separated at a flow rate of 300

µL/min with a solvent system of H2O with 20 mM ammonium formate and 0.2% formic

acid as Solvent A, and 90 % ACN with 20 mM ammonium formate with 0.2 % formic

acid as solvent B. The initial gradient was 100 % Solvent B with a linear gradient to 80

% Solvent B at 2 minutes, followed by a decrease to 20 % Solvent B by 11.0 minutes, up

to 100 % Solvent B at 11.5 min, and holding at 100 % Solvent B until minute 15. For

each amino acid, the transitions monitored are listed below with collision energies. For

all experiments, the capillary voltage was set to 5.0 kV with a capillary temperature of

320 °C, a vaporizer temperature of 125 °C, a sheath gas of 15, auxiliary gas of 10, and

2.2 sweep gas. The MS was set to parallel reaction monitoring (PRM) mode at a
99
resolution of 35,000, a 1×106 automatic gain control (AGC) target, a maximum IT of 100

ms, and an isolation window 1.0 m/z, with the selected fragmentation areas below

selected.

100
Table 4.1: Validation table for GC/MS identification of amino acids

Collision Collision
Precursor Product Precursor Product
Compound Energy Compound Energy
(m/z) (m/z) (m/z) (m/z)
(V) (V)
Gly 76.04 76.04 10 Glu 148.06 84.04 25
Ala 90.05 90.05 10 Met 150.06 104.05 25
Ser 106.05 60.04 25 Glu-13C5 153.08 88.06 25
Ser-13C3 109.06 62.05 25 Lys-13C6 153.13 89.1 25
Pro 116.07 70.06 25 His 156.08 110.07 25
Val 118.09 72.08 25 His-13C6 162.1 115.09 25
Thr 120.07 102.05 25 Phe 166.09 120.1 25
Pro-13C5 121.09 74.08 25 Arg 175.12 70.06 25
Val-13C 5 123.1 76.09 25 Phe-13C9 175.12 128.1 25
Ile/Leu 132.1 86.09 25 Arg-13C6 181.14 74.08 25
Asp 134.04 74.02 25 Tyr 182.08 136.08 25
Asp-13C4 138.06 76.03 25 Tyr-13C9 191.11 144.1 25
Ile/Leu-
13C 138.12 91.11 25 Cystine 241.03 151.98 25
6
Lys 147.11 84.08 25

4.2.2.3 Conversion to amino acid profile

Single amino acid (AA)concentrations were determined using the standard curve for each

AA. Each AA was converted from a micromolar concentration to a milligram amount

using its molecular weight. For each sample the AA masses were summed to determine

to total AA mass detected for each sample. Each AA was individually divided by the total

mass resulting in each sample’s AA profile (AA mg/ (AA mg))*100. Also, for each

sample the total AA mass detected was divided by the initial sample mass to generate the

%AA value included in Table 4.4-Table 4.8. This value represents an underestimate for

101
protein content of each sample since tryptophan was not quantified. MATLAB® was

used for all calculation.

4.2.2.4 Stock proteins and amino acid profiles from literature

Purified proteins controls, referred to as “stock proteins” were used as an additional

validation of the amino acid profile analysis. Bovine serum albumin (BSA) was sourced

from KSE Scientific (item 98-101p, Durham, NC)). The amino acid profile used for

comparison was derived from the amino acid sequence for BSA as reported by (Peters,

1995). Purified chicken myosin (Huffman et al., 2012), was also used as a calibration

standard. The amino acid sequence reported by Buttkus (1967) was used as a reference

for the amino acid profile of myosin. In both cases, the molecular mass of each amino

acid was used to determine the profile for each residue in proportion to the total number

of residues in the protein (AA mg/ (AA mg))*100. These two stock references along

with all references found to be acceptable amino acid profile comparisons are found in

Table 4.3. Only references citing the amino acid profile of the whole or part of a single

macroalga were used. Appropriate transformations into the units AA mg/ (AA mg) were

conducted when necessary.

102
4.3 Results and discussion

4.3.1 Extraction analysis

4.3.1.1 Proximate analysis

Table 4.2: Proximate analysis for the Alaria and Saccharina samples used in the protein
extraction experiments (see Figure 4.1) compared to a report by Schiener et al., (2014)
on seasonal variation of brown kelp species.

Experimental analysis (wet wt.) Schiener, 2015 (dw.)

Alaria Saccharina Alaria Saccharina
% moisture 12.4% 7.2% NR NR
% fat 4.4% 4.5% NR NR
% protein (Dumas) 8.2% 6.3% 9.4-12% 5.3-9.9%
Sum 25.0% 18.1% - -
carbohydrates NR1 NR 65-78% 60-80%
Ash NR NR 8-12% 2-3%
1NR indicates data not reported by the respective source.

The data summarized in Table 4.1 shows the biomass proximate composition of the

Alaria and Saccharina samples studied for protein extraction. The values determined

experimentally for MC, FC, and PC are comparable to the data reported by (Schiener et

al., 2014). Schiener et al. (2014) reported dry weight values while the analysis conducted

in our laboratory used freeze-dried kelp which may lead to differences in the amount of

residual moisture explaining variations in protein content. An important conclusion from

the work of Schiener et al. (2014) was that the reported values are affected by season,

thus the variability was expected.

103
4.3.1.2 Analysis of recovered solids fraction (pellet):

In the experimental design described in Figure 4.1, two major sample groups from this

study are included: neutral (samples adjusted to pH 7, blue boxes) and alkali (samples

extracted with 2.5 N NaOH, orange boxes). The pellet characteristics from each group

were visibly different. The neutralized pellets were less-compact, watery, and the cellular

matrix was visible and heterogeneous. In contrast, the samples from alkali treatment

exhibited dense pellets that were sandy, dry, and homogenous. The mass of the pellet

(wet weight) was found to be statistically (p<0.001) larger for the neutral samples

compared to the alkali group. The pellet mass collected from Saccharina samples was

more variable and slightly greater across all conditions when compared to those from

Alaria. Saccharina samples generally formed less-compact pellets as well. This was

attributed to the grinding technique and the difference in final particle size. The Alaria

sample were ground before their reception with different equipment than is available at

the authors laboratory. As an objective comparison of the solids, the dried weights were

collected and are shown below in Figure 4.2.

Moisture content (MC) analysis of the pellets showed that the neutral (avg. 91%) group

had a significantly (p=0.0044) higher moisture content than the alkali group (avg. 85%).

The variation in MC was an indication of the solids’ ability to hold water. Alkali

treatment diminished the solids material's ability to hold water.

104
 10
Neutral samples Alkali samples
9 A B

Solid fraction dry matter (g)

8
7
6
5
4
3
2
1
0
Water buffer pH7 Enzyme Enzyme 10% NaOH buffer pH enzyme Enzyme
(2hr) (2hr) pH7 (2hr) pH7 (18hr) (2hr) 14 (2hr) pH14 (2hr) ph14
(18hr)

Saccharina Alaria

Figure 4.2: Pellet dry matter collected for all conditions tested showing pH7 or “neutral”
samples on the left and 2.5 N NaOH or “alkali” samples on the right for Saccharina and
Alaria. Initial mass was approximately 10 grams for all samples. Capital letters indicate a
difference between sample groups (α<0.05). Each bar represents 1 run.

The dry solid weights collected from the pellets were presented in Figure 4.2 showing

the neutral samples leaving a significantly (p<0.0001) larger pellet solids mass than the

alkali samples across all conditions. Variation between the species was low for 7 out of 8

runs. The initial dry mass solids were approximately 10 grams, thus the alkali samples,

on average, lost about 60% of their mass to the solvent while the neutral samples lost

about 34% of their mass, on average. Furthermore, due to the neutralization, the alkali

samples also contained higher salt content. In situations where pellet mass retention is

critical, this is an important observation as these additional salts will not contribute to

valorization in feed or biofuel applications.

105
4.3.1.3 Analysis of recovered supernatant and soluble protein

The analysis of the supernatant was focused on the materials extracted; thus, total solids

(Figure 4.2) and extracted protein (Figure 4.3) are presented.

The results showed significantly (p<0.0001) greater solids extracted from the alkali group

than the neutral group across all conditions. It is important to consider that the final

volume of the alkali group was doubled during neutralization. The data presented were

not normalized; the data is presented as it was collected.

12%

10%
Alkali samples B
Supernatant % solids

8%

6%
Neutral samples
4%
A

2%

0%
Water buffer pH7 Enzyme Enzyme 10% NaOH buffer pH enzyme Enzyme
(2hr) (2hr) pH7 (2hr) pH7 (18hr) (2hr) 14 (2hr) pH14 (2hr) ph14
(18hr)

Saccharina Alaria

Figure 4.3: Supernatant % solids for all conditions determined via drying at 105 °C for
17 hours showing pH7 or “neutral” samples on the left and 2.5 N NaOH or “alkali”
samples on the right for Saccharina and Alaria. Initial mass was approximately 10 grams
for all samples. Capital letter indicates a difference between sample groups (α<0.05).
Each bar represents 1 run.

106
Analysis of protein concentration in the supernatant showed significantly (p=0.005) more

protein extracted from the alkali group than the neutral group across all conditions. Also,

the Alaria samples were significantly more concentrated in the neutral (p=0.027) and

alkali (p=0.0317) groups than the Saccharina samples. An unexpected outcome was

observed in that diminishing protein concentrations were observed with enzyme addition

and extended treatment in the Alaria alkali group.

The protein concentrations were multiplied by the supernatant volumes collected to

determine a protein yield from the initial 10 g of kelp; Figure 4.4 displays these values.

Please note BCA protein determination was not an absolute measure of protein content as

it is based on a standard protein’s affinity to the BCA dye, which may be different than

the kelp protein’s affinity. Furthermore, these values did not account for processing loss

that may be associated with downstream operations such as drying and packaging.

107
 1200
Alkali samples
1046 B
1000
825
extracted protein (mg)
800 724
625
600
Neutral samples
A 408
400 321
294
218 226
159 182
200 111 117
46 52 53
0
Water buffer pH7 Enzyme Enzyme 10% NaOH buffer pH enzyme Enzyme
(2hr) (2hr) pH7 (2hr) pH7 (18hr) (2hr) 14 (2hr) pH14 (2hr) ph14
Saccharina Alaria (18hr)

Figure 4.4: Extracted protein collected for all conditions tested showing pH7 or “neutral”
samples on the left and 2.5 N NaOH or “alkali” samples on the right for Saccharina and
Alaria. Initial mass is approximately 10 grams for all samples. Values are the product of
the BCA protein content times the supernatant volume. Capital letter indicates a
difference between sample groups (α<0.05). Each bar represents 1 run.

4.3.2 Amino acid analysis

The amino acid (AA) profile results from the present analysis along with AA profiles

from an extensive literature review of macroalgae were presented in Table 4.3-4.8. These

tables are first sorted so essential amino acids (EAA) appear in the top rows followed by

non-essential amino acids, then alphabetized separately. The tables are separated by

phyla with brown macroalgae presented in Table 4.3-4.5, red macroalgae presented in

Table 4.6, and green macroalgae presented in Table 4.7. Table 4.3 also contains an

analysis of stock proteins described in section 4.4.2. The results show agreement with the

108
literature for the stock proteins providing confidence in the hydrolysis and LC/MS

analysis.

AA% values corresponding to the ratio of the sum of the AA per unit mass of the

seaweed as a percent are presented in Table 4.3-4.8. This value was reported not as

protein content since tryptophan was not detected, but rather as a detected AA content.

There also appears to be appreciable loss during the analysis based on the low recovery

(~80%) observed for the two pure stock proteins. However, this measure was used as

relative protein concentration within similar genera. Each genus with greater than 1

sample analyzed in the present study was presented in Table 4.9; the average and

standard deviation of the %AA of each genus was displayed for direct comparison.

4.3.2.1 Ochrophyta amino acid profiles

Results from genus Laminaria as well as data from two published sources are displayed

in Table 4.3 (Biancarosa et al., 2017, Manns et al., 2017). Low variability was observed

between the two commercial samples. The largest difference among these samples was

with His and Cys. Focusing on the EAE, comparison to the literature data shows

differences in Ile/Leu and Met residue concentrations. Ile/Leu are branched AA important

in neonatal growth and are of interest for human nutrition and muscle growth (Karlsson,

2004). Met is an EAAacid in animals and the limiting AA in poultry diets (Bunchasak,

2009). Aside from Gly, there was not a notable difference in the non-essential amino

acids. Cys values were also low which may be attributed to its destruction during

hydrolysis (Biancarosa et al., 2017).

109
A broader picture of the Ochrophyta group, reporting on Alaria, Macrocystis, Eualaria,

and Nereocystis, was reported in Table 4.4-Table 4.6. Harvest month (April, May, June)

was provided for Alaria samples as well as the algae part (blade or stipe). Given the lack

of replication, the analysis did not have sufficient power to conclude a null effect of these

variables. There are minor fluctuations in the AA% that may indicate harvest date affects

protein content, like the results shared by (Manns et al., 2017) when studying the brown

kelp Laminaria and Saccharina. Met for the blade samples varies the greatest which may

provide justification for replication of a similar study. With only one reported AA profile

found in literature, this represents the most extensive analysis of the genus Alaria.

The next genus, Macrocystis, also has only been reported once previously. There was

very little difference between the two analyses. There were no literature reports of

Eualaria, and Nereocystis. Nereocystis was high in Met which should encourage further

investigation.

Table 4.5 focused on the genus Saccharina which was one of the more heavily studied

seaweeds with 5 reported profiles from two different sources. Like Alaria, the month and

algae part were collected for some Saccharina samples. As with Alaria, no trend across

time for EAA were observed. Stipe samples showed a slight difference in Met, this may

justify further investigation. The present study reported higher levels of Met than

literature. Asp, Glu, and Ala were the highest AA for samples that were measured,

comparable to literature

110
4.3.2.2 Rhodophyta amino acid profiles

The red algae (phylum: Rhodophyta) with genera Palmaria and Porphyra were presented

in Table 4.7. There were no literature reports identified of Palmaria AA profiles. Met

was the only residue noticeably different among the two Palmaria samples analyzed.

Porphyra has been extensively studied in the past with four profiles previously reported

(Biancarosa et al., 2017; Bjarnadóttir et al., 2018) Among the commercial samples

studied, Met was again the most variable across samples and literature. The literature

sources report lower Met concentrations. It is unlikely these differences can be solely

attributed to differences in samples, instead hydrolysis and analysis may explain a portion

of the variability. Both genera of the red algae show potentially high Met concentrations

which is a promising indictor for further investigation. Overall, values among samples

and literature agree for both genera.

4.3.2.3 Chlorophyta amino acid profiles

AA profiles of Ulva have been well-characterized with 5 profiles identified in previous

reports (Biancarosa et al., 2017; Shuuluka et al., 2013). Three literature profiles were

successful in quantifying Trp and showed a high level of this AA in their Ulva samples.

The present study reported lower His concentrations than in previous data reported by the

literature. The largest differences in the EAA values between the two samples was in Lys,

which was lower than some of the literature values. These same literature reports also

showed higher amounts of Phe and lower amounts of Pro.

4.3.2.4 Differences among phyla and discussion

Referring to Table 4.9, comparing across all genera from the present analysis, Met

showed the most variability. Identifying the genera and growth conditions that maximize
111
Met should be the focus of further investigations. Both brown and red algae have the

potential to be a source of Met which may add value to the macroalgae as human or

animal feed. Among the remaining EAA, variability within genera was low as well as the

variability across genera and phyla. This remains true for non-essential AA as well.

Macroalgae are presently a staple food in certain cultures and for certain cuisines. The

results presented suggested there may be more consistency in the AA present than

previously expected. The analytical techniques should be considered as a potential source

of variability when comparing profiles over time. The ability to fully quantify Trp and

Cys may require adjustments to macroalgae AA profiles.

112
Table 4.3: List of amino acid references used for comparison and validation of the
macroalgae samples and purified protein controls.

Phyla Genus Source 1 ID Source 2 ID

(Biancarosa et al.,
Ochrophyta Alaria [1] (Manns et al., 2017) [7]
2017)
(Biancarosa et al.,
Ochrophyta Saccharina [1] (Manns et al., 2017) [7]
2017)
Ochrophyta Macrocystis (Ortiz et al., 2009) [3]
Ochrophyta Eualaria Not found
Ochrophyta Nereocystis Not found
(Biancarosa et al.,
Ochrophyta Laminaria [1] (Manns et al., 2017) [7]
2017)
(Bjarnadóttir et al.,
Rhodophyta Palmaria [8]
2018)
(Biancarosa et al.,
Rhodophyta Porphyra [1]
2017)
(Biancarosa et al., (Shuuluka et al.,
Ulva [1] [9]
Chlorophyta 2017) 2013)
BSA Peters,1995 [4]
Myosin (Buttkus, 1967) [6]

113
Table 4.4: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Laminaria and stock protein samples

Laminaria Known controls

AA B.E. Cml. Literature Ctl. Lit. Ctl. Lit.
LA-BC-5-B LA-CH-B LA-lit[1] LA-lit[7]-2 LA-lit[7]-3 LA-lit[7]-7 LA-lit[8]-8 BSA-ctl(1) BSA-lit[4] MYO-ctl(3) MYO-lit[6]

His 0.85 1.53 3.60 1.50 1.50 1.70 2.30 3.43 3.42 2.02 2.09
Ile/Leu 6.72 5.61 6.60 11.00 8.40 11.30 11.30 7.94 12.96 7.78 14.46
Lys 5.49 5.43 5.00 5.50 4.20 5.20 5.90 11.72 11.20 11.66 11.17
Met 3.78 7.15 2.10 2.00 1.60 2.30 2.30 2.14 0.77 11.34 2.95
Phe 5.18 4.62 4.60 4.90 3.70 5.20 5.50 6.43 5.79 4.17 4.00
Pro 5.12 6.49 4.90 4.50 4.70 5.00 5.10 4.38 4.18 1.95 2.27
Trp 0.00 0.00 0.00 1.90 2.30 3.10 2.30 0.00 0.53 0.00 0.70
Ala 12.21 10.14 16.60 13.50 15.50 7.80 7.40 7.95 5.32 7.98 6.24
Arg 5.38 4.50 4.80 4.80 4.00 5.00 5.10 5.20 5.20 6.03 6.41
Asp 12.90 12.38 11.90 12.40 9.50 14.00 14.50 10.79 9.33 9.74 10.16
Cys 0.44 1.12 14.70 0.00 0.00 0.00 0.00 1.36 5.50 0.14 0.94
Glu 13.79 13.79 5.30 14.80 26.50 13.40 12.90 17.10 14.98 21.09 20.34
Gly 10.17 8.79 1.80 5.20 4.00 5.40 5.90 2.43 1.58 3.78 2.66
Ser 5.38 5.51 4.70 4.50 3.60 5.40 5.50 4.04 3.82 3.66 3.87
Thr 6.20 6.43 5.40 4.90 3.90 5.90 5.90 6.14 5.25 3.77 4.39
Tyr 2.38 2.73 2.90 3.40 2.60 3.50 3.50 4.39 4.70 2.06 2.93
Val 4.02 3.77 5.10 5.10 4.00 5.40 5.50 4.57 5.47 2.82 4.42
AA% 4.23 7.27 - - - - - 79.58 - 83.33 -

114
Table 4.5: Amino acid profile (AA mg/Σ(AA mg)) * 100, Alaria, Macrcytis, Eularia, and Nereocytis species

Alaria Macrocystis Eualaria Nereocystis

Blue Evolution Commercial Lit. B.E. Lit. Cml. Cml
AL-P-5-
AL-W-4-S AL-P-5-S AL-P-6-S AL-P-4-B B AL-P-6-B AL-HME-B AL-AK-B AL-lit[1] MA-W-4-S MA-lit[3] EL-AK-7-B NE-ME-W

His 1.52 1.69 1.57 1.51 0.99 1.52 1.36 1.54 1.40 1.59 1.37 1.65 1.04
Ile/Leu 4.95 5.97 4.70 4.97 3.64 4.72 6.01 5.50 8.60 5.22 7.17 5.57 4.58
Lys 6.69 5.96 7.39 6.56 3.22 4.79 5.75 4.57 5.20 5.19 2.72 5.09 5.15
Met 3.86 4.45 5.56 3.11 6.81 19.03 7.34 10.21 1.30 6.95 9.41 3.18 17.17
Phe 3.51 4.81 3.62 4.44 2.89 3.86 4.07 4.14 3.60 4.04 4.99 4.82 3.72
Pro 3.87 4.48 4.32 5.30 2.73 4.40 7.05 4.14 3.40 4.85 0.01 4.72 4.02
Trp 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Ala 9.60 11.25 7.17 8.42 6.83 7.91 11.08 7.58 13.30 11.51 5.45 11.58 11.45
Arg 3.85 5.11 3.91 4.17 3.30 4.41 4.63 4.82 3.40 5.02 8.00 5.78 4.61
Asp 13.66 12.07 15.41 16.28 7.62 10.20 11.55 9.80 11.90 12.25 11.33 14.03 10.91
Cys 1.72 0.67 1.89 2.01 0.26 0.47 0.70 0.27 0.00 0.98 1.93 0.21 0.45
Glu 17.40 14.85 14.07 14.27 8.91 13.88 13.35 11.01 25.80 14.12 15.47 15.32 10.75
Gly 9.10 9.72 8.73 9.08 5.80 6.82 10.09 9.37 0.00 8.49 5.63 10.95 9.85
Ser 6.01 5.86 7.31 6.42 3.63 5.35 5.62 4.81 4.80 6.26 7.03 6.79 4.97
Thr 7.61 7.15 7.33 7.80 3.19 7.47 5.60 3.75 4.70 7.56 6.23 5.40 5.72
Tyr 2.92 1.99 3.11 2.05 1.91 1.60 1.78 15.96 2.90 1.94 3.61 2.02 2.53
Val 3.73 3.96 3.90 3.62 2.09 3.59 4.02 2.52 4.90 4.03 9.65 2.89 3.07

AA% 3.73 3.35 3.97 2.42 6.38 5.42 9.03 12.58 - 5.02 - 10.59 4.13

115
 Table 4.6: Amino acid profile (AA mg/Σ(AA mg)) * 100, Saccharina species

Saccharina
Literatur
Blue Evolution Commercial e
SA-W-4- SA-W-5- SA-W-6-
B B B SA-W-4-S SA-W-5-S SA-W-6-S SA-P-4-B S—–—-B SA-AK-0-B S—–—-B SA-lit[1] SA-lit[7]-2 SA-lit[7]-7 SA-lit[7]-2 SA-lit[7]-7

His 1.63 1.80 1.36 1.74 1.62 1.26 1.76 1.18 1.13 1.47 4.40 1.70 2.00 1.60 1.70
Ile/Le
u 6.54 6.75 6.19 5.35 5.78 7.20 6.96 5.52 7.08 6.25 7.90 11.30 11.80 12.60 9.30
Lys 6.00 5.74 5.43 5.89 6.27 5.45 6.19 4.98 5.19 5.45 5.90 5.20 5.40 6.00 4.60
Met 6.35 5.29 6.40 6.23 3.19 4.81 0.00 9.40 4.49 4.05 2.40 2.20 2.30 1.80 1.60
Phe 5.21 5.44 5.28 4.36 4.35 5.38 5.45 4.58 5.96 5.10 5.20 5.10 5.40 6.10 4.90
Pro 5.47 5.12 6.04 4.85 5.19 5.05 5.55 5.09 5.65 5.68 4.50 4.50 5.00 4.90 4.40
Trp 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10.8 10.2 10.2 12.5 10.7
Ala 2 8.73 9.41 7 4 8 3 10.09 11.33 8.86 11.00 12.90 6.80 12.30 7.20
Arg 5.52 5.76 6.19 5.02 4.16 5.50 6.29 5.23 5.28 5.59 4.80 4.50 5.20 5.40 4.90
12.0 12.5 12.9 13.5 14.5 11.3 12.8
Asp 1 0 9 7 3 8 5 11.97 11.11 13.63 13.40 12.20 13.40 11.70 11.40
Cys 0.28 0.52 0.40 0.87 2.12 0.52 0.32 0.52 0.49 0.49 13.80 1.50 2.70 0.00 2.70
12.9 15.2 13.3 13.8 14.5 13.0 14.5
Glu 4 0 4 7 2 2 9 14.60 13.00 14.51 5.60 15.70 12.70 13.40 21.20
Gly 8.02 9.03 9.43 8.78 9.27 9.96 9.62 9.18 9.75 9.50 1.60 5.20 6.10 6.30 7.80
Ser 5.42 5.62 5.98 5.76 6.22 5.51 5.40 5.82 5.26 6.18 5.00 4.20 5.70 4.50 4.90
Thr 5.79 5.32 6.37 8.10 6.94 5.02 7.62 6.27 6.33 6.65 5.30 4.90 6.10 4.40 4.70
Tyr 3.77 3.10 1.88 1.57 2.10 3.14 1.99 2.09 3.68 2.91 3.10 3.40 3.90 3.60 3.70
Val 4.21 4.08 3.31 3.78 3.49 4.22 4.68 3.47 4.27 3.68 6.00 5.20 5.40 5.60 5.00

AA% 7.27 5.95 11.19 4.88 3.86 5.14 7.30 7.92 13.42 3.77 - - - - -

116
Table 4.7: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Palmaria and Porphyra species

Palmaria Porphyra
AA B.E. Cml. Cml Lit.
P–—-B P—–—-W PO–—-W PO–—-P PO-lit[1]-- PO-lit[1]-- PO-lit[1]-- PO-lit[8]--

His 1.55 1.56 1.39 1.33 4.10 3.70 3.70 4.10

Ile/Leu 5.70 5.99 5.18 6.71 8.00 7.70 7.50 14.90
Lys 5.05 6.91 6.77 6.00 6.50 6.20 7.20 0.90
Met 10.67 4.63 9.91 0.00 1.30 1.90 1.20 0.00
Phe 4.52 4.23 4.17 4.74 4.70 4.00 4.60 4.70
Pro 4.27 4.67 5.21 5.95 5.00 4.80 4.90 2.50
Trp 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.20
Ala 10.71 10.13 13.42 12.94 11.40 13.00 10.90 5.60
Arg 5.17 6.05 4.72 4.76 6.90 6.30 6.60 2.70
Asp 11.95 11.61 10.59 11.31 10.70 10.90 12.00 5.70
Cys 0.40 0.63 0.37 0.29 10.50 12.60 11.50 1.70
Glu 13.96 12.22 10.68 12.16 7.00 6.20 6.70 2.00
Gly 9.08 10.59 9.69 10.59 1.40 1.40 1.40 8.80
Ser 5.68 5.31 4.61 5.16 5.70 5.40 5.40 5.80
Thr 5.00 8.06 7.70 7.82 5.90 5.80 6.00 11.50
Tyr 3.00 2.31 2.03 4.02 4.20 3.30 3.50 7.90
Val 3.28 5.10 3.56 6.23 6.80 6.70 6.90 9.70

AA% 4.12 4.23 10.78 18.42 - - - -

117
Table 4.8: Amino acid profile (AA mg/Σ(AA mg)) * 100, for Ulva species

Ulva
AA Cml. Lit.
UL–—-B UL–—-B UL-lit[1]-- UL-lit[1]-- UL-lit[1]-- UL-lit[9]-- UL-lit[9]--

His 0.51 1.27 4.00 4.20 8.80 7.80 10.70

Ile/Leu 5.58 5.00 7.30 8.00 0.00 1.10 0.40
Lys 2.50 6.17 5.40 5.50 11.80 12.30 14.20
Met 4.34 3.96 1.80 2.20 6.30 5.60 6.20
Phe 5.25 3.77 4.90 5.60 10.90 9.40 9.00
Pro 4.94 4.85 7.30 4.70 1.50 1.50 1.60
Trp 0.00 0.00 0.00 0.00 6.40 6.10 5.90
Ala 14.04 13.40 9.20 8.40 6.80 5.20 6.70
Arg 5.09 4.67 5.20 6.40 3.30 4.60 3.60
Asp 13.84 13.84 14.60 12.10 1.70 1.40 1.80
Cys 0.38 0.66 13.20 13.50 2.00 2.20 2.10
Glu 13.08 14.48 5.90 6.40 17.20 13.00 12.30
Gly 11.33 10.57 1.30 1.80 3.50 3.10 3.70
Ser 6.75 5.01 5.00 5.50 4.00 3.30 4.00
Thr 6.73 6.61 5.80 5.50 5.00 5.00 7.70
Tyr 2.05 1.85 2.50 3.50 3.60 4.30 5.30
Val 3.62 3.90 6.50 6.40 3.70 3.70 4.20

AA% 10.26 5.77 - - - - -

118
Table 4.9: Average amino acid content (AA mg/ Σ (AA mg)) * 100 for blue evolution and commercial samples separated by phyla
and species where more than two samples were measured

Chlorophyta Rhodophyta Ochrophyta

Ula Palmaria Porphyra Laminaria Alaria Saccharina
Avg stdev Avg stdev Avg stdev Avg stdev Avg stdev Avg stdev

His 0.89 0.54 1.30 0.37 1.36 0.04 1.19 0.48 1.46 0.21 1.50 0.25
Ile/Leu 5.29 0.41 5.29 1.00 5.94 1.08 6.16 0.79 5.06 0.78 6.36 0.66
Lys 4.33 2.60 6.03 1.24 6.38 0.54 5.46 0.05 5.62 1.36 5.66 0.43
Met 4.15 0.27 10.90 8.87 4.96 7.01 5.46 2.39 7.55 5.16 5.02 2.46
Phe 4.51 1.05 3.97 0.36 4.45 0.40 4.90 0.39 3.92 0.59 5.11 0.52
Pro 4.89 0.06 4.34 0.46 5.58 0.53 5.81 0.97 4.54 1.25 5.37 0.37
Trp 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Ala 13.72 0.45 10.79 0.93 13.18 0.34 11.17 1.46 8.73 1.72 10.31 1.16
Arg 4.88 0.29 5.33 1.01 4.74 0.03 4.94 0.62 4.27 0.59 5.46 0.61
Asp 13.84 0.00 11.26 0.50 10.95 0.50 12.64 0.37 12.07 2.93 12.65 1.07
Cys 0.52 0.20 0.54 0.13 0.33 0.06 0.78 0.48 1.00 0.75 0.65 0.54
Glu 13.78 0.99 11.49 1.03 11.42 1.05 13.79 0.00 13.47 2.55 13.96 0.83
Gly 10.95 0.54 10.22 0.52 10.14 0.64 9.48 0.97 8.59 1.49 9.25 0.55
Ser 5.88 1.23 5.14 0.24 4.89 0.39 5.45 0.09 5.63 1.10 5.72 0.33
Thr 6.67 0.08 6.89 1.65 7.76 0.08 6.32 0.16 6.24 1.84 6.44 0.95
Tyr 1.95 0.14 2.42 0.15 3.03 1.41 2.55 0.25 3.92 4.90 2.62 0.79
Val 3.76 0.20 4.08 1.44 4.89 1.89 3.90 0.18 3.43 0.72 3.92 0.44
n 2 2 2 2 8 10

119
4.4 Conclusions

For both Saccharina and Alaria, protein yield is considerably higher after alkali treatment

(2.5 N NaOH) compared to enzymatic treatment. A 2.5 N NaOH treatment decreases the

biomass water-holding capacity of the insoluble materials and increases the protein and

non-protein material solubility. The mass and properties of the insoluble fractions were

affected by the type of solvent. The large amounts of NaOH needed to reduce the

biomass protein sufficiently were associated with neutralization costs and salt disposal.

Economic analysis of the process inputs would be needed to justify the use of 2.5 N

NaOH. This analysis would also require more details about the component distribution in

the resulting fractions. The amino acid analyses showed, there was low sample-sample

and phyla-phyla variability among all amino acids except Met. The observed variability

present for Met should justify further investigation into macroalgae amino acid and

digestibility since it is an EAA for humans and of economic importance to animal feed.

120
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 5. Chapter 5 Experimental fractionation system including
a process model, experimental procedure, and
associated mass balances used for novel food
evaluation
Abstract

With a growing population and many unsustainable food practices, innovation in food

systems is needed. The world is full of organisms that could benefit humans and the earth

through cultivation for food. To make novel sources of nutrients more appealing to

consumers, fractionation processes can be used to extract proteins for development into

high-value ingredients. Development of non-food products such as biofuel or animal feed

as co-products were also considered by investigating the remaining biomass composition.

The objectives were to create an experimental fractionation system with associated mass

balance for component quantification after fractionation and to employ this system to

improve the understanding of macroalgae and BSFL fractionations. Standard operations

included extraction, separation, and drying while defatting, desalting, and other process

parameters were studied as explanatory variables. Solids, moisture, and protein were

quantified experimentally to characterize process parameters. Explicitly defined metrics

combine these values with mass balance relationships to normalize across various process

parameters and are used to inform future scale-up and valorization.

The experimental fractionation system was found to be repeatable with 95% confidence

intervals for mass balance derived metrics for moisture and solids balances having a

maximum range of 6%. The solids component was the most variable and may require

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updated procedures. The experimental fractionation system identified multiple

explanatory variables that have minimal effect on process metrics of interest while

identifying that the NaOH concentration and exposure time influenced the extraction and

separation significantly. NaOH continually extracts protein as time extends while

reducing the total mass and water-holding capacity of the unextracted biomass. SDS-

PAGE of the resulting extracts shows how the proteins degrade over time in NaOH. An

unbiased comparison of biomass and process parameters was achieved through an

experimental fractionation system employing mass balance relationships that provides a

robust characterization of both the extracted and unextracted biomass.

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5.1 Introduction

Regional human cuisine has consistently evolved to meet the needs of communities with

the nutrient sources available locally. Historically and presently, nutrition availability is

often insufficient, not meeting the needs of many people across the globe. Scientific

advancements and improved agricultural practices have allowed food production to keep

up with population growth (Johnson, 2000). However, a significant amount of the food

produced globally is lost along the supply chain or at the consumer level (ENEP et al.,

2021), while many people lack sufficient access to the food that is produced leading to

hunger (O’Hara & Toussaint, 2021). The available land and resources to produce new

food are finite leading to limitations in our growth (Utuk & Daniel, 2015). Exacerbating

these challenges are the effects of climate change causing higher temperatures and

dramatic weather events reducing crop yields (Hasegawa et al., 2018; Tito et al., 2018).

The global food system must continue to innovate to provide sufficient nutrients for

human populations in the face of these challenges. It must do this in a manner that is not

only resilient in the face of these challenges but also sustainable such that environmental

damage is reduced during the process.

The agriculture sector of the global economy is responsible for approximately 20% of

global greenhouse gas emissions (GHGE; Bezner Kerr et al., 2022), while up to 10% are

associated with emissions caused by food waste decomposing in landfills (ENEP et al.,

2021). Along with contributing directly to climate change, food production is associated

with other environmental impacts including a reduction in biodiversity, contamination,

and eutrophication of local waterways, destruction of marine ecosystems, and

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deforestation (Recanati et al., 2015; Thrupp, 2000; Withers et al., 2014). Furthermore,

sustainable systems must consider balancing economic and social aspects with the goal of

equitable food distribution and supply chains (Muscat et al., 2020). To address these

challenges, this work focuses on two emerging sustainable raw materials and the

development of a generalized experimental approach for protein extraction and biomass

fractionation.

The remaining sections in this introduction provide context for the experimental work

through a targeted literature review including (1) a justification for the two novel

biomasses used for experimentation, (2) a discussion of sustainability-driven food

development, and (3) an overview of the generic and systematic fractionation system

developed for this research.

5.1.1 Emerging raw materials for sustainable food development

Insects, and algae (macro- and micro-) are considered foods of the future due to

characteristics expected to lead to improved resiliency and sustainability in the food

system such as low land and water use and minimal net GHGE associated with their

cultivation and processing (Parodi et al., 2018). These classes of organisms are highly

diverse. Macroalgae (seaweed) are made up of more than 6,500 known species

worldwide (Garza et al., 2005). Microalgae are even more numerous and diverse with

estimates of 200,000 to 1 million species worldwide (Koyande et al., 2019; Matos, 2019).

While only a small number of these species have been cleared by governmental agencies

for sale as food (Barros de Medeiros et al., 2021), this diversity represents an immense

potential for novel food discovery. When considering insects, estimates suggest there are

between 1,700 and 2,100 different edible species consumed by humans around the world
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with a similarly small group being regulated currently (Govorushko, 2019). The diversity

within these classes of organisms represents a potential for mankind to be able to produce

novel sustainable biomolecules for food as well as other applications such as feed, fuel,

plastics, pharmaceuticals, and cosmetics (Morin-Crini et al., 2019; Vanthoor-Koopmans

et al., 2013). One insect and two macroalgae were analyzed as part of the present research

during the development of a generic and systematic fractionation experiment.

The insect used was the Black Soldier Fly larvae (Hermetia illuscens; BSFL) acquired

from a North American company that produces animal feed. BSFL enhances food

systems by providing a link in circular economies by recycling waste into homogenous

biomass for feed and other byproducts, (Cadinu et al., 2020; Ojha et al., 2020). While the

BSFL is not recognized as food in the United States or Europe it is being studied due to

its environmental benefits and is currently used as animal feed and pet food. The Black

Soldier Fly has been studied as feed for swine (Veldkamp & Bosch, 2015), poultry

(Cullere, et al., 2016; Schiavone et al., 2017), and fish (Belghit et al., 2019; Irungu et al.,

2018) with many promising results. This previous research represents a strong

foundational knowledge of the rearing practices and nutritional profile of the BSFL.

Their nutritional aspects suggest BSFL has the potential to be a valuable stock for human

foods and ingredients due to an appealing vitamin and mineral content, especially

calcium, and the presence of all essential amino acids (Wang & Shelomi, 2017; Finke,

2013; Müller et al., 2017; Spranghers et al., 2017). Many insects share appealing

nutritional qualities while crickets have been sold in food products in the USA

(Lähteenmäki-Uutela et al., 2021). BSFL were included in these experiments for their

remarkable ability to break down and digest organic material. This leads to high
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cultivation rates and provides a more sustainable alternative to landfilling and

composting for the high levels of food and animal waste produced globally (Perednia et

al., 2017). This high efficiency is also expected to help the land use challenges, for

instance, one acre of production space dedicated to mealworms production, a similar

insect to BSFL, can yield approximately eleven times the protein from the same land

dedicated to cattle production (Do et al., 2020; Oonincx & de Boer, 2012). Previous

research has found differences in composition and fatty acid profiles when BSFL and

other insects are fed on diets of differing compositions (Barragan-Fonseca et al., 2017;

Spranghers et al., 2017; St-Hilaire et al., 2007; Yoong et al., 2016), providing a challenge

for producers hoping to use the BSFL as both a waste reduction strategy and a feedstock

for a novel product. This reality of variable biomass composition exacerbates the

challenges discussed with high biomass diversity and increases the need for a systematic

way for fractionation evaluation. A brief discussion of macroalgae will reveal similar

benefits and challenges.

Commercial macroalgae samples were acquired to perform screening experiments and to

better understand the limitations of the experimental fractionation system under

development. Macroalgae are considered sustainable sources of nutrients like insects, but

for different reasons. Both sources require minimal land usage, while macroalgae

typically requires minimal water when grown in an ocean. These organisms may produce

a reduction in GHGE over traditional foods. Insects prevent carbon emissions when fed

waste diverted from a landfill but often require energy to maintain growth conditions and

thus are associated with a carbon footprint (Goldstein et al., 2017; Rust et al., 2020).

Macroalgae are photosynthetic and capture CO2 directly from the atmosphere. Food from
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macroalgae starts with a significant negative carbon footprint, but the retrieval,

processing, and transportation all contribute to a net positive carbon product. Both insects

and macroalgae have the potential for their products to become carbon negative as

electrification increases and fossil fuel use is minimized (Rinker et al., 2021). Macroalgae

also provide broad environmental benefits to the local areas where cultivation takes

place. While macroalgae grow, they continually filter the local ocean water removing

organic and nitrogenous compounds, but they will concentrate inorganic molecules such

as iodine and heavy metals like arsenic. While very few reported acute illnesses have

been attributed to fresh seaweed consumption, negative long-term effects have been

reported (Cheney, 2016). Hazards are a reality in the production of many foods and

should be taken seriously. When fractionation processes are conducted there is a risk that

heavy metals or carcinogens are concentrated, thus continual monitoring will be needed.

The presence of regulations and increased monitoring at these scales will likely lead to

safer products than fresh seaweed due to quality control practices. Their ability to filter

the water allows seaweed farms to be strategically placed to combat high nitrogen levels

associated with farming runoff (Murphy et al., 2015). Furthermore, during their growth,

seaweeds may provide temporary shelter for marine life, rejuvenate local ecosystems, and

eventually lead to increased fish stocks among other benefits (Bren Smith, 2019).

Seaweeds and insects can provide unique and important roles to benefit the environment

so increasing their demand as food or another material is considered advantageous. Like

insects, the composition of farmed seaweed is dependent on factors during cultivation.

Changes in season are the major driver of composition variability (Renaud & Luong-Van,

2006).
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The previous discussion outlines the benefits of future food sources, specifically BSFL

and macroalgae, in terms of their environmental impact and nutritional benefits.

Additional reviews of these topics are available in the literature (Anankware et al., 2018;

FAO, 2021; Henchion et al., 2017; Hurtado et al., 2019; Kouřimská & Adámková, 2016;

Matufi & Choopani, 2020; Parodi et al., 2018; Smetana et al., 2021; Torres-Tiji et al.,

2020), however cultivation of these raw materials is just the starting point for evaluating

a novel food product.

5.1.2 Sustainability-driven food development

Macroalgae and insects will not likely become common food products in their raw or

minimally processed (dried and/or ground) forms. Even in areas such as southeast Asia

where entomophagy has strong historical and cultural relevance, younger generations are

increasingly rejecting insects as their food (Müller, 2019). Even though insects are more

sustainable than many alternatives, their benefits are not realized because demand is

decreasing. Researchers in western cultures, where entomophagy is less common, have

studied this issue in the hopes of promoting insect foods. A common finding among the

investigations is that consumers are less turned off by familiar products that contain

insects than by foods that include whole insects. (Lammers et al., 2019; Mishyna et al.,

2020; Sogari et al., 2018). These findings provide one strategy for increasing the

sustainability of the food system; essentially make food products out of the most

sustainable materials possible but make them look familiar and appealing. An additional

strategy is to educate consumers about the benefits of their new foods; research does

suggest that better information about the positive attributes (sustainability, nutrition, and

safety) of algae may encourage consumption (Lähteenmäki-Uutela et al., 2021; Palmieri

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& Forleo, 2021). The current epitome of familiar food mimicry combined with successful

educational marketing is the plant-based meat revolution sparked by Impossible Foods ™

and Beyond Meat™. They have created burgers and other products that resemble meat

without the need for animals. Both burger products are associated with lower

environmental impacts than their animal-based counterpart, according to life cycle

assessments (LCA) performed on the products (Heller & Keoleian, 2018; Khan et al.,

2019). The key difference between these burgers and previous veggie burgers is how

much they taste and feel like the meat version. If their success continues and they can

displace a portion of the future meat demand from the less sustainable animal-based

products, they will be positively affecting the sustainability of the food system overall.

Successful food analogs require ingredients derived from raw materials after refinement.

Currently, meat analogs are made from soy or pea protein which have benefited from

significant academic and industrial development over the years (Sumner et al., 1981;

Yasumatsu et al., 1972). To achieve the potential environmental benefits of novel food

materials, ingredients with similarly favorable properties must be achieved. An additional

strategy for sustainable food development is to efficiently produce ingredients that

exhibit favorable properties. Efficient production of food ingredients is necessary to

capitalize on the environmental benefits of novel raw materials. The field of bioprocess

engineering provides an established approach to evaluating processes in terms of

efficiency and more broadly, sustainability (Carvalho et al., 2006; Jiménez-González &

Woodley, 2010). These authors discuss important considerations for sustainable

processes. At the systems level, holistic LCA evaluations are necessary but when

focusing on the process, the authors point to the importance of maximizing process
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efficiency as an approach to enhance sustainability. In the context of food ingredients and

in particular protein ingredients, an efficient process must achieve high yields and

desirable physicochemical properties. An ingredient that does not express desirable

properties will have limited success during its development into familiar foods.

Meanwhile, low yields or poor separation will result in low profitability and high waste.

During the development of a food ingredient, identifying product properties facilitates

desirability, but to achieve sustainability, efficiency for the overall biomass must be

considered. Sustainability is driven by life cycle thinking (Jiménez-González & Woodley,

2010); so, the final strategy to develop sustainable food is to consider the whole biomass

during processing. Previous research aimed at achieving an efficient biorefinery system

from biomass can be used as a model for identifying the utility of the materials that do

not find success in the food industry. Biorefinery is an approach to fractionation that

results in a biofuel product along with other materials suitable for the food, feed,

chemical and cosmetic industries and is studied as an approach to valorizing

biomolecules from biomass (Cruce & Quinn, 2019; Greene et al., 2020). Biorefinery

studies have focused on a bulk macromolecule such as BSFL studies aiming to optimize

lipid extraction for biofuel production using response surface methodologies; with little

focus on other chemical constituents of the BSFL (Su et al., 2019; H. Wang et al., 2017).

Biorefinery research has also characterized fatty acid and mineral profiles to understand

the value of Ulva biomass (Bikker et al., 2016). When animal feed was the subject of

previous literature, protein quantification and amino acid profiles are reported; research

that is available for BSFL (Huang et al., 2019) and many macroalgae genera (Mouritsen

et al., 2018; Tabarsa et al., 2012). Alternatively, broad assessments of up to six

133
potentially valuable fractions have been conducted on Ulva (Prabhu et al., 2020). These

diverse attempts at fractionation point to a need for a more systematic approach. For bulk

macromolecules or specific smaller molecules, successful quantification and monitoring

can be used to establish material flow analyses which are critical for future LCA-type

analyses (Allesch & Brunner, 2017; Guo et al., 2021). Thus, determining the

composition of the biomass and downstream fractions is a major focus for the following

generic and systematic experiment.

5.1.3 Generic and systematic fractionation system for food ingredient development.
A generic experimental system to evaluate fractionation must be sufficiently versatile to

handle a wide array of factors including different inputs and processing conditions. As

discussed in the previous section this experiment must also effectively evaluate a wide

array of response variables including component quantifications and fraction properties.

The implementation of systematic approaches to factor selection and fractionation

evaluation is an important step in accelerating the adoption of novel materials. Evaluating

fractionation processes is no different than evaluating any process where an experiment is

used to determine the effects factors have on response variables by assessing variability.

The two main approaches to process evaluation include 1) identifying the factors that

influence response variability (i.e. sensitivity analysis) and 2) optimizing factor

parameters for a specific application (Uy & Telford, 2009). The purpose of the present

work is to create an experiment that can evaluate relevant factors associated with biomass

fractionation. This will ensure that future optimization results are “global” and robust

within reasonable bounds (Nakai, 1981). Many insects and macroalgae species, as well as

most emerging biomasses, are not positioned for optimization evaluation due to a large
134
and mostly unknown experimental design space controlling their fractionation. The

experiments described in this research identify and explore this design space to facilitate

future experimentation. As described previously, fractionations and biorefinery systems

attempt to produce materials for a wide range of applications. To understand the impacts

of the processing factors, a similarly wide-ranging set of evaluations or response

variables are needed.

Fractionations are made up of multiple operations and are controlled by multiple

parameters where each output may be used for a specific application or go on to be

processed further into an ingredient or novel product. The inputs and outputs to the

experiment are biomass as defined by Vassilev et al., (2010) as a “contemporaneous

(non-fossil) and complex biogenic organic-inorganic solid…”. A definition that can

apply to all raw materials used for food, feed, and biofuel. This overlap has caused a

dilemma at the intersection of agriculture and bioenergy concerning the handling of raw

materials. A balance among biomass applications is important but experts in the field

point to food as the priority (Muscat et al., 2020). Identifying experimental procedures

that allow for evaluations of all outputs across multiple applications is an important

quality for this experiment to achieve.

Creating a generalized experiment requires the synthesis of a broad array of factors and

evaluations along with considerations for their time and resource use. The long-term goal

is to maximize the number of experimental evaluations, i.e. response variables, conducted

on the resulting fractions from each run. If selected properly, each response variable

would provide additional insight into the value of one or more fractions. Meanwhile,

135
identifying the most fundamental analytics, those relevant to multiple applications, is an

essential step in building a generalized approach. Focusing on fundamental evaluations

while understanding long-term goals of optimization will result in generic and systematic

experiments. The following three sections will discuss operations included in the

fractionation process along with the evaluations considered and eventually used for

experimental evaluation.

5.1.3.1 Identifying fractionation operations

A fraction enriched in proteins will be the main target of this food-focused fractionation

experiment but understanding the composition of all output fractions is also a major goal.

There are numerous examples of fractionation and protein extraction processes and

protocols described in the literature for a wide array of biomass types (Barka, Amira,

Francis, et al., 2018; Boye et al., 2010; Bußler et al., 2016; Cavonius, 2016; Cavonius et

al., 2015; Chiong et al., 2016; Kulkarni & Nikolov, 2018; Miron et al., 2019; Möller et

al., 2022; Oliveira et al., 2012; Smets et al., 2020). Identification of the fundamental

operations most common among this diverse collection of literature, was needed to

synthesize a generalized fractionation protocol. The core operations controlling protein

extraction were identified as mixing with an aqueous solution and separation. Together

mixing and separation facilitate the fractionation of a biomass through the extraction of

protein and other soluble materials from the remaining material (pellet in the case of

centrifugation). Mixing and separation are ubiquitous in bench-scale and industrial-scale

operations. Separation techniques such as centrifugation and filtration have been shown

to have sufficient scalability (Cui, 2005; Maybury et al., 2000). However, filtration

presented additional challenges for the development of mass-balance relationships due to

136
the material and moisture collected in the filter, especially at small scales. For this reason,

the experiments described below used centrifugation for separation. Centrifugation itself

was not identified as a major variable of interest, instead, the mixing step where various

extraction aids (solutes) and conditions (time, temperature, mass: vol ratio, etc.) were the

main focus of the experiment (see Table 5.2 for active variables in the present study).

Using standard laboratory equipment, mixing can be conducted in tubes as small as 1.5

ml centrifuge tubes or up to 4 L flasks before requiring what would be considered pilot-

scale equipment. Centrifuging and otherwise clarifying large batches presents additional

challenges due to equipment availability. There are no bench-top centrifuges that can

separate large volumes in a reasonable time. Centrifugation was limited to 50 ml tubes or

525 ml bottles which are common in many labs and now serve as the experimental unit

for this fractionation experiment.

Drying via freeze drying (with no additional active variables) was conducted for all

experimental runs as a way to safely store samples for further analysis such as SDS-

PAGE. Drying of protein ingredients as an operation has been studied in the past

(Claussen et al., 2007) and will need to be evaluated during scale-up. Extraction and

separation process efficiency should be established before drying is evaluated as drying

influences physicochemical functionality more than mass distributions. Drying also

provides essential information needed to establish mass balance relationships.

Variable operations, those not conducted for each run, include defatting before mixing

and desalting. Preliminary experiments and literature searches suggested that defatting

has a significant influence on the properties of the outputs as well as on the sustainability

137
of the process overall (Lie-Piang et al., 2021; Ursu et al., 2014). Defatting is also

important for runs utilizing NaOH to reduce saponification. A desalting step was added

out of necessity as NaOH and high salt conditions required neutralization and/ or solute

removal. Dialysis was used in the present study to remove material smaller than 1 kDa

which mostly includes salts and sugars. It was an effective, user-friendly, and low-cost

solution to desalting in this experiment. Optimizations before scale-up may use

alternative chemical (precipitation) or physical (ultrafiltration) techniques to separate

salts and other small molecules (Abdollahi et al., 2019; Alonso-Miravalles et al., 2019;

Boye et al., 2010; Fetzer et al., 2019; Veide Vilg & Undeland, 2017; Wingfield, 2016).

Defatting via Soxhlet and desalting via dialysis were each considered as binary process

variables i.e. optional steps.

In summary, the fractionation process identified for the generalized wet fractionation

experiment contains five operations. Sequentially, the first operation is defatting and will

be an optional factor. Mixing and centrifugation create biomass fractionation. These

operations are the core of the experiment with multiple variables identified to augment

the mixing step. Desalting may follow separation to remove small molecules from the

aqueous fraction to concentrate proteins and is also optional. Drying is the final operation

to prepare fractions for storage and further analysis. These five operations make up the

fractionation process detailed in the Methods section, Figure 5.2.

5.1.3.2. Fractionation evaluation and practical limitation

Each evaluation performed on a fraction requires a portion of its mass which creates a

physical limitation on the number of analyses that can be performed. These limitations

138
can be overcome by increasing sample mass, but this is associated with tradeoffs such as

reduced throughput and replication plus additional experimental costs. Furthermore, for

many novel biomasses, the sample mass available may be small requiring strategic

analysis. In other cases, the state of an output fraction is not conducive to a particular

quantification technique. Standard protocols should be used for constituent

quantification, when possible, but sometimes modifications are needed. A

complementary approach is to utilize mass-balance relationships to determine indirect

component mass values. Mass balance relationships have been underutilized in extraction

research in the past. They were used to calculate solids and moisture content for extracted

fractions (Araújo et al., 2018), an application expanded upon in the present research.

Collecting data to evaluate process efficiency is the priority when allocating fraction

mass for evaluations. Process efficiency will be evaluated with “process metrics”, a

collective term for ratios that normalize fractionation mass and component mass

outcomes, such as yield or purity. These ratios may be comprised of total fraction masses

and specific chemical constituents, such as protein. General definitions for different types

of metrics are described in Sec. 5.2.1.5. While these definitions allow for a many metrics

to be conceived, experimental limitations required that specific metrics of interest be

identified; these are presented in Sec. 5.2.1.6. Once a satisfactory assessment of the

process efficiency can be established, additional fraction properties may be assessed to

better valorize one or more of the output fractions.

The general approach to property evaluation was to build from fundamental to specific

while considering sample mass requirements, preparation time, and experimental cost.

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After quantification of bulk component masses (protein, lipids, carbohydrates, ash,

moisture), previous food, feed, and fuel research has aimed for higher resolution

compositional quantification. A fatty acid analysis is important for animal nutrition and

may determine the biomass's suitability for alternative applications. However, in the

present research fatty acid profiling was considered a low priority, due to the presence of

past knowledge (Ewald et al., 2020; Tabarsa et al., 2012), highly efficient commercial

procedures already in place (Russin et al., 2011), and a high variability depending on

biomass cultivation conditions (Ewald et al., 2020; Renaud & Luong-Van, 2006).

Mineral profiles were considered a low priority as well but should be given more

attention for BSFL and macroalgae fractionation in the future. The literature has regularly

reported on biomass mineral profiles for insects and macroalgae (Finke, 2013; Meng et

al., 2022; Parjikolaei et al., 2016; Tabarsa et al., 2012; Wang & Shelomi, 2017) but

typically these types of analyses are less available on a resulting fraction after a

separation (Bikker et al., 2016). Technical expertise is required for performing mineral

profiling which increases the cost substantially. Mineral profiles are similar to fatty acid

profiles in that they also depend on cultivation (Ewald et al., 2020; Renaud & Luong-

Van, 2006). The variability indicates that the fatty acid and mineral profile are not

fundamental attributes of the biomass and will need to be continually monitored in

outputs after a process is established industrially. From the perspective of a food-first

fractionation, lipids and minerals are less likely to drive valorization, instead,

carbohydrates and proteins drive biomass value (Sadhukhan et al., 2019).

The carbohydrate components are not the focus of the experimental work presented

despite their potential to add value. The primary reason is that proteins are in greater
140
demand from a global food security perspective (de Boer & Aiking, 2011). Also, current

protein sources such as animal-based products are more damaging to the environment as

discussed in the first two sections. The main carbohydrate in insects is chitin which has

been identified as a molecule with promise in multiple applications (Tharanathan et al.,

2010). However, chitin makes up a small portion of the BSFL except for in its latest life

stage (Rehman et al., 2022) making it a poor candidate to drive material utilization for

circular economy concepts discussed previously. Carbohydrates are often highly specific

to macroalgal genera; thus, their recovery may require deliberate protocols or specific

downstream processing (Stiger-Pouuvreau et al., 2016). While some specific applications

exist for macroalgae carbohydrates, it will be assumed that the bulk component will be

utilized as a biofuel feedstock (Kawai & Murata, 2016). This leaves the protein

component as the main focus of the present research; however, limitations must also be

considered.

5.1.3.2.1 Evaluation of the protein component

Full evaluation of the protein component requires dynamic analysis through application-

specific properties referred to as functionalities which can be nutritional or

physicochemical (Foegeding et al., 2017). Decisions about the inclusion of a particular

functionality evaluation will follow the same strategy where fundamentals were

prioritized over specifics. Quantification of the total protein in resulting fractions and the

effect of processing parameters (explanatory variables) on the protein efficiencies of the

process is the first priority. This can be done with minimal biomass requirements to

potentially narrow down variables of interest for more robust assessments of

functionality. Quantifying bulk protein and amino acid profiles requires minimal sample

141
size making them feasible from a technical perspective. Determination of bulk protein

content using spectrophotometric analysis requires minimal sample volumes and this

approach is described in the Methods (Sec. 5.2.1.4.3). Amino acid profiles were not

completed for the present study due primarily to analysis cost. Once amino acid profiles

are completed the next step is to determine protein digestibility to better understand the

nutritional quality of the extracted proteins. Protein digestibility of bulk protein has been

investigated in BSFL due to its current use as an animal feed (Cullere, Tasoniero,

Giaccone, Miotti-Scapin, Claeys, de Smet, et al., 2016; Do et al., 2020; Huang et al.,

2019; Marono et al., 2015; Mohamad-Zulkifli et al., 2019; Schiavone et al., 2017). These

studies provide a promising base for future human studies. Early investigations into in

vitro digestibility of Ulva and Gracelaria protein isolates have been conducted (Kazir et

al., 2019). In vitro analyses are used to gain preliminary information and to avoid the use

of live animal testing. Once bulk protein and nutritional aspects are sufficiently

understood, physicochemical properties and functionalities require significant

investigation to facilitate product development.

Physicochemical functionalities are critical to valorizing protein ingredients. The protein

content, amino acid profiles, and protein profiles will all influence how more dynamic

functionalities present themselves in protein ingredients. In the present study, sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized as a

fundamental assessment to characterize a protein fractions’ molecular weight distribution.

SDS-PAGE has also been used to monitor protein hydrolysis (Alarcón et al., 2002).

Identification of unique proteins due to certain processing conditions or observations

about hydrolysis can be used to hypothesize differences in functionalities for later

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evaluations. SDS-PAGE can be conducted with very small sample quantities making it

ideal for early-stage analyses.

As processing conditions are narrowed down and fractionation volumes are scaled up,

functional evaluation will be investigated more deliberately. Three critical

physicochemical functionalities will be discussed briefly; however, this is not meant to be

an exhaustive list and specific analyses based on desired applications will need to be

identified. This would include emulsification assessments designed to determine the

amount of oil that can be incorporated and stabilized in a protein solution (Sengupta et

al., 2019; Teuling et al., 2019), and foaming assessments which are designed to evaluate

a protein solution's ability to hold air (Amagliani et al., 2021; Binks et al., 2007;

Schwenzfeier et al., 2013). Both emulsification and foaming require substantially larger

sample sizes than most quantification techniques. When performing these

physicochemical functionalities, the influence of protein concentration, pH, and salt

concentrations should be included for a complete evaluation. The final physicochemical

functionality included in the discussion is gelation which should also be evaluated across

the conditions previously mentioned (Kharlamova et al., 2018). However, gelation is

more complex and can be induced by enzymes and heat (Tang, 2007). Gelation is the

process of proteins unfolding, aggregating, then coalescing to form a semi-solid and is

regularly evaluated in the literature (Chronakis, 2001; Kharlamova et al., 2018; Maltais et

al., 2005; Mishyna et al., 2019; Xiong & Brekke, 1989). Evaluations of gel formation

also include the incorporation of lipids (Alejandre et al., 2019; Dickinson & Hong, 1995)

and carbohydrates (Benjakul et al., 2002; Chen et al., 2016; Harnsilawat et al., 2006).

Once a gel formation is identified and can be created at a macro-level, texture analysis
143
can be studied to evaluate the gel and compare it to current products (Shand et al., 2007).

These interactions of proteins with other major constituents play an important role in the

protein’s success as a meat analog or confectionary ingredient.

Physicochemical functionality assessment can begin qualitatively at the lab bench while

conducting quantification assessments. For example, foam formation has been observed

after using a vortex to mix a supernatant. Films form due to similar interactions that lead

to gelation and have been observed in small test tubes after drying. These types of

observations can guide evaluations by identifying certain processing decisions that lead

to or inhibit the property. Regardless, to fully evaluate these properties, larger sample

sizes are needed. As discussed previously, large batches are costly or require more

biomass than is available so confidence in the conditions used to create a large batch is

important. Optimizations based on separation yields are one approach to adding

confidence before undertaking a scale-up for detailed functionality assessment. The

Methods section describes a procedure that is appropriate for 50 ml tubes or 525 ml

bottles. The former is ideal for material-based optimizations while the latter should then

be sufficient to conduct preliminary physicochemical analysis.

5.1.4 Rationale and objectives

The previous discussions provide background for the development of the objectives and

experiments presented in this chapter. This chapter provides a uniform experimental

procedure for the evaluations of hypotheses concerning the influence of processing

parameters on material separation and physicochemical properties after biomass

fractionation. The development of a generic and systematic fractionation experiment

required broad considerations from published research and laboratory experiences.

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Multiple experiments were conducted using a consistent approach allowing for the

evaluation of the procedures and the completion of specific objectives. Specific process

metrics were defined which incorporate measurements, mass balance relationships, and

assumptions necessary for normalized evaluations of various factors. The

physicochemical properties of fractions, assessed with water-holding capacity and SDS-

PAGE protein profiles, were coupled to these process metrics. Coupling process metrics

with fraction physicochemical properties in a defined generic and systematic

fractionation experiment provides the means for comprehensive evaluations.

This chapter is organized around two broad objectives relating to a generic and

systematic fractionation experiment. Specific objectives were established to guide the

evaluation of Black Soldier Fly larvae (BSFL) and the green macroalgae, Ulva, against

various explanatory variables associated with fractionation. The following objectives

were addressed during the completion of this research:

1. Develop an experimental fractionation system with associated mass balance

relationships for the evaluation of organic biomass fractionation

1.1. Evaluate the repeatability of the separation and drying operations and conduct a

sensitivity analysis.

1.2. Characterize the deionized water fractionation of the BSFL and macroalgae by

evaluating moisture, solids, and protein separation.

2. Use a uniform experimental approach to gain an improved understanding of

fractionation as applied to macroalgae and the BSFL, as well as the physicochemical

properties of the fractions from the process.

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 2.1. Conduct a sensitivity analysis for BSFL fractionation to identify factors that

influence moisture, solids, and protein separation

2.2. Evaluate the fractionation of Ulva using different extraction solutions including

deionized water, phosphate-buffered saline, and sodium hydroxide.

2.3. Evaluate the effects of sodium hydroxide exposure time, sodium hydroxide

concentration, and high-salt extraction conditions on BSFL fractionation.

5.2 Materials and Methods

5.2.1 Fractionation process analysis

The fractionation process analysis relies on the creation of metrics to monitor mass

change during the process and on physicochemical analysis to understand the properties

of select outputs. The experimental designs used to evaluate process parameters or

decision points are described in the following section (5.2.2) however, the explanatory

(see Table 5.2 ) and response variables (metrics and physicochemical properties) are

outlined within this section. The following sub-sections describe (1) definition of terms,

(2) theoretical equations used to relate inputs and outputs based on mass balance

relationships, (3) a specific description of the experimental process and associated mass

balance relationships relating inputs to intermediates and outputs, (4) experimental data

collection procedures including analytical quantification methods and protein profiling,

(5) general approach to defining process metrics, and (6) specific metrics of interest used

for analyses.

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5.2.1.1 Fractionation terminology

During each run, a biomass sample undergoes a series of unit operations resulting in

fractions, each with a unique chemical composition. The collection of operations defines

the process under evaluation i.e., a multi-step wet-fractionation performed in centrifuge

tubes (50 ml) or bottles (525 ml). Before describing the analysis, commonly used words

require specific definitions for clarity, these include input, output, intermediate group,

fraction, component, separation yield, and process metric. Inputs (I, mass units) describe

any materials, including the biomass, that enter the process at any time. Outputs (O, mass

units) describe any material that exits an operation and does not enter another operation

within the process boundary. Intermediates exist between operations and will be

referenced in intermediate groups. Intermediate group (G) refers to any fraction resulting

from a similar operation and may include outputs. The word “fraction” is used in multiple

contexts. The primary usage will be as biomass fraction (F), defined as a mass value

describing the initial biomass and any biomass-containing intermediates and outputs

throughout the process. Sec. 5.2.1.3 contains a process flow diagram, Figure 5.2,

describing the process in which all fractions are colored gray. In this context, the word

fraction alone will often be used instead of biomass fraction. As mentioned earlier, each

fraction has a unique chemical composition that can be grouped into sets of components.

The chemical species making up a fraction can be grouped into components based on

their similarities and the needs of an analysis. For the present analysis, two sets of

components will be used: a low-resolution set containing moisture and solids (C2, see

Sec. 5.2.1.3) and a higher resolution set to analyze macromolecular species (C1, see Sec.

5.2.1.2). While components represent a mass value, they are typically expressed as mass
147
fractions (X) describing the ratio of a component mass to the fraction mass it is a part of

(Toledo et al., 2018). When mass fractions are referenced, either mass fraction or

component mass fraction will be used to distinguish them from biomass fraction

described above. Different groups or sets of component mass fractions can be defined in

future research if two criteria are met: (1) the sum of all mass fractions in a set equals

one, and (2) each chemical species is grouped into only one component. The term

separation yield (Y) is used to describe the ratio of post-separation biomass fractions

mass or component mass to the total mass or component mass before separation. This

term was used to describe the mass distribution of fractions after the defatting and

separation operations. Separation yields will be used during the mass balance discussion

in Sec. 5.2.1.3 while a more general definition for yield is provided in Sec. 4.2.1.5 during

the discussion of process metrics. Process metrics are normalized mass ratios created

through direct measurement or mass balance relationships. They are used as response

variables in statistical evaluations of the process (see Sec. 5.2.2). Due to the complexity

of fractionation processes, a large number of metrics may be defined depending on the

needs of an analysis. For the experiments described herein, metrics of interest are detailed

in Sec. 5.2.1.6, Table 5.3.

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Table 5.1: Summary of terms used for fractionation analysis
Term Abbreviation Definition/ description
“input”, I Process input, includes the entire process (mass)
“output”, O Process output, includes the entire process (mass)
“intermediate G A group containing all fractions resulting from a
group” similar operation
“fraction”, F Fraction, any input intermediate, or output that contains
the biomass. (mass)
“component”, C1 or A group of similar chemical species making up a
C2 fraction (mass)
“mass fraction” X A mass ratio of a component to the fraction it is a part
of. Subscripts will be used to identify components and
fractions, if necessary. (mass ratio)
“separation yield” 𝑌𝑎,𝑐 A mass ratio of two fractions or a component of two
𝑏 fractions used to describe separation outcomes. “a”
identifies a post-separation fraction; “b”, a pre-
separation fraction; and “c”, a specific component, if
applicable. (mass ratio)
“process metrics” see Sec. 5.2.1.5 Ratios of either fractions or component masses to other
fractions or component masses. General definitions and
specific metrics are described in Sec. 5.2.1.5 &
5.2.1.65, respectively. (mass ratio)

5.2.1.2 Theoretical fractionation mass balance relationships

Before describing the specifics of the process used for experimental evaluation, a

simplified fractionation is presented to establish key concepts. The theoretical

fractionation process presented in Figure 5.1 is based on a dry-fractionation process

described in the literature (Pelgrom, 2015; Schutyser & van der Goot, 2011). This

theoretical process is used to establish fundamental mass balance relationships before

solvents and intermediates are introduced with the wet-fractionation process described in
149
the next section. The material balance relationships will allow for additional fraction and

component quantification.

Figure 5.1: Theoretical dry-fractionation process shows the transformation of a biomass

into n generic fractions after a separation process. Each fraction is labeled with an Fi as
unique identifier, where i is a count of fractions. Inputs (I) and outs (O) are identified and
make up the overall process mass balance.

For any biomass flour input, F0, a fractionation process will create two or more fractions

(Fi, Fi+1, …Fn). The accompanying mass balances associated with Figure 5.1 can be

written in terms of inputs and outputs or as a summation of the fractions on either side of

the separation operation. The process boundary is not explicitly shown in Figure 5.1 but

only contains the separation operation. Since there is no accumulation or intermediate

fractions, the following equation can be defined based on the Law of Conservation of

Mass:

∑𝐼 = ∑𝑂 (5.1)

Where ΣI=the sum of inputs, ΣO= the sum of the outputs.

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To explicitly identify biomass fractions, Fi was chosen as a numbering convention where

i is the fraction count. For the biological systems under consideration, the biomass and

fractions are made up of major macromolecular components (water, w; protein, p; lipids,

l; ash, a; carbohydrates, h). Together the mass fractions (X) of these components can be

grouped into a set, C1 = {Xw, Xp, Xl, Xa, Xh}. By definition, the sum of the components

in this set will equal 1, as illustrated:

1 = 𝑋𝑤 + 𝑋𝑝 + 𝑋𝑙 + 𝑋𝑎 + 𝑋ℎ (5.2)

The set of component mass fractions can be applied to any biomass fraction according to

the following relationship, F0 is shown as an example:

𝐹0 = 𝐹0 𝑋𝑤 + 𝐹0 𝑋𝑝 + 𝐹0 𝑋𝑙 + 𝐹0 𝑋𝑎 + 𝐹0 𝑋ℎ (5.3)

Component mass fractions are also used to relate the distribution of a component in the

outputs to the same component in the inputs as indicated by the following expression

using protein as an example:

𝐹0 𝑋𝑝 = 𝐹𝑖 𝑋𝑝 + 𝐹𝑖+1 𝑋𝑝 + ⋯ + 𝐹𝑛 𝑋𝑝 (5.4)

Based on these relationships, for an input F0, with a set of fractions as outputs, made up

of a set of components C1, the following equation is true for 1-step separation processes:

𝐹0 = ∑𝑘=𝐶1 ∑𝑛𝑖=1 𝐹𝑖 𝑋𝑘 (5.5)

Where k is an index for set C1 and i is an index of the output fractions in the process

described in Figure 5.1. The relationships described in Equation 5.5 provide the basis for

the process analysis conducted in this research. These relationships can be applied to any

151
set of components that meet the criteria outlined in Sec. 5.2.1.1. Equation 5.5 provide the

basis for a multi-step fractionation process to be described.

5.2.1.3 Process description and specific mass balance relationships

Rational for the inclusion of each operation in the fractionation process is detailed in the

introduction Sec. 5.1.3. The overall process established for experimental analysis is

presented in Figure 5.2 and includes five operations creating up to twelve unique

fractions from a ground organic biomass flour. An organic solvent and an aqueous

solution are used which make up the remaining inputs and outputs. The process is

controlled by 5 decisions points (D#) that correspond to explanatory variables described

in Table 5.2 and described in the experimental design section (5.2.2). The remainder of

this section will provide specific mathematical relationships describing material flows

and three explicit assumptions (A#) needed to establish the process metrics defined in

Sec. 4.2.1.6.

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Figure 5.2: Experimental fractionation scheme featuring twelve possible unique fractions
created from five operations with parameters dependent on five process decision points
(D1-5). Each fraction is labeled with an Fi as a unique identifier. Inputs (I) and outs (O)
transverse the process boundary, are identified, and make up the overall process mass
balance. All operations and fractions not identified as input or output are within the
process boundary. Intermediate groups (G1-5) identify groups of fractions that are
outputs of the same operation. Dotted lines indicate operations and associated fractions
that are included as explanatory variables.

Table 5.2: Decision points corresponding to “D” labels in Figure 5.2. Each decision
point represents one or more explanatory variables that may be manipulated during
experiments. The variables and variable types are included in the table.

D Variable description
1 Categorical, Biomass type
2 Binary, Defatted/ non-defatted
Solution variables:
3 a. Categorical, solute type: diH2O, NaCl, NaOH
b. Continuous, solute concentration
Mixing variables:
a. Continuous, biomass: solution ratio, Yr*
4 b. Continuous, temperature
c. Continuous, time
d. Binary, Homogenized/ non-homogenized
5 Binary, desalted/ non-desalted
*
Yr always represents the ratio of aqueous solution volume to F0 even when defatting is
conducted. When defatted weights are recorded, Equation 5.7 is used to determine the Y r

153
The nature of multi-step processes is that intermediates exist as the output from one

operation and the input to another operation. The concept of intermediate groups will be

used to augment Equation 5.5 for single operations when the process inputs (I) and

outputs (O) cannot be used. The fractions making up each intermediate group are

identified in Figure 5.1. Equation 5.6 provides an example of this for the first operation,

defatting:

Fo = ∑k=C1 ∑ni=1 G1k Xi = G1 (5.6)

Where k is an index in the set C1 of macromolecular components, and i is a count of

fractions in G1. The mass balance for the defatting operation appears in Equation 5.6, but

an assumption is needed to relate the individual fractions to one another. Assumption A1

– During the defatting operation, there exists some mass transfer of non-lipid material

into the lipid fraction, F2, as well as a small amount of solvent that is not removed from

either fraction in G1. For the purposes of this analysis, these small quantities of

materials are assumed to be negligible. As the process is scaled or if pressing is used

instead of solvent extraction, this assumption would require reevaluation. Relying on this

assumption, the following relationship between a separation yield (𝑌2, ) is defined as the
0

ratio defatted flour (F2) to the input flour (F0) as described by Equation 5.7:

𝐹2
𝑌2 , = (5.7)
0 𝐹0

Given this assumption (A1) and that the analytical Soxhlet method was used for lipid

determination and defatting (see Sec. 5.2.1.4.1), 𝑌2, is equivalent to the mass fraction of
0

non-lipid materials in F0: (1-Xl ).

154
The mixing and separation operations together facilitate extraction. Since defatting is

optional, I1 will be used to represent the input to mixing; F0 or F2 may enter mixing

depending on D2. Equation 5.8 is based on the relationships described in Equations 5.5

and 5.6 to relate the mass of mixing inputs, to the slurry fraction (F3), and to the mass of

fractions after separation (G3):

I1 + I3−n = F3 = G3 (5.8)

Where I3-n represents the aqueous solution used for extraction.

Similar to how the defatted flour can be related to the initial flour with the lipid fraction,

it is also possible to describe ratios to relate each fraction after separation (G3) to the

slurry (F3). These relationships also apply to a component within a defined component set

such as C1 or C2. The component set C2 includes mass fractions for moisture (w),

biomass solids (bs), and solute solids (ss); C2=[Xw, Xbs, Xss], which equals 1. Solids (s)

will refer to the sum of bs and ss. Using the component set C2, 9 ratios for each fraction

in G3 (low density, wet; supernatant, wet; pellet, wet) and components in C2 are captured

in the following equation:

FG3j 𝑋𝐶2𝑖
Y𝐺3𝑗 = (5.9)
𝐶2𝑖 F3 𝑋𝐶2𝑖
𝐹3

In this context, Y is a separation yield relating a specified fraction (j) of G3 to the slurry

(F3) for a specified component (i) of the set C2. Equation 5.9 can be written to describe

the total fraction weights by omitting the component mass fraction term (XC2). The values

for moisture and solids are determined from the results of the drying operation. During

155
runs where solutes are used, differentiating between solute solids (ss) and biomass solids

(bs) is difficult.

There is a desire to understand the mass of biomass solids in a post-separation fraction

from an economic perspective and to complete mass balance relationships for process

evaluation. There are no rapid cost-effective ways to determine the ratio of solids type, so

an alternative approach based on fundamental chemical solubility is described here to

gain an estimate of the two values. Assumption A2 – The separation operation does not

change the concentration of added ionic solutes in the solution that exists in the resulting

fractions. This implies that the separation yields for the moisture component (w) can be

used for the solute solids component (ss), the following equation defines this explicitly.

YG3iw = 𝑌G3iss (5.10)

𝐹3 𝐹3

Using these relationships, the ss component mass of a post-separation fraction can be

calculated by multiplying these coefficients by the sum of the solute input masses as

shown in the following expression:

𝐹𝐺3j 𝑋𝑠𝑠 = ∑ 𝐼3−𝑛 × 𝑌𝐺3j,𝑤 (5.11)

𝐹3

Where ΣI3-n is the total mass of all solutes added with the aqueous solution and j is used

to identify specific fraction in G3. With the mass of the solute solids determined for each

fraction in G3, the biomass solids (bs) can be determined via difference. The total solids

content (s) of a given fraction is a value that can readily be determined from

experimentation so the following equation can be used to determine biomass solids.

𝐹𝐺3𝑖 𝑋𝑠 −𝐹𝐺3𝑖 𝑋𝑠𝑠

𝑌𝐺3𝑖𝑏𝑠, = (5.12)
𝐹3
𝐹3
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With equations 5.9-5.12, the biomass solids (bs) can be estimated for any post-separation

fraction.

The mass balances associated with the drying operation incorporate the final assumption.

Assumption A3 – The drying process results in no residual moisture in the G5 fractions

and only water is evaporated. Explicitly: G5iXw=0, and G3iXs=G5iXs.This assumption is

used when relating known macromolecular component masses (C1) of a fraction in G3 or

G5 to its counterpart in the other. Separation yields for the drying of each fraction can be

established using the format shown in Equation 5.9.

The final decision (D5) is concerned with the inclusion of a desalting step. This is a

binary variable and is used for NaOH and high salt samples to concentrate protein before

drying. Since there is no quantifiable component that remains constant during desalting

(tie material) it is not possible to relate F7 or F 11 back to F0. Only protein content (or

purity, see Sec. 5.2.1.5) will be discussed in the context of desalted samples. The next

section will describe the experimental procedures used to collect fraction and component

masses.

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5.2.1.4 Experimental data collection of masses and components

Figure 5.3: Data collection flow chart for the extraction, desalting, and drying operations.
Masses and volumes are identified and numbered with W/ V in boxes outlined in black,
processes are numbered corresponding to Figure 5.2 in dark gray boxes, unmeasured
intermediates are named in light gray boxes, and additional analytics are identified in
black boxes nearest the intermediate fraction the analysis is conducted on. Analytical
measurements are identified for moisture content (MC), protein content (via BCA), and
SDS-PAGE.

158
The experimental protocol for mass (W) and volume (V) data collection used to

determine the mass of each fraction and select component masses s are detailed in Figure

5.3. This section will describe the protocols associated with Figure 5.3 while referencing

the mass balance equation presented in the previous section as needed to determine the

masses for each fraction presented in Figure 5.2. The analytical methodology for

component quantification is included in this section as well. The total fraction masses and

moisture, solids, and protein mass fractions were the focus of the present analysis.

The protocol begins with the defatting operation (not shown, see Sec. 5.2.1.4.1) which is

an explanatory variable and not conducted on all runs. Either a whole biomass flour (F0)

or a defatted flour (F2) was used as the biomass, I1 will be used to represent the initial

flour mass regardless of the defatting operation. The initial flour (I1) is determined by

difference:

𝐼1 = 𝑊2 − 𝑊1 (5.13)

The remaining inputs are the aqueous solution including the solvent (water) and variable

solutes. For this bench-scale procedure, solutions were premade on a molarity basis so

separating the mass contribution of the solutes and solvent is required. Solutes mass is

determined first:

𝐼≥4 = ∑𝑖 𝑉𝑠 ∗ 𝑀𝑖 (5.14)

Where I≥4 represents the total solute mass present and the aqueous solution volume, V s is

the volume of the solution used (ml), and Mi is the molarity (mmol/mL) of each solute in

the aqueous solution. Equation 5.14 is then used in the determination of the water weight

(I3) added from the solution.

159
𝐼3 = (𝑊4 − 𝑊3 ) − 𝐼≥4 (5.15)

The approach described in Eq. 5.14 and 5.15 is used to accommodate solutions with

known molarity since chemicals were premade. Next, the slurry (F3) mass was

determined via mass balance (Equation 5.8) as it was not weighed directly:

𝐹3 = 𝐼1 + 𝐼3 + 𝐼>4 (5.16)

Where the initial flour (I1) represents the mass of F0 or F2 depending on D2 (defatting).

This value is the sum of inputs that enter the mixing (lateral shaking 120 RPM) and

separation processes conducted via centrifugation (2,000xg, 4°C, 1 h).

Post-separation fraction mass collection (F[4-6, 9, 10, and 12]), excluding the desalting

process, will be described first. A graduated cylinder with 0.1 ml readability was placed

on a scale and tared. A metal screen, pre-weighed with a weigh boat (W5), was then

placed on top of the cylinder before slowly decanting the aqueous supernatant through

the screen. Additional material that was floating above the aqueous fraction was moved

to the screen which collectively is referred to as “low density” (F4), then the screen was

moved back to the weigh boat and the weight was recorded (W6). The weigh boat,

screen, and low-density fraction was then placed in a fume hood for 2 days then a

desiccator to dry before reweighing (W7). The mass (W8) and volume (V1) of the

aqueous fraction were recorded after the removal of the screen. The moisture content of

the aqueous fraction was determined using a modified moisture content analysis

described (Sec. 5.2.1.4.1) and the dried samples were saved for further analysis. The

resulting solids fraction X5,s was then multiplied by the mass (W8/ F5) to determine the

total extracted solids, F10. The pellet, wet (F6) remained in T1 (W1) after decanting,

160
which was weighed (W9), then freeze-dried and reweighed (W10) to determine the

moisture content. The following equations describe these actions mathematically:

Low-density material:

𝐹4 = 𝑊6 − 𝑊5 (5.17)

𝐹9 = 𝑊7 − 𝑊5 (5.18)

Aqueous fraction:

𝐹5 = 𝑊8 (5.19)

𝐹10 = 𝐹5 𝑋10/5 × (5.20)

Where X5,s represents the solids mass fraction of the supernatant (F5).

Solids fraction:

𝐹6 = 𝑊8 − 𝑊1 (5.21)

𝐹12 = 𝑊9 − 𝑊1 (5.22)

Desalting was conducted via dialysis which results in changes to the fraction moisture

and mass. These changes were not tracked experimentally. Only the protein fraction of

the final dry weight (F11) was measured and will be included as a metric of interest, Sec.

4.2.1.6.

5.2.1.4.1 Moisture, Fat, and solids content of fractions

The moisture content of the biomass flours was determined by methods presented by

Belluco et al., (1983), while fraction moisture content was determined with modification.

Whole flours were measured in triplicate where 2.5-3.5g were measured on pans of

161
known weight and recorded. The pans were then placed in a drying oven set to 110°C for

17 h. After drying the pans were moved to a desiccator for a minimum of 4 h and then

weighed. For the supernatant (F5), the analysis was performed in triplicate after

separation using a vacuum centrifuge. For each replicate, 1 ml of sample was pipetted

into a weighed centrifuge tube, then dried under vacuum at 60 °C for 17 hours. The low-

density materials (F4 ) were dried directly in the pan they were collected in, and for the

low-density materials, the dish was left in a fume hood for 2 days and then place in a

desiccator overnight before reweighing. The pellet fraction (F6) was weighed, frozen then

freeze-dried. The weights of the empty tube (W1) and the dried was vacuum drying at 60

°C for 48 hours were used to determine the dried mass.

Fat content was determined according to (ISO, 1973) using hexane as a solvent in a

Soxhlet extractor. Dried samples from the moisture content analysis were used. After

conducting Soxhlet, the defatted flour was collected from multiple runs and mixed before

being used in the fractionation experiment.

5.2.1.4.2 Protein concentration of liquid intermediates and reconstituted products.

For each sample, the protein concentration of the supernatant, wet (F5, Figure 5.2) was

measured via the Bicinchoninic Acid (BCA) protein assay according to Smith et al.,

(1985) with modification to accommodate for NaOH extraction solutions. For samples

containing NaOH, an acidified phosphate buffer (0.5 M HCl, 0.5 M Na2HPO4) was used

to neutralize NaOH samples between pH 7-8. The buffer and sample were combined in

discrete ratios using trial and error until neutralization was identified. This approach was

taken to reduce the volume and mass changes associated with titration before the

colorimetric assay. A known concentration of bovine serum albumin was used to create a
162
standard curve and absorption measurements at 562 nm were conducted. Dried products

were reconstituted in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl 10

mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and then diluted across the linear dynamic

range, (n=4). All measurements used in the above analyses were the average of, at

minimum, three dilutions within the linear dynamic range. Dialyzed samples were dried

and then reconstituted with a known weight and volume before BCA analysis was

performed to determine protein content.

5.2.1.4.3 Protein Profiles via SDS-PAGE and zymogram

Standard electrophoretic analysis of the samples was conducted with sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with modifications of the

method described by Updike et al., (2006). Samples were dissolved in a dissociation

buffer (sample buffer, 8 M urea, 2M thiourea, 60 mM Tris buffer, pH 6.8, containing 2%

SDS, 15% glycerol, 350 mM DTT, and 0.1% bromophenol blue) overnight a 25 °C with

agitation, then diluted with the dissociation buffer as needed. Approximately 15 μl of

sample dilutions were loaded onto each lane of a 12.5% denaturing resolving gel (30: 0.8,

acrylamide: N, N’- bis-methylene acrylamide) with a 3% stacking gel containing 1 %

SDS. The proteins were resolved at 100 V cm-1 until the dye front reached the bottom of

the gel. Gels were stained with Coomassie Brilliant Blue G-250 overnight and then

destained overnight with 10% acetic acid. Gels were imaged using near-infrared with

automatic exposure with an Azure Biosystems imager (Dublin, CA, USA).

5.2.1.5 General definitions for process metrics

The data collected, as described in the previous section (5.2.1.4), can be processed in

multiple ways depending on the needs of a specific analysis. The present work aimed to
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be as general as possible, but specificity was still required to produce the data analysis

presented in the Results& Discussion section. Explicit definitions for the specific metrics

of interest are presented in the following section (5.2.1.6). This section contains general

definitions allowing for the creation of additional specific metrics as needed. These

general definitions below are based on metrics used in fractionation experiments found in

the literature.

The first metric, yield, refers to a mass ratio of a selected component in a post-separation

fraction to a starting material, in most cases the biomass flour, F0. One exception to this is

for moisture yield where the total moisture in the system is used as the denominator

instead (Soto-Sierra et al., 2018).

𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑜𝑟 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 "A"

𝑦𝑖𝑒𝑙𝑑, % = × 100 (5.23)
𝑖𝑛𝑝𝑢𝑡

Efficiencies are similar to yield, but the denominator is replaced with a specific

component of the initial biomass. This means for the same fraction component, yield and

efficiency will correlate.

𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 "𝐴"
𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦, % = × 100 (5.24)
𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 "A" 𝑖𝑛 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

Where component “A” refers to any measurable component of interest.

Purity refers to a ratio for a single component’s mass in a fraction to the same fraction’s

total mass (Möller et al., 2022; Juul et al., 2021). “Content” will often be used to express

purity in writing i.e., the protein content is x%. Purity/ content is an alternative way of

expressing component mass fractions as a percent. This concept is express in the equation

below where FD refers to a dried fraction in G5, referencing Figure 5.2.

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 𝐶𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑚𝑎𝑠𝑠 𝑖𝑛 𝐹𝐷
𝑃𝑢𝑟𝑖𝑡𝑦, % = × 100 (5.25)
𝑀𝑎𝑠𝑠 𝑜𝑓 𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛, 𝐹𝐷

The water-holding capacity of the pellet (WHC) or remaining, unextracted biomass, is a

physicochemical property similar to a purity metric concerning F6 but describes the ratio

of moisture to biomass solids in the insoluble, wet fraction, see Table 5.3 for specific

equation. The amount of water the pellet holds is an indicator of structural changes to the

biomass and has effects on the extraction of other components into the aqueous phase.

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5.2.1.6 Metrics of interest:

Table 5.3: Process metrics with their explicit definition referring to Figure 5.2 and the
rationale for why each metric was included in the research.

Metrics Equation (Figure 5.2) rationale

Supernatant/ Extract metrics (F5, F10, F11)

𝐹5 𝑋𝑝 Used for economic and

Protein yield
𝐹0 environmental predictions

𝐹5 𝑋𝑝 Used to compare among

Protein efficiency
𝐹0 𝑋𝑝 biomass

𝐹5 𝑋𝑝 Indicator of food value,

Protein purity and if additional
𝐹10 processing is needed

Dialyzed protein 𝐹5 𝑋𝑝
1 kDa retention
purity 𝐹11

Raw protein measure (no

Supernatant BCA F5, BCA measurement influence of volume
measurement)

Moisture yield of the 𝐹5 𝑋𝑤 Determinant for drying

supernatant 𝐹3 𝑋𝑤 energy demand

Pellet/ insoluble biomass metrics (F4, F6)

Non-protein, non- (𝐹4+6 ) − (𝐹4+6 𝑋𝑝 + 𝐹4+6 𝑋𝑠𝑠 )
additives yield in Proxy biofuel metric
pellet 𝐹3 𝑋𝑠
𝐹6 𝑋𝑤 Raw measurement of the
Solids yield in pellet 𝐹3 𝑋𝑤 pellet weight
Water-holding 𝐹6 𝑋𝑤 Indicator of molecular
capacity of the pellet
𝐹6 𝑋𝑠 structure
(WHC)

5.2.2 Experimental designs and statistical analysis

The data reported in the present study was collected as part of multiple smaller

investigations using a uniform experimental set-up with the expectation of post-hoc

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statistical analysis. All computations and data organization to determine metrics of

interest were conducted using MATLAB. As the analyses are described, a distinction will

be made between hypothesis-driven analyses and retrospective analyses. The

retrospective analyses provide information about the repeatability of the system across

various explanatory variables (process parameters). Active explanatory variables are

listed in Table 5.2. Some sub-experiment includes one-factor-at-a-time analysis while

other variables will only be considered as co-variables. This experimental system has

been used for 88 different runs; select conditions were replicated but many runs represent

a unique set of process parameters.

All computations to determine the metrics of interest (Sec. 5.2.1.6) were conducted using

MATLAB. Figures were generated using MATLAB and Microsoft Excel.

5.2.2.1 Analysis 1 – Retrospective process analysis of the experimental fractionation

system.

The purpose of the retrospective process analysis is to determine the magnitude of

experimental error associated with the separation and drying operations and to identify

correlating factors. Mass balance principles, established in Sec. 5.2.1, describes how the

sum of the fractions after separation (G2) must equal the inputs before separation,

represented by the slurry mass (F3). Four mass balances derived equalities (Equations

5.26, 5.28, 5.30, and 5.32) based on Figure 5.2 were identified which relate the total

mass (F3) and select component masses (C2) before and after separation. Equations 5.27,

5.29, 5.31, and 5.33 detail the measurements used to determine the fraction masses, based

on Figure 5.3. The following relationships were used to evaluate the process operations.

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1. “Total wet weight” balance
F3= F4 + F5 + F6 (5.26)
(W2-W1) + (W4-W3) = (W6-W5) + (W8) + (W9-W1) (5.27)

2. “Moisture balance”
F3Xw= F4 Xw + F5 Xw + F6 Xw (5.28)
I3+ F0Xw = (W6-W7) + (W8)R1 + (W9-W10) (5.29)
• Where R1 is the moisture content of the supernatant

3. “Additives balance”
I>3= I>3* Xw,F4 + I>3* Xw,F5+ I>3* Xw,F6 (5.30)
• Where was I>3 determined from the solution recipe
I>3 = (W6-W7) 𝑌𝐹4,𝑤 + ((W8)R1)𝑌𝐹5,𝑤 + (W9-W10)) 𝑌𝐹6,𝑤 (5.31)
𝐹3 𝐹3 𝐹3

• Where I>3 is determined from the solution recipe

• Where Y denotes separation yields described in Equations 5.9-5.11

4. “Dry solids balance”

F3Xs= F9 + F10 + F12 (5.32)
(F0-F1)+I>3=(W7-W5)+W8(1-R1)+W10-W1 (5.33)
• Where I>3 is determined from the solution recipe; and the C1 content of the
supernatant
The “total wet weight” relationship refers to all materials (biomass, solutes, solvent)

added to a run during mixing. The total wet weight was used to evaluate the effectiveness

of mass collection after separation. Mass collection after the drying operation was

evaluated with the “moisture balance” which refers to the moisture component of the

slurry mass, the “additives balance” which refers to added solutes introduced as part of

the extraction solution, and the “solids balance” which refers to the total solids

component of the slurry mass. The methodology for the determination of the moisture

and solids content was described in Sec. 5.2.1.4.1 while the additives determination relied

on the moisture quantification and Equations 5.9-5.11.

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These four relationships were evaluated using JMP’s “distribution” and “fit model”

functions. Four normalized response variables, also referred to as: “difference metrics”

(%), were calculated for each run (N=88) by subtracting the output side from the input

side and dividing by the input side. The values were then multiplied by 100 and

represented as a percentage. The range and 95% confidence interval (CI) were reported

for the four resulting response variables. The four response variables were also evaluated

with ANOVA using the process parameters (Table 5.3) as explanatory variables in a top-

down model. The null hypothesis was that no explanatory variables are significant

(α=0.05). This analysis provides diagnostic information for the mass collection

procedures associated with separation and drying.

5.2.2.2 Analysis 2 – Deionized water fractionation among four biomasses (BSFL, Alaria,
Ulva, and soy)

Deionized water (diH2O) extraction is a special case allowing the system to be evaluated

with fewer assumptions than when solutes are involved. Di extraction was conducted

more than any other process parameter, nearly a third of the total runs, as it was used as a

control/ reference across multiple batches. The analysis of Di samples will be considered

retrospective as well. The response variables of interest include the metrics described in

Table 5.3, except dialyzed purity. Biomass type will be the main explanatory variable

with solution: biomass ratio, vessel size, and temperature are included as co-variables.

While the stated null hypothesis is that all metrics for all biomasses are equivalent

identifying the differences in variability for each metric among biomasses is the main

purpose of this analysis.

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5.2.2.3 Analysis 3 – Retrospective sensitivity analysis for BSFL process decisions

A preliminary sensitivity analysis to identify which factors drive differences in select

metrics describing BSFL fractionation was performed. During the development of the

experimental fractionation system and the completion of independent objectives to

evaluate the use of NaOH during extraction, other explanatory variables were evaluated.

After multiple fractionation experiments, sufficient data were available to compare the

effects of five explanatory variables (homogenization, NaOH, solution volume (ml),

defatting, temperature 18-37 C) and a random variable (batch). A top-down approach to

developing an ANOVA model was used where insignificant (α=0.05) factors were

removed for four metrics (protein yield, protein purity, WHC, and solids yield in the

pellet). The effect size was reported with the F ratio for significant explanatory variables

for the four process metrics. A disclaimer must be made for this analysis as there was not

a deliberate experimental design; however, the value of this analysis is in its ability to

begin narrowing down potential variables for future experiments. All statistical analysis

was conducted with JMP.

A preliminary analysis of NaOH concentration is also included here due to the strong

effect of NaOH concentration on the metrics, identified by the ANOVA. No statistical

analysis was conducted but data are plotted and discussed. All samples have an n=1

except diH2O (n=4) and 0.1 M NaOH (n=3).

5.2.2.4 Experiment 1 – Ulva fractionation with different extraction solution conditions

An independent experiment aimed at producing a preliminary evaluation of Ulva

fractionation under five different extraction solution conditions was conducted. The

170
conditions tested were diH2O at a 1:10 biomass solution (ratio), diH2O at a 1:40 ratio

(osmotic shock), phosphate-buffered saline (PBS), 0.05 M NaOH, and 0.5 M NaOH. The

latter three conditions were also at a 1:10 ratio. The 1:40 ratio and the 0.05 M NaOH

conditions were tested in duplicate while the other three were tested with an n=4. The

statistical analysis treated each condition as a discrete using ANOVA and Tukey Honest

Significant Difference (HSD) post-hoc test to determine differences among groups using

JMP (α=0.05).

5.2.2.5 Experiment 2 – BSFL fractionation with different solutions and versus NaOH
exposure time

Experiment 2 aimed to understand the effect NaOH exposure time and high-salt

conditions (3 M NaCl) had on BSFL fractionation metrics and the extract’s (F11 or F12)

protein profile. The present evaluation of NaOH exposure time was based on earlier

experiments (Sec. 3.3.1) where nitrogen was monitored with time. The experiment to be

described was conducted with a direct protein quantification method, Bicinchoninic Acid

(BCA). The effect of NaOH exposure time was tested using 0.75 M NaOH from 0 to 24

hours with ten-time points total. Metrics of interest (Sec. 5.2.1.6) were evaluated to better

understand protein, moisture, and solids separation after extended exposure to NaOH.

Fractionation runs were considered the experimental unit and each time point was

conducted in duplicate.

A second aim was to evaluate different extraction solutions (diH 2O, 3 M NaCl, and 0.75

M NaOH) on BSFL fractionation metrics and the extract’s (F11 or F12) protein profile.

Extraction of the biomass samples using NaOH was conducted after defatting, while the

biomass samples extracted with water and salt did not include the defatting operation. 3

171
M salt was included based on previous literature showing high protein solubility in BSFL

under this condition (Bußler et al., 2016). Extraction solutions were evaluated using

ANOVA with Tukey HSD, fractionations were conducted in duplicate.

5.3 Results and Discussion:

5.3.1 Analysis 1 – Retrospective process analysis of the experimental fractionation

system
A benefit of conducting fractionation experiments inside centrifuge tubes was the ability

to measure material and fraction masses directly. As the scale of the experiment

increases, the ease of measuring mass decreases along with sample throughput. Thus,

smaller scales were used to thoroughly evaluate fractionation operations. Before

macromolecular components (i.e. protein) were evaluated, moisture and solids balances

across the separation and drying operation were analyzed, as described in Sec. 5.2.2.1. In

theory, the relationships described by Equations 5.26, 5.28, 5.30, and 5.32 apply to all

runs, independent of the initial biomass or extraction parameters. Deviations from zero in

any of the four “difference metrics” (see Sec. 5.2.6.1) indicated the presence of error in

the mass collection procedures associated with either the separation or drying operation.

Some error was expected but it required quantification to evaluate repeatability so future

experiments can be improved.

The response variables (difference metrics), derived from Equations 5.26-5.33 and

described in the experimental design section (5.2.6.1), were plotted in Figure 5.4. The

results of a paired t-tests and ANOVA for each of the calculated difference metrics (total

wet weight, moisture, additive / non-biomass solutes, and solids balances) were presented

in Table 5.4. Eighty-eight runs were included in the analysis of each response variable.
172
The normalized differences of the total mass (“total wet weight”) represent the error in

the mass collection procedures associated with the separation operation. For the “total

wet weight” variable (Equation 5.26), the difference ranged from -2.2% to 6.0%, with

just one value exceeding ±5%. A 95% confidence interval (CI) of -0.66% to -1.2%

suggests that only small differences between the fraction masses collected and the total

slurry mass are expected in future runs. These data suggest the mass collection techniques

related to the separation operation were repeatable.

Next, the moisture, additives, and solids components were analyzed to evaluate the

procedures associated with the drying operation. The moisture analysis follows the same

logic as the total wet weight metric, described above, but was applied to Equations 5.28

and 5.29 and normalized to the moisture component of the slurry. The CI for the moisture

balance was -2.2 to 0.93. The additives balance relies on the same measurements used to

compute the moisture balance but was important to consider since the normalization

factor was different. When compared to the moisture component, the additives CI was

similarly small at -1.6 to -0.062%. These two components suggest repeatability in the

drying operation and related mass collection techniques. The solids balance, normalized

to the slurry solids component, expressed greater variability with a high overall range and

a CI of -0.38% to -6.5%.

Along with studying the raw distribution of each response variable, ANOVA was

conducted to identify process explanatory variables (Table 5.2) that drove variability in

the four response variables. ANOVA factors included biomass type, solute type, volume,

defatting, and solute concentration while batch was included as a random effect. The

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“significant factors” are presented in Table 5.4. The ANOVA suggested that changing

the centrifuge bottle type (vessel) and the biomass influenced the variability as seen in

Table 5.4. The variability among biomasses may be caused by different compositions

that hold moisture differently. Beyond the factors identified through ANOVA, drying

variability might be reduced through other means. Instead of relying on volume and

molarity, the solution should be measured in molality or solutes and water could be added

directly by mass.

Overall, the operations appeared to be under control with the balance of the total solids

exhibiting the most spread and largest confidence interval of any response variable

distribution. The variability may be explained by the number of measurements required

to determine the values and the relatively small input masses being used. Future

experiments may be improved by considering these results.

Table 5.4: Summary statistics for retrospective analysis of mass collection after
separation and drying, N=88. Difference metrics (Sec. 5.2.2.1) were presented as
normalized percentages (fractionC-slurry) C/slurryC)*100). Where C refers to component.

Component Mean Range P value, 95% Confidence Significant factors

interval α=0.05
Total wet -0.92% -6.0: 2.2% <0.0001 -1.2: -0.66% –
weight

Moisture -1.5% -10.3: <0.0001 -2.2: -0.93% Solute concentration

4.3%

Additives -1.1% -9.1: 2.2% <0.0001 -1.6: -0.062% Solution type, Solute
concentration
Total solids 3.5% -31.4: 0.0141 -0.38%: -6.5% Biomass type
44.7%
Vessel volume
1
refers to non-biomass solutes introduced as part of the extraction solution
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Figure 5.4: Retrospective analysis of mass collection after separation and drying (N=88),
Box plot of differences metrics (Sec. 5.2.2.1) for “total wet weight” and components
presented as normalized percentages (fractionC-slurryC) /slurryC)*100). Where C refers to
component.

5.3.2 Analysis 2 – Deionized water fractionation among four biomasses (BSFL, Alaria,
Ulva, and soy)
Deionized water (diH2O) extractions serve an important purpose as an additional means

of validation to the experimental fractionation system and as a baseline control for the

fractionation of each biomass. The diH2O runs do not include added solutes and were all

mixed for 1 hour. The variability in the solids fraction was reduced when only diH 2O

extractions are included, in the analysis (n=22) as seen in Figure 5.5 as opposed to the

results described in Sec. 5.3.1. As expected, the solids difference metric still presents

175
variability (CI of -0.94 to 7.5%), but the range was approximately half, indicating that the

most variable runs contained additives. Next, process metrics are described and a

comparison among biomass types (BSFL, Alaria, Ulva, and soy) was presented in Figure

5.6.

Figure 5.5: Retrospective analysis of mass collection for deionized water extractions
after separation and drying (N=22), Box plot of differences metrics (Sec. 5.2.2.1) for
“total wet weight” and components presented as normalized percentages (fractionC-
slurryC) /slurryC)*100). Where C refers to component.

The data collected describing the effect of biomass type on eight process metrics were

presented in Figure 5.6. The metrics presented in Plot I contain various representations

of the extracted protein. BCA (mg/ml) represents the unnormalized values from the BCA

protein quantification data. The BCA values were multiplied by the extracted volume (the

unnormalized numerator of Moisture yield in supernatant metric) to produce an extracted

176
protein mass. The other three metrics (yield, efficiency, and purity) in Plot I express

ratios that normalize the extracted protein mass against the original biomass, the protein

component of the original biomass, or the total extracted material; respectively (see. Sec.

5.2.1.5). The metrics presented in Plots II and III describe the moisture and solids

separation. Moisture yield in supernatant describes the ratio of the amount of water in the

supernatant to the total water added. Non-protein, non-additive yield in the pellet is a

yield metric describing the biomass solids in the pellet that are not protein as a ratio to the

initial biomass since there were no additives used in this section. Solids yield in the pellet

is a similar metric that represents the ratio of all unextracted solids mass to the initial

biomass; this metric does not rely on mass balance relationships. The water-holding

capacity of the pellet (WHC) describes the ratio of moisture to dry solids in the pellet

fraction (F6). Past researchers have identified that reduced WHC is associated with

structural damage to the polymeric materials and leads to more efficient drying on

account of less water (Du et al., 2018). Tukey HSD statistical analysis was used to

determine differences among biomass for each metric, except BCA.

Biomass type was the main factor in the ANOVA to evaluate diH 2O extraction which

showed no influence from co-variables [biomass: solution (w/v) ratio, vessel size, and

temperature (18 - 37  C), α=0.05] for the process metrics presented in Figure 5.6.

Different letters within each metric indicate differences (α=0.05) among the biomass

types. Deionized water results in low protein yields and efficiency for all four biomasses

studied. Soy not only has the highest protein yield, but it also has the lowest solids yield

in the pellet. The low solids retention explains why BSFL presents a higher protein purity

in its extract despite extracting less than half the protein. The two macroalgae (Alaria and
177
Ulva) exhibit very similar metrics only differing in protein efficiency, solids yield in

pellet, and WHC. While the protein efficiency is higher for Ulva, most of the protein

remained unextracted for both biomasses. The macroalgae present very high WHC likely

due to their high carbohydrate content (Afonso et al., 2019; Postma et al., 2018). WHC is

also closely related to the moisture yield in the supernatant metric which is

approximately 50% for the macroalgae. The high WHC may affect protein extraction and

will have implications for drying efficiencies.

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Figure 5.6: Process metric and measurements for distilled water fractionation of four
different biomass (Alaria, n=4; Ulva, n=4; BSFL, n= 12; Soy, n=2); Tukey HSD
pairwise comparisons, α=0.05, different letters indicate difference within metric category

179
5.3.3 Analysis 3 – Retrospective sensitivity analysis for BSFL process decisions
During the development of this fractionation system, multiple variables were tested to

determine their influence on future experiments. There were 5 two-level, and one four-

level variable that were conducted on BSFL and included in the ANOVA. The data came

from five batches and is used to estimate which explanatory variables influence the

process metrics the greatest. The explanatory variables and their levels tested for this

analysis were displayed in

Table 5.5. The body of the table shows F ratios for individual ANOVA models

conducted on each metric. The F ratios are only present if the effect is significant,

α=0.05. The F-ratio is a measure of the variability each effect is responsible for in the

ANOVA model. Larger F-ratios are an indication that an explanatory variable influences

a given metric more, relative for each metric, F ratios should not be compared among

metrics.

Table 5.5: Preliminary sensitivity analysis of BSFL explanatory variables (N=28); table
body contains ANOVA F-ratio values representing a relative effect size, “n.s” means
non-significant effect (α=0.05
Solution
Metric Homogenization NaOH volume Defat Temp. Batches
Variable 10, 30, 40,
Yes/ no 0/ 0.75 M Yes/ no 20/ 37 °C 1-5
levels 525 ml
Protein yield n.s. 112.21 5.34 7.36 n.s. n.s.
Protein
n.s. 7.4249 13.26 4.787 n.s. n.s.
Purity
WHC n.s. 21.9 6.6 4.2 n.s. n.s.
Insoluble
n.s 4.39 12.01 29.10 n.s. n.s.
solids yield

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The results in Table 5.4 revealed that protein yield was influenced by the inclusion of

NaOH, solution volume, and biomass defatting. Of the variables tested, NaOH has the

most influence on protein yield, while the solution volume and defatting accounted for

less relative variation in the model. The observed effect of solution volume may impact

future experiments with a focus on scale-up. The protein purity of the extracted fraction

does not follow the same trends as protein yield with solution volume exhibiting the

greatest variability. The F-ratios for protein purity were close for the three significant

variables. The addition of NaOH resulted in more protein being extracted, but also the

solubilization of non-protein material, so the protein content (purity) of the extract is not

affected greatly.

The water-holding capacity of the pellet (WHC) increased with the addition of NaOH

and was influenced less by the solution volume and the defatting operation. The results

from the solids yield in the pellet ANOVA indicated that defatting had the greatest effect

on the metric. This outcome is expected since the defatting operation occurred before

extraction and yield metrics were normalized to the whole biomass; thus, there would be

less insoluble material in the slurry. The sensitivity analysis results should provide

preliminary guidance for future experiments. The analysis presented in

Table 5.5 indicated that homogenization and temperature change up to 37 °C did not

influence any of the process metrics. Sodium hydroxide is the most influential factor in

protein extraction thus it was investigated in more detail.

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The influence of changing sodium hydroxide concentration on BSFL fractionation was

the focus of independent experiments and the results are presented in Figure 5.7. These

preliminary results revealed noteworthy trends to be considered in future experiments.

The trends displayed in Figure 5.7 were for non-defatted larvae and are independent of

the sensitivity analysis data. The NaOH concentration range was 0.05-2.5 M and included

diH2O as the “0 M NaOH” with extraction time held at 1 h. A statistical analysis of the

outcomes was not included due to the lack of replicates. For two conditions (0 and 0.1

M), error bars were included and added to Figure 5.7; note the error bars are behind the

markers in both cases. The protein yield increased slowly as NaOH concentration were

increased from 0 and peaked at a concentration of 1.0 M then decreased when

concentration was increased to 2.5 M. The decrease in yield at the highest NaOH

concentration may be due to the degradation of proteins, such as unfolding or peptide

formation, disrupting the BCA analysis. Furthermore, the large amounts of acid required

to neutralize the 2.5 M sample may have influenced the results. Further investigation into

protein quantification of highly alkali solutions may be needed if these conditions are

pursued in future experiments. Lacking an improvement in yield, it is unlikely that 2.5 M

NaOH would be used commercially due to cost and hazards. The results in Figure 5.7

indicate that protein purity increased significantly at NaOH concentrations greater than

0.05 M, but decreased gradually with greater concentrations. This trend was expected and

suggests diminished protein extraction returns with increased NaOH concentration, while

the additive mass continued to increase.

The WHC of the pellet increased at a low NaOH concentration (< 0.05 M) but decreased

as concentration was increased to 2.5 M. This decrease in pellet WHC as the [NaOH] is
182
increased was attributed to the pellet fraction containing an increased NaOH content and

biomass degradation. In conclusion, 0.75 M NaOH was identified as a reasonable balance

between yield, purity, and NaOH usage for future BSFL studies (see Sec. 5.3.5).

60 350

50 300

WHC %, (dotted line, X)

250
40
Metric, %

200
30
150
20
100
10 50

0 0
0 0.5 1 1.5 2 2.5
NaOH, M

BSFL protein yield BSFL protein purity

Non-protein, non-additve Pellet WHC %

Figure 5.7: The relationship between, protein yield, protein purity; non-protein, non-
additives pellet yield, and WHC% as a function of NaOH concentration in molarity used
during extraction for 1 hour at 20 °C. Error bars for all metrics on 0 M (diH2O, n=4), and
0.1 M NaOH (n=3), remaining concentrations had 1 replication.

5.3.4 Experiment 1 – Ulva fractionation with different solution conditions

The fractionation of Ulva was investigated (n=16) using five extraction conditions

designed to study the effect of diH2O, low salt (PBS), and two concentrations of NaOH

on the process metrics (Sec. 5.2.1.6). All runs were mixed for 1 h at the same

183
temperature. As illustrated in Figure 5.8, the magnitudes for seven metrics were analyzed

for five conditions: diH2O (1:10), diH2O (1:40), PBS, 0.05 M NaOH, and 0.5 M using

ANOVA. Each group was considered discrete and compared using Tukey HSD with

different letters to indicate statistical differences in Figure 5.8, α=0.05.

Differences in protein yield and efficiency were found; the 0.5 M NaOH and the diH 2O

(1:40) groups were statistically similar and greater than the other three groups. The diH2O

(1:40) may also be referred to as osmotic shock and was expected to produce a greater

yield as compared to the diH2O (1:10) condition (Veide Vilg & Undeland, 2017). The 0.5

M NaOH condition produced the highest protein purity value but was not statistically

different from the two diH2O conditions. The BCA mg/ml value was not normalized to

the initial biomass but the analysis demonstrates the concentration difference in the

supernatant due to osmotic shock.

The Moisture yield in the supernatant is greatly affected by changes to the biomass:

solution ratio. Aside from the osmotic shock condition (diH2O 1:40), the trends in the

moisture yield are all small with the 0.05M NaOH conditions being the next greatest.

This is the inverse trend of the WHC metric, but a statistical difference was not observed

for WHC.

The influence of the extraction conditions on solids yield in the pellet metric was

illustrated in Figure 5.8II. The low solids yield in the osmotic shock condition indicates

it solubilized the most biomass materials. After the protein and solutes content was

normalized using the non-protein, non-additives yield metric, similar trends were

observed.

184
 25
I diHwater
Di 2O (1:10) diHwater
Di 2O (1:40) PBS 0.05 M NaOH 0.5M NaOH

20
b
a a,b a a

15
Metric, %

10 A
a,b a,b b b

5
b
a a,b a a

0
Protein yiel d Protein efficency Protein puri ty BCA (mg/ml)

Di
diHwater
2O (1:10) diHwater
Di 2O (1:40)
Di water
diH 2O (1:10) Di
diHwater
2O (1:40) PBS II PBS 0.05 M NaOH
100 0.05 M NaOH 0.5M NaOH 0.5M NaOH
b a a a 1000 b
a,b
III
80
a a
800
b
c a a a a
Metric, %

a,b a,b
60 a a a
600

b
40
400

20 200

0 0
Moi sture yield in non-protein, non- Solids yield in pellet WHC of pellet
supernatant additves in pellet

Figure 5.8: Process metrics describing Ulva fractionation experiment in three plots: Plot
I contain supernatant protein metrics, II contains moisture and solids related metrics, III
contains % WHC of the pellet. Error bars show standard error. Tukey HSD pairwise
comparisons, α=0.05, different letters indicate difference within metric category

185
5.3.5 Experiment 2 – BSFL fractionation with different solutions and versus NaOH
exposure time
Experimental design details for the results to be presented are described in Sec. 5.2.2.5. A

comparison of the process metrics of interest used for the evaluation of diH 2O, 3 M

NaOH, and select 0.75 M NaOH time points are presented in Figure 5.9. The

relationships of select process metrics with NaOH exposure time (0-24 h) were presented

in Figure 5.10. Protein profiles from these fractionations are presented in Figures 5.11

and Figure 5.12.

5.3.5.1 Process analysis of BSFL fractionation in different solutions and versus NaOH
exposure time

As expected, extraction with 0.75 M NaOH provided the greatest protein yield and

protein efficiency (α=0.05). Extraction using the 3 M NaCl solution produced the lowest

protein yield, contrary to protein solubility results reported by Bußler et al., (2016).

Although the process metrics for extraction using a 3 M NaCl solution were not

significantly different from metrics for di water, differences in protein profile were

observed and explored in more detail later. The protein purity of the 3 M NaCl condition

was approximately 1% due to the large amount of salt in the solution. When the salts and

other small molecules were removed, with 1Kda dialysis before drying, the protein purity

was 20.5% ± 2.5% (±std. error). This result was similar to the protein purity after

extraction using diH2O. The 3 M NaCl solution was not an effective extraction aid for

increasing protein yield from the BSFL biomass.

When comparing the influence of NaOH exposure time on protein yield and protein

purity, the trends did not match. The protein yield continued to increase with time, while

the purity reaches a maximum at hour 12 before slightly decreasing to the 24 h value.
186
These trends are presented in both Figure 5.10. This divergence of protein yield and

protein purity was attributed to an increase in the solubility of non-protein material as

time progresses. The solids yield in pellet metric shows an approximately linear decrease

in the unextracted biomass solids. At a point between hours 6 and 24, the solubilization

of non-protein material becomes greater than the solubilization of protein leading to

reduced purities. The significance of this inflection point will depend on the target

products and co-products. If the extraction of non-protein material is of little concern to

the application, extending extraction time may be of interest as a maximum protein

extraction efficiency of just below 83% was achieved after 24 hours in 0.75 NaOH. Also,

as expected the 0 h sample has the lowest protein yield and purity while having the

highest pellet solids retention (highest solids yield in pellet).

A difference in the water-holding capacity of the pellet (WHC) was identified between

the diH2O and 3 M salt conditions (α=0.05). Extraction with the 3 M NaCl solution

provided a higher WHC; although NaCl was not expected to contribute to water-holding

while contributing to the mass of solids in the pellet. When comparing the WHC of the

diH2O and 3 M NaCl samples to the NaOH extracted samples, the effects of defatting

before NaOH extraction must be considered. Since fats were about a third of the BSFL

mass and were hydrophobic, their contribution to the water-holding ability was minimal

while adding mass to the insoluble pellet. A more appropriate demonstration of WHC

was illustrated in Figure 5.10 where a relative effect of NaOH exposure time on WHC

was displayed. The influence of mixing time on WHC was similar to protein purity, the

magnitude increased during the first 6 h, then decreases as mixing times were extended to

24 h. These relative changes to WHC were driven by the solubilization of the biomass
187
during mixing and a variable release of water into the supernatant during centrifugation.

The increase in WHC of the pellet during the first 6 h of extraction correlated with a

decrease in solids yield in the pellet during the same period. The decrease in WHC after 6

hours correlated with an increase in supernatant moisture yield. On the molecular level,

the NaOH was likely altering the structure of the biomass so that interactions with water

were favorable during early mixing times. As time progressed from 0 to 6 h and as

materials were solubilized (reduction in solids pellet yield), the remaining pellet appeared

to have superior water-holding since the moisture yield in the supernatant remained

mostly constant (0-6 h). Then during the longer NaOH solution exposure times (6-24 h),

the NaOH solution was likely degrading and solubilizing the structures that had

contributed to WHC initially, resulting in the observed decrease. Based on these

observations, WHC was viewed as a valuable property for monitoring complex

interactions of biomass and water during fractionation experiments. When the

interactions are combined with evaluations of the moisture and solids yields, an improved

understanding of the fractionation was possible.

The influence that dialysis (1 kDa) had on the protein purity of the supernatant is

displayed in Figure 5.10. The inclusion of the dialysis operation increased the protein

purity by approximately 50% for the 0 h samples and 12% for the 12 h samples. These

results suggest that changes to the molecular size distribution in the supernatant

continued to occur during exposure to NaOH. Future experiments might employ a

particle size analysis to further elucidate the observed phenomena, but the use of 1 kDa

dialysis tubing helped establish hypotheses for future work. The solids yield in the pellet

is at its highest after the 0 h condition. This observation was an indication that minimal
188
material had been solubilized. The solubilized materials at the zero-hour time point were

likely small polar molecules (salts and sugars). The greatest change in protein purity from

dialysis occurred at 0 h due to the minimal protein extraction, minimal biomass

degradation and solubilization, and the contribution of the NaOH solute to the mass of

the undialyzed extract. Much smaller changes in purity were observed after12 and 24 h of

extraction due to the additional time NaOH had to break down complex molecules and

structures that, even when solubilized, remain larger than 1 kDa. Future work should also

consider using Nitrogen analysis in addition to spectrophotometric protein assays. Protein

quantification with BCA was considerably more variable for the NaOH samples after

dialysis than for samples that were neutralized before drying. These results may be

attributed to irreversible changes in the proteins during the drying operation with reduced

sugar and salt and no additives, which have been shown to improve drying quality

(Shaviklo et al., 2010). These changes could lead to less reliable solubility and eventually

to the greater variability in BCA for the dialyzed samples.

Most metrics follow complex trends over time. Exponential, logarithmic, parabolic, and

two-phase linear relationships may provide adequate fits to several of the metrics

discussed. Modeling the process metrics was not the objective of this analysis as

optimization requirements have not yet been established. Furthermore, other variables

such as temperature and NaOH concentration, which may exhibit important interaction

effects with time will need to be included in future models. Understanding active

interaction effects are critical before pursuing global optimizations. The results presented

here show the interconnected nature of the process metrics of interest. These results can

189
be leveraged for future experimental design to establish reasonable variable bounds and

accelerate future ingredient development.

190
 100
I Di
diHwater
2O 3M NaCl NaOH 0.5 h NaOH 6 h NaOh 24 h
90 d
80
70
c
60
Metric, %

50
40 c
b
d
30
d
c a a
20
b a a
10 a a b
0
Protein yiel d Protein efficency Protein puri ty BCA (mg/ml)

100 Di water 3M NaCl

Di
diHwater
2O
II a a
NaOH 0.5 h NaOH 6 h
90 a 3M NaCl NaOh 24 h
80 b b b NaOH 0.5 h 900
NaOH 6 h 800 III d
70
NaOh 24 h
b 700 c
60
Metric, %

600 e
50 c
500
40 d
400
30
e 300 b
a
20 200
10 100
0 0
Moi sture y ield in Solids yield in pellet WHC of pellet
super natant

Figure 5.9: Process metrics describing BSFL fractionation experiment in three plots: Plot
I illustrates supernatant protein metrics, II contains moisture- and solids-related metrics,
and III contains WHC. All NaOH samples have undergone defatting as described in Sec.
5.2.1. Error bars show standard error and statistical differences among groups were
determined with Tukey HSD pairwise comparisons, α=0.05, different letters indicate
difference within metric category

191
 80

70

60

50
Metric, %

40

30

20

10
Protein yield Protein purity Dialysed purity
0
0 5 10 15 20 25 30
Time, h

100 900
90 800
80

WHC % (dotted line, X)

700
70
600
Metric, %

60
500
50
400
40
30
300
20 200
10 100
Moisture yield in supernatant Solids yield in pellet WHC of pellet%
0 0
0 5 10 15 20 25 30
Time, h

Figure 5.10: NaOH versus time process metrics. top) protein-based process metrics,
bottom) moisture and solids process metrics.

192
5.3.5.2 SDS-PAGE protein characterization of di water, NaCl, and NaOH extracts.

The samples described in the previous section also underwent protein characterization

with the extracts from the diH2O, 3 M NaCl, and NaOH time series fractionations were

characterized with SDS-PAGE. The results presented in Figure 5.11 present the

molecular weight (MW) distribution of extracts collected with diH 2O and NaOH extracts

after neutralization. The lanes are not normalized based on protein concentrations but

were each loaded to maximize visibility. The diH2O extract contains a large buildup of

approximately 8 kDa MW proteins compared, relative to a common 20 kDa band, to the

NaOH samples. The 0 and 1 h NaOH extracts present more discrete bands with less

smearing than the 6, 12, and 24 h extracts. The 0 h extract had a ~60 kDa band that was

not present in the 1 h lane. The 6, 12, and 24 h lanes have a low resolution which may be

attributed to hydrolysis caused by the extended NaOH exposure. For the NaOH extracts,

light intensity migrated lower in the lanes as the exposure time extends suggesting that

NaOH exposure results in hydrolysis.

The results presented in Figure 5.12 showed dialyzed samples which were compared to

neutralized samples in Figure 5.11. Unlike the neutralized samples, the dialyzed samples

underwent dialysis in 1 kDa tubing before being dried and prepared for SDS-PAGE. The

neutralized 0 and 1 h NaOH extracts show more discrete banding than the dialyzed

sample. This is an unexpected result, possibly caused by additional exposure time in

NaOH before dialysis could be initiated. The 12 and 24 h samples are more consistent

between the two treatments, suggesting the breakdown products already present were less

susceptible to changes while undergoing dialysis. The 3 M NaCl condition shows unique
193
bands (80, 85 kDa) not in the diH2O lane. It was difficult to achieve a properly loaded

sample for the dialyzed 3 M NaCl extract. The dialyzed 3M NaCl sample was loaded at a

concentration of 100 mg/ml to produce a light lane, 5 times the diH2O loading. The

dialyzed NaCl extract was determined to have a protein purity of 21.2±2.4% (±std. error)

so the lane should be heavily overloaded. This might be attributed to irreversible changes

that occurred while in the high ionic strength extraction solution. However, unique bands

in the 3 M NaCl lane may justify the use of NaCl for fractionations in future

investigations aimed at identifying unique functionality.

Short NaOH exposure, on the other hand, may be a promising condition for future

ingredient development. Ursu et al., (2014) suggested that NaOH treatment was

detrimental to food functionality. While Queiroz et al., (2021) demonstrated superior

foaming capacity when extracting BSFL protein with 0.25 M NaOH than without. The

observations described as part of Experiment 2 (Figure 5.9-5.11) suggest the amount of

protein and the characteristics of the protein will be influenced by time. Future

investigations may work to understand the influence of other process variables, such as

NaOH concentration and temperature, will have on process metrics of interest, protein

profiles, and protein functionalities.

194
Figure 5.11: SDS-PAGE gel (12.5% acrylamide, 0.75 mm) with deionized water, and
NaOH samples (0, 1, 6, 12, and 24 h) between two protein ladders. Loadings were not
normalized by protein concentration.

Figure 5.12: SDS-PAGE gel (12.5% acrylamide, 0.75 mm) with deionized water,
dialyzed 3M NaCl and dialyzed NaOH samples (0, 2, 6, 12, and 24 h) with protein
ladders. Loadings were not normalized by protein concentration.

195
5.4 Conclusions

A process model, associated procedures, and mass balance relationships were developed

and presented as an experimental fractionation system to evaluate novel food sources.

The experimental data collected was transformed into various metrics which were used to

assess repeatability and evaluate multiple experimental objectives. Mass loss after the

separation operation was evaluated for all runs with a normalized metric demonstrating a

95% confidence interval (CI) of -1.2 to -0.66%, indicating repeatability. The drying

operation was also found to repeatably conserve mass using a similar normalized metric

but showed greater variability in the solids balance (CI of 0.38%: -6.5%) which

correlated with biomass type and solution volume (α=0.05). The ability to quantify the

repeatability of these operations and identify correlating factors provides a basis for

continual improvement to this experimental fractionation system.

The next analysis aimed to compare the variability in the process metrics for runs that

used only deionized water (diH2O) during extraction. Extraction with diH2O showed low

variability for all process metrics across each biomass studied and significant differences

(α=0.05) among biomass types (Alaria, Ulva, BSFL, and soy) were identified for

multiple metrics that describe the protein, moisture, and solids separation.

A preliminary sensitivity analysis for Black Soldier Fly larvae (BSFL) fractionation was

conducted. The analysis showed the sodium hydroxide (NaOH) concentration factor had

the greatest effects while solution volume and defatting also influenced BSFL

fractionation across four metrics (protein yield, protein purity, WHC, and insoluble solids

yield; α=0.05). Meanwhile, homogenization, temperature (20-37 °C), and batch

196
variability did not significantly affect the BSFL fractionation metrics analyzed. A series

of increasing NaOH concentration runs were also analyzed which identified a potential

intersection point between protein purity and protein yield between 0.5 and 1 M NaOH.

The maximum protein extraction efficiency observed was 58% when using 1 M NaOH

for one hour. These results provide a baseline and set of procedures to monitor process

metrics of interest against several factors which can be used in future experiments to

identify interaction effects.

Two discrete experiments were described and conducted, each with a specific objective.

The first was to identify differences in the fractionation of the macroalgae, Ulva, when

using different solution conditions. Osmotic shock (diH2O 1:40) showed a small but

significant (α=0.05) increase in protein efficiency over the diH2O (1:10) condition but

increases water usage by four times. The protein efficiency for Ulva extractions was low,

less than 20% for all conditions, suggesting alternative approaches to fractionation should

be investigated to increase efficiency.

The final experiment aimed to understand the influence NaOH exposure time and a high

salt (3 M NaCl) solution had on BSFL biomass fractionation. The 3 M NaCl condition

was not successful at increasing the protein efficiency of BSFL when compared to diH2O

extraction, however, unique protein bands were identified with SDS-PAGE in the high

salt extract. NaOH exposure time influenced all process metrics (α=0.05), the protein

profile, and the visual appearance of BSFL extracts. Protein efficiency reached its

maximum of 83% after 24 h but was associated with a tradeoff in the fraction’s protein

purity, which decreased, as greater non-protein solids were co-extracted. Extracting non-

197
protein solids has implications for extract purification and the valorization of the

unextracted materials. There were further tradeoffs associated with the protein profiles of

NaOH extracts. The protein profiles of BSFL extracts show increasing degradation and

peptide formation as NaOH extraction time progresses, an important consideration when

developing future experiments to evaluate BSFL protein physicochemical functionality.

The experimental fractionation system provides a reliable approach for the evaluation of

biomass fractionation outcomes and the ability to test a wide array of factors. Once

applications for fractionation products and co-products are identified, a similar system

can be used for optimization analyses that include component separation and

physicochemical properties.

198
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 Chapter 6 Conclusions
Sustainable innovations are critical to the long-term success of agricultural systems. In

this dissertation, two diverse classes of organisms: insects and algae, were evaluated as

nutritious and environmentally friendly sources of nourishment for humans. While their

cultivation is associated with low carbon, land, and/or water footprints, the impact of the

processing required for their transformation into appealing ingredients is not well known.

The nutritional understanding of macroalgae was improved after amino acid profiles of

twenty-nine macroalgae samples were determined in one of the largest studies on

seaweeds available. Two genera were profiled for the first time: Eularia and Nereocystis.

Trends in the amino acid profiles suggest less difference between phyla than previously

reported. Methionine presents the most variability; an important consideration as

methionine is an essential amino acid for humans and some livestock animals. The amino

acid profiles presented provide fundamental knowledge for the nutrition field while

improving understanding of macroalgae protein for food developers.

While evaluating the extraction of commercial Black Soldier Fly larvae (Hermetia

illuscens; BSFL) proteins, enzymatic activity was observed. This was an unexpected

observation as many protocols employ a blanching step during harvesting to eliminate

enzymatic activity. However, the commercial BSFL used in the analysis were only dried

which did not fully deactivate the enzymes. Active proteases were extracted with

deionized water or PBS which may provide a marketable product after future

investigation. The BSFL are highly efficient bioconverters due to their high enzyme

expression, future investigations might capitalize on this natural enzyme source by

214
optimizing harvesting conditions. The enzymes were also found to auto-digest BSFL

proteins which may increase the bioavailability of the protein as feed and food.

Controlling the proteolytic activity of the biomass is also important for food

development. As proteins are converted into peptides, their solubility and chemical

properties change influencing their physicochemical functionality.

Multiple experiments aiming to evaluate protein extraction of the BSFL and macroalgae

were conducted. Early experiments led to the conclusion that NaOH was the most

effective processing aid for the extraction of protein. Washing BSFL with NaOH three

times was sufficient to remove all detectable protein. Extending mixing times did not

result in an increase in the nitrogen extracted when using deionized water while nitrogen

extraction continually increased with NaOH up to 24 h. During an experiment to evaluate

enzymatic-assisted-extraction (EAE) of brown macroalgae, NaOH alone resulted in

greater protein extraction than samples that underwent an enzyme treatment targeting

carbohydrates. Identifying optimal extraction parameters is important, however, in the

pursuit of the most sustainable processes in the future, a broader examination of biomass

fractionation was needed.

The experiments described above did not track all biomass materials through each

operation so overall process efficiency was not adequately assessed. Updates to early

experimental procedures were paired with explicit mass balance relationships and

accompanying assumptions used to define process metrics. Process metrics include yield,

efficiency, and purity which can be applied to measured or mass balance derived

component masses. This system was found to repeatably conserve mass after separation

215
and drying operations through the evaluation of metrics derived from fundamental mass

balance relationships. The system was then used to evaluate diH2O extraction of various

biomass types. Process metrics defined to evaluate process efficiency were used to

characterize differences between biomass type. The system successfully identified

significant differences (α=0.05) in the protein, and biomass material separation among

BSFL, two macroalgae species (Ulva and Alaria), and soy flour. After an initial

development phase, the experimental system was used for two independently designed

experiments.

In the first experiment Ulva fractionation using variable aqueous solution parameters was

evaluated. The 0.5 M NaOH condition extracted more Ulva protein than the PBS,

osmotic shock, and the 0.05 M NaOH conditions, but it was only able to remove 20% of

the biomass’ protein. This indicates that expanded process parameters or alternative

treatments will be needed to facilitate protein extraction in future investigations. The final

experiment identified tradeoffs associated with the fractionation of BSFL using NaOH

and mixing for up to 24 h. Protein efficiency reached 83% after 24 h in NaOH but greater

non-protein solids were also solubilized resulting in reduced protein content of the BSFL

extract. The extended mixing with NaOH also resulted in protein degradation observable

after 1 h with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

NaOH exposure time influenced all process metrics, the protein profile, and the visual

appearance of BSFL extracts

The experimental system developed herein has identified critical process parameters and

provides a systematic, unbiased, and repeatable approach to fractionation evaluation.

216
Sustainability is an ongoing process but systems that couple material balances with

output properties are needed to facilitate positive development.

217
 Chapter 7 Future works
With promising results and low variability seen in macroalgae amino acid profile,

research into the digestibility differences among phyla and genera is needed. For all novel

food products, post-processing amino acid digestibility analysis is important to better

understand the benefits of an extract removed from its biological system.

The BSFL are strong producers of proteases, but they are known to produce an

assortment of other digestive enzymes. Studying the processing steps and conditions that

lead to the inactivation of these enzymes may provide insights for future applications.

The fractionation system requires minor procedural improvements to reduce variability

during the solids determination. The fractionation system has wide applicability and

specific studies will depend on desired application and biomass of interest. One facet of

the system that requires further investigation is the use of large-volume centrifuge bottles.

The procedure would remain the same except improvements to the desalting step are

needed to increase throughput. Filtration could replace dialysis for example. The small

volumes primarily used in the present work do not allow for detailed food functionality

evaluations. The small volumes were used since they provide high throughput allowing

for replication and a wider array of process parameters to be studied. Larger batches and

more detailed functional analysis may help identify the appropriate application for each

fraction while informing scale-up expectations.

218
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248
 Appendix A: Images from preliminary BSFL experiments

Figure A.1: Processing of raw BSFL from collection to dried flour. BSFL are gown in
large bins (A) at waterman farms. They were transported to Dr. Heldman’s lab (B).
Thermal blanching was then conducted in a makeshift hot water bath (C). Photo D shows
the enzymatic activity associated with BSFL and the results of grinding without heat
treatment. The cleaned, inactivated, and frozen product is shown in photo E. The final
image (F) shows ground BSFL during the sieving process to obtain a uniform flour prior
to protein extraction.

249
Figure A.2: Ground BSFL flour suspend in aqueous solvents after different treatment.
Image A ground flour was mixed vigorously by hand with 20x its mass in water then left
to settle at 4 °C for 24 hours. Image B demonstrates the extraction process for different
solvents: Alkali (1M NaOH), di water, and salt (0.5 M NaCl) water after shaking in a
room temperature water bath for 24 hours.

Figure A.3: “Soldier Croutons.” A product created by a student group using BSFL flour
for the American Society of Baking product development competition.

250
 Appendix B: Fractionation system data management
and analysis code
The first 44 variables in “data” are logged in an excel document.

filepath= "fracdataV6.”lsx";
%read file as a table
factors=readtable(filep”th,"ReadVariableN”mes",t”ue,"S”
e”t","fac”ors");
masses=readtable(filep”th,"ReadVariableN”mes",t”ue,"s”e
e“", "ma”ses");
concentrationsractions=readtable(filep”th,"ReadVariable
N”mes",t”ue,"S”e”t","concentrat”ons");
designs=readtable(filep”th,"ReadVariableN”mes",t”ue,"S”
e”t","de”ign");

data=join(factors,masses);
data=join(data,concentrationsractions);
dataset=data;
%dataset is original data

%%%%%% Section 2
%Determine Fraction and Compon251fficisess %%
%%%DEFINIT–ON - SOLIDS = Biomass Solids

%%out of order
outoforderSuperproteinmg=data.x23_M_supernatant_ml.*dat
a.x30_C_SupWet_BCA_mgml;
fr_insolublewet_solid=(data.x25_M_T1andPelletDry_g-
data.x18_M_T1_g)./(data.x24_M_T1andPelletWet_g-
data.x18_M_T1_g);
InputSolution=data.x21_M_T2andSolvent_g-
data.x20_M_T2_g;
%C–F3 - Biomass slurry, wet (slurry)
%mass calculations
data.x47_C_slurry_solids=data.x19_M_T1andBiomass_g-
data.x18_M_T1_g;
data.x46_C_slurry_additives=data.x28_M_tot_solutes_g+da
ta.x29_M_tot_enzymes_g;

251
data.x45_C_slurry_moisture=(InputSolution-
data.x46_C_slurry_additives)%+((data.x35_fr_flour_
moisture.*data.x47_C_slurry_solids));
data.x48_C_slurry_total=InputSolution +
data.x47_C_slurry_solids;

%H–F4 - low density wet

data.x70_H_lowdensitywet_moisture=(data.Screen_residue_
wet-data.Screen_residue_Dry);
data.x71_H_lowdensitywet_additives=(data.x46_C_slurry_a
dditives.*(data.x70_H_lowdensitywet_moisture./data
.x45_C_slurry_moisture));
data.x72_H_lowdensitywet_solids =
data.Screen_residue_Dry-data.Screen_Boat;
data.x73_H_lowdensitywet_total= data.Screen_residue_–et
- data.Screen_Boat;
%I–F6 - Solids (pellet when 3 phase separation)
data.X74_D_solidswet_moisture=(data.x24_M_T1andPelletWe
t_g-data.x25_M_T1andPelletDry_g);
data.X75_D_solidswet_additives=(data.x46_C_slurry_addit
ives.*data.X74_D_solidswet_moisture./(data.x45_C_s
lurry_moisture));
data.X76_D_solidswet_solids=(data.x25_M_T1andPelletDry_
g-data.x18_M_T–_g -
data.X75_D_solidswet_additives);
data.X77_D_solidswet_total=data.x24_M_T1andPelletWe–_g
- data.x18_M_T1_g;

%D F6 +F4 - insoluble materials Pellet wet fraction

(insolublewet)
data.X49_D_insolublewet_moisture=(data.x24_M_T1andPelle
tWet_g-
data.x25_M_T1andPelletDry_g)+data.x70_H_lowdensity
wet_moisture;
data.X50_D_insolublewet_additives=(data.x46_C_slurry_ad
ditives.*(data.X49_D_insolublewet_moisture./data.x
45_C_slurry_moisture))+data.x71_H_lowdensitywet_ad
ditives;
data.X51_D_insolublewet_solids=(data.x25_M_T1andPelletD
ry_g-data.x18_M_T–_g -
data.X50_D_insolublewet_additives) +
data.x72_H_lowdensitywet_solids;

252
data.X52_D_insolublewet_total=data.x24_M_T1andPelletWe–
_g -
data.x18_M_T1_g+data.x73_H_lowdensitywet_total;
%E –12 - Pellet 253fficienionn (insolubledry)
data.x53_E_insolubledry_additives(:)=data.X50_D_insolub
lewet_additives;
data.x54_E_insolubledry_protein=((data.x47_C_slurry_sol
ids.*data.x34_E_BiomassProtein)-
(outoforderSuperproteinmg./1000));
data.x55_E_insolubledry_lipid(:)= 0;
data.x56_E_insolubledry_ash(:)=0;
data.x57_E_insolubledry_remainder=data.X51_D_insolublew
et_solids-(data.x53_E_insolubledry_additives +
data.x54_E_insolubledry_protein);
data.x58_E_insolubledry_total=data.x25_M_T1andPelletDry
_g-data.x18_M_T1_g;
%F F5- Soluble WET
data.x59_F_solublewet_moisture=data.x23_M_supernatant_m
l.*(1-data.x33_fr_solublewet_solid);
data.x60_F_solublewet_additives=data.x46_C_slurry_addit
ives.*(data.x59_F_solublewet_moisture./data.x45_C_
slurry_moisture);
data.x61_F_solublewet_solids=
(data.x23_M_supernatant_ml.*data.x33_fr_solublewet
_solid)-data.x60_F_solublewet_additives;
data.x62_F_solublewet_total=data.x22_M_supernatant_g;
%G –10 - Soluble DRY
data.x63_G_solubledry_additives(:)=data.x60_F_solublewe
t_additives;
data.x64_G_solubledry_protein=outoforderSuperproteinmg.
/1000;
data.x65_G_solubledry_lipid(:)=0;
data.x66_G_solubledry_ash(:)=0;
data.x67_G_solubledry_remainder=data.x61_F_solublewet_s
olids-(data.x63_G_solubledry_additives +
data.x64_G_solubledry_protein);
data.x68_G_solubledry_total=data.x22_M_supernatant_g.*d
ata.x33_fr_solublewet_solid;

%%%%% initial flour ma253fficier253fficfatt

data.x78_initial_flour=data.x47_C_slurry_solids./data.d
efat_correct;

253
data=join(data, designs);

%%%%% Metrics

metrics=removevars(factor‘,
{'x11_E_Enzym’_’i','x9_E_BufferpH_’o’t','x10_E__sa
lt_’mo‘', 'x15_E_EnzymeMassMG_’on‘',
'x16_E_RPM’cat'})

metrics.m21_protein_yield=data.x64_G_solubledry_protein
*100./data.x78_initial_flour;
metrics.m22_protein_efficency =
data.x64_G_solubledry_protein*100./(data.x36_fr_fl
our_protein.*data.x78_initial_flour);
metrics.m23_protein_purity=data.x64_G_solubledry_protei
n*100./data.x68_G_solubledry_total;
metrics.m24_WHC_pellet=data.X49_D_insolublewet_moisture
*100./data.X51_D_insolublewet_solids;
metrics.m25_moistureyield_pellet=
data.X49_D_insolublewet_moisture*100./
data.x45_C_slurry_moisture;
metrics.m29_moistureyield_super=
data.x59_F_solublewet_moisture *100./
data.x45_C_slurry_moisture;
metrics.m26_BCA_super=data.x30_C_SupWet_BCA_mgml;
metrics.m27_nPnSbiomasssolids_pellet_yield=
(data.x58_E_insolubledry_total-
data.X50_D_insolublewet_additives-
data.x54_E_insolubledry_protein)*100./data.x78_ini
tial_flour;
metrics.m28_solids_pellet_yield=data.x58_E_insolubledry
_total*100./data.x78_initial_flour;
metrics.m30_solids_super_yield=
data.x68_G_solubledry_total./data.x78_initial_flou
r;

254
 Appendix C: Author permission, Chapter 2

From: Serventi, Luca <[email protected]>

Sent: Thursday, August 4, 2022 3:55 PM
To: Caminiti, Jeffrey T. <[email protected]>; Amoafo, Omega
<[email protected]>; Badiger, Aishwarya S. <[email protected]>
Subject: Re: New foods chapter in dissertation

Yes,
The publisher says it is OK.

Luca

From: Badiger, Aishwarya S. <[email protected]>

Sent: Monday, August 1, 2022 4:48 AM
To: Caminiti, Jeffrey T. <[email protected]>; Serventi, Luca
<[email protected]>; [email protected]
Subject: Re: New foods chapter in dissertation

Hi Jeff,

I don’t have any issues with this.

Best,
Aishwarya

From: Caminiti, Jeffrey T. <[email protected]>

Sent: Monday, 1 August 2022 7:05 am
To: Serventi, Luca <[email protected]>; Amoafo, Omega
<[email protected]>; Badiger, Aishwarya S. <[email protected]>
Subject: New foods chapter in dissertation

Good afternoon/ morning everyone,

I am reaching out about obtaining permission to use our Understanding New Foods: Plant
Based chapter in my dissertation as my literature review. I read through the authorship agreement
and saw there is a reuse clause from the publisher, but I also wanted to make sure it was okay
with each of you.

Please reply all if possible,

Thank you! It was great working with everyone!

Jeff Caminiti

255
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