DocuSign Envelope ID: FCA4F54E-C6FE-40FB-A5FC-944C7FB527E7

GRAS Notice (GRN) No. 1142 with amendments

https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory

April 19, 2023

RECEIV~D
Dr. Paulette Gaynor
APR 2 0 2023
Office of Food Additive Safety (HFS-200)
Center for Food Safety and Applied Nutrition (CFSAN)
Food and Drug Administration OFFICE OF FOOD ADDITIVE SAFETY
5001 Campus Drive
College Park, MD
20740 USA

Dear Dr. Gaynor:

Re: GRAS Notice for Oubli Fruit Sweet Protein Derived from Komagataella pha/fii

In accordance with 21 CFR §170 Subpart E consisting of§§ 170.203 through 170.285, Oobli, Inc. [202
Cousteau Place, Suite 210, Davis, CA, 95618 USA], as the notifier, is submitting one hard copy and one
electronic copy (on CD), of all data and information supporting the company's conclusion that Oubli Fruit
Sweet Protein, manufactured from a genetically engineered strain of Komagataella phaffii, is GRAS on the
basis of scientific procedures, for use as a sweetening agent in conventional food and beverage products.
These food uses of Oubli Fruit Sweet Protein are therefore not subject to the premarket approval
requirements of the Federal Food, Drug and Cosmetic Act. Information setting forth the basis for Oobli's
GRAS conclusion, as well as a consensus opinion of an independent panel of experts, also are enclosed for
review by the agency.

I certify that the enclosed electronic files were scanned for viruses prior to submission and are thus
certified as being virus-free using Symantec Endpoint Protection 12.1.5.

Should you have any questions or concerns regarding this GRAS notice, please do not hesitate to contact
me at any point during the review process so that we may provide a response in a timely manner.

Sincerely,
l rOocuSigned by:

~ D6AD848049D24FB...

Jason Ryder, Ph.D.

Chief Technology Officer
Oobli, Inc.
[email protected]
GRAS NOTICE FOR OUBLI FRUIT
SWEET PROTEIN DERIVED FROM
KOMAGATAELLA PHAFFII

SUBMITTED TO:
Office of Food Additive Safety (HFS-200)
Center for Food Safety and Applied Nutrition (CFSAN)
Food and Drug Administration
5001 Campus Drive
College Park, MD
20740 USA

SUBMITTED BY:
Oobli, Inc.
202 Cousteau Place, Suite 210
Davis, CA
95618 USA

DATE:
18 April 2023

Oobli, Inc.
18 April 2023 1
GRAS Notice for Oubli Fruit Sweet Protein Derived from
Komagataella phaffii
TABLE OF CONTENTS
PART 1. §170.225 SIGNED STATEMENTS AND CERTIFICATION........................................................................... 5
1.1 Name and Address of Notifier .................................................................................................. 5
1.2 Common Name of Notified Substance ..................................................................................... 5
1.3 Conditions of Use...................................................................................................................... 6
1.4 Basis for GRAS........................................................................................................................... 7
1.5 Availability of Information ........................................................................................................ 7
1.6 Freedom of Information Act, 5 U.S.C. 552 ................................................................................ 7

PART 2. §170.230 IDENTITY, METHOD OF MANUFACTURE, SPECIFICATIONS, AND PHYSICAL OR

TECHNICAL EFFECT................................................................................................................................. 8
2.1 Identity of the Ingredient.......................................................................................................... 8
2.1.1 Oubli Fruit Sweet Protein ............................................................................................ 8
2.1.2 Brazzein........................................................................................................................ 9
2.2 Manufacturing Process ...........................................................................................................11
2.2.1 Description of the Manufacturing Process ................................................................11
2.2.2 Information on the Production Microorganism ........................................................12
2.3 Product Specifications and Batch Analyses ............................................................................16
2.3.1 Product Specifications ...............................................................................................16
2.3.2 Batch Analysis ............................................................................................................17
2.3.3 Mineral Profile ...........................................................................................................17
2.4 Stability ...................................................................................................................................18
2.5 Technical Effect.......................................................................................................................18
2.5.1 Mechanism of Action of Brazzein ..............................................................................18
2.5.2 Sensory Studies on Oubli Fruit Sweet Protein ...........................................................19

PART 3. §170.235 DIETARY EXPOSURE .............................................................................................................20

3.1 Estimated Dietary Consumption of Oubli Fruit Sweet Protein...............................................20
3.1.1 Methodology .............................................................................................................20
3.1.2 Intake Estimates for Oubli Fruit Sweet Protein .........................................................20
3.1.3 Dietary Intake Estimates of Minerals from Oubli Fruit Sweet Protein ......................22
3.1.4 Summary and Conclusions.........................................................................................22

PART 4. §170.240 SELF-LIMITING LEVELS OF USE.............................................................................................23

PART 5. §170.245 EXPERIENCE BASED ON COMMON USE IN FOOD BEFORE 1958 .........................................24

PART 6. §170.250 NARRATIVE AND SAFETY INFORMATION.............................................................................25

6.1 Introduction ............................................................................................................................25
6.2 Safety of the Production Strain...............................................................................................26
6.3 Safety of Oubli Fruit Sweet Protein ........................................................................................27
6.3.1 Digestibility of OFSP and Brazzein .............................................................................27
6.3.2 Mutagenicity/Genotoxicity Studies on Oubli Fruit Sweet Protein ............................30
6.3.3 Studies in Animals......................................................................................................32

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18 April 2023 2
 6.3.4 Bioinformatics Assessment of Brazzein and Residual Host Cell Proteins
within Oubli Fruit Sweet Protein ...............................................................................34
6.4 GRAS Panel Evaluation............................................................................................................40
6.5 Conclusion...............................................................................................................................41

PART 7. §170.255 LIST OF SUPPORTING DATA AND INFORMATION ................................................................42

List of Appendices
Appendix A Residual Host Cell Proteins Within OFSP

Appendix B GRAS Panel Statement

List of Figures and Tables

Figure 2.1.2-1 Sequence Coverage from Tandem Mass Spectrometry Peptide Mapping Analysis
of Oobli’s Brazzein-53 ........................................................................................................ 10
Figure 2.1.2-2 Deconvoluted MS1 Spectrum of Oobli’s Brazzein-53 ........................................................ 10
Figure 2.1.2-3 Three-Dimensional Structure of Brazzein .......................................................................... 11
Figure 2.2.1-1 Manufacturing Process for OFSP ....................................................................................... 12
Figure 2.2.2.2-1 Streaked Yeast Extract Peptone Dextrose Plates with Hygromycin, Zeocin, or
Geneticin Selection Markers of the Production and Wild-type
Komagataella phaffii Strain ............................................................................................... 15
Figure 2.2.2.2-2 Colony Polymerase Chain Reaction Results of the Production Strain ............................... 15
Figure 6.3.1.1-1 Results of In Vitro Digestibility Study with OFSP with Simulated Gastric Fluid ................. 27
Figure 6.3.1.1-2 Results of In Vitro Digestibility Study with OFSP with Pepsin in Simulated Gastric
Fluid and with Pancreatin in Simulated Intestinal Fluid .................................................... 28

Table 1.3-1 Summary of the Individual Proposed Food Uses and Use Levels for OFSP in the
United States........................................................................................................................ 6
Table 2.1.1-1 Summary of the Abundance of Residual Komagataella Proteins in OFSP........................... 8
Table 2.2.2.1-1 Summary of GRAS Notices for Food Ingredients Produced Through Fermentation
of Komagataella phaffii ..................................................................................................... 13
Table 2.2.2.1-2 Taxonomic Identity of Komagataella phaffii ..................................................................... 13
Table 2.3.1-1 Product Specifications for OFSP......................................................................................... 16
Table 2.3.2-1 Summary of the Batch Analysis for 3 Production Batches of OFSP ................................... 17
Table 2.4-1 Summary of the Analysis for Proximates and Microbiological Parameters of OFSP
Following Storage for 15 Months at Ambient Conditions (Temperature: 23.5°C,
Relative Humidity: 40%)..................................................................................................... 18
Table 3.1.2-1 Summary of the Estimated Daily Intake of OFSP from Proposed Food Uses in the
United States by Population Group (2017-2018 NHANES Data)........................................ 21
Table 3.1.2-2 Summary of the Estimated Daily Per Kilogram Body Weight Intake of OFSP from
Proposed Food Uses in the United States by Population Group (2017-2018
NHANES Data) .................................................................................................................... 21
Table 3.1.3-1 Comparison of Mineral Intake from OFSP and Corresponding Tolerable Upper
Limit Values as Established by NASEM .............................................................................. 22
Table 6.3.1.2-1 Summary of In Silico Digestion Using PeptideCutter with Pepsin (pH 1.3 and >2.0)......... 29

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18 April 2023 3
Table 6.3.1.2-2 Summary of In Silico Digestion Using PeptideCutter with Trypsin .................................... 29
Table 6.3.4.1-1 Summary of the Allermatch Results of Brazzein within OFSP (Full-Length
Sequence Alignment) ......................................................................................................... 36
Table 6.3.4.1-2 Summary of the AllergenOnline (Version 21) Results of Residual Komagataella
Proteins in OFSP ................................................................................................................. 37
Table 6.3.4.1-3 Assessment of the Allergenicity Potential of Brazzein within OFSP Using AlgPred........... 39
Table 6.3.4.1-4 Assessment of the Allergenicity Potential of Residual Host Cell Proteins from
Komagataella phaffii Using AlgPred .................................................................................. 39

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

GRAS Notice for Oubli Fruit Sweet Protein Derived from

Komagataella phaffii
PART 1. §170.225 SIGNED STATEMENTS AND CERTIFICATION
In accordance with Title 21 of the Code of Federal Regulations (CFR) §170 Subpart E consisting of §170.203
through 170.285, Oobli, Inc. (“Oobli”) hereby informs the United States (U.S.) Food and Drug Administration
(FDA) that the intended uses of its Oubli Fruit Sweet Protein Product, derived from Komagataella phaffii and
containing ≥35% brazzein by weight (hereinafter “Oubli Fruit Sweet Protein,” or “OFSP”). The intended uses
of OFSP as a sweetener in various conventional food and beverage products as described in Section 1.3
below are not subject to the premarket approval requirements of the Federal Food, Drug, and Cosmetic Act
based on Oobli’s view that these notified uses of OFSP are Generally Recognized as Safe (GRAS). In addition,
as a responsible official of Oobli, the undersigned hereby certifies that all data and information presented in
this GRAS Notice represent a complete and balanced submission that is representative of the generally
available literature as described in Section 6.1. Oobli considered all unfavorable as well as favorable
information that is publicly available and/or known to Oobli and that is pertinent to the evaluation of the
safety and GRAS status of OFSP as a food ingredient for addition to conventional food and beverage
products, as described herein.

Signed,

(""f DocuSigned by:

4/18/2023
J D6AD848049D2J

Jason Ryder, Ph.D. Date

Chief Technology Officer
Oobli, Inc.
[email protected]

1.1 Name and Address of Notifier

Oobli, Inc.
202 Cousteau Place, Suite 210
Davis, CA
95618 USA

1.2 Common Name of Notified Substance

Oubli fruit sweet protein (OFSP)

Oubli fruit sweet protein

Oubli fruit sweet protein product

Oubli fruit sweet protein ingredient

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

1.3 Conditions of Use

Oobli's OFSP is intended for use as a sweetening agent in various conventional foods and beverages. A
summary of the food categories and use levels in which OFSP is intended for use is provided in Table 1.3-1
below. Food uses are organized according to 21 CFR §170.3 (U.S. FDA, 2022a).

OFSP is not intended for use in infant formula or infant food products, and the proposed food categories do
not include food uses that are subject to the oversight by the United States Department of Agriculture
(USDA) and the USDA Food Safety Inspection Service.

Table 1.3-1 Summary of the Individual Proposed Food Uses and Use Levels for OFSP in the
United States
Food Category Food Usesa OFSP Use Levels
(21 CFR §170.3) (mg/100 g)
(U.S. FDA, 2022a)
Beverages, alcoholic Cocktail drinks (pre-packaged) 7
Beverages and Beverages Bases, Packaged water-based beverages 22
non-alcoholic Non-milk-based meal replacement beverages and protein drinks 2
Chewing Gum Chewing gum 99
Coffee and Tea Ready-to-drink coffee beverages 7
Ready-to-drink tea beverages 9
Dairy Product Analogs Milk analogs 4
Non-dairy yogurts 13
Frozen Dairy Desserts and Mixes Ice cream 23
Frozen yogurt 23
Frozen milk desserts and bars 24
Fruit and Water Ices Edible ices 24
Sherbet 30
Sorbet 31
Grain Products and Pastas Cereal bars, granola bars, energy, protein, and meal replacement bars 30
Granola 27
Milk Products Packaged milk-based beverages 6
Yogurt 12
Yogurt drinks 9
Processed Fruits and Fruit Juices Packaged fruit drinks, nectar, and fruit-based smoothies 16
Snack Foods Fruit-based bars (without granola) 36
Soft Candy Truffles 47
Gummy bear 62
CFR = Code of Federal Regulations; OFSP = Oubli Fruit Sweet Protein.
a OFSP is intended for use in unstandardized products and products with standards of identity, as established under 21 CFR §130
to 169, do permit its addition.

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18 April 2023 6
1.4 Basis for GRAS
Pursuant to 21 CFR §170.30 (a)(b) (U.S. FDA, 2022b), Oobli has concluded that the intended uses of OFSP as
described herein are GRAS on the basis of scientific procedures.

1.5 Availability of Information

The data and information that serve as the basis for this GRAS Notice will be sent to the U.S. FDA upon
request or will be available for review and copying at reasonable times at the offices of:

Oobli, Inc.
202 Cousteau Place, Suite 210
Davis, CA
95618 USA

Should the U.S. FDA have any questions or additional information requests regarding this GRAS Notice,
Oobli will supply these data and information upon request.

1.6 Freedom of Information Act, 5 U.S.C. 552

It is Oobli’s view that all data and information presented in Parts 2 through 7 of this GRAS Notice do not
contain any trade secret, commercial, or financial information that is privileged or confidential; therefore,
all data and information presented herein are not exempted from the Freedom of Information Act,
5 U.S.C. 552.

Oobli, Inc.
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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

PART 2. §170.230 IDENTITY, METHOD OF MANUFACTURE,

SPECIFICATIONS, AND PHYSICAL OR TECHNICAL EFFECT
2.1 Identity of the Ingredient
2.1.1 Oubli Fruit Sweet Protein

OFSP is produced through a strain of K. phaffii that has been genetically engineered to express a gene
encoding for brazzein- 53; brazzein is the component of OFSP that provides the ingredient's sweetening
properties and is discussed in greater detail under Section 2.1.2 below. OFSP is comprised of primarily
protein (ca. 80%) which contains the active constituent brazzein (ca. 40%) with remaining balance of host
cell proteins carried over from the fermentation process of K. phaffii (ca. 40%), followed by ash (ca. 5%),
moisture (ca. 5%), carbohydrates (ca. 10%), and fat (ca. 0.3%).

A proteomics assessment of OFSP was performed to investigate the identity of the host cell proteins
originated from the production organism. Protein solutions (1 mg/mL) were prepared from three
production batches of OFSP (Lot Nos. OFSPB53-304, OFSPB53-215, and OFSPB53-538), digested by trypsin,
and analyzed using liquid chromatography with tandem mass spectrometry (LC- MS/MS), where peptides
were then reassembled into the complete protein sequence. The database search tool MSFragger was used
for peptide identification using the brazzein 53-amino acid sequence and the K. phaffii proteome data for
brazzein protein identification and host cell protein identification, respectively. The results were returned
using Scaffold Proteome Software. Data were reported on the basis of total spectral count, defined as the
total number of spectra identified for a protein, yielding a semiquantitative measure of protein abundance
in proteomic studies. An approximate 1% spectra count cut-off criteria was used for identifying host cell
proteins in the final product. As shown in Table 2.1.1-1, five host cell proteins were identified that met this
1% cut-off value in the proteomics analysis, accounting for up to 13.2% of the total protein fraction of OFSP.
The remaining total protein fraction are comprised of residual host cell proteins that were below the 1%
cut-off value. Of the total protein in OFSP, the average brazzein content was reported to be 46.78% of the
total spectrum, with the balance of 53.22% to be residual host cell proteins derived from the K. phaffii
production strain. The complete list of residual host cell proteins and their average values in the three
production batches of OFSP is provided in Appendix A.

Table 2.1.1-1 Summary of the Abundance of Residual Komagataella Proteins in OFSP

Identified Protein Accession Number Protein Length Manufacturing Lot No. Average %
(amino acids) OFSPB53- OFSPB53- OFSPB53- of total
304 (% of 215 (% of 538 (% of spectrum)
total total total
spectrum) spectrum) spectrum)
Brazzein protein P56552 53 50.04 39.02 51.29 46.78
Fusion protein, identical to C4R0U2 128 6.06 5.87 6.78 6.23
Rpl40Bp OS=Komagataella
phaffii (strain GS115 / ATCC
20864) OX=644223
GN=PAS_chr2-1_0486 PE=4
SV=1
Transaldolase C4R245 324 2.86 3.25 3.34 3.15
OS=Komagataella phaffii
(strain GS115 / ATCC 20864)
OX=644223 GN=PAS_chr2-
2_0337 PE=3 SV=1

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

Table 2.1.1-1 Summary of the Abundance of Residual Komagataella Proteins in OFSP

Identified Protein Accession Number Protein Length Manufacturing Lot No. Average %
(amino acids) OFSPB53- OFSPB53- OFSPB53- of total
304 (% of 215 (% of 538 (% of spectrum)
total total total
spectrum) spectrum) spectrum)
Vacuolar aspartyl protease C4R6G8 410 1.77 1.19 1.8 1.59
(Proteinase A)
OS=Komagataella phaffii
(strain GS115 / ATCC 20864)
OX=644223
GN=PAS_chr3_1087 PE=3
SV=1
Peptide hydrolase C4QYZ6 509 1.18 0.95 1.54 1.22
OS=Komagataella phaffii
(strain GS115 / ATCC 20864)
OX=644223 GN=PAS_chr1-
4_0611 PE=3 SV=1
S-adenosylmethionine C4R5U7 384 0.42 1.67 0.94 1.01
synthase OS=Komagataella
phaffii (strain GS115 / ATCC
20864) OX=644223
GN=PAS_chr3_0876 PE=3
SV=1
OFSP = Oubli Fruit Sweet Protein.

2.1.2 Brazzein

Brazzein protein is the characterizing component of OFSP. Brazzein is naturally present in the fruit of the
West African plant Pentadiplandra brazzeana Baillon, commonly referred to as oubli fruit (Caldwell et al.,
1998). Natural brazzein has been found to occur in the oubli fruit in 2 isoforms: major isoform with
54 amino acids (~80%) and minor isoform with 53 amino acids (~20%) (Neiers et al., 2021). The amino acid
sequence of the major isoform (referred to as "brazzein - 54") is listed in the UniProt database under
Accession No. P56552. Sensory analyses have reve aled that the minor isoform (referred to as "brazzein -53")
is sweeter than the major form (i.e., brazzein-54) (Poirer et al., 2012). The brazzein characterizing the OFSP
is the minor isoform (brazzein-53), which has a monoisotopic mass of 6,357.734 Da in its native
conformation. The amino acid sequence of Oobli's brazzein -53 is shown below.

DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY

The identity of the active brazzein constituent of OFSP has been demonstrated via tandem mass
spectrometry (MS/MS) peptide mapping and intact protein mass spectrometry (IPMS). As shown in
Figure 2.1.2- 1, the peptide map of Oobli's brazzein -53, obtained by tryptic digest peptide mapping,
demonstrates a high degree of sequence coverage (~96%) with 15 exclusive unique peptides matching the
amino acid sequence to native, plant-based brazzein-53 sequence. The IPMS- measured mass of Oobli's
brazzein-53 is 6,357.741 Da and can be matched to the calculated mass of 6,357.734 Da for brazzein-53 in
its native conformation (see Figure 2.1.2-2). These data indicate that brazzein-53 produced by Oobli is
substantially equivalent to native brazzein.

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Figure 2.1.2-1 Sequence Coverage from Tandem Mass Spectrometry Peptide Mapping Analysis of
Oobli’s Brazzein-53

I I I
I I I I
I I I I
I I I I
I I I I

DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY

Figure 2.1.2-2 Deconvoluted MS1 Spectrum of Oobli’s Brazzein-53

6357.741
100

90

80

]::- 70
"iii
C
2 60
s
Q)
>
~ 50
ai
a:::
40

30

20

10
2521_. 354
2000 4000 6000 8000 10000 12000 14000 16000 18000
Mass

The three-dimensional (3D) structure of brazzein in solution was determined by proton nuclear magnetic
resonance spectroscopy at pH 5.2 and 22°C (Caldwell et al., 1998). The 3D structure of brazzein is shown in
Figure 2.1.2-3 (Caldwell et al., 1998). The protein contains 1 α-helix, 3 strands of antiparallel β-sheet, and
4 disulfide bonds, which confer stability to the chemical structure. The protein structure of brazzein is
unique such that it does not share any similarity to other sweet-tasting proteins with known structures,
including monellin and thaumatin (Caldwell et al., 1998; Picone and Temussi, 2012).

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Figure 2.1.2-3 Three-Dimensional Structure of Brazzein

2.2 Manufacturing Process

2.2.1 Description of the Manufacturing Process

The manufacturing process of OFSP complies with current Good Manufacturing Practice (cGMP) and Hazard
Analysis and Critical Control Points (HACCP) principles. A schematic of the production process is provided in
Figure 2.2.1-1 and is described below.

The manufacturing process for OFSP uses precision fermentation to produce the naturally sweet brazzein
protein. As a first step, inoculum is prepared from working cell banks using multiple seed propagation steps.
Next, the resulting bolus of cells is used to inoculate the production fermentation process. Both the seed
propagation and production fermentation steps are monitored for cell growth, culture purity, protein titer,
glucose, nitrogen, phosphate, and ethanol. Process variables including pH, temperature, and dissolved
oxygen are monitored and controlled throughout the precision fermentation process.

Upon reaching the end of fermentation, the whole cell broth is processed to recover, separate, and purify
OFSP. First, a solid–liquid separation process separates the wet biomass from the fermentation supernatant
containing brazzein. This supernatant is then concentrated, pH-adjusted, and further purified using a
sequence of filtration, chromatography, and diafiltration steps. The resulting concentrated OFSP solution is
then dried into a powder consisting of at least 35% (w/w) brazzein in addition to sodium salts used as
stabilizing agents from the purification process.

All raw materials and processing aids, filtration aids, and pH adjusters used in the fermentation and
recovery processes for OFSP are safe and suitable standard ingredients that meet predefined quality
standards used in the food/enzyme industry. The raw materials conform to either the specifications set out
in the Food Chemicals Codex (11th edition) or to other applicable regulatory standards. The raw materials
are food-grade, of high purity and quality (Aunstrup et al., 1979), and suitable for their intended use,
i.e., they are either food-grade and GRAS or of high-quality chemical or pharmaceutical grades
(United States Pharmacopeia [USP], National Formulary [NF], or American Chemical Society [ACS] grades)
from approved suppliers.

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Figure 2.2.1-1 Manufacturing Process for OFSP

Fermentation

J
Solid-Liquid
Separation

J
Concentration

J
I Chromatography I

J
Diafiltration

J
Drying

OFSP = Oubli Fruit Sweet Protein.

2.2.2 Information on the Production Microorganism

2.2.2.1 Parental (Host) Organism

The parental (host) organism of OFSP is K. phaffii. Current laboratory strains of K. phaffii are from lineages
isolated from oak and chestnut trees and have been deposited in the culture collection at the
Northern Regional Research Laboratories (NRRL). The genome of K. phaffii was sequenced in 2009
(De Schutter et al., 2009) and has been extensively employed in the production of food ingredients and
pharmaceutical products (Ahmad et al., 2014). It is estimated that approximately 17% of total recombinant
products produced in 2009 were obtained from K. phaffii production organisms. The available evidence
demonstrates that K. phaffii is a well characterized microorganism with an established history of safe use in
food production. A summary of GRAS Notices pertaining to food ingredients obtained through fermentation
of K. phaffii that were filed without objection from the U.S. FDA is provided in Table 2.2.2.1-1 below.

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Table 2.2.2.1-1 Summary of GRAS Notices for Food Ingredients Produced Through Fermentation of
Komagataella phaffii
GRN No. Substance Intended Use
737 Soy leghemoglobin preparation from a For use at levels up to 0.8% soybean leghemoglobin protein to
strain of Pichia pastoris optimize flavor in ground beef analogue products intended to be
cooked.
967 Soluble egg-white protein produced by Intended for use as a substitute for egg-white protein in foods
Komagataella phaffii strain GSD-1209 containing eggs; and as a source of protein in nutritional
powders and drinks; bars; and certain snack foods at levels in
accordance with current good manufacturing practices
(excluding infant formula, or in any products under the
jurisdiction of the United States Department of Agriculture).
1001 Myoglobin preparation from a strain of Intended to impart flavor and aroma at levels up to
Pichia pastoris expressing the myoglobin 2% myoglobin in ground meat and poultry analogue products.
gene from Bos taurus

The safe strain lineage of K. phaffii NRRL Y-11430 is largely based on the recognized non-pathogenic and
non-toxigenic nature of this strain and the fact that the species itself has not been implicated with any
known adverse effects throughout its extensive use in food production. In addition, proteins originating
from a production strain derived from K. phaffii NRRL Y-11430 have been demonstrated to lack allergenic
and toxigenic potential (Jin et al., 2018; Reyes et al., 2021). Based on the Joint FAO/WHO Expert Committee
on Food Additives (JECFA) criteria for safe strain lineage, K. phaffii NRRL Y-11430 has been considered to be
a safe strain lineage suitable to serve as a host organism for food production (FAO/WHO, 2020). As
summarized in Table 2.2.2.1-1, a number of food ingredients, including soy leghemoglobin,
bovine myoglobin, and hen egg ovomucoid, produced using K. phaffii NRRL Y-11430 as a host organism
currently have GRAS status under GRAS Notice (GRN) 737, 1001, and 967, respectively (U.S. FDA 2018,
2021a,b).

OFSP is produced by precision fermentation using a strain of K. phaffii that has been genetically engineered
to express genes encoding for the biosynthesis of brazzein. The production strain is derived from K. phaffii
BG10, which originates from the well-characterized host organism K. phaffii NRRL Y-11430. The taxonomic
identity of K. phaffii is shown in Table 2.2.2.1-2 below. The host organism, K. phaffii BG10, has been
demonstrated to be genomically similar to K. phaffii NRRL Y-11430 and does not contain any native plasmids
or antibiotic resistance genes.

Table 2.2.2.1-2 Taxonomic Identity of Komagataella phaffii

Kingdom IFungi
Phylum IAscomycota
Class ISaccharomycetes
Order ISaccharomycetales
Family IPhaffomycetaceae
Genus IKomagataella
Species Iphaffii

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2.2.2.2 Construction of the Production Organism

The production strain for OFSP was derived from K. phaffii BG10 through a series of transformations with
different expression constructs to enable the biosynthesis of brazzein. The promoter and terminator within
the expression cassette are native sequences of K. phaffii. The gene encoding for brazzein was synthesized
de novo and codon-optimized from Pentadiplandra brazzeana (UniProtKB P56552). Brazzein expression
cassettes were stably integrated into the genome, where no plasmids or antibiotic resistance genes were
present in the production strain. Therefore, no plasmids or antibiotic resistance genes were expected to be
transferred to non-related microorganisms or the final product. All gene sequences were assembled into a
cloning plasmid, where the expression cassette was transformed into K. phaffii. The plasmid was selected
using zeocin, geneticin, or hygromycin. The plasmid was cured by re-streaking the colony 3 times and was
confirmed by negative selection on both selection and non-selection plates, as well as validated by colony
polymerase chain reaction (PCR). A glycerol stock of the production strain and wild-type strain was streaked
on yeast extract peptone dextrose (YPD) agar plates with the antibiotics hygromycin, zeocin, and geneticin,
with YPD agar plates without antibiotics used as a positive control. On the plates with the selection markers,
there was no indication of growth of the production strain or wild-type strain (see Figure 2.2.2.2-1),
indicating that the strain does not contain any plasmids with selective genes. The absence of plasmids was
also confirmed with colony PCR using primers to amplify the selective genes used for plasmid selection (i.e.,
encoding for resistance to hygromycin, geneticin, or zeocin (see Figure 2.2.2.2-2).

Furthermore, as the introduced genes were obtained de novo by DNA synthesis and were not derived
directly from the source organism, there was no risk of introducing unintended or undesirable genes from
the source organism into the production strain. The genetic stability of the production strain was confirmed
by Sanger sequencing at the beginning and end of the fermentation process. Additionally, bioinformatics
searches conducted using the amino acid sequence of brazzein encoded by the inserted gene confirm that
there are no significant sequence homology to known protein toxins or to known allergens (see
Section 6.3.4 for further details).

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18 April 2023 14
Figure 2.2.2.2-1 Streaked Yeast Extract Peptone Dextrose Plates with Hygromycin, Zeocin, or Geneticin
Selection Markers of the Production and Wild-type Komagataella phaffii Strain

Figure 2.2.2.2-2 Colony Polymerase Chain Reaction Results of the Production Strain

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18 April 2023 15
2.3 Product Specifications and Batch Analyses
2.3.1 Product Specifications

Food-grade product specifications have been established for OFSP. Specification limits have been
established for the proximate parameters (e.g., moisture, total protein, fat, ash, and carbohydrates), as well
as for brazzein as a percentage of the total mass of the ingredient as well as the percentage of the total
protein, heavy metals (arsenic, cadmium, lead, mercury), and microbiological contaminants. The methods of
analysis for each parameter follow internationally recognized methods (e.g., Association of Official
Analytical Collaboration [AOAC]). The brazzein content is measured using a validated internal method based
on high-performance liquid chromatography. See Table 2.3.1-1 for details.

Table 2.3.1-1 Product Specifications for OFSP

Specification Parameter Specification Limit Method of Analysis
Moisture (% w/w) <10% AOAC 950.46
Total protein (% w/w) >70% AOAC 950.23
Brazzein purity (% of total protein) >40% Calculation (brazzein mass/total protein)
Total brazzein (% total mass w/w) >35% Internal method (HPLC)
Fat by fatty acid profile (%) <1% AOAC 996.06
Asha (% w/w) <10% AOAC 945.46
Carbohydrates (% w/w) <15% Calculated
Heavy Metals
Arsenic <0.5 ppm AOAC 2015.01
Cadmium <0.5 ppm AOAC 2015.01
Lead <0.5 ppm AOAC 2015.01
Mercury <0.5 ppm AOAC 2015.01
Microbiological Contaminants
Aerobic plate count <10,000 CFU/g AOAC OMA
Total coliforms <10 CFU/g AOAC OMA
Escherichia coli <10 CFU/g AOAC OMA
Yeast <10 CFU/g AOAC OMA
Mold <10 CFU/g AOAC OMA
Salmonella spp. Not detected in 10 g AOAC OMA
Listeria Not detected in 10 g AOAC OMA
AOAC = Association of Official Analytical Collaboration; CFU = colony-forming units; HPLC = high-performance liquid
chromatography; OFSP = Oubli Fruit Sweet Protein; OMA = Official Methods of Analysis; ppm = parts per million.
a The ash content of the ingredient is composed of primarily sodium and lesser amounts of other minerals.

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2.3.2 Batch Analysis

Analysis of three non-consecutive production batches of OFSP demonstrates that the manufacturing
process as described in Section 2.2 produces a consistent product that meets the established specification
limits (see Table 2.3.2-1).

Table 2.3.2-1 Summary of the Batch Analysis for 3 Production Batches of OFSP
Specification Parameter Specification Limit Manufacturing Lot
OFSPB53-304 OFSPB53-231 OFSPB53-061
Moisture (% w/w) <10% 2.82 2.29 2.53
Total protein (% w/w) >70% 87.47 88.43 88.5
Brazzein Purity (% of total protein) >40% 57.2 44.8 48.2
Total Brazzein (% total mass w/w) >35% 50.1 39.7 42.7
Fat by fatty acid profile (%) <1% 0.12 0.03 0.76
Ash (% w/w) <10% 7.25 4.73 4.79
Carbohydrates (% w/w) <15% 2.34 4.52 3.42
Heavy Metals
Arsenic (ppm) <0.5 ppm 0.03 0.10 0.11
Cadmium (ppm) <0.5 ppm 0.02 0.006 <0.001
Lead (ppm) <0.5 ppm 0.05 0.01 <0.01
Mercury (ppm) <0.5 ppm 0.047 0.041 <0.005
Microbiological Contaminants
Aerobic plate count (CFU/g) <10,000 CFU/g 190 330 40
Coliforms (CFU/g) <10 CFU/g <10 <10 <10
Escherichia coli (CFU/g) <10 CFU/g <10 <10 <10
Yeast (CFU/g) <10 CFU/g <10 <10 <10
Mold (CFU/g) <10 CFU/g <10 <10 <10
Salmonella spp. Not detected in 10 g ND ND ND
Listeria Not detected in 10 g ND ND ND
CFU = colony-forming units; ND = not detected; OFSP = Oubli Fruit Sweet Protein; ppm = parts per million.
Note: <10 = less than the reporting limit as noted.

2.3.3 Mineral Profile

The mineral profile of three batches of OFSP (Lot Nos. OFSPB53-304, OFSPB53-231, and OFSPB53-061) was
determined using AOAC 2015.01. OFSP contains, on average, approximately 17,700 parts per million (ppm)
sodium, 2,408 ppm magnesium, 151 ppm manganese, 151 ppm iron, 1,560 ppm calcium, 108 ppm zinc, 73
ppm copper, 1 ppm molybdenum, 0.6 ppm nickel, 83 ppm phosphorus, and 10 ppm potassium. Based on
the proposed food uses of the OFSP, the resulting dietary exposures to these minerals in final consumers
are not expected to pose a safety concern (see Section 3.1.3).

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2.4 Stability
The shelf-life stability of OFSP was investigated under ambient temperature at 23.5°C and 40% relative
humidity. OFSP powder (Lot No. OFSPB53-101) was heat-sealed in mylar foil bags at room temperature and
stored for up to 12 months. Samples were taken at 0, 4, 6, 9, 12, and 15 months for analysis of aerobic plate
count, coliforms, Escherichia coli, yeast, mold, Salmonella spp., Listeria, as well as moisture, total protein,
fat by fatty acid, ash, and carbohydrates. As shown in Table 2.4-1, brazzein content of Oobli’s ingredient
remained stable over the storage periods, with no significant changes in any other measured parameter,
including the proximate and microbiological profile, indicating that the shelf-life of OFSP is at least
15 months. There were no significant changes in the brazzein content of the ingredient over the 15-month
storage period.

Table 2.4-1 Summary of the Analysis for Proximates and Microbiological Parameters of OFSP
Following Storage for 15 Months at Ambient Conditions (Temperature: 23.5°C,
Relative Humidity: 40%)
Parameter Specification Limit Timepoint
0 Months 4 Months 6 Months 9 Months 12 Months 15 Months
Moisture (% w/w) <10% 7.33 7.74 8.28 8.94 8.45 7.21
Total protein (% w/w) >70% 91.3 88.47 87.74 87.16 88.00 90.63
Brazzein purity >40% 60.2 NM NM 63.1 NM 61.7
(% of total protein)
Total brazzein >35% 55 NM NM 55 NM 56
(% total mass w/w)
Fat by fatty acid profile <1% 0.09 0.1 0.02 0.02 0.02 0.03
(g/100 g)
Ash (% w/w) <10% 3.09 3.1 3.06 3.27 3.34 3.59
Carbohydrates (% w/w) <15% <0.1 0.59 0.9 0.61 0.19 <0.1
Aerobic plate count (CFU/g) <10,000 CFU/g 5,400 4,700 2,500 8,500 1,900 1,600
Total coliforms (CFU/g) <10 CFU/g <10 <10 <10 <10 <10 <10
Escherichia coli (CFU/g) <10 CFU/g <10 <10 <10 <10 <10 <10
Yeast (CFU/g) <10 CFU/g <10 50 <10 <10 <10 <10
Mold (CFU/g) <10 CFU/g <10 <10 290 <10 <10 <10
Salmonella spp. (/10 g) Not detected in 10 g ND ND ND ND ND ND
Listeria (/10 g) Not detected in 10 g ND ND ND ND ND ND
CFU = colony-forming units; ND = not detected; NM = not measured; OFSP = Oubli Fruit Sweet Protein.

2.5 Technical Effect

2.5.1 Mechanism of Action of Brazzein

The protein characterizing the OFSP is the 53-amino acid brazzein, which is comprised of 1 α-helix (21–29),
3 strands of antiparallel β-sheet (strand I: 5–7; strand II: 44–50; strand III: 34–39), and 4 disulfide bonds
(see Figure 2.1.2-3). It has been previously reported that brazzein binds 536–545 residues of T1R3 (Jiang
et al., 2004). However, recent studies using in silico docking modeling have demonstrated that the primary
binding of brazzein is to the G protein-coupled receptor (GPCR), T1R2, and stabilized by T1R3 (Walters and
Hellekant, 2006; Belloir et al., 2017). These taste receptors are composed of the Venus flytrap motif (VFTM),
cysteine-rich domain, and transmembrane domain, and are present in both oral and extraoral tissues, such
as in the gastrointestinal tissue (Walters and Hellekant, 2006; Laffitte et al., 2014; Belloir et al., 2017; Kim et

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al., 2022). Binding to GPCR yield a taste signal through the GPCR signal transmission mechanism (Kim et al.,
2022). Using high-resolution structure data and models, Kim et al. (2022) demonstrated that brazzein binds
between the cysteine-rich domain of T1R2 and T1R3 to form a heterocomplex to elicit a sweet taste
response.

The binding mechanism of brazzein to T1R2 and T1R3 at the cysteine-rich domain is similar to other known
sweet proteins such as thaumatin and monellin, despite the differences in their protein structure
(Caldwell et al., 1998; Kim et al., 2022). Other commonly consumed small molecule sweetening agents, such
as sucrose, aspartame, neotame, saccharin, cyclamate, and sucralose, also bind to the T1R2 and T1R3
receptors, although on different regions, such as the pocket cavity of the VFTM of T1R2 and T1R3 (sucrose),
amino terminal domain of T1R2 (aspartame, neotame, saccharin), transmembrane region of T1R3
(cyclamate), and amino terminal domain of both T1R2/T1R3 (sucralose) (Kim et al., 2022). Laffitte et al.
(2014) reported that the sweet taste receptors, T1R2 and T1R3, expressed on enteroendocrine cells in the
gastrointestinal (GI) tract, especially in the small intestine and colon (Kojima and Nakagawa, 2011), are
involved in glucose sensing, expression of glucose transporters, and maintenance of glucose homeostasis.
However, as discussed in Sections 6.3.1 and 6.3.3, brazzein is expected to denature under the highly acidic
conditions of the stomach, thereby losing both its characteristic 3D protein structure as well as its ability to
bind to the T1R2 and T1R3 receptors encountered within the GI tract. In addition, as discussed in
Section 6.3.1.2 of in silico digestibility, the primary protein sequence is expected to be readily broken down
into smaller peptides of less than 11 amino acids in length, which would even further reduce or eliminate its
ability to bind to or stimulate these receptors. In summary, while brazzein is able to maintain its
characteristic 3D structure and elicit a sweet response in the more favorable conditions within the mouth, it
is likely both denatured and degraded under the much less favorable conditions within the GI tract, thus
rendering both protein and resulting peptides unable to bind or elicit a biological response. Furthermore,
when compared against sugars and other small molecule sweeteners which continue to stimulate T1R2 and
T1R3 receptors while traveling through the GI tract, the consumption of brazzein as a sweetening agent
represents a potential net reduction in overall stimulation of these receptors and any associated negative
feedback loops.

2.5.2 Sensory Studies on Oubli Fruit Sweet Protein

Oobli intends to market OFSP, as a sweetening agent in conventional food and beverage products. As
reported in the scientific literature, brazzein has a sweetness potency between 500 and 2,000 times that of
a 10% or 2% sucrose solution, respectively (Izawa et al., 1996). The sweet taste of brazzein remains stable
over pH 2.5 to 8 and upon heating up to 80°C for 4.5 hours or 98°C for 2 hours (Stone and Oliver, 1969).
Oobli has determined the relative sweetness intensity of OFSP against a 5% sucrose solution, which has a
designated sweetness intensity of 100. Five concentrations of OFSP (brazzein purity: 79%), i.e., 0.001, 0.003,
0.005, 0.007, and 0.01%, were prepared and given to a trained panel (N=8). Based on the results of this
study, Oobli’s OFSP was determined to have a sweetness intensity of 750 times that of a 5% sucrose
solution.

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PART 3. §170.235 DIETARY EXPOSURE
3.1 Estimated Dietary Consumption of Oubli Fruit Sweet Protein
3.1.1 Methodology

An assessment of the anticipated intake of OFSP as an ingredient under the intended conditions of use was
conducted using data available in the 2017-2018 cycle of the U.S. National Center for Health Statistics’
National Health and Nutrition Examination Survey (NHANES) (CDC, 2022a,b; USDA, 2022). Consumption
data from individual dietary records, detailing food items ingested by each survey participant, were collated
by computer and used to generate estimates for the intake of OFSP by the U.S. population. Estimates for
the daily intake of OFSP represent projected 2-day averages for each individual from Day 1 and Day 2 of
NHANES 2017-2018; these average amounts comprised the distribution from which mean and percentile
intake estimates were determined. Mean and percentile estimates were generated incorporating survey
weights in order to provide representative intakes for the entire U.S. population. “Per capita” intake refers
to the estimated intake of OFSP averaged over all individuals surveyed, regardless of whether they
consumed food products in which OFSP is proposed for use, and therefore includes individuals with “zero”
intakes (i.e., those who reported no intake of food products containing OFSP during the 2 survey days).
“Consumer-only” intake refers to the estimated intake of OFSP by those individuals who reported
consuming food products in which the use of OFSP is currently under consideration. Individuals were
considered “consumers” if they reported consumption of 1 or more food products in which OFSP is
proposed for use on either Day 1 or Day 2 of the survey.

The estimates for the intake of OFSP were generated using the maximum use level indicated for each
intended food use, as presented in Table 1.3-1, together with food consumption data available from the
2017-2018 NHANES datasets. The results for these assessments are presented in Section 3.1.2.

3.1.2 Intake Estimates for Oubli Fruit Sweet Protein

A summary of the estimated daily intake of OFSP from proposed food uses is provided in Table 3.1.2-1 on an
absolute basis (mg/person/day) and in Table 3.1.2-2 on a body weight basis (mg/kg body weight/day).

The percentage of consumers was high among all age groups evaluated in the current intake assessment;
more than 83.8% of the population groups consisted of consumers of food products in which OFSP is
currently proposed for use (see Table 3.1.2-1). Children 6 to 11 years old had the greatest proportion of
consumers, at 95.7%. The consumer-only estimates are more relevant to risk assessments, as they
represent exposures in the target population. Only the consumer-only intake results are discussed herein.

Among the total population (2 years and older), the mean and 90th percentile consumer-only intakes of
OFSP were determined to be 89 and 199 mg/person/day, respectively. Of the individual population groups,
male adults were determined to have the greatest mean and 90th percentile consumer-only intakes of OFSP
on an absolute basis, at 119 and 260 mg/person/day, respectively, while children 2 to 5 years old had the
lowest mean and 90th percentile consumer-only intakes, at 35 and 76 mg/person/day, respectively
(see Table 3.1.2-1).

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Table 3.1.2-1 Summary of the Estimated Daily Intake of OFSP from Proposed Food Uses in the
United States by Population Group (2017-2018 NHANES Data)
Population Group Age Group Per Capita Intake (mg/day) Consumer-Only Intake (mg/day)
(Years) Mean 90th Percentile % n Mean 90th Percentile
Children 2 to 5 32 70 92.0 412 35 76
Children 6 to 11 54 115 95.7 642 56 117
Female teenagers 12 to 19 64 137 91.4 403 71 143
Male teenagers 12 to 19 88 174 91.8 394 95 183
Female adults 20 and older 65 162 83.8 1,793 78 172
Male adults 20 and older 102 252 85.2 1,660 119 260
Total population 2 and older 77 181 86.5 5,304 89 199
n = sample size; NHANES = National Health and Nutrition Examination Survey; OFSP = Oubli Fruit Sweet Protein.

On a body weight basis, the total population (2 years and older) mean and 90th percentile consumer-only
intakes of OFSP were determined to be 1.30 and 2.88 mg/kg body weight/day, respectively. Among the
individual population groups, children 2 to 5 years old were identified as having the highest mean and
90th percentile consumer-only intakes of any population group, at 2.03 and 4.25 mg/kg body weight/day,
respectively. Female adults had the lowest mean and 90th percentile consumer-only intakes, at 1.05 and
2.21 mg/kg body weight/day, respectively (see Table 3.1.2-2).

Table 3.1.2-2 Summary of the Estimated Daily Per Kilogram Body Weight Intake of OFSP from
Proposed Food Uses in the United States by Population Group (2017-2018 NHANES
Data)
Population Group Age Group Per Capita Intake Consumer-Only Intake
(Years) (mg/kg bw/day) (mg/kg bw/day)
Mean 90th Percentile % n Mean 90th Percentile
Children 2 to 5 1.87 4.20 92.4 408 2.03 4.25
Children 6 to 11 1.64 3.41 95.7 640 1.72 3.43
Female teenagers 12 to 19 1.06 2.18 91.5 397 1.16 2.25
Male teenagers 12 to 19 1.37 2.72 91.7 391 1.50 2.96
Female adults 20 and older 0.88 2.09 83.8 1,779 1.05 2.21
Male adults 20 and older 1.13 2.82 85.1 1,647 1.33 3.11
Total population 2 and older 1.12 2.63 86.5 5,262 1.30 2.88
bw = body weight; n = sample size; NHANES = National Health and Nutrition Examination Survey; OFSP = Oubli Fruit Sweet
Protein.

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3.1.3 Dietary Intake Estimates of Minerals from Oubli Fruit Sweet Protein

As discussed in Section 2.3.3, OFSP contains measurable amounts of minerals such as sodium, magnesium,
manganese, iron, calcium, and zinc. The dietary intakes of the minerals present in OFSP were estimated
based on the highest estimated intake of 260 mg/day of OFSP by male adults (90th percentile). As shown in
Table 3.1.3-1 below, the estimated intakes of minerals present in the OFSP based on its proposed conditions
of use are well below the respective upper limit as established by the National Academies of Sciences,
Engineering, and Medicine (NASEM) [formerly Institute of Medicine (IOM)]. For sodium, the proposed uses
of OFSP contributes up to 5 mg/day to the American diet, which is well below the chronic disease risk
reduction intake established by the NASEM, which sets the limit for sodium for individuals 14 years and
older at 2,300 mg/day. For the other minerals, the contribution of these minerals to the diet based on the
proposed food uses of OFSP are negligible and therefore, it is not anticipated that dietary consumption of
the minerals present in OFSP would pose any safety concern.

Table 3.1.3-1 Comparison of Mineral Intake from OFSP and Corresponding Tolerable Upper Limit
Values as Established by NASEM
Mineral Average Mineral Composition of Mineral Intake from OFSP UL
OFSP (mg/kg) (mg/day)a (mg/day)b
Calcium 1,560 0.41 2,500
Copper 73 0.02 10,000
Iron 151 0.04 45
Magnesium 2,408 0.63 350
Manganese 151 0.039 11
Molybdenum 1.1 0.0003 2,000
Nickel 0.59 0.0002 1.0
Phosphorus 83 0.02 4,000
Potassium 10 0.003 ND
Sodium 17,700 4.6 ND
Zinc 108 0.03 40
NASEM = National Academies of Sciences, Engineering, and Medicine; ND = not detected; OFSP = Oubli Fruit Sweet Protein; UL =
tolerable upper limit intake.
a Calculated based on consumption of 260 mg/day of OFSP by male adults (90th percentile).
b Value represent the tolerable upper intake level for male adults (age ≥18 years) as established by NASEM (2019).

3.1.4 Summary and Conclusions

Consumption data and information pertaining to the intended food uses of OFSP were used to estimate the
per capita and consumer-only intakes of this ingredient for specific demographic groups and for the total
U.S. population. There were a number of assumptions included in the assessment which render exposure
estimates suitably conservative. For example, it has been assumed in this exposure assessment that all food
products within a food category contain OFSP at the maximum specified level of use. In reality, the levels
added to specific foods will vary depending on the nature of the food product and it is unlikely that OFSP
will have 100% market penetration in all identified food categories.

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

PART 4. §170.240 SELF-LIMITING LEVELS OF USE

No known self-limiting levels of use are associated with OFSP.

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

PART 5. §170.245 EXPERIENCE BASED ON COMMON USE IN FOOD

BEFORE 1958
Not applicable.

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18 April 2023 24
PART 6. §170.250 NARRATIVE AND SAFETY INFORMATION
6.1 Introduction
The subject of this GRAS Notice is OFSP, a novel protein-based sweetener manufactured by Oobli through
microbial fermentation of a bioengineered strain of K. phaffii. The production strain was derived from the
safe strain lineage K. phaffii NRRL Y-11430, a non-pathogenic and non-toxigenic species, and was genetically
engineered to express the gene encoding for brazzein. The gene encoding for brazzein was identified from
the naturally occurring plant source, Pentadiplandra brazzeana, within a gene databank and was
synthesized de novo prior to introduction into the host organism through standard biotechnology
techniques. The production strain underwent fermentation and the resulting expressed brazzein was
concentrated, purified, and processed into a powder consisting of ≥70% total protein (w/w) and
≥35% brazzein (w/w). OFSP is demonstrated to be absent of chemical (e.g., heavy metals) and
microbiological impurities that would raise a safety concern. Therefore, the safety assessment of OFSP is
focused on 2 aspects: the safety of the production organism and the safety of the ingredient itself. The
safety assessment of OFSP followed the principles described by Pariza and Johnson (2001) and Sewalt et al.
(2016) that are commonly employed in the evaluation of microbially-derived enzyme preparations. Under
this safety paradigm, elements such as characterization of the production organism and the genetic
modification steps to obtain this organism were evaluated, as well as any available toxicological studies on
the resulting ingredient (i.e., OFSP), as well as bioinformatics evaluation of the characterizing protein,
brazzein, within the OFSP. These elements are discussed in further detail in the sections that follow.

To facilitate the safety assessment of OFSP, a comprehensive search of the scientific literature was
conducted through March 2023 to identify publications on the safety of brazzein and the plant source
(Pentadiplandra brazzeana). The search was limited to articles with full texts within peer-reviewed scientific
journals and the following databases were accessed: Adis Clinical Trials Insight, AGRICOLA, AGRIS, Allied &
Complementary Medicine™, BIOSIS® Toxicology, BIOSIS Previews®, CAB ABSTRACTS, Embase®, Foodline®:
SCIENCE, FSTA®, MEDLINE®, NTIS: National Technical Information Service, Toxicology Abstracts, and
ToxFile®. One publication was identified in which mice were provided a 3M-brazzein solution in the drinking
water over 15 weeks and obesity-related endpoints were observed (Kim et al., 2020). No significant effects
on adiposity hypertrophy, glucose homeostasis, insulin resistance, or inflammation were observed
throughout the study period. However, this study did not evaluate standard toxicological endpoints such as
those described in Organisation for Economic Co-operation and Development (OECD) Test Guideline 408
and did not report on the purity of brazzein. This study was therefore not considered to be relevant to the
safety discussion of OFSP. A second study was identified which discussed a series of toxicological studies
investigating whether OFSP containing its active constituent, brazzein, has any genotoxic or systemic toxicity
potential (Lynch et al., 2023). These studies included a bacterial reverse mutation test (OECD Test Guideline
471), an in vitro mammalian micronucleus test (OECD Test Guideline 487), and a 90-day repeated-dose oral
toxicity study in rats (OECD Test Guideline 408) (OECD, 2016, 2018, 2020). All studies were conducted in
accordance with Good Laboratory Practices (GLP) and appropriate OECD Test Guidelines with OFSP meeting
specifications of Section 2.3. These studies are discussed in Section 6.3. The studies described by Lynch et al.
(2023) served as the pivotal evidence of safety to support OFSP as described herein.

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6.2 Safety of the Production Strain
The safety of Oobli’s production strain used in the production of OFSP was assessed using the principles
commonly employed in the safety evaluation of microbially-derived enzyme preparations (Pariza and
Johnson, 2001; Sewalt et al., 2016; FAO/WHO, 2020). This approach included an evaluation of the
pathogenicity, toxigenicity, and antimicrobial resistance of the production strain, as well as the genetic
modification steps to generate the production strain. These elements are discussed in further detail herein.

The production strain, K. phaffii P-BRZ-013, was derived from the host organism K. phaffii strain BG10,
which is a derivative of K. phaffii NRRL Y-11430, a Biosafety Level 1 organism. Strain BG10 and strain
NRRL Y-11430 are genomically similar (see Section 2.2.2.1 for further information on the history of these
strains) and have been well documented in GRNs 737, 967, 1001, and 1056 (U.S. FDA, 2018, 2021a,b,
2022c). K. phaffii (formerly referred to as Pichia pastoris) has an established history of safe use in food
production, particularly in the production of food enzymes and other food ingredients. In the U.S., P.
pastoris is permitted for use as a source of protein in broiler feed at levels up to 10% (21 CFR §573.750 –
U.S. FDA, 2022d). In the European Union, K. phaffii (known as Komagataella pastoris) has qualified
presumption of safety status for use in food production on the basis that it is incapable of producing toxic
metabolites under standard conditions of food processing. K. phaffii (formerly referred to as P. pastoris) is
not listed as a pathogen by the European Commission, National Institute of Allergy and Infectious Diseases,
or the U.S. FDA (EC, 2000; NIAID, 2018; U.S. FDA, 2022e). A search of the PubMed database indicates that
there have not been any pathogenic or toxigenic reports associated with this species in the scientific
literature. The totality of evidence indicates that K. phaffii (formerly referred to as P. pastoris) is a non-
pathogenic and non-toxigenic species, a viewpoint that is generally recognized by the scientific community
given its extensive use as a source organism for the production of food ingredients. Therefore, the publicly
available information on K. phaffii strains BG10 and NRRL Y-11430, as documented in previous GRAS Notices
for various food ingredients and food enzymes that received “no questions” from the FDA, demonstrates
that these species are non-pathogenic, non-toxigenic, and have an extensive history of safe use in food
production; these species would therefore serve as a safe and suitable source organism for the production
of brazzein.

As described in Section 2.2.2.2, Oobli’s production strain has been bioengineered to express the gene
encoding for brazzein using standard biotechnology techniques. The production strain has been
demonstrated to be absent of antibiotic resistance genes, and it does not contain any remaining expression
plasmids. The synthetic gene encoding for brazzein is the only exogenous genetic material introduced to
K. phaffii BG10. This gene is well characterized and has been demonstrated through bioinformatics means
to express the protein (brazzein) and does not confer any pathogenic or toxigenic factor into the host
organism. Oobli has demonstrated the production strain to be genetically stable after fermentation.
Therefore, it is concluded that Oobli’s production strain, K. phaffii P-BRZ-013, meets the criteria for a safe
and suitable source organism as described by Pariza and Johnson (2001).

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6.3 Safety of Oubli Fruit Sweet Protein
6.3.1 Digestibility of OFSP and Brazzein

6.3.1.1 In Vitro Digestibility of OFSP

Two in vitro digestibility studies have been conducted using OFSP containing brazzein. In the first study, the
methodology described by Thomas et al. (2004) was employed. The digestion assay was performed with
simulated gastric fluid (SGF) at pH 2 with pepsin/OFSP ratios of 5 and 10 U/µg of OFSP. The digestion of the
sample was run for 0.5, 1, 2, 5, 10, 20, and 30 minutes. An aliquot from each digestion time point was then
run on a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. As shown in
Figure 6.3.1.1-1 below, OFSP was not digested at any tested pepsin concentration at up to 30 minutes of
incubation.

A second 2-step in vitro digestibility study was conducted using a methodology described by
Brodkorb et al. (2019) with SGF at pH 2 with pepsin at 10 U/µg of OFSP and sequentially with simulated
intestinal fluid (SIF) at pH 7 with a mixture of pancreatic enzymes (pancreatin), including trypsin,
chymotrypsin, amylase, lipase, and colipase, at enzyme concentrations of 0.5 U/µg of OFSP for up to
180 minutes. Samples were digested in SGF with pepsin for 30 minutes, pH adjusted to pH 7 and introduced
into SIF with pancreatin. These samples then were obtained after 10, 30, 60, 90, 120, and 180 minutes. As
shown in Figure 6.3.1.1-2, OFSP was partially digested after 180 minutes. Based on a semiquantitative
analysis from the SDS-PAGE gel, after 180 minutes, approximately 70% of OFSP was digested by pancreatin.
The findings of these studies suggest that OFSP containing brazzein is partially digested under simulated
gastric followed by intestinal conditions.

Figure 6.3.1.1-1 Results of In Vitro Digestibility Study with OFSP with Simulated Gastric Fluid

.J!2. _____o_FS._P_ _ __ .J!2. ____,a: O.:..FS;:..:P_ _ __

Pepsin + + + + + + ++ + + + + Pepsin + + + + + + ++ + + + +
Time (min) O 30 O 30 30 O 0.5 1 2 5 10 20 30 Time (min) O 30 O 30 30 O 0.5 1 2 5 10 20 30

75 75
BSA ► BSA ►
50 50
37
Pepsin ►
37
Pe psi n ►

25 25
- 20 20

15
15
10
OFSP ► OFSP ► 10
5
s
Digested BSA [ Digested BSA [
L __ _ _ _ _ _ _ _ _ _ _ _ _ __ _ J

lOU pepsin /ug of OFSP SU pepsin /ug of OFSP

BSA = bovine serum albumin; min = minutes; OFSP = Oubli Fruit Sweet Protein.

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Figure 6.3.1.1-2 Results of In Vitro Digestibility Study with OFSP with Pepsin in Simulated Gastric Fluid
and with Pancreatin in Simulated Intestinal Fluid

Pepsin
Pepsin +
+ Panaeatin

Time (min)
Pepsin
0 --------
Pancreatin OFSP (no OFSP)

30 10 30 60 90 120180180180 ladder

75
so
37
Pepsin ►

25
20

15

10
OFSP ►
5

min = minutes; OFSP = Oubli Fruit Sweet Protein.

6.3.1.2 In Silico Digestibility of Brazzein

The digestibility of brazzein, the active protein within OFSP, was investigated using an in silico model,
PeptideCutter, under 2 conditions with pepsin (pH 1.3 and >2.0). A summary of the model results is
provided in Table 6.3.1.2-1. The PeptideCutter predictions suggest that brazzein is broken down by pepsin
into peptides that are up to 11 amino acids in length; the majority of the peptide digests are less than
11 amino acids in length, suggesting that brazzein is readily digested.

Following modeling of protein digestion using pepsin, the digests obtained from each condition were
evaluated with PeptideCutter using trypsin to further simulate digestion conditions within the GI tract.
Peptides that were less than 5 amino acids in length were not predicted to be digested by trypsin. The
remaining digests (DKCKKVYENY, PVSKCQL, DKHARSGEC, and DEKRNLQCICD) that were 10, 7, 9, and
11 amino acids in length, respectively, were predicted to be readily digested by trypsin as the digests were
less than 5 amino acids in length (see Table 6.3.1.2-2). Considering that small peptides <5 amino acids in
length were identified following trypsin digestion, these peptides were not further considered for
bioinformatics evaluation for allergenicity and/or toxigenicity potential. As discussed in Section 6.3.4,
bioinformatics assessment of the full-length sequence of brazzein did not identify any concern for
allergenicity and/or toxigenicity. Overall, the PeptideCutter modeling results suggest that brazzein is
digested under GI conditions and intact protein would not be present in the intestinal tract.

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Table 6.3.1.2-1 Summary of In Silico Digestion Using PeptideCutter with Pepsin (pH 1.3 and >2.0)
Position of Cleavage Cleaving Enzyme Resulting Peptide Sequence Peptide Length Peptide Mass
Site (amino acids) (Da)
10 Pepsin (pH>2) DKCKKVYENY 10 1289.469
17 Pepsin (pH1.3) PVSKCQL 7 773.946
Pepsin (pH>2)
22 Pepsin (pH>2) ANQCN 5 548.571
23 Pepsin (pH>2) Y 1 181.191
26 Pepsin (pH1.3) DCK 3 364.417
Pepsin (pH>2)
27 Pepsin (pH1.3) L 1 131.175
Pepsin (pH>2)
36 Pepsin (pH1.3) DKHARSGEC 9 1002.070
Pepsin (pH>2)
37 Pepsin (pH1.3) F 1 165.192
Pepsin (pH>2)
38 Pepsin (pH>2) Y 1 181.191
49 Pepsin (pH>2) DEKRNLQCICD 11 1336.500
50 Pepsin (pH>2) Y 1 181.191
52 Pepsin (pH>2) CE 2 250.270
53 end of sequence Y 1 181.191

Table 6.3.1.2-2 Summary of In Silico Digestion Using PeptideCutter with Trypsin

Position of Cleavage Cleaving Enzyme Resulting Peptide Sequence Peptide Length Peptide Mass
Site (amino acids) (Da)
DKCKKVYENY
2 Trypsin DK 2 261.278
4 Trypsin CK 2 249.328
5 Trypsin K 1 146.189
10 end of sequence VYENY 5 686.719
PVSKCQL
4 Trypsin PVSK 4 429.517
7 end of sequence CQL 3 362.444
DKHARSGEC
2 Trypsin DK 2 261.278
5 Trypsin HAR 3 382.423
9 end of sequence SGEC 4 394.400
DEKRNLQCICD
2 Trypsin DK 2 261.278
5 Trypsin HAR 3 382.423
9 end of sequence SGEC 4 394.400

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It is generally recognized that the normal fate of dietary proteins is digestion into small peptides and
individual amino acids that are absorbed into the systemic circulation via transcellular or paracellular routes
(EFSA, 2021). Proteins that are not fully digested or are partially digested travel to the large intestine where
they are ultimately fermented by the gut microbiota (Portune et al., 2016; Joye, 2019). In the large
intestine, where the microbiota concentration is much higher and the transit time is longer than in the small
intestine, the remaining protein is broken down to peptides and amino acids via extracellular bacterial
proteases and peptidases (Macfarlane et al, 1986). As part of the weight-of-evidence for the safety
discussion of a protein, the resistance of a protein to partial or complete digestion is relevant, as it suggests
that the protein could have the potential to elicit localized toxic effects by microbial fermentation (i.e., in
the gut) or could be absorbed as an intact protein that may elicit toxic effects in the systemic circulation
and/or allergenic effects (EFSA, 2021). It is noted that only a small number of food proteins have been
demonstrated to be capable of causing adverse effects with respect to allergy or toxicity when consumed
(Markell et al., 2017). It should be noted that dietary proteins are denatured at low pH (1.5 to 3.5) that
occur in the stomach, thus unfolding their 3D structure to yield the polypeptide chain, thereby impairing the
protein’s function (Callahan et al., 2022). Therefore, it would be expected that brazzein within OFSP,
following consumption, would not be present in an active form in the stomach and would be digested under
gastric and/or intestinal conditions.

As discussed in Sections 6.3.3 and 6.3.4, the potential systemic toxicity of OFSP was investigated in a 90-day
dietary toxicity study conducted in accordance with OECD Test Guideline 408 (OECD, 2018), and a
bioinformatics assessment was conducted to investigate the allergenicity potential of brazzein. The results
of the 90-day dietary toxicity study conducted with OFSP demonstrate that the intact brazzein protein and
any potential peptide digests do not pose any safety concern to final consumers based on the absence of
adverse effects in any toxicologically relevant test endpoint at the highest tested dose of 1,000 mg/kg body
weight/day (see Section 6.3.3 for further details). Additionally, OFSP was demonstrated to have a low
potential for allergenicity based on the results of an in silico investigation following internationally
recognized in silico guidelines established by FAO/WHO (2001) and Codex Alimentarius (2009)
(see Section 6.3.4 for further details).

6.3.2 Mutagenicity/Genotoxicity Studies on Oubli Fruit Sweet Protein

Bacterial Reverse Mutation Test

The potential mutagenicity of OFSP was investigated in a bacterial reverse mutation assay with
Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA in the presence
and absence of metabolic activation (Lynch et al., 2023). The study was conducted in accordance with the
OECD Test Guideline 471 (OECD, 2020) and the Principles of GLP (OECD, 1998). The main study was
conducted using the plate incorporation method. OFSP was tested in triplicate at concentrations of
1.58, 5.0, 15.8, 50, 158, 500, 1,580, and 5,000 μg/plate with and without metabolic activation. A
confirmatory test using the pre-incubation method was performed using the same strains and
concentrations as the main study. All strains received distilled water as the negative control. The
compounds used as positive controls in the assays conducted in the absence of metabolic activation
included 2-nitrofluroene (2-NF), sodium azide (NaN3), 9-aminoanthracene (9-AA), or 4-nitroquinoline
1-oxide (4-NQO). Assays conducted in the presence of metabolic activation used 2-aminoanthracene (2-AA)
or benzo(a)pyrene as the positive control. The number of revertant colonies in the positive controls were
greater than 2 times (or greater than 3 times in the case of strains TA1535 and TA1537) those reported in
the negative controls.

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OFSP did not induce any biologically relevant, concentration-related, or statistically significant increases in
revertant colony numbers compared with vehicle control counts in either the presence or absence of
metabolic activation. The mean number of revertant colonies of the vehicle controls were all within the
historical vehicle control ranges. Based on the results of the bacterial reverse mutation assay, OFSP is
concluded to have no mutagenic potential.

In Vitro Mammalian Cell Micronucleus Test

The genotoxic potential of OFSP was investigated in an in vitro mammalian cell micronucleus test (Lynch et
al., 2023). The study was conducted in accordance with OECD Test Guideline 487 (OECD, 2016). Human
peripheral blood lymphocytes were collected from whole blood samples via venous puncture from healthy,
non-smoking donors. An initial test for cytotoxicity of OFSP was conducted with and without metabolic
activation at concentrations of 15.6, 31.3, 62.5, 125, 250, 500, 750, 1,000, 2,500, and 5,000 μg/mL.
Cytotoxicity was assessed via cytokinesis block proliferation index, which was used to determine the
proportion of cytostasis (the inhibition of cell growth of treated cultures in comparison to control cultures).
The short-term experiment was conducted in duplicate at concentrations of 62.5, 125, 250, 500, 1,000, and
2,000 μg/mL with and without metabolic activation for 4 hours. The long-term experiment was conducted in
duplicate with the same concentrations of OFSP without metabolic activation for 24 hours. The compounds
used as positive controls in the assays conducted in the absence of metabolic activation included
methylmethanesulfonate (MMS; clastogenic control) and colchicine (aneugenic control). Assays conducted
in the presence of metabolic activation used cyclophosphamide as the positive clastogenic control. The S9
liver microsomal fraction was obtained from male Sprague-Dawley rats induced with phenobarbital/
β-naphthoflavone. The positive controls induced biologically relevant and statistically significant increases in
the percentage of micronucleated cells compared with vehicle controls, with mean values within historical
positive control ranges.

In the initial cytotoxicity test, OFSP did not induce excessive cytotoxicity (≤30% cytostasis); hence, the
maximum concentration tested in the short-term and long-term experiments was 2,000 µg/mL, the highest
recommended by OECD Test Guideline 487 (OECD, 2016). In the short-term experiment, a statistically
significant increase in the percentage of micronucleated cells was observed at the highest concentration of
2,000 µg/mL with metabolic activation when compared with vehicle controls. However, the number of
micronucleated cells was within the range of the historical negative control; therefore, this increase was
regarded as not biologically relevant. In the long-term experiment, no biologically relevant increases in the
percentage of micronucleated cells were reported at any of the concentrations analyzed. Based on the
results of this study, OFSP was concluded to have no clastogenic or aneugenic potential in human peripheral
blood lymphocytes, as there were no biologically relevant increases in the percentage of micronucleated
cells at any of the concentrations analyzed compared with vehicle controls.

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6.3.3 Studies in Animals

6.3.3.1 Systemic Toxicity Studies with Oubli Fruit Sweet Protein

A 90-day oral toxicity study was conducted with in accordance with OECD Test Guideline 408 (OECD, 2018)
and the Principles of GLP (OECD, 1998) (Lynch et al., 2023). Adult CRL: Sprague-Dawley® CD® IGS rats
(n=10/sex/group) were provided OFSP in the diet at concentrations of 0 (control diet), 250 (low-dose),
500 (mid-dose), and 1,000 (high-dose) mg/kg body weight/day for 90 days. The animals in all study groups
were provided access to water and food ad libitum. The doses were selected based on the results of 14-day
oral repeated-dose study that served as a dose range–finding study in which no treatment-related
toxicological effects were reported at the highest dose tested (nominally 1,000 mg/kg body weight/day).

The dietary concentrations of OFSP achieved the targeted nominal intake values of 250, 500, and
1,000 mg/kg body weight/day (actual values: 0, 245, 490, and 978 mg/kg body weight/day and 0, 245, 493,
and 985 mg/kg body weight/day in males and females, respectively). The general condition of the animals
was assessed 3 times a day. Weekly measurements were conducted in all animals for body weight and food
consumption. Ophthalmological examinations were conducted before the study and at Week 13. In the final
week of the dose administration period, urinalysis was conducted. At the end of the study period,
hematology, blood chemistry, and pathological examinations (i.e., organ weight measurement, macroscopic
and histopathological examinations) were conducted. The incidence of alopecia was well dispersed
throughout the control and treatment groups of both sexes. This finding was due to the type of feeding
equipment used per the guidelines in the protocol. There were no treatment-related toxicological effects on
body weights, body weight gain, food consumption, functional observation battery (i.e., sensorimotor
function, grip strength, and locomotor activity), ophthalmological findings, hematological and blood
chemistry parameters, urinalysis findings, organ weights, macroscopic, and histopathological findings. No
mortality was reported.

Functional observational battery (FOB) tests and motor activity (MA) tests were conducted prior to dose
administration and monthly thereafter. A statistically significant interaction with time, but not treatment,
was reported for the FOB tests; however, this was an expected finding as the animals are more active in the
initial sessions relative to the later ones.

Hematological evaluations revealed the following statistically significant findings: decreased mean cell
hemoglobin (MCH) and platelet counts in the top 2 dose group females, a slight decrease in lymphocyte
counts in high-dose females, and a slight increase in activated partial thromboplastin time (APTT) time in
top dose females only. There were no statistically significant changes in any of the male dose groups. The
decrease in MCH was not of toxicological significance since all values remained within the normal historical
control range. The decrease in platelet count was considered of no toxicological significance as in each
group, including the controls, the values reported were above the historical control range and no decreases
were noted in males. Likewise, the slight increase in APTT time in high-dose females was slight, not seen in
males, and not accompanied by any changes in prothrombin time (PT).

Statistically significant changes in clinical chemistry parameters included increased aspartate

aminotransferase (AST) in high-dose females, slightly increased total protein in low- and high-dose females,
and increased total cholesterol and low-density lipoprotein in mid-dose males. The AST values in the high-
dose females remained within the historical control range and were not accompanied by any statistically
significant increases in other enzymes associated with liver function (i.e., alanine transaminase, alkaline
phosphatase, SDH, and gamma-glutamyl transferase). In addition, there was no evidence of any increase in
AST in males or changes in liver histopathology indicative of an adverse effect of treatment in either sex.

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The weights of the epididymides were slightly decreased in the treated males, with relative-to-body weight
values achieving statistical significance in the low- and high-dose groups and relative-to-brain weight values
achieving statistical significance in the top 2 dose groups. There were no statistically significant effects on
the absolute weights of the epididymides. In addition, pituitary gland weights (absolute and relative-to-body
weight) were slightly increased in mid-dose males, prostate gland weights (absolute and relative-to-body)
slightly increased in the low- and high-dose groups, and the thyroid parathyroid weights minimally
increased in low-dose males. None of these changes were observed in females.

The few statistically significant changes reported from the FOB, hematology, clinical chemistry, and organ
weight analyses did not show a dose-dependent increase and did not correlate with any adverse
histopathological findings.

Under the conditions of this 90-day dietary toxicity study conducted in compliance with appropriate test
guidelines (OECD Test Guideline 408) and GLP, in the absence of test article–related adverse effects in both
male and female rats at any tested dose, the no-observed-adverse-effect level (NOAEL) for OFSP was
concluded to be 978 mg/kg body weight/day in males and 985 mg/kg body weight/day in females, the
highest tested dose (OECD, 1998, 2018). Based on the reported NOAEL and the highest estimated dietary
intake of 4.25 mg/kg body weight/day (90th percentile in children 2 to 5 years of age), an approximately 230-
fold margin of exposure (MOE) can be estimated. This suggests that there are no safety concerns of OFSP
under its proposed conditions of use as described in Section 1.3.

6.3.3.2 Other Brazzein Studies

Kim et al. (2020) investigated the effects of exposure to brazzein on obesity, metabolic disorder, and
inflammation markers in 7-week old C57BL/6J mice (N=20). Animals were provided a 3M-brazzein solution
in the drinking water for 15 weeks. The 3M-brazzein solution was produced by Kluyveroyces lactis and was
reported to be a 6.5 kDa protein that is 22,500 times sweeter than a 1% sucrose solution, and
approximately 6,000 times sweeter than a 10% sucrose solution on a weight basis. The food and water
intake were monitored daily, and body weights were measured weekly. A glucose tolerance test and insulin
tolerance test were performed after fasting for 12 hours. Blood samples were collected from the tail vein at
0, 15, 30, 60, and 120 minutes after glucose or insulin injection, and blood glucose was measured. All
animals were terminated at the end of the study and blood samples were collected for biochemical analysis.
Inguinal and epididymal white adipose tissue and liver were collected for analysis.

In addition, animals were administered 3M-brazzein by intraperitoneal (i.p.) injection, and blood glucose
and insulin levels were measured and compared to animals administered glucose by i.p. injection after
12 hours of fasting. The authors reported that blood glucose levels increased in the animals administered
glucose, but not in animals administered 3M-brazzein. No insulin response was reported in animals
receiving 3M-brazzein.

Following consumption of 3M-brazzein in the drinking water for 15 weeks, no significant changes in body
weight, food intake, water intake, total calorie intake, body fat accumulation, glucose homeostasis, insulin
response, or inflammation biomarker (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α) were reported
compared to the control group receiving a normal chow diet. The levels of insulin and c-peptide were higher
in the animals receiving sucrose than 3M-brazzein, which was not significantly higher compared to the
control. No effects on insulin resistance, or insulin signaling (as measured by mRNA expression in the
liver insulin pathway of IRS2 and GLUT4) were reported in animals receiving 3M-brazzein. The findings of
this study suggest that brazzein, following consumption, does not impact any obesity-related endpoint, and
does not elicit an insulin response.

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6.3.4 Bioinformatics Assessment of Brazzein and Residual Host Cell Proteins within
Oubli Fruit Sweet Protein

6.3.4.1 Allergenicity

The Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) and
Codex Alimentarius have recommended a stepwise approach to the allergenicity evaluation of proteins in
which the source of the protein of interest is first evaluated then the protein itself is assessed for sequence
homology to known allergenic proteins (FAO/WHO, 2001; Codex Alimentarius, 2009). The protein of interest
is considered to share significant sequence homology if a set of 80-amino acid length sequences (segments
1–80, 2–81, 3–82, etc.) derived from the full-length amino acid sequence shares more than 35% identity
with a known allergen, or an exact match of 6 contiguous amino acids, in which case possible cross-
reactivity between the proteins may exist (FAO/WHO, 2001; Codex Alimentarius, 2009). The sliding window
of 80 amino acids correspond with a typical domain size of a protein, such that a single protein domain may
contain epitopes that mediate antibody binding. In cases where sequence homology is not identified, the
protein of interest is not considered to contain potential allergenicity and unlikely to be cross-reactive to
known allergens (Codex Alimentarius, 2009). In theory, 6 to 8 contiguous amino acids may represent a
minimum length of linear immunoglobulin E (IgE) binding epitopes and T cell epitopes from peanut allergens
(Ara h 1 and Ara h 2) (Herman et al., 2009; Ladics, 2019; Abdelmoteleb et al., 2021); however, its usefulness
in predicting potential allergenicity is unclear, as these matches have been known to produce false positives
(Ladics, 2019). The allergenicity potential of brazzein and five residual host cell proteins in OFSP was
investigated using the step-wise approach described by FAO/WHO (2001) and Codex Alimentarius (2009).
Sequence homology searches were conducted against the curated databases of AllergenOnline and
Allermatch. In addition, a support vector machine (SVM) search with AlgPred was conducted with the same
brazzein and the five residual host cell proteins. The results of the searches are summarized below.

To assess whether the brazzein (i.e., brazzein-53) within OFSP shares amino acid sequence homology to
putative allergens that would suggest potential for allergenic cross-reactivity, a search of the AllergenOnline
database (Version 21) maintained by the Food Allergy Research and Resource Program of the University of
Nebraska-Lincoln was conducted using the approaches described by FAO/WHO (2001) and Codex
Alimentarius (2009). The 80-amino acid alignment searches were conducted using default settings (percent
identity >35%) and the FASTA36 algorithm. No matches between brazzein and putative allergens were
identified sharing greater than 35% identity, indicating that brazzein would be unlikely to have any
allergenic cross-reactivity. Despite the potential for false positives, a search for exact 8-amino acid matches
was performed with the amino acid sequence of brazzein against the AllergenOnline database. No 8-amino
acid exact matches were identified.

A full-length sequence homology search was also conducted with brazzein against the AllergenOnline
database using default settings. One match to a putative allergen from peach (Prunus persica) with
>50% identity was identified. The corresponding E-value was 0.76 and bit-score was 24.6. It has been
reported in the scientific literature that allergenic cross-reactivity between proteins sharing less than
50% similarity is rare and typically requires >70% identity (Aalberse, 2000). In addition, the biological
relevance of identified matches in in silico allergenicity assessment is typically inferred through the identity
score, E-value, and bit-score of the match. Pearson (2013) reported that an E-value of less than 0.001 and
bit-scores greater than 40 can be reliably used to infer homology; matches meeting these criteria may be
considered significant. In a recent bioinformatics publication on a comprehensive allergenicity assessment
of the proteomes of novel microorganisms, Abdelmoteleb et al. (2021) demonstrated that an E-value less
than 10-7 reflects a functional similarity between two proteins and likely suggests a biologically relevant
similarity for allergenic cross-reactivity potential. Therefore, considering that the identified match in the

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full-length search was less than 70% identity with an E-value and bit-score of 0.76 and 24.6, respectively,
this finding was not considered to be significant and is not likely to be reflective of allergenic cross-reactivity
of brazzein.

Additional in silico allergenicity searches were performed using Allermatch, which is maintained by a
research group at Wageningen University. The Allermatch database is composed of known allergenic
proteins from the UniProtKB allergen database,1 the WHO and International Union of Immunological
Societies list of allergen nomenclature,2 and the Comprehensive Protein Allergen Resource database.3 The
80-amino acid alignment searches were conducted using default settings (percent identity >35%) and the
FASTA algorithm. Similar to the search with AllergenOnline, no matches sharing greater than 35% identity
were identified with brazzein and putative allergens from the Allermatch database. An 8-amino acid exact
match search was also performed, and no 8-amino acid exact matches were identified. A full-length search
of the amino acid sequence of brazzein was also conducted using default settings against the Allermatch
database.

The results of this search indicated that brazzein shares >50% identity with 20 sequences from the
Allermatch database (see Table 6.3.4.1-1). Despite these putative allergens sharing >50% identity, the
statistical significance of the sequence homology is very low based on the E-values and bit-scores of the
matches. Given that the all of the E-values of the significant hits from the full-length search of the
Allermatch database were above 0.001 (the lowest E-value was 1.1) and that all of the bit-scores were
below 40 (the highest bit-score was 24.1) these alignments with greater than 50% identity are not
considered significant sequence alignments.

1 https://www.uniprot.org/docs/allergen.
2 http://www.allergen.org/.
3 https://comparedatabase.org/.

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DocuSign Envelope ID: D3944DA8-99CC-4988-BC67-059E84B8E185

Table 6.3.4.1-1 Summary of the Allermatch Results of Brazzein within OFSP (Full-Length Sequence Alignment)
Protein Species % Identitya % Similaritya E-value Bit-score Amino Amino Accession No.
Acid Acid
Overlap Length
Basic form of pathogenesis related Prunus persica 60.0 73.3 1.1 24.1 15 161 GenBank XP_007199020
protein 1
PREDICTED: lysozyme C, milk isozyme Equus asinus 53.3 73.3 17 20.0 15 148 GenBank XP_014705584
Phospholipase A1 2 Vespa affinis 72.7 72.7 28 20.3 11 301 UniProt PA12_VESAF
Venom allergen 5.02 Vespa crabro 58.3 75.0 31 19.6 12 202 UniProt VA52_VESCR
Venom allergen 5.01 Vespa crabro 58.3 75.0 31 19.6 12 202 UniProt VA51_VESCR
Alcohol dehydrogenase Cochliobolus lunatus 54.5 81.8 38 20.1 11 352 UniProt Q2HYZ7_COCLU
Unnamed protein product, partial Dermatophagoides pteronyssinus 53.8 69.2 50 18.2 13 129 GenBank CAD38378
Unnamed protein product, partial Dermatophagoides pteronyssinus 53.8 69.2 50 18.2 13 129 GenBank CAD38381
Unnamed protein product, partial Dermatophagoides pteronyssinus 53.8 69.2 50 18.2 13 129 GenBank CAD38383
Unnamed protein product, partial Dermatophagoides pteronyssinus 53.8 69.2 50 18.2 13 129 GenBank CAD38382
Unnamed protein product, partial Dermatophagoides pteronyssinus 53.8 69.2 50 18.2 13 129 GenBank CAD38379
36 kda allergen {peptide 143-115} Blattella germanica 57.1 71.4 74 15.3 7 25 GenBank AAB29345
Phospholipase A1 1 Dolichovespula maculate 63.6 72.7 76 18.8 11 300 UniProt PA11_DOLMA
Phospholipase A1 1 Vespa affinis 63.6 72.7 76 18.8 11 301 UniProt PA11_VESAF
Phospholipase A1 Vespa crabro 63.6 72.7 76 18.8 11 301 UniProt PA1_VESCR
Phospholipase A1 2 Dolichovespula maculate 63.6 72.7 76 18.8 11 303 UniProt PA12_DOLMA
Phospholipase A1 Vespa velutina 63.6 72.7 77 18.8 11 304 UniProt PA1_VESVE
PREDICTED: parvalbumin beta Crocodylus porosus 50.0 83.3 80 17.3 12 109 GenBank XP_019397705
Procalin Triatoma protracta 55.6 88.9 83 17.7 9 151 UniProt PRCLN_TRIPT
Hydrophobic seed protein Glycine max 60.0 70.0 95 16.6 10 80 UniProt HPSE_SOYBN
OFSP = Oubli Fruit Sweet Protein.
a Percent identity refers to the ratio of the number of matching residues to the total length of the alignment. Percent similarity counts “similar” residues (usually amino acids) in

addition to the identical ones.

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Brazzein was co-purified with other proteins from its K. phaffii host cell organism in OFSP. Using methods
described in Section 2.1.1, five host cell proteins were identified to be co-purified with brazzein. The
allergenicity potential of the five host cell proteins was investigated using the stepwise approach described
above with AllergenOnline. Of the five host cell proteins, two of the proteins—transaldolase
(UniProt: C4R245) and vacuolar aspartyl protease (UniProt: C4R6G8)—shared a number of matches to
putative allergens in the 80-amino acid sliding window search (see Table 6.3.4.1-2). No matches were
identified for the other three proteins. The identified matches suggest significant sequence homology to
allergenic proteins from fungal species such as Fusarium proliferatum, Pencillium chrysogenum,
Cladosporium cladosporioides, Rhizopus oryzae, and Aspergillus fumigatus, as well as mosquito
(Aedes aegypti) and wild boar (Sus scrofa). The matches to the proteins from fungal sources are likely
matches to respiratory allergenic proteins, while the matches to proteins from mosquito and wild boar are
likely dermal and oral allergens, respectively. No matches were identified to major food allergens that are of
relevance to final consumers. While allergenic reactions to mosquitoes have been documented, mosquito
allergy is quite rare and it is expected that the prevalence of mosquito allergy is rare in the global
population (Arias-Cruz et al., 2006; González Diaz et al., 2010). In the U.S., the prevalence of mosquito
allergy is estimated to be in the range of 1 to 10 per 10 million people (Kausar, 2018). Similarly, respiratory
allergy from fungal sources is rare and is estimated to impact up to 6% of the general population (Horner et
al., 1995). Therefore, although the identified matches may be suggestive of a significant sequence homology
with potential for allergenic cross-reactivity, under the proposed conditions of use of OFSP, the allergenic
risk would be low. Furthermore, the allergenic potential of residual host cell proteins from production
strains derived from K. phaffii NRRL Y-11430 and BG10 have been previously discussed (Jin et al., 2018;
Reyes et al., 2021) as part of the GRAS determination of soy leghemoglobin and bovine myoglobin derived
from a genetically engineered strain of K. phaffii (see GRNs 737 and 1001).

No proteins native to Komagataella have been identified as allergenic concerns. Considering that Oobli’s
production strain is derived from K. phaffii BG10, and the genetic modifications to the host organism are
well characterized and do not pose any allergenic concern, it is not expected that any native residual
proteins from the host organism would pose a risk for allergenicity.

Table 6.3.4.1-2 Summary of the AllergenOnline (Version 21) Results of Residual Komagataella
Proteins in OFSP
Protein Species Best % # Hits Full Alignment
Identity >35% E-value % Identity Length
Transaldolase
Transaldolase Fusarium proliferatum 81.30% 245/245 1.5e-098 72.40% 322
Transaldolase Penicillium chrysogenum 75.00% 245/245 1e-090 66.00% 324
Transaldolase Cladosporium cladosporioides 73.80% 245/245 2.6e-090 66.30% 323
Vacuolar Aspartyl Protease (Proteinase A)
Aspartyl Rhizopus oryzae 63.70% 307/331 2.3e-088 48.50% 410
endopeptidase
Lysosomal aspartic Aedes aegypti 61.70% 276/331 1.3e-066 47.40% 340
protease
Pepsin A Sus scrofa 53.80% 152/331 4.5e-054 39.50% 324
Aspergillopepsin i Aspergillus fumigatus 36.27% 25/331 7.1e-022 29.10% 357
OFSP = Oubli Fruit Sweet Protein.

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The allergenicity potential of brazzein and the five residual host cell proteins was also considered through a
search using AllerTOP (version 2.0),4 a bioinformatics tool for prediction of allergenicity. The method is
based on auto cross covariance transformation of protein sequences into uniform equal-length vectors as
developed by Wold et al. (1993). AllerTOP predicted brazzein to be a “probable allergen,” with the nearest
protein being the antigen five precursor from Glossina morsitans (Accession No. ADD19985.1). With the
exception of vacuolar aspartyl protease (UniProt No. C4R6G8) and transaldolase (UniProt No. C4R245),
AllerTOP predicted the other three residual host cell proteins to be “probable non-allergens.” Vacuolar
aspartyl protease was predicted to be a “probable allergen” with the nearest protein to be transaldolase
from Homo sapiens (UniProt No. P37837), while transaldolase was predicted to be a “probable allergen”
with the nearest protein to be pollen allergen Sec c 4 from Secale cereale (Accession No. CAH92627.1).

In addition to the in silico searches against AllergenOnline and Allermatch, the allergenicity potential of
brazzein and the five residual host cell proteins was investigated using a SVM analysis from AlgPred.5
Information on the sensitivity, specificity, and error rate of AlgPred, as well as the results of the analysis for
brazzein, are summarized in Table 6.3.4.1-3. The results for the five residual host cell proteins are presented
in Table 6.3.4.1-4. The results of AlgPred identified mixed results: the brazzein protein was predicted to be a
non-allergen based on algorithms for IgE epitopes, the Motif Alignment and Search Tool, and allergen
representative peptides, and was predicted to be a potential allergen based on SVM analysis of the
amino acid composition and dipeptide composition. Similar results were identified for the five residual host
cell proteins. AlgPred has been used previously for the allergenicity assessment of soy leghemoglobin
obtained from a genetically engineered strain of P. pastoris, which has GRAS status for use in meat-
analogue products as described in GRN 737 (U.S. FDA, 2018). The notifier reported that the allergenicity
assessment using AllergenOnline,6 similar as described above, was “more than adequate to demonstrate
that both soy leghemoglobin and the Pichia proteins within LegH Prep have little or no allergenic potential.”
Furthermore, the notifier stated that SVM-based analysis is controversial, as the reliability of this method is
questionable. AlgPred predicted the soy leghemoglobin to be a potential allergen; however, it was noted
that this methodology has a high false positive rate. The applicant stated that AlgPred identified 46% of all
proteins in the SwissProt to be potential allergens, even after all known allergens and related proteins were
removed (Saha and Raghana, 2006; Impossible Foods Inc., 2018).

4 https://www.ddg-pharmfac.net/AllerTOP/index.html.
5 http://crdd.osdd.net/raghava/algpred/index.html.
6 http://www.allergenonline.org/.

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Table 6.3.4.1-3 Assessment of the Allergenicity Potential of Brazzein within OFSP Using AlgPred
Algorithm Result Sensitivity Specificity Error Rate Analysis
(True Allergen) (True Non-Allergen) (False Allergen) Type
IgE epitopes The protein sequence 10.84% 98.25% 1.75% Sequence
does not contain motif
experimentally proven
IgE epitope
Motif Alignment and Non-allergen 22.05% 86.68% 13.32% Sequence
Search Tool (MAST) motif
Allergen representative Non-allergen 66.56% 97.97% 2.03% Sequence
peptides (ARP) motif
Support vector machine Potential allergen 84.21% 56.07% 43.93% Amino acid
(SVM) amino acid composition
composition
Support vector machine Potential allergen 84.83% 61.09% 38.91% Amino acid
(SVM) dipeptide Composition
composition
IgE = immunoglobulin E; OFSP = Oubli Fruit Sweet Protein.

Table 6.3.4.1-4 Assessment of the Allergenicity Potential of Residual Host Cell Proteins from
Komagataella phaffii Using AlgPred
Protein Algorithm Result
Peptide hydrolase IgE epitopes The protein sequence does not contain experimentally
(UniProt No. C4QYZ6) proven IgE epitope.
MAST Non-allergen
ARP Non-allergen
SVM amino acid composition Allergen
SVM dipeptide composition Allergen
Fusion protein IgE epitopes The protein sequence does not contain experimentally
(UniProt No. C4R0U2) proven IgE epitope.
MAST Non-allergen
ARP Non-allergen
SVM amino acid composition Potential allergen
SVM dipeptide composition Potential allergen
S-adenosylmethionine synthase IgE epitopes The protein sequence does not contain experimentally
(UniProt No. C4R5U7) proven IgE epitope.
MAST Non-allergen
ARP Non-allergen
SVM amino acid composition Non-allergen
SVM dipeptide composition Non-allergen
Vacuolar aspartyl protease IgE epitopes The protein sequence does not contain experimentally
(UniProt No. C4R6G8) proven IgE epitope.
MAST Non-allergen
ARP Non-allergen
SVM amino acid composition Allergen
SVM dipeptide composition Allergen
Transaldolase IgE epitopes The protein sequence does not contain experimentally
(UniProt No. C4R245) proven IgE epitope.
MAST Non-allergen

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Table 6.3.4.1-4 Assessment of the Allergenicity Potential of Residual Host Cell Proteins from
Komagataella phaffii Using AlgPred
Protein Algorithm Result
ARP Non-allergen
SVM amino acid composition Allergen
SVM dipeptide composition Allergen
ARP = Allergen Representative Peptides; IgE = immunoglobulin E; MAST = Motif Alignment and Search Tool; SVM = support vector
machine.

The results of the above-described in silico allergenicity assessment has been published by Lynch et al.
(2023). The publicly available information sufficiently demonstrates that the brazzein protein within OFSP
and presence of residual Komagataella proteins do not contain any inherent allergenicity potential and
would pose a low risk for allergenicity in final consumers.

6.3.4.2 Toxigenicity

To determine whether brazzein within OFSP shares significant sequence homology with known protein
toxins, sequence homology searches were conducted using the Basic Local Alignment Search Tool (BLAST)
with the amino acid sequence of brazzein-53 and the residual Komagataella proteins (from Table 2.1.1-1)
against protein sequences obtained from a curated databases of 7,683 animal venom proteins and toxins7
maintained by UniProt. The BLAST searches were conducted using algorithm (word size of 6 and expect
threshold of 0.001) and scoring parameters (BLOSUM62 scoring matrix with default gap costs and
composition adjustments). Sequences were considered to share structural homology/similarity based on
the criteria described by Pearson (2013). Pearson (2013) reported that “homologous sequences that share
more than 40% identity are very likely to share functional similarity,” and that an E-value of <0.001 can
reliably be used to infer sequence homology (Pearson, 2013). Alternatively, the bit-score can be used to
infer homology and is considered to be more reliable indicator of significant sequence homology. A
bit-score of 50 is “almost always significant,” while a bit-score of 40 is only significant (E-value <0.001) in
searches of protein databases with less than 7,000 entries (Pearson, 2013).

Based on the above criteria, no significant similarity to any of the venom proteins and toxins were identified
(E-values >0.0015) in the homology searches with brazzein and the residual Komagataella proteins. These
findings suggest that brazzein and the presence of any residual Komagataella protein within OFSP does not
harbor any toxigenic potential based on its amino acid sequence.

6.4 GRAS Panel Evaluation

Oobli has concluded that OFSP is GRAS for use in conventional food and beverage products, as described in
Section 1.3, on the basis of scientific procedures. This GRAS conclusion is based on data generally available
in the public domain pertaining to the safety of OFSP, as discussed herein, and on consensus among a panel
of experts (the GRAS Panel) who are qualified by scientific training and experience to evaluate the safety of
food ingredients. The GRAS Panel consisted of the following qualified scientific experts: Professor Joseph
Baumert (University of Nebraska-Lincoln); Professor Emeritus George C. Fahey, Jr. (University of Illinois);
and Professor Emeritus Michael W. Pariza (University of Wisconsin-Madison).

The GRAS Panel, convened by Oobli, independently and critically evaluated all data and information
presented herein, and also concluded that OFSP is GRAS for use in conventional food and beverage products

7 UniProt release Oct, 2017; available at: https://www.uniprot.org/program/Toxins.

Oobli, Inc.
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as described in Section 1.3, based on scientific procedures. A summary of data and information reviewed by
the GRAS Panel, and evaluation of such data as it pertains to the proposed GRAS uses of OFSP, is presented
in Appendix B.

6.5 Conclusion
Oobli intends to manufacture the Oubli Fruit Sweet Protein for use as a sweetener in various food and
beverage products. Oobli produces brazzein through precision fermentation using a genetically engineered
host strain of Komagataella phaffii. The production strain has been derived from the safe strain lineage
K. phaffii NRRL Y-11430, a non-pathogenic and non-toxigenic species, that has been genetically engineered
to express the gene encoding for brazzein. The gene encoding for brazzein was identified from the naturally
occurring plant source, Pentadiplandra brazzeana, synthesized de novo, and introduced to the host
organism through standard biotechnology techniques. OFSP is manufactured in accordance with cGMP and
HACCP using appropriate food-grade raw materials and processing aids. The final product consists of
~35% total brazzein (w/w), ~80% total protein (w/w), ~5% moisture (w/w), ~5% ash (w/w), and ~10%
carbohydrates (w/w) and does not contain levels of heavy metals and microbiological contaminants that
could pose a safety concern.

Oobli has conducted a series of in silico, in vitro, and in vivo studies to address safety concerns of
allergenicity and toxicity of brazzein within OFSP and residual host cell proteins, as well as in vitro and in
silico digestibility assays, which have indicated that the majority of the protein is digested in simulated
intestinal conditions. Additionally, in silico analyses for potential allergenicity and toxigenicity of brazzein
and residual proteins from K. phaffii in the final ingredient have been assessed. The results of these studies,
collectively, demonstrate that brazzein within OFSP and any residual proteins from the production strain
lack allergenicity potential. The OFSP has also been subject to the standard battery of toxicology studies to
investigate its mutagenic/genotoxic profile and systemic toxicity. These studies included a bacterial reverse
mutation test (OECD Test Guideline 471), in vitro micronucleus test (OECD Test Guideline 487), and a 90-day
dietary toxicity study in rats (OECD Test Guideline 408). All studies were conducted in accordance with
appropriate OECD Test Guidelines and GLP (OECD, 1998, 2016, 2018, 2020). Further, the toxigenicity
potential of brazzein within OFSP investigated through an in silico approach against a curated database of
animal venom proteins and toxins suggest the proteins also lack toxigenicity potential, which is consistent
with the results of the 90-day dietary toxicity study in rats.

The use levels of OFSP range between 2 and 99 mg/100 g in the different food categories in the U.S. Using
information within the NHANES database, the resulting mean and 90th percentile intakes is approximately
1.30 mg/kg body weight/day and 2.88 mg/kg body weight/day, respectively. The findings from these studies
demonstrate OFSP to be non-mutagenic and non-genotoxic, and the NOAEL from the 90-day study at the
highest dose tested was approximately 978 mg/kg body weight/day in males and 985 mg/kg body
weight/day in females. Based on the highest estimated dietary intake of 4.25 mg/kg body weight/day
(90th percentile in children 2 to 5 years of age), the MOE is estimated to be 230-fold, suggesting that OFSP,
under its proposed conditions of use, does not pose any safety concerns to end consumers.

Based on the technical and scientific data and information presented herein, Oobli has concluded that the
OFSP produced from a genetically engineered strain of K. phaffii is GRAS for use in conventional food and
beverage products on the basis of scientific procedures. OFSP therefore may be marketed and sold for its
intended purpose in the U.S. without the promulgation of a food additive regulation under Title 21,
Section 170.3, of the Code of Federal Regulations.

Oobli, Inc.
18 April 2023 41
PART 7. §170.255 LIST OF SUPPORTING DATA AND INFORMATION
Aalberse RC (2000). Structural biology of allergens. J Allergy Clin Immunol 106(2):228-238.
DOI:10.1067/mai.2000.108434.

Abdelmoteleb M, Zhang C, Furey B, Kozubal M, Griffiths H, Champeaud M, et al. (2021). Evaluating potential
risks of food allergy of novel food sources based on comparison of proteins predicted from genomes
and compared to www.AllergenOnline.org. Food Chem Toxicol 147:111888 [13pp].
DOI:10.1016/j.fct.2020.111888.

Ahmad M, Hirz M, Pichler H, Schwab H (2014). Protein expression in Pichia pastoris: recent achievements
and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301-5317.
DOI:10.1007/s00253-014-5732-5.

Arias-Cruz A, Avitia-Valenzuela E, Gonzalez-Diaz SN, Galindo-Rodriguez G (2006). Epidemiology of mosquito

bite allergy in the Centre of Allergy and Clinical Immunology of Monterrey, Mexico. J Allergy Clin
Immunol 117(2):S128 [abstract 500]. DOI:10.1016/j.jaci.2005.12.512.

Aunstrup K, Andersen O, Falch EA, Nielsen TK (1979). Production of microbial enzymes (chapter 9). In:
Peppler, HJ, Perlman D, editors. Microbial Technology: Volume 1, 2nd edition, pp. 282-309.

Belloir C, Neiers F, Briand L (2017). Sweeteners and sweetness enhancers. Curr Opin Clin Nutr Metab Care
20(4):279-285. DOI:10.1097/MCO.0000000000000377.

Brodkorb A, Egger L, Alminger M, Alvito P, Assunção R, Ballance S, et al. (2019). INFOGEST static in vitro
simulation of gastrointestinal food digestion. Nat Protoc 14(4):991-1014. DOI:10.1038/s41596-018-
0119-1.

Caldwell JE, Abildgaard F, Dzakula Z, Ming D, Hellekant G, Markley JL (1998). Solution structure of the
thermostable sweet-tasting protein brazzein. Nat Struct Biol 5(6):427-431. DOI:10.1038/nsb0698-
427.

Callahan A, Leonard H, Powell T (2022). Protein digestion and absorption. In: Nutrition: Science and
Everyday Application, v. 2.0: PressBooks (Creative Commons Attribution-NonCommercial 4.0
International License). Available at:
https://openoregon.pressbooks.pub/nutritionscience2e/chapter/6d-protein-digestion-absorption/.

CDC (2022a). National Health and Nutrition Examination Survey (NHANES): 2017-2018. Hyattsville (MD):
Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS).
Available at: https://wwwn.cdc.gov/nchs/nhanes/continuousnhanes/default.aspx?BeginYear=2017
[updated July 2022].

CDC (2022b). National Health and Nutrition Examination Survey (NHANES): 2017-2018 – Dietary Data.
Hyattsville (MD): Centers for Disease Control and Prevention (CDC), National Center for Health
Statistics (NCHS). Available at:
https://wwwn.cdc.gov/nchs/nhanes/Search/DataPage.aspx?Component=Dietary&CycleBeginYear=2
017 [updated July 2022].

Oobli, Inc.
18 April 2023 42
Codex Alimentarius (2009). Foods Derived from Modern Biotechnology, 2nd edition. (Codex Alimentarius).
Geneva, Switzerland: World Health Organization (WHO) / Rome, Italy: Food and Agriculture
Organization of the United Nations (FAO). Available at:
https://www.fao.org/3/a1554e/a1554e00.htm.

De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, et al. (2009). Genome sequence of
the recombinant protein production host Pichia pastoris. Nat Biotechnol 27(6):561-566 [plus
supplementary data]. DOI:10.1038/nbt.1544.

EC (2000). Directive 2000/54/EC of 18 September 2000 on the protection of workers from risks related to
exposure to biological agents at work (seventh individual Directive within the meaning of Article
16(1) of Directive 89/391/EEC). Off J Eur Communities 43(L262):21-45. Available at: https://eur-
lex.europa.eu/legal-content/EN/TXT/?qid=1579115863802&uri=CELEX:32000L0054 [current
consolidated version: 24/06/2020].

EFSA (2021). Statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of
genetically modified plants (EFSA Panel on Genetically Modified Organisms/GMO) (Question no:
EFSA-Q-2020-00314, adopted: 26 November 2020, published: 12 January 2021 by European Food
Safety Authority). EFSA J 19(1):6350 [16pp]. DOI:10.2903/j.efsa.2021.6350. Available at:
https://www.efsa.europa.eu/en/efsajournal/pub/6350.

FAO/WHO (2001). Evaluation of Allergenicity of Genetically Modified Foods. Report of a Joint FAO/WHO
Expert Consultation on Allergenicity of Foods Derived from Biotechnology, 22-25 January 2001.
Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) / Geneva, Switzerland:
World Health Organization (WHO). Available at:
https://www.who.int/publications/m/item/evaluation-of-allergenicity-of-genetically-modified-
foodsreport-of-a-joint-fao-who-expert-consultation-on-allergenicity-of-foods-derived-from-
biotechnology.

FAO/WHO (2020). Chapter 9: Principles related to specific groups of substances. Section 9.1.4.2: Enzymes.
In: Principles and Methods for the Risk Assessment of Chemicals in Food. (Environmental Health
Criteria, No. 240). Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) /
Geneva, Switzerland: World Health Organization (WHO), International Programme on Chemical
Safety (IPCS). Available at: https://www.who.int/publications/i/item/9789241572408;
https://cdn.who.int/media/docs/default-source/food-safety/publications/section9-1-4-2-
enzymes.pdf?sfvrsn=e238e86e_2 [section 9.1.4.2. revised from 2009 edition].

González Diaz SN, Cruz AA, Sedó Mejía GA, Rojas Lozano AA, Valenzuela EA, Vidaurri Ojeda AC (2010).
Prevalencia de reacciones secundarias por picadura del mosquito aedes aegypti en el centro
Regional de Alergia e Inmunología Clínica del Hospital Universitario, Monterrey, Nuevo León
[Prevalence of reactions secundary to mosquito bites Aedes aegypti at en el Regional Center of
Allergy and Clinical Immunology, University Hospital, de Monterrey, Nuevo Leon]. Rev Alerg Mex
57(2):37-43 [Spanish].

Herman RA, Song P, Thirumalaiswamysekhar A (2009). Value of eight-amino-acid matches in predicting the
allergenicity status of proteins: an empirical bioinformatic investigation. Clin Mol Allergy 7:9 [7pp].
DOI:10.1186/1476-7961-7-9.

Horner WE, Helbling A, Salvaggio JE, Lehrer SB (1995). Fungal allergens. Clin Microbiol Rev 8(2):161-179.
DOI:10.1128/CMR.8.2.161.

Oobli, Inc.
18 April 2023 43
Impossible Foods Inc. (2018). GRAS Notification for Soy Leghemoglobin Protein Preparation Derived from
Pichia Pastoris. (GRN 737 with Studies dated 2018). Submitted by Redwood City (CA): Impossible
Foods Inc. to College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food
Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 [Jul. 23,
2018 - FDA response - no questions].

Izawa H, Ota M, Kohmura M, Ariyoshi Y (1996). Synthesis and characterization of the sweet protein
brazzein. Biopolymers 39(1):95-101. DOI:10.1002/(sici)1097-0282(199607)39:1<95::aid-
bip10>3.0.co;2-b.

Jiang P, Ji Q, Liu Z, Snyder LA, Benard LM, Margolskee RF, et al. (2004). The cysteine-rich region of T1R3
determines responses to intensely sweet proteins. J Biol Chem 279(43):45068-45075 [plus
supplementary figures]. DOI:10.1074/jbc.M406779200.

Jin Y, He X, Andoh-Kumi K, Fraser RZ, Lu M, Goodman RE (2018). Evaluating potential risks of food allergy
and toxicity of soy leghemoglobin expressed in Pichia pastoris. Mol Nutr Food Res 62(1):1700297
[13pp]. DOI:10.1002/mnfr.201700297.

Joye I (2019). Protein digestibility of cereal products. Foods 8(6):199 [14pp]. DOI:10.3390/foods8060199.

Kausar MA (2018). A review on respiratory allergy caused by insects. Bioinformation 14(9):540-553.

DOI:10.6026/97320630014540.

Kim H, Kang J, Hong S, Jo S, Noh H, Kang BH, et al. (2020). 3M-brazzein as a natural sugar substitute
attenuates obesity, metabolic disorder, and inflammation. J Agric Food Chem 68(7):2183-2192.
DOI:10.1021/acs.jafc.0c00317.

Kim TY, Woo EJ, Yoon TS (2022). Binding mode of brazzein to the taste receptor based on crystal structure
and docking simulation. Biochem Biophys Res Commun 592:119-124.
DOI:10.1016/j.bbrc.2022.01.004.

Kojima I, Nakagawa Y (2011). The role of the sweet taste receptor in enteroendocrine cells and pancreatic β-
cells. Diabetes Metab J 35(5):451-457. DOI:10.4093/dmj.2011.35.5.451.

Ladics GS (2019). Assessment of the potential allergenicity of genetically-engineered food crops. J

Immunotoxicol 16(1):43-53. DOI:10.1080/1547691X.2018.1533904.

Laffitte A, Neiers F, Briand L (2014). Functional roles of the sweet taste receptor in oral and extraoral
tissues. Curr Opin Clin Nutr Metab Care 17(4):379-385. DOI:10.1097/mco.0000000000000058.

Lynch B, Wang T, Vo T, Tafazoli S, Ryder J (2023). Safety evaluation of Oubli fruit sweet protein (brazzein)
derived from Komagataella phaffii, intended for use as a sweetener in food and beverages. Toxicol
Res Appl 7 [21pp]. DOI:10.1177/23978473231151258.

Macfarlane GT, Cummings JH, Allison C (1986). Protein degradation by human intestinal bacteria. J Gen
Microbiol. 132(6):1647-56. DOI: 10.1099/00221287-132-6-1647.

Oobli, Inc.
18 April 2023 44
Markell LK, Wezalis SM, Roper JM, Zimmermann C, Delaney B (2017). Incorporation of in vitro digestive
enzymes in an intestinal epithelial cell line model for protein hazard identification. Toxicol In Vitro
44:85-93. DOI:10.1016/j.tiv.2017.06.018.

NASEM (2019). Appendix J: Dietary Reference Intakes: Summary tables. In: Dietary Reference Intakes for
Sodium and Potassium. (National Academies of Sciences, Engineering, and Medicine/NASEM; Health
and Medicine Division/HMD, Food and Nutrition Board/FNB, Health and Medicine Division,
Committee to Review the Dietary Reference Intakes for Sodium and Potassium). Washington (DC):
National Academies Press (NAP). Available at:
https://nap.nationalacademies.org/read/25353/chapter/28.

Neiers F, Belloir C, Poirier N, Naumer C, Krohn M, Briand L (2021). Comparison of different signal peptides
for the efficient secretion of the sweet-tasting plant protein brazzein in Pichia pastoris. Life (Basel)
11(1):46 [12pp, plus supplementary figure]. DOI:10.3390/life11010046.

NIAID (2018). NIAID Emerging Infectious Diseases/ Pathogens. Bethesda (MD): National Institutes of Health
(NIH), National Institute of Allergy and Infectious Diseases (NIAID). Available at:
https://www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens [content last
reviewed on July 26, 2018].

OECD (1998). OECD Principles of Good Laboratory Practice. (Series on Principles of Good Laboratory Practice
and Compliance Monitoring, No. 1 [ENV/MC/CHEM(98)17]). Paris, France: Organisation for
Economic Co-operation and Development (OECD), Environment Directorate, Chemicals Group and
Management Committee, OECD Environmental Health and Safety Publications.
DOI:10.1787/9789264078536-en. Available at: https://doi.org/10.1787/9789264078536-en [as
revised in 1997].

OECD (2016). In vitro mammalian cell micronucleus test. In: OECD Guidelines for the Testing of Chemicals.
(OECD Guideline, No. 487) [updated & adopted: 29 July 2016]. Paris, France: Organisation for
Economic Co-operation and Development (OECD). DOI:10.1787/9789264264861-en. Available at:
https://doi.org/10.1787/9789264264861-en.

OECD (2018). Repeated dose 90-day oral toxicity study in rodents. In: OECD Guidelines for the Testing of
Chemicals. (OECD Guideline, No. 408) [updated & adopted: 27 June 2018]. Paris, France:
Organisation for Economic Co-operation and Development (OECD). DOI:10.1787/9789264070707-
en. Available at: https://doi.org/10.1787/9789264070707-en.

OECD (2020). Bacterial reverse mutation test. In: OECD Guidelines for the Testing of Chemicals. (OECD
Guideline, No. 471) [updated & adopted: 26 June 2020]. Paris, France: Organisation for Economic
Co-operation and Development (OECD). DOI:10.1787/9789264071247-en. Available at:
https://doi.org/10.1787/9789264071247-en.

Pariza MW, Johnson EA (2001). Evaluating the safety of microbial enzyme preparations used in food
processing: update for a new century. Regul Toxicol Pharmacol 33(2):173-186.
DOI:10.1006/rtph.2001.1466.

Pearson WR (2013). An introduction to sequence similarity ("homology") searching. Curr Protoc

Bioinformatics 42(1):3.1.1-3.1.8 [Chapter 3, Unit 3.1]. DOI:10.1002/0471250953.bi0301s42.

Oobli, Inc.
18 April 2023 45
Picone D, Temussi PA (2012). Dissimilar sweet proteins from plants: oddities or normal components? Plant
Sci 195:135-142. DOI:10.1016/j.plantsci.2012.07.001.

Poirier N, Roudnitzky N, Brockhoff A, Belloir C, Maison M, Thomas-Danguin T, et al. (2012). Efficient

production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.
J Agric Food Chem 60(39):9807-9814. DOI:10.1021/jf301600m.

Portune KJ, Beaumont M, Davila A-M, Tomé D, Blachier F, Sanz Y (2016). Gut microbiota role in dietary
protein metabolism and health-related outcomes: The two sides of the coin. Trends Food Sci
Technol 57(B):213-232. DOI:10.1016/j.tifs.2016.08.011

Reyes TF, Chen Y, Fraser RZ, Chan T, Li X (2021). Assessment of the potential allergenicity and toxicity of
Pichia proteins in a novel leghemoglobin preparation. Regul Toxicol Pharmacol 119:104817 [15pp,
plus supplementary tables]. DOI:10.1016/j.yrtph.2020.104817

Saha S, Raghava GPS (2006). AlgPred: prediction of allergenic proteins and mapping of IgE epitopes. Nucleic
Acids Res 34(Web Server issue):W202-W209. DOI:10.1093/nar/gkl343.

Sewalt V, Shanahan D, Gregg L, La Marta J, Carrillo R (2016). The Generally Recognized as Safe (GRAS)
process for industrial microbial enzymes. Ind Biotechnol 12(5):295-302. DOI:10.1089/ind.2016.0011.

Stone H, Oliver SM (1969). Measurement of the relative sweetness of selected sweeteners and sweetener
mixtures. Food Sci 34(2):215-222. DOI:10.1111/j.1365-2621.1969.tb00922.x

Thomas K, Aalbers M, Bannon GA, Bartels M, Dearman RJ, Esdaile DJ, et al. (2004). A multi-laboratory
evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of
novel proteins. Regul Toxicol Pharmacol 39(2):87-98. DOI:10.1016/j.yrtph.2003.11.003.

U.S. FDA (2018). Agency Response Letter GRAS Notice No. GRN 737 [Soy leghemoglobin preparation from a
strain of Pichia pastoris, Redwood City (CA): Impossible Foods Inc.], Silver Spring (MD): U.S. Food and
Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of
Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 [Jul. 23,
2018 - FDA response - no questions].

U.S. FDA (2021a). Agency Response Letter GRAS Notice No. GRN 967 [Soluble egg-white protein produced by
Pichia pastoris strain “DFB-003”, South San Francisco (CA): Clara Foods Co.]. Silver Spring (MD): U.S.
Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN),
Office of Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=967 [Sep. 9,
2021 - FDA response - no questions].

U.S. FDA (2021b). Agency Response Letter GRAS Notice No. GRN 1001 [Myoglobin preparation from a strain
of Pichia pastoris expressing the myoglobin gene from Bos Taurus, Boston (MA): Motif FoodWorks,
Inc.]. Silver Spring (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and
Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=1001 [Dec. 3,
2021 - FDA response - no questions].

Oobli, Inc.
18 April 2023 46
U.S. FDA (2022a). Part 170—Food additives. Section §170.3—Definitions. In: U.S. Code of Federal
Regulations (CFR). Title 21: Food and Drugs (Food and Drug Administration). Washington (DC): U.S.
Government Printing Office (GPO). Available at: https://www.govinfo.gov/app/details/CFR-2022-
title21-vol3/CFR-2022-title21-vol3-sec170-3.

U.S. FDA (2022b). Part 170—Food additives. Section §170.30—Eligibility for classification as generally
recognized as safe (GRAS). In: U.S. Code of Federal Regulations (CFR). Title 21: Food and Drugs (Food
and Drug Administration). Washington (DC): U.S. Government Printing Office (GPO). Available at:
https://www.govinfo.gov/app/details/CFR-2022-title21-vol3/CFR-2022-title21-vol3-sec170-30.

U.S. FDA (2022c). Agency Response Letter GRAS Notice No. GRN 1056, β-lactoglobulin produced by
Komagataella phaffii strain “yRMK-66”, Rehovot, Israel: Remilk Ltd.]. Silver Spring (MD): U.S. Food
and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of
Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=1056
[pending as of: Jul. 25, 2022].

U.S. FDA (2022d). Part 573—Food additives permitted in feed and drinking water of animals. §573.750—
Pichia pastoris dried yeast. In: U.S. Code of Federal Regulations (CFR). Title 21: Food and Drugs. (U.S.
Food and Drug Administration). Washington (DC): U.S. Food and Drug Administration (U.S. FDA),
U.S. Government Printing Office (GPO). Available at: https://www.govinfo.gov/app/details/CFR-
2022-title21-vol6/CFR-2022-title21-vol6-sec573-750.

U.S. FDA (2022e). Part 317—Qualifying pathogens. §317.2—List of qualifying pathogens that have the
potential to pose a serious threat to public health. In: U.S. Code of Federal Regulations (CFR). Title
21: Food and Drugs. (U.S. Food and Drug Administration). Washington (DC): U.S. Government
Printing Office (GPO). Available at: https://www.govinfo.gov/app/details/CFR-2022-title21-
vol5/CFR-2022-title21-vol5-sec317-2.

USDA (2022). What We Eat in America: National Health and Nutrition Examination Survey (NHANES): 2017-
2018. Riverdale (MD): U.S. Department of Agriculture (USDA). Available at:
https://www.ars.usda.gov/northeast-area/beltsville-md-bhnrc/beltsville-human-nutrition-research-
center/food-surveys-research-group/docs/wweianhanes-overview/#release [last modified:
08/09/2022].

Walters DE, Hellekant G (2006). Interactions of the sweet protein brazzein with the sweet taste receptor. J
Agric Food Chem 54(26):10129-10133. DOI:10.1021/jf062359y.

Wold S, Jonsson J, Sjöström M, Sandberg M, Rännar S (1993). DNA and peptide sequences and chemical
processes mutlivariately modelled by principal component analysis and partial least-squares
projections to latent structures. Anal Chim Acta 277(2):239-253. DOI: 10.1016/0003-2670(93)80437-
P.

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Protein ID Organism Description Average percentage of 3 lots of OFSP
(OFSPB53-304, OFSPB53-215, OFSPB53-538)

P56552 Brazzein 46.78

C4R0U2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Fusion protein, identical to Rpl40Bp 6.23
C4R245 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Transaldolase 3.15
C4R6G8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Vacuolar aspartyl protease (Proteinase A) 1.59
C4QYZ6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Peptide hydrolase 1.22
C4R5U7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 S-adenosylmethionine synthase 1.01
C4R2P5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Long chronological lifespan protein 2 0.97
C4R7E5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Thioredoxin 0.91
C4R7F9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Golgi-localized protein with homology to gamma-adaptin 0.91
C4QW42 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Chitin deacetylase, together with Cda1p involved in the biosynthesis ascospore wall 0.83
component
C4QWG6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, identical to Rpl2Bp 0.72

C4R3H3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.68
C4R8X7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Superoxide dismutase [Cu-Zn] 0.61
C4R2G3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 FK506-binding protein 0.56
C4R537 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein that binds to cruciform DNA structures 0.5
C4QY12 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Suppressor protein STM1 0.47
C4QZY8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 RNAse 0.47
C4R9F6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.47
C4QVG3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.41
C4R312 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Dihydrolipoyl dehydrogenase 0.42
C4QW56 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.39
C4QYU3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Phosphotransferase 0.39
C4QZU2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cobalamin-independent methionine synthase, involved in amino acid biosynthesis 0.39

C4R6V3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear protein required for transcription of MXR1 0.39
C4R708 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Tetradecameric mitochondrial chaperonin 0.38
C4R1K7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein that binds tRNA and methionyl-and glutamyl-tRNA synthetases (Mes1p and 0.33
Gus1p)
C4R1R2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Ribosomal protein 51 (Rp51) of the small (40s) subunit 0.33
C4R5R5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.33
P52710 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Carboxypeptidase Y 0.31
C4QYT0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Vacuolar proteinase B (YscB), a serine protease of the subtilisin family 0.31
C4QYX3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) subunit, essential for control of translational 0.3
accuracy
C4R074 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit of Elongator complex, which is required for modification of wobble 0.3
nucleosides in tRNA
C4R2Z2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein chaperone involved in regulation of the HSP90 and HSP70 functions 0.3

C4R3J1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear protein that binds to RNA and to Mex67p, required for export of poly(A)+ 0.3
mRNA from the nucle
C4R3X5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Primary component of eisosomes 0.3
C4R5M4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial intermembrane space cysteine motif protein 0.3
C4R5P6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Succinate--CoA ligase [ADP-forming] subunit alpha, mitochondrial 0.3
C4R686 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutathione reductase 0.3
C4R282 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein with seven cysteine-rich CCHC zinc-finger motifs, similar to human CNBP 0.28

C4R887 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATPase involved in protein folding and nuclear localization signal (NLS)-directed 0.27
nuclear transport
C4QW48 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.25
C4QZ51 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.24
C4R2U6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.25
C4R3T2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Major of three pyruvate decarboxylase isozymes 0.25
C4R430 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 6-phosphogluconate dehydrogenase, decarboxylating 0.25
C4R7U6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytochrome c oxidase subunit 0.25
C4R9E0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Alcohol dehydrogenase 0.25
C4QVU1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Thiol oxidase required for oxidative protein folding in the endoplasmic reticulum 0.25

C4QVY8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Translation initiation factor eIF4G, subunit of the mRNA cap-binding protein complex 0.22
(EIF4F)
C4QW09 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Fructose 1,6-bisphosphate aldolase, required for glycolysis and gluconeogenesis 0.22

C4QWA6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.22
C4QWH9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Isocitrate dehydrogenase [NADP] 0.22
C4QYE8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 40S small ribosomal subunit 0.22
C4QYV1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Primary component of eisosomes 0.22
C4QYY3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutamyl-tRNA synthetase, cytoplasmic 0.22
C4R1P7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Acetyl-coenzyme A synthetase 0.22
C4R1R9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Long chain fatty acyl-CoA synthetase with a preference for C12:0-C16:0 fatty acids 0.22

C4R2R2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.22
C4R736 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nucleolar protein that binds nuclear localization sequences 0.22
C4QW46 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutamate dehydrogenase 0.19
C4QY91 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.19
C4R0Q7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Major exo-1,3-beta-glucanase of the cell wall, involved in cell wall beta-glucan 0.2
assembly
C4R184 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.19
C4R715 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.19
C4R8Z2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Coatomer subunit beta 0.19
C4R1C9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Ribosomal protein L15 0.19
C4QV89 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Heat shock protein that cooperates with Ydj1p (Hsp40) and Ssa1p (Hsp70) 0.2
C4R2P3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Methionine aminopeptidase 2 0.17
C4QV16 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl4Ap 0.17

C4QY72 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.16
C4QZ89 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.16
C4R0B4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Calnexin 0.17
C4R0P1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glyceraldehyde-3-phosphate dehydrogenase 0.16
C4R3H8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Enolase I, a phosphopyruvate hydratase that catalyzes the conversion of 2- 0.16
phosphoglycerate to phosph
C4R4Y8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATP synthase subunit alpha 0.17
C4R5F9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytochrome c oxidase assembly protein/Cu2+ chaperone 0.16
C4R5T3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein with a potential role in actin cytoskeletal organization 0.17
C4R5X1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Homoserine kinase, conserved protein required for threonine biosynthesis 0.16

C4R6D0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Translation initiation factor eIF-4B, has RNA annealing activity 0.17
C4R6L9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytochrome c, isoform 1 0.17
C4R6U5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Putative transcription factor involved in regulating the response to osmotic stress 0.16

C4R852 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Tyrosine--tRNA ligase 0.17
C4R8P5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic isoleucine-tRNA synthetase, target of the G1-specific inhibitor 0.16
reveromycin A
C4R1X8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial 0.17
C4QZC2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Phosphatidylglycerol/phosphatidylinositol transfer protein 0.16
C4R1C2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Key endocytic protein involved in a network of interactions with other endocytic 0.16
proteins
C4R8L5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 GTP-binding protein 0.17
Q92448 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATP-dependent 6-phosphofructokinase subunit alpha 0.14
C4QVL4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 1,3-beta-glucanosyltransferase 0.14
C4QWH2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Vacuolar proteinase B (YscB), a serine protease of the subtilisin family 0.14
C4QY74 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit of cleavage factor I 0.14
C4QYQ8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit of the heterodimeric FACT complex (Spt16p-Pob3p) 0.14
C4R0W4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.14
C4R201 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Aminopeptidase 0.13
C4R262 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Actin-binding protein of the cortical actin cytoskeleton 0.14
C4R461 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Constituent of the mitochondrial inner membrane presequence translocase (TIM23 0.14
complex)
C4R4H3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Ornithine aminotransferase 0.14
C4R4V8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 cAMP-dependent protein kinase regulatory subunit 0.14
C4R5W4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.14
C4R6N7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein kinase involved in the response to oxidative and osmotic stress 0.14
C4R7R0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 One of six ATPases of the 19S regulatory particle of the 26S proteasome 0.14
C4QXJ0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Beta subunit of the translation initiation factor eIF2 0.14
C4R885 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.14
C4QVB5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Probable di-and tri-peptidase 0.14
C4QVX0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Delta subunit of the coatomer complex (COPI), which coats Golgi-derived transport 0.14
vesicles
C4R300 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nucleoside diphosphate kinase 0.14
C4R646 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Transcription factor involved in cell-type-specific transcription and pheromone 0.14
response
C4QXR4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of the holoenzyme form of RNA polymerase transcription factor TFIIH 0.14

C4QV80 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Dihydrolipoyl transsuccinylase, component of the mitochondrial alpha-ketoglutarate 0.11
dehydrogenase
C4QW50 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein involved in negative regulation of transcription of iron regulon 0.11
C4QXW3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4QYF5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of mRNP complexes associated with polyribosomes 0.11
C4QZB0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Elongation factor 1-alpha 0.11
C4QZE7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl7Ap 0.11
and has similarit
C4QZS3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATPase involved in protein import into the ER, also acts as a chaperone to mediate 0.11
protein folding i
C4R0N8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R155 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Presumed helicase required for RNA polymerase II transcription termination and 0.11
processing of RNAs
C4R2S0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial 0.11
C4R2X4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial import inner membrane translocase subunit TIM50 0.11
C4R5A8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Part of actin cytoskeleton-regulatory complex Pan1p-Sla1p-End3p 0.11
C4R5K2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic glyoxalase II 0.11
C4R6F1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R6Z0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit 0.11
C4R7F0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein interacting with poly(A)-binding protein Pab1p 0.11
C4R7I8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R7T7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl13Bp 0.11

C4R7T1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R9C0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.11
C4R5T7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R650 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic mRNA cap binding protein 0.11
C4R6Y3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit 0.11
C4R901 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 NADPH--cytochrome P450 reductase 0.11
C4R8L6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 GTPase-activating protein (RhoGAP) for Rho3p and Rho4p 0.11
C4QVA4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R8S3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 One of six ATPases of the 19S regulatory particle of the 26S proteasome 0.11
C4QVQ4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Peptidyl-prolyl cis-trans isomerase 0.11
C4QXM0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.11
C4R0E2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Methionyl-tRNA synthetase, forms a complex with glutamyl-tRNA synthetase 0.11
(Gus1p) and Arc1p
C4R2S5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Peroxidase 0.11
C4R4M5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Alanine--tRNA ligase 0.11
C4R7L8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein that interacts with Spt6p and copurifies with Spt5p and RNA polymerase II 0.11

C4R8X6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutamine synthetase 0.11
C4QWN4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.11
C4QZ92 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 60S ribosomal protein L36 0.11
Q9P4D1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Actin 0.08
C4QXU3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Essential phosphoprotein component (P150) of the COPII coat of secretory pathway 0.08
vesicles
C4QXU4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.09
C4QZ09 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Actin assembly factor, activates the Arp2/3 protein complex that nucleates branched 0.08
actin filaments
C4R2Q1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Type II HSP40 co-chaperone that interacts with the HSP70 protein Ssa1p 0.08
C4R348 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 One of several homologs of bacterial chaperone DnaJ, located in the ER lumen 0.08

C4R3T7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, identical to Rpl42Ap and 0.08
has similarity to r
C4R3X8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATPase involved in protein folding and the response to stress 0.08
C4R4K7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Putative dihydrokaempferol 4-reductase 0.08
C4R796 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear type II J heat shock protein of the E. coli dnaJ family 0.08
C4R938 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein disulfide isomerase, multifunctional protein resident in the endoplasmic 0.08
reticulum lumen
C4R4X4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Elongation factor Tu 0.08
C4QZ47 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4QZA7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Vacuolar protein sorting-associated protein 27 0.08
C4R1H5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4QY71 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit 0.08
C4R760 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Major ADP/ATP carrier of the mitochondrial inner membrane 0.08
C4R5L9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4R4X1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ADP-ribosylation factor GTPase activating protein (ARF GAP) 0.08
C4R339 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Pyruvate carboxylase 0.08
C4R2D3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.09
C4R7G7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4R547 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Tryptophan synthase 0.08
C4R6C8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4R7R8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytosolic leucyl tRNA synthetase, ligates leucine to the appropriate tRNA 0.08
C4QWQ9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Rho GDP dissociation inhibitor involved in the localization and regulation of Cdc42p 0.08

C4R0T7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Ribosomal protein 59 of small subunit, required for ribosome assembly and 20S pre- 0.08
rRNA processing
C4R1A7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Chorismate synthase 0.08
C4R3C8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cysteine desulfurase involved in iron-sulfur cluster (Fe/S) biogenesis 0.08
C4R5Z3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4R641 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutamate decarboxylase 0.08
C4R6N9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Translational elongation factor 3, stimulates the binding of aminoacyl-tRNA (AA- 0.08
tRNA) to ribosomes
C4R3E7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 60S ribosomal protein L20 0.08
C4R0V9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Peroxiredoxin 0.08
C4R1T4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit of the HIR complex, a nucleosome assembly complex 0.09
C4QX18 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear protein involved in asymmetric localization of ASH1 mRNA 0.08
C4R376 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.08
C4R707 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Histone H2A 0.08
C4R0M7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Histone H2B 0.08
C4QWE4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Aspartate aminotransferase 0.05
C4QZS2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial ribosomal protein of the small subunit 0.06
C4R1D1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Enzyme of 'de novo' purine biosynthesis 0.05
C4R2H4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Guanylate kinase, converts GMP to GDP 0.05
C4R6J5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear envelope protein, interacts with GDP-bound Gsp1p and with proteins of the 0.06
nuclear pore
C4QVA2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.06
C4QVT8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Fatty acid synthase subunit beta 0.05
C4QZT4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.06
C4QZU0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Anthranilate synthase, catalyzes the initial step of tryptophan biosynthesis, forms 0.05
multifunctional
C4R306 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Phosphomannomutase 0.05
C4R570 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Aconitate hydratase, mitochondrial 0.05
C4QVA8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.05
C4QWS5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Diadenosine 5',5''-P1,P4-tetraphosphate phosphorylase II (AP4A phosphorylase) 0.06

C4R099 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glucose-6-phosphate 1-dehydrogenase 0.05
C4QXV1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl34Bp 0.06

C4QXZ1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 MAP kinase kinase kinase of the HOG1 mitogen-activated signaling pathway 0.05

C4R4A2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Non-specific serine/threonine protein kinase 0.05
C4QW14 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Regulatory subunit of type 1 protein phosphatase Glc7p 0.05
C4R4J6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 5'-3' exoribonuclease 1 0.06
C4QXF2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic aspartyl-tRNA synthetase, homodimeric enzyme 0.05
C4R259 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Co-chaperone that stimulates the ATPase activity of the HSP70 protein Ssc1p 0.05

C4R2H3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Thioredoxin peroxidase, acts as both a ribosome-associated and free cytoplasmic 0.05
antioxidant
C4R3R8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Arginine biosynthesis bifunctional protein ArgJ, mitochondrial 0.05
C4QW19 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 RNA-binding protein that carries poly(A)+ mRNA from the nucleus into the cytoplasm 0.06

C4QW75 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.06
C4QWX1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein that interacts with Cdc48p and Npl4p, involved in recognition of 0.05
polyubiquitinated proteins
C4QXG9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.06
C4QYF6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) 0.05

C4QZG8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Hsp70 (Ssa1p) nucleotide exchange factor, cytosolic homolog of Sil1p, which is the 0.05
nucleotide exchan
C4QZL6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of the mitochondrial alpha-ketoglutarate dehydrogenase complex, 0.06
which catalyzes a key step
C4R0T4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 dUTPase, catalyzes hydrolysis of dUTP to dUMP and PPi 0.06
C4R2M5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.06
C4R3G3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Carnitine acetyl-CoA transferase present in both mitochondria and peroxisomes, 0.06
transfers activated a
C4R410 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.05
C4R4E1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial intermembrane space protein 0.05
C4R526 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit Va of cytochrome c oxidase 0.05
C4R546 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Carboxypeptidase 0.06
C4R8G8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.05
C4R3I6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl19Ap 0.05
and has similari
C4QXH9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.05
C4R2U4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit 0.05
C4R2N9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Adenosine kinase, required for the utilization of S-adenosylmethionine (AdoMet) 0.05

C4QWM6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 6-phosphogluconolactonase-like protein 0.06
C4QXL0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Alpha aminoadipate reductase 0.06
C4R3P1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glucose-repressible protein kinase involved in signal transduction during cell 0.06
proliferation in resp
C4R8H9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Acetylornithine aminotransferase 0.05
C4R577 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.05
C4QYG2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.06
C4R566 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Phosphatidylinositol/phosphatidylcholine transfer protein 0.06
C4QV38 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 rRNA-processing protein 0.05
C4R0S8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial alcohol dehydrogenase isozyme III 0.05
C4R666 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nexin-1 homolog required for localizing membrane proteins 0.06
C4R3Z9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of the chromatin assembly complex (With Rlf2p and Msi1p) 0.05
C4R6X3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit beta of the cytosolic chaperonin Cct ring complex, related to Tcp1p 0.05

C4QXS0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 RNA helicase in the DEAH-box family involved in the second catalytic step of splicing, 0.03
exhibits ATP
C4R0G3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoskeletal protein binding protein required for assembly of the cortical actin 0.03
cytoskeleton
C4QXW1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Acetyl-CoA carboxylase, biotin containing enzyme 0.03
C4R1M6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Allantoicase, converts allantoate to urea and ureidoglycolate 0.03
C4R0F5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R444 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.03
C4R4W6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R7J7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Methionine and cysteine synthase (O-acetyl homoserine-O-acetyl serine 0.03
sulfhydrylase)
C4QZ37 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Mitochondrial respiratory chain complexes assembly protein RCA1 0.03
C4R4T7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Essential component of the nuclear pore complex 0.03
C4R722 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 E3 ubiquitin protein ligase 0.03
C4R0G5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 3-isopropylmalate dehydratase 0.03
C4QZQ0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Isocitrate dehydrogenase [NAD] subunit, mitochondrial 0.03
C4QVD6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 High osmolarity signaling protein SHO1 0.03
C4QVD4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein implicated in polar growth, functionally redundant with Boi1p 0.03
C4QWG1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QWI0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QWK1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 GTPase 0.03
C4QWU8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the large (60S) ribosomal subunit 0.03
C4QYN4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 40S ribosomal protein subunit 0.03
C4QZE9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R007 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nicotinate phosphoribosyltransferase 0.03
C4R048 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R0N1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R101 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Putative benzil reductase 0.03
C4R1A2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Integral membrane component of endoplasmic reticulum-derived COPII-coated 0.03
vesicles
C4R1H2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R2Z5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein of the SUN family (Sim1p, Uth1p, Nca3p, Sun4p) that may participate in DNA 0.03
replication
C4R453 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein required for proper cell fusion and cell morphology 0.03
C4R5D9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytidine deaminase 0.03
C4R5R4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic response regulator, part of a two-component signal transducer that 0.03
mediates osmosensing
C4R7U9 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nucleolar GTP-binding protein 2 0.03
C4R7Z0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R8R1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Thioredoxin 0.03
C4QZ61 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Small nuclear ribonucleoprotein G 0.03
C4QWN1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Xylulokinase, converts D-xylulose and ATP to xylulose 5-phosphate and ADP 0.03

C4QZV6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Alpha-1,4 glucan phosphorylase 0.03
C4R5F1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Transcription initiation factor IIE subunit beta 0.03
C4R382 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QZ53 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R5Z8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QWV4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R781 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Copper-binding protein of the mitochondrial inner membrane 0.03
C4R1K3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Putative transporter, member of the sugar porter family 0.03
C4R4N5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R922 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Myo-inositol transporter with strong similarity to the minor myo-inositol transporter 0.03
Itr2p
C4QZF4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Putative kinase, suppressor of GTPase mutant, similar to bovine rhodopsin kinase 0.03

C4R7M5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Adenylate cyclase, required for cAMP production and cAMP-dependent protein 0.03
kinase signaling
C4QVL2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R1V5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Subunit 2 of the ubiquinol cytochrome-c reductase complex 0.03
C4R8Z0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Nuclear protein related to mammalian high mobility group (HMG) proteins 0.03

C4QVK2 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Deoxyhypusine synthase, catalyzes formation of deoxyhypusine 0.03
C4QV10 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Cytoplasmic inorganic pyrophosphatase (PPase) 0.03
C4QZP0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Fructose-2,6-bisphosphatase, required for glucose metabolism 0.03
C4R0C5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Probable cochaperone, regulates activity of Cyr1p (Adenylyl cyclase) 0.03
C4R107 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Bisphosphate-3'-nucleotidase, involved in salt tolerance and methionine biogenesis 0.03

C4R1W5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Vacuolar membrane protein involved in vacuolar polyphosphate accumulation 0.03

C4R1J7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R4N4 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Aspartic beta semi-aldehyde dehydrogenase 0.03
C4R2S1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Catalase 0.03
C4R6K6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QWZ3 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Argininosuccinate lyase, catalyzes the final step in the arginine biosynthesis pathway 0.03

C4R194 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Glutamine-fructose-6-phosphate amidotransferase 0.03
C4QZA6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein ROT1 0.03
C4R0D6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QW98 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R130 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein involved in error-free postreplication DNA repair 0.03
C4R5L7 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Large subunit of carbamoyl phosphate synthetase 0.03
C4R498 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R3Q6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Arginyl-tRNA synthetase 0.03
C4R5B6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R5R6 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein kinase, phosphorylates the alpha-subunit of translation initiation factor eIF2 0.03
(Sui2p)
C4QXA5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 ATP-dependent 6-phosphofructokinase subunit beta 0.03
C4R241 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Metalloprotease subunit of the 19S regulatory particle of the 26S proteasome lid 0.03

C4R310 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of the RSC chromatin remodeling complex 0.03
C4QWZ5 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R162 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4QY47 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Ribosomal RNA-processing protein 8 0.03
C4R389 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
C4R8A0 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Phosphatidylinositol transfer protein (PITP) 0.03
C4R7T8 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Protein component of the small (40S) ribosomal subunit 0.03
C4R024 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Malate dehydrogenase 0.03
C4R4Q1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Kynureninase 0.03
C4R476 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Component of the commitment complex 0.03
C4R7E1 Komagataella phaffii (strain GS115 / ATCC 20864) OX=644223 Uncharacterized protein 0.03
GRAS Panel Evaluation of Oubli Fruit Sweet Protein (Brazzein)
for Uses in Conventional Food and Beverage Products
6 December 2022

INTRODUCTION
Oobli, Inc. convened a panel of independent scientists (the GRAS Panel), qualified by their scientific training
and relevant national and international experience in the safety evaluation of food ingredients, to conduct a
critical and comprehensive assessment of data and information pertinent to the safety of the company’s
Oubli fruit sweet protein (hereinafter referred to as “brazzein”) and to determine whether the intended
uses of brazzein in conventional food and beverage products as a sweetener as described in Table A-1
would be Generally Recognized as Safe (GRAS) based on scientific procedures.

A GRAS Panel was selected and convened in accordance with the U.S. Food and Drug Administration (FDA)’s
draft guidance for industry on Best Practices for Convening a GRAS Panel (U.S. FDA, 2017). Oobli confirms
that prior to convening the GRAS Panel, all reasonable efforts were made to identify and select a balanced
GRAS Panel with expertise in appropriate scientific disciplines deemed necessary for the safety evaluation of
brazzein, and efforts were placed on identifying conflicts of interest or relevant appearance issues that
would potentially bias the outcome of the deliberations of the GRAS Panel; no such conflicts of interest or
appearance of conflicts were identified. The GRAS Panel received reasonable honoraria as compensation for
its time, and honoraria provided to the GRAS Panel were not contingent upon the outcome of the GRAS
Panel’s deliberations. The GRAS Panel consisted of the below-signed qualified scientific experts: Professor
Joseph Baumert (University of Nebraska-Lincoln); Professor Emeritus George C. Fahey, Jr. (University of
Illinois); and Professor Emeritus Michael W. Pariza (University of Wisconsin-Madison).

The GRAS Panel, independently and collectively, critically evaluated a comprehensive package of publicly
available scientific data and information compiled from the literature and summarized in a dossier titled
“Documentation Supporting the GRAS Use of Oubli Fruit Sweet Protein (Brazzein) in Conventional Food and
Beverage Products” (dated 11 November 2022), which included an evaluation of available scientific data and
information, both favorable and unfavorable, relevant to the safety of the intended food and beverage uses
of brazzein. This information was prepared in part from a comprehensive search of the scientific literature
conducted through December 2022 and included information characterizing the identity and purity of the
ingredient, the manufacture of the ingredient, product specifications, supporting analytical data, intended
conditions of use, estimated exposure under the intended uses, and the safety of brazzein. A summary of
the information critically evaluated by the GRAS Panel is presented below.

SUMMARY AND BASIS FOR GRAS

Brazzein is a naturally occurring sweet protein present in the fruit of the West African Oubli plant
(Pentadiplandra brazzeana). The sweet taste of brazzein remains stable over pH 2.5 to 8 and upon heating
up to 80°C for 4.5 hours or 98°C for 2 hours (Stone and Oliver, 1969). Brazzein binds to the taste receptors
T1R2 and T1R3, with the binding mechanism similar to other sweet proteins such as thaumatin (Walters and
Hellekant et al., 2006; Belloir et al., 2017; Kim et al., 2022). Two isoforms of brazzein exist in nature: major
isoform with 54 amino acids (~80%) and minor isoform with 53 amino acids (~20%) (Neiers et al., 2021).
Oobli manufactures the brazzein 53-amino acid isoform through precision fermentation of a genetically
modified (GM) strain of Komagataella phaffi (formerly Pichia pastoris) that has been codon-optimized to
improve protein expression using standard biotechnology practices. The identity of the active constituent of
Oobli’s brazzein has been demonstrated to be the 53-amino acid isoform and substantially equivalent to the
naturally occurring protein via MS/MS peptide mapping and intact protein mass spectrometry. The protein
has a sweetness potency of approximately 750 times that of a 5% sucrose solution on a weight-weight basis
as determined by Oobli in a sensory panel.

The GRAS Panel critically evaluated the manufacturing process for brazzein. Oobli’s brazzein is produced
through precision fermentation of a GM strain of K. phaffi and follows standard fermentation processes that
are closely controlled and monitored. At the end of fermentation, the whole cell broth is processed to
recover, separate, and purify the brazzein ingredient. These downstream processing steps include solid-
liquid separation steps to separate the wet biomass from the fermentation supernatant containing brazzein.
This supernatant is then concentrated and further purified using a sequence of filtration, chromatography,
and diafiltration steps. The resulting concentrated brazzein solution is then dried into a powder consisting of
at least 30% (w/w) brazzein. The manufacturing process complies with current Good Manufacturing Practice
(cGMP) and Hazard Analysis and Critical Control Points (HACCP) principles. All materials used in the
manufacturing process are food-grade and of high purity and quality, and have been concluded to be GRAS
and suitable for their intended purpose.

The GRAS Panel noted that the production strain is derived from K. phaffii BG10, which originates from the
well-characterized host organism, K. phaffii NRRL Y-11430. K. phaffii NRRL Y-11430 is recognized as a non-
pathogenic and non-toxigenic microorganism that has not been implicated with any known adverse effects.
K. phaffi NRRL Y-11430 is suitable for use in food production as a production organism. The GRAS Panel
noted that proteins originating from this strain has been demonstrated to lack allergenic and toxigenic
potential (Jin et al., 2018; Reyes et al., 2021). Oobli’s production strain derived from K. phaffii BG10 is
genetically modified to express copies of brazzein-encoding genes under the presence of promoter and
terminator genes from K. phaffii. The gene encoding for brazzein was identified from a publicly available
protein sequence database and synthesized de novo. The brazzein expression cassettes were stably
integrated into the K. phaffii genome and demonstrated that no plasmids or antibiotic resistance genes
were present in the production strain by plate streaking and colony polymerase chain reaction (PCR). The
genetic stability of the production strain was confirmed by Sanger sequencing before and after the
fermentation process.

Food-grade specifications have been established for brazzein. These specifications include limits for the
proximate profile, brazzein content, heavy metals, and microbial contaminants. All analytical methods are
validated and fit-for-purpose. The GRAS Panel reviewed the results of 4 production batches of brazzein and
concluded that the manufacturing process yields a consistent product that conforms to the established
product specifications. The mineral profile of the ingredient is well characterized and based on the
proposed food uses of the brazzein ingredient, the resulting dietary exposures to these minerals in final
consumers were well below the corresponding Institute of Medicine’s upper limit for each mineral, as
available. Therefore, the mineral content of brazzein would not pose a safety concern to the final consumer
under the conditions of intended use.

The GRAS Panel reviewed the data related to the shelf-life stability of Oobli’s brazzein, which indicate no
significant change in the brazzein content or proximates and microbiological profile when the ingredient is
stored at 23.5°C and 40% relative humidity for up to 9 months.
Brazzein is intended for use as a food ingredient in a variety of conventional foods and beverages as a
sweetener (see Table A-1). The GRAS Panel reviewed data related to the estimated dietary intakes of
brazzein in the U.S. population under the described conditions of use. The dietary intake of the ingredient
was estimated using consumption data from the 2017-2018 cycle of the National Health and Nutrition
Examination Survey (NHANES) combined with the maximum use level of the ingredient for each intended
use. Among the total population (2 years and older), the mean and 90th percentile consumer-only intakes of
brazzein were determined to be 89 and 199 mg/person/day, respectively. On a body weight basis, the total
population (2 years and older) mean and 90th percentile consumer-only intakes of brazzein were
determined to be 1.30 and 2.88 mg/kg body weight/day, respectively. Of the individual population groups,
male adults were determined to have the greatest mean and 90th percentile consumer-only intakes of
brazzein on an absolute basis, at 119 and 260 mg/person/day, respectively, equivalent to 1.33 and 3.11
mg/kg body weight/day, respectively. Children 2 to 5 years old had the lowest mean and 90th percentile
consumer-only intakes of 35 and 76 mg/person/day, respectively, equivalent to 2.03 and 4.25 mg/kg body
weight/day, respectively.

The GRAS Panel reviewed scientific information supporting the safety of brazzein and the production
organism, K. phaffi strain NRRL Y-11430 BG10, using the principles described by Pariza and Johnson (2001)
and Sewalt et al. (2016). This included information on characterization of the production organism and the
genetic modification steps to obtain the production organism, as well as in vitro digestibility studies on
brazzein, in silico assessments of allergenicity and toxigenicity potential of the protein, as well as published
product-specific pivotal toxicology studies (Lynch et al., 2023). These elements are discussed as follows.

The GRAS Panel reviewed information on the production strain demonstrating that the organism is derived
from the host organism K. phaffii strain BG10, a derivative of K. phaffii NRRL Y-11430, a Biosafety Level 1
organism. This host organism is a non-pathogenic and non-toxigenic species with an established history of
safe use in food production, and therefore, considered to be a safe and suitable source organism for the
production of brazzein. The GRAS Panel noted that the genetic modification steps to obtain the production
strain is well characterized and does not introduce any exogenous factors into the production organism that
would pose a safety concern. The introduced genes encode for brazzein and does not encode for any
genetic element that would pose a pathogenic, allergenic, or toxigenic risk. The GRAS Panel concluded that
there are no safety concerns with the production organism used to produce Oobli’s brazzein.

The GRAS Panel reviewed the results of two in vitro digestibility studies conducted with Oobli’s brazzein.
The first study followed the methods described by Thomas et al. (2004), and the results indicate that
brazzein was not digested at any tested pepsin concentration up to 30 minutes of incubation. The second
digestibility study was conducted using the methodology described by Brodkorb et al. (2019) and Atallah et
al. (2020) at enzyme concentrations of 0.05, 0.25, and 0.5 U/µg of brazzein for up to 120 minutes. Brazzein
was partially digested after 10 minutes at concentrations of 0.05, 0.25, and 0.5 U/µg. Based on a
semiquantitative analysis from the SDS-PAGE gel, after 120 minutes, approximately 26%, 46%, and 67% of
brazzein was digested by pancreatin concentrations of 0.05, 0.25, and 0.5 U/µg, respectively. The GRAS
Panel concluded that brazzein is partially digested under simulated intestinal conditions.

A comprehensive search of the scientific literature was conducted through December 2022 to identify
publications on the safety of brazzein and the plant source (Pentadiplandra brazzeana). The search was
limited to articles with full texts within peer-reviewed scientific journals and the following databases were
accessed: Adis Clinical Trials Insight, AGRICOLA, AGRIS, Allied & Complementary Medicine™, BIOSIS®
Toxicology, BIOSIS Previews®, CAB ABSTRACTS, Embase®, Foodline®: SCIENCE, FSTA®, MEDLINE®,
NTIS: National Technical Information Service, Toxicology Abstracts, and ToxFile®. Two publications were
identified (Kim et al., 2020; Lynch et al., 2023).
In the first study, mice were provided a 3M-brazzein solution in the drinking water over 15 weeks and
obesity-related endpoints were observed (Kim et al., 2020). No significant effects on adiposity hypertrophy,
glucose homeostasis, insulin resistance, or inflammation were observed throughout the study period. The
GRAS Panel noted that this study did not evaluate standard toxicological endpoints such as those described
in Organisation for Economic Co-operation and Development (OECD) Test Guideline 408 and did not report
on the purity of brazzein or its source. Therefore, the GRAS Panel did not consider this study to be relevant
to the safety discussion of Oobli’s brazzein.

In the second study by Lynch et al. (2023) described the results of a bacterial reverse mutation test, an in
vitro micronucleus test, and a 90-day oral (dietary) toxicity study in rats that were conducted with Oobli’s
brazzein. The studies were conducted in accordance with appropriate OECD Test Guidelines and Principles
of Good Laboratory Practice (GLP).

In the bacterial reverse mutation test, brazzein did not induce any biologically relevant, concentration-
related, or statistically significant increases in revertant colony numbers at test concentrations up to 5,000
µg/plate compared with vehicle control counts in either the presence or absence of metabolic activation. In
the in vitro micronucleus test, a statistically significant increase in the percentage of micronucleated cells
was observed at the highest concentration of 2,000 µg/mL with metabolic activation when compared with
vehicle controls. However, the number of micronucleated cells was within the range of the historical
negative control; therefore, this increase was regarded as not biologically relevant. In the long-term
experiment, no biologically relevant increases in the percentage of micronucleated cells were reported at
any of the concentrations analyzed. Based on the results of these two in vitro studies, the GRAS Panel
concluded that brazzein does not have any mutagenic or clastogenic or aneugenic potential.

In the 90-day oral (dietary) toxicity study, adult Sprague-Dawley rats (10/sex/group) were provided brazzein
in the diet at concentrations of 0 (control diet), 250 (low-dose), 500 (mid-dose), and 1,000 (high-dose)
mg/kg body weight/day for 90 days. The dietary concentrations of brazzein achieved the targeted nominal
intake values of 250, 500, and 1,000 mg/kg body weight/day (actual values: 0, 245, 490, and 978 mg/kg
body weight/day and 0, 245, 493, and 985 mg/kg body weight/day in males and females, respectively). With
the exception of statistically significant changes in hematology, clinical chemistry, and organ weight
biomarkers, no treatment-related toxicological effects on body weights, body weight gain, food
consumption, functional observation battery (i.e., sensorimotor function, grip strength, and locomotor
activity), ophthalmological findings, urinalysis, macroscopic, and histopathological findings. No mortality
was reported in any test group. The statistically significant changes in hematology, clinical chemistry, and
organ weight parameters were within the historical control range for the testing laboratory, or they
occurred in a non-dose-related manner, or were not associated with any adverse histopathological findings.
Therefore, the GRAS Panel concluded that these findings were not toxicologically relevant. The NOAEL was
concluded to be 978 mg/kg body weight/day in males and 985 mg/kg body weight/day in females, the
highest tested dose. Based on the reported NOAEL and the highest estimated dietary intake of 4.25 mg/kg
body weight/day (90th percentile in children 2 to 5 years of age), an approximately 230-fold margin of
exposure (MOE) can be estimated.

The GRAS Panel reviewed a proteomics assessment of the final brazzein ingredient using LC-MS/MS, which
indicated that the ingredient contains 6 host cell proteins each at levels above 1%, accounting for up to
5.6% of the final ingredient. The amino acid sequence of brazzein and the residual host cell proteins were
investigated for their allergenicity potential using the stepwise approach described by FAO/WHO (2001) and
Codex Alimentarius (2009), as well as their toxigenicity potential using the Animal Toxin Annotation Project
maintained in the UniProtKB database. The allergenicity searches were conducted against the curated
databases of AllergenOnline and Allermatch, and a support-vector machine (SVM) search was conducted
with AlgPred. No matches between brazzein and putative allergens were identified sharing greater than
35% identity or 8-amino acid exact matches, indicating that brazzein would be unlikely to have any
allergenic cross-reactivity. In the full-length amino acid searches, brazzein shared >50% identity with a
number of sequences from the AllergenOnline and Allermatch databases. However, the GRAS Panel noted
that the corresponding E-values and bit-scores suggest the findings to not be statistically significant, and
may not be suggestive of allergenic cross-reactivity. In the -amino acid sliding window searches with the
residual host cell proteins, two proteins shared a number of matches to putative allergens from fungal
species such as Fusarium proliferatum, Pencillium chrysogenum, Cladosporium cladosporioides,
Rhizopus oryzae, and Aspergillus fumigatus, as well as mosquito (Aedes aegypti) and wild boar (Sus scrofa).
The identified matches were to respiratory fungal allergens, or dermal allergens (from mosquitoes), or oral
allergens (from wild boars). The GRAS Panel noted that no matches were identified to major food allergens
that are of relevance to final consumers, and the allergenic potential of residual host cell proteins from
production strains derived from K. phaffii NRRL Y-11430 and BG10 have been previously discussed in the
scientific literature (Jin et al., 2018; Reyes et al., 2021). Considering that Oobli’s production strain is derived
from K. phaffii BG10, and the genetic modifications to the host organism is well characterized and do not
pose any allergenic concern, the GRAS Panel concluded that any native residual proteins from the host
organism would pose a low risk for allergenicity. The SVM searches using AlgPred predicted mixed results
for brazzein and the residual host cell proteins. The GRAS Panel noted that AlgPred has been used
previously for the allergenicity assessment of soy leghemoglobin obtained from a genetically modified strain
of P. pastoris as described in GRN 737 (U.S. FDA, 2018), and concluded that the reliability of the SVM-based
analysis is controversial due to reliability of the method to accurately predict the allergenicity potential of a
protein. Based on the totality of evidence, the GRAS Panel concluded that brazzein and the residual host cell
proteins in the final ingredient pose a low risk for allergenicity to final consumers. Collectively, the results of
the in silico assessment of allergenicity along with the absence of any adverse effects in the 90-day dietary
toxicity study demonstrate that the intact protein and any potential undigested peptides of Oobli’s brazzein
do not pose any safety or allergenicity concern to final consumers.

In addition to the allergenicity searches, brazzein and the residual host cell proteins were investigated for
significant sequence homology with known protein toxins using BLAST. A curated database of known animal
venom proteins and toxins maintained by UniProt was searched against the amino acid sequence of
brazzein and the residual host cell proteins using default parameters. Sequences were considered to share
structural homology/similarity based on the criteria described by Pearson (2013). No significant similarity to
any of the venom proteins and toxins were identified (E-values >0.0015) in the homology searches with
brazzein and the residual Komagataella proteins. These findings suggest that brazzein and any residual
Komagataella protein does not harbor any toxigenic potential based on its amino acid sequence.

Based on the technical and scientific data and information presented herein, Oobli, Inc. has concluded that
the Oubli fruit sweet protein (brazzein) produced from a genetically modified strain of Komagataella phaffii
is GRAS for use in conventional food and beverage products on the basis of scientific procedures. Brazzein
therefore may be marketed and sold for its intended purpose in the U.S. without the promulgation of a food
additive regulation under Title 21, Section 170.3 of the Code of Federal Regulations.
CONCLUSIONS OF THE GRAS P.ANEL
We, the GRAS Panel, have, independently and collectively, critica lly evaluated the data and information
summarized above and conclude that the proposed uses of brazzein as an ingredient in conventional foods
and beverages, meeting appropriate food-grade specifications and manufactured consistent with current
Good Manufacturing Practice, are Generally Recognized as Safe (GRAS) based on scientific procedures.

It is our opinion that other qualified experts would concur with these conclusions.

' II

Professor Joseph Baumert, Ph.D. Date

University of Nebraska-Lincoln

Professor Erne • .. s George C. Fahe , Jr., P Date

I I
University of Illinois

January 23 2023
Professor Emeritus Michael W. Pariza, Ph.D. Date
University of Wisconsin-Madison
REFERENCES
Atallah N, Deracinois B, Boulier A, Baniel A, Jouan-Rimbaud Bouveresse D, Ravallec R, et al. (2020). In vitro
assessment of the impact of industrial processes on the gastrointestinal digestion of milk protein
matrices using the INFOGEST Protocol. Foods 9(11):1580 [20pp, plus supplementary figure].
DOI:10.3390/foods9111580.

Belloir C, Neiers F, Briand L (2017). Sweeteners and sweetness enhancers. Current Opinion in Clinical
Nutrition and Metabolic Care: 20(4):279-285. DOI:10.1097/MCO.0000000000000377

Brodkorb A, Egger L, Alminger M, Alvito P, Assunção R, Ballance S, et al. (2019). INFOGEST static in vitro
simulation of gastrointestinal food digestion. Nat Protoc 14(4):991-1014. DOI:10.1038/s41596-018-
0119-1.

Codex Alimentarius (2009). Foods Derived from Modern Biotechnology, 2nd edition. (Codex Alimentarius).
Geneva, Switzerland: World Health Organization (WHO) / Rome, Italy: Food and Agriculture
Organization of the United Nations (FAO). Available at:
https://www.fao.org/3/a1554e/a1554e00.htm.

FAO/WHO (2001). Evaluation of Allergenicity of Genetically Modified Foods. Report of a Joint FAO/WHO
Expert Consultation on Allergenicity of Foods Derived from Biotechnology, 22-25 January 2001.
Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) / Geneva, Switzerland:
World Health Organization (WHO). Available at:
https://www.who.int/publications/m/item/evaluation-of-allergenicity-of-genetically-modified-
foodsreport-of-a-joint-fao-who-expert-consultation-on-allergenicity-of-foods-derived-from-
biotechnology

Jin Y, He X, Andoh-Kumi K, Fraser RZ, Lu M, Goodman RE (2018). Evaluating potential risks of food allergy
and toxicity of soy leghemoglobin expressed in Pichia pastoris. Mol Nutr Food Res 62(1):1700297
[13pp]. DOI:10.1002/mnfr.201700297.

Kim H, Kang J, Hong S, Jo S, Noh H, Kang BH, et al. (2020). 3M-brazzein as a natural sugar substitute
attenuates obesity, metabolic disorder, and inflammation. J Agric Food Chem 68(7):2183-2192.
DOI:10.1021/acs.jafc.0c00317.

Kim TY, Woo EJ, Yoon TS (2022). Binding mode of brazzein to the taste receptor based on crystal structure
and docking simulation. Biochem Biophys Res Commun. 592:119-124.
DOI:10.1016/j.bbrc.2022.01.004.

Lynch B, Wang T, Vo T, Tafazoli S, Ryder J (2023). Safety evaluation of oubli fruit sweet protein (brazzein)
derived from Komagataella phaffi, intended for use as a sweetener in food and beverages. Toxicol
Res Appl 7 [21pp]. DOI:10.1177/23978473231151258.

Neiers F, Belloir C, Poirier N, Naumer C, Krohn M, Briand L (2021). Comparison of different signal peptides
for the efficient secretion of the sweet-tasting plant protein brazzein in Pichia pastoris. Life (Basel)
11(1):46 [12pp, plus supplementary figure]. DOI:10.3390/life11010046.

Pariza MW, Johnson EA (2001). Evaluating the safety of microbial enzyme preparations used in food
processing: update for a new century. Regul Toxicol Pharmacol 33(2):173-186.
DOI:10.1006/rtph.2001.1466.
Pearson WR (2013). An introduction to sequence similarity ("homology") searching. Curr Protoc
Bioinformatics 42(1):3.1.1-3.1.8 [Chapter 3, Unit 3.1]. DOI:10.1002/0471250953.bi0301s42.

Reyes TF, Chen Y, Fraser RZ, Chan T, Li X (2021). Assessment of the potential allergenicity and toxicity of
Pichia proteins in a novel leghemoglobin preparation. Regul Toxicol Pharmacol 119:104817 [15pp,
plus supplementary tables]. DOI:10.1016/j.yrtph.2020.104817

Sewalt V, Shanahan D, Gregg L, La Marta J, Carrillo R (2016). The Generally Recognized as Safe (GRAS)
process for industrial microbial enzymes. Ind Biotechnol 12(5):295-302. DOI:10.1089/ind.2016.0011.

Stone H, Oliver SM (1969). Measurement of the relative sweetness of selected sweeteners and sweetener
mixtures. Food Sci 34(2):215-222. DOI:10.1111/j.1365-2621.1969.tb00922.x

Thomas K, Aalbers M, Bannon GA, Bartels M, Dearman RJ, Esdaile DJ, et al. (2004). A multi-laboratory
evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of
novel proteins. Regul Toxicol Pharmacol 39(2):87-98. DOI:10.1016/j.yrtph.2003.11.003.

U.S. FDA (2017a). Best Practices for Convening a GRAS Panel: Guidance for Industry: Draft Guidance. (Docket
Number: FDA-2017-D-0085). College Park (MD): U.S. Food and Drug Administration (U.S. FDA),
Center for Food Safety and Applied Nutrition (CFSAN), Center for Veterinary Medicine (CVM).
Available at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/draft-
guidance-industry-best-practices-convening-gras-panel [November 2017].

U.S. FDA (2018). Agency Response Letter GRAS Notice No. GRN 737 [Soy leghemoglobin preparation from a
strain of Pichia pastoris, Redwood City (CA): Impossible Foods Inc.], Silver Spring (MD): U.S. Food and
Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of
Food Additive Safety. Available at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 [Jul. 23,
2018 - FDA response - no questions].

Walters DE, Hellekant G (2006). Interactions of the Sweet Protein Brazzein with the Sweet Taste Receptor.
Agric Food Chem. 54(26): 10129–10133. DOI:10.1021/jf062359y.
Table A-1 Summary of the Individual Proposed Food Uses and Use Levels for Brazzein in the United
States
Food Category Food Usesa Purified Brazzein
(21 CFR §170.3) Use Levels (mg/100 g)
(U.S. FDA, 2021b) Based on 750x
Sweetness Potency
Beverages, alcoholic Cocktail drinks (pre-packaged) 7
Beverages and Beverages Bases, Packaged water-based beverages 22
non-alcoholic Non-milk-based meal replacement beverages and protein drinks 2
Chewing Gum Chewing gum 99
Coffee and Tea Ready-to-drink coffee beverages 7
Ready-to-drink tea beverages 9
Dairy Product Analogs Milk analogs 4
Non-dairy yogurts 13
Frozen Dairy Desserts and Mixes Ice cream 23
Frozen yogurt 23
Frozen milk desserts and bars 24
Fruit and Water Ices Edible ices 24
Sherbet 30
Sorbet 31
Grain Products and Pastas Cereal bars, granola bars, energy, protein, and meal replacement 30
bars
Granola 27
Milk Products Packaged milk-based beverages 6
Yogurt 12
Yogurt drinks 9
Processed Fruits and Fruit Juices Packaged fruit drinks, nectar, and fruit-based smoothies 16
Snack Foods Fruit-based bars (without granola) 36
Soft Candy Truffles 47
Gummy bear 62
CFR = Code of Federal Regulations.
a Brazzein is intended for use in unstandardized products and products with standards of identity, as established under 21 CFR

§130 to 169, do permit its addition.

GRAS Notice (GRN) 1133 Amendments

From: Jason Ryder

To: Kampmeyer, Christopher
Cc: Ali Wing; Naomi Sachs; Tina Wang
Subject: [EXTERNAL] Re: Questions for GRN 001142
Date: Wednesday, December 13, 2023 12:15:31 PM
Attachments: image013.png
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GRAS Notice No. 001142_Technical Review Questions and Responses_121323-1.pdf

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you
recognize the sender and know the content is safe.

Dear Mr. Kampmeyer,

Attached please find Oobli, Inc.'s responses to the technical questions from the FDA's review
of GRAS Notice No. 001142 shared on December 4, 2023. Note that we've set forth the FDA
team's questions, followed by our responses.

Wishing you and the team a joyous and restful holiday season. We look forward to hearing
from you again soon.

Best,

Jason

--
Jason Ryder
CTO & Co-Founder

510.684.5610
[email protected]
Oobli.com

Engage with us!

On Mon, Dec 4, 2023 at 1:52 PM Kampmeyer, Christopher

<[email protected]> wrote:

Dear Dr. Ryder,

We noted some questions during our review of GRAS Notice No. 001142—please see the
attachment in this email.

We respectfully request a response within 10 business days. If you are unable to complete
the response within that time frame, please contact me to discuss further options. Please do
not include any confidential information in your responses. Thank you in advance for your
attention to our comments.

Best regards,

Chris

Chris Kampmeyer, M.S.

Regulatory Review Scientist

Office of Food Additive Safety

Center for Food Safety and Applied Nutrition

U.S. Food and Drug Administration

[email protected]
December 4, 2023
Dear Dr. Ryder:
During our review of GRAS notice (GRN) 001142, submitted by Oobli, Inc. (Oobli; “the
notifier”), regarding the intended use of brazzein produced by K. phaffii, we noted the following
questions. We respectfully request a response to these questions within 10 business days. If you
are unable to complete the response within that timeframe or have questions, please contact me
to discuss via email at [email protected].
Chemistry Questions
1. Please state whether any of the raw materials used in the fermentation are major
allergens or are derived from major allergens. If any of the raw materials used are major
allergens or are derived from major allergens, please discuss why these materials do not
pose a safety concern.

2. On p. 11 of notice, the final brazzein product is described to contain sodium salts that are
used as stabilizing agents. Please identify the sodium compounds used in the production
of brazzein. In addition, please provide a statement that all materials used in the
manufacturing process are approved for their respective uses via a regulation in Part 21
of the U.S. Code of Federal Regulations, are the subject of an effective food contact
notification, or are GRAS for that use in the U.S.

3. The specifications on p. 16 of the notice include a limit of <0.5 mg/kg for arsenic,
cadmium, lead, and mercury. We note that the results of the analyses (p. 17) of three
batches of the notified substance for these heavy metals are well below the specified
limit. We note that specifications help to ensure that the ingredient is being
manufactured in accordance with good manufacturing practices. In addition, FDA's
recent "Closer to Zero" initiative focuses on reducing dietary exposure to heavy metals
from food. We request that specifications for heavy metals be as low as possible and
consistent with the methods used and the results obtained from the batch analyses.

4. The structure depicted in Figure 2.1.2-3 (p. 11) appears to be the 54-amino acid isoform
of brazzein that includes a N-terminal pyroglutamic acid. Please confirm whether the
structure presented in the notice corresponds to the subject of the notice, des-
pyroglutamic acid brazzein, or provide an amended figure with the correct structure.
Toxicology Questions
1. Is the sweetening effect of brazzein transient, similar to polysaccharide-based
sweeteners, or are effects on taste receptors sustained for a significant duration? Please
note that such activity has been reported in sensory studies of other protein-based
sweeteners, such as miraculin.

Background information in the GRN that provides the basis for the following
questions: A BLASTp of the brazzein sequence (PDB ID: 7W8E_A) shows that it has 100%
identity with defensin-like protein.

Figure 6.3.1.1.-1 shows that the OFSP band is persistent throughout the entire period of
incubation, indicating that brazzein is resistant to pepsin digestion at pH 2.0. Additionally, Figure
6.3.1.1.-2 shows that only about OFSP is not fully digested by pancreatin at pH 7.0 after a
significant duration (180 min). Since brazzein has four disulfide bonds and disulfide bonds are
difficult to hydrolyze even in acidic pH, it is understandable why brazzein may be resistant to
digestion by pepsin in acidic pH. Brazzein from Pentadiplandra brazzeana is apparently also
thermostable (PMID 7957951).

Since you state that the OFSP has >35% brazzein by weight, implying that there are significant
amounts of other proteins, such as P. pastoris proteins, it is not clear what the digestion
percentage represents, that of brazzein or other proteins in OFSP.

FDA notes that pepsin preferentially cleaves certain peptide bonds and not all. Hence, it is not
clear that the in silico prediction of digestibility by PeptideCutter can be accurately applied to
brazzein, which is resistant to pepsin digestion and nearly resistant to pancreatin digestion.
PeptideCutter should more accurately predict pepsin digestibility for proteins that are denatured
into a linearized/nearly linearized form following consumption with all/most of the peptide bonds
exposed to pepsin action.

2. Since brazzein has 100% amino acid identity with defensin-like protein, does brazzein
have potential “defensin-like” function that could be cause for a safety concern to the
consumers, especially because brazzein is not digested in the GI tract following oral
consumption? Please provide an explanation.

3. The stability of brazzein raises some questions that need to be addressed. In the
literature, there are reports that resistance of proteins to gastric digestion and thermal
stability may be correlative indicators for potential allergenic risk (e.g., PMID: 9631091,
30134536, FAO/WHO (2001)). However, other recent publications conclude that protein
stability is relevant for allergenicity of some proteins, but not for all (e.g.,
PMIDs: 31063834, 33473251, 21906650). Please provide a scientific narrative that
addresses why the stability of brazzein (digestion and thermal) is not a cause for a safety
concern when consumed orally.
December 13, 2023

Chris Kampmeyer
Regulatory Review Scientist
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
U.S. Food and Drug Administration

Re: GRAS Notice No. 001142 - Technical Review Questions & Answers

Dear Chris:
We are writing to respond to the technical questions from the FDA’s review of GRAS Notice No. 001142
attached to your email on December 4, 2023. Below please find Oobli, Inc.’s responses to the review
team’s questions. Note that we’ve set forth the FDA team’s questions, followed by our responses.
Yours sincerely,
Jason

Jason Ryder, Ph.D.

CTO & Founder, Oobli, Inc.
510.684.5610
[email protected]

CC: Ali M. Wing, CEO, Oobli

Naomi Sachs, CFO, Oobli
Tina Wang, Oobli
_____________________________________________________________________________________
Chemistry Questions

QUESTION 1: Please state whether any of the raw materials used in the fermentation are major allergens
or are derived from major allergens. If any of the raw materials used are major allergens or are derived
from major allergens, please discuss why these materials do not pose a safety concern.

RESPONSE 1: The raw materials used in the fermentation media are not major allergens or derived from
major allergens. Oobli obtains a Certificate of Analysis for each incoming raw material to ensure that they
are absent of major allergens prior to use in the production process.

QUESTION 2: On p. 11 of notice, the final brazzein product is described to contain sodium salts that are
used as stabilizing agents. Please identify the sodium compounds used in the production of brazzein. In
addition, please provide a statement that all materials used in the manufacturing process are approved
for their respective uses via a regulation in Part 21 of the U.S. Code of Federal Regulations, are the subject
of an effective food contact notification, or are GRAS for that use in the U.S.

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

RESPONSE 2: The sodium compounds used as stabilizing agents in the final brazzein products (OFSP) are
composed of sodium acetate and sodium chloride. All materials used in the manufacturing process are
approved for their respective uses with respect to Part 21 of the U.S. Code of Federal Regulations and are
GRAS for their respective use in the U.S.

QUESTION 3: The specifications on p. 16 of the notice include a limit of <0.5 mg/kg for arsenic, cadmium,
lead, and mercury. We note that the results of the analyses (p. 17) of three batches of the notified
substance for these heavy metals are well below the specified limit. We note that specifications help to
ensure that the ingredient is being manufactured in accordance with good manufacturing practices. In
addition, FDA's recent "Closer to Zero" initiative focuses on reducing dietary exposure to heavy metals
from food. We request that specifications for heavy metals be as low as possible and consistent with the
methods used and the results obtained from the batch analyses.

RESPONSE 3: The specifications of heavy metals have been revised to 0.1 ppm for cadmium, lead, and
mercury, and 0.2 ppm for arsenic.

QUESTION 4: The structure depicted in Figure 2.1.2-3 (p. 11) appears to be the 54-amino acid isoform of
brazzein that includes a N-terminal pyroglutamic acid. Please confirm whether the structure presented in
the notice corresponds to the subject of the notice, des-pyroglutamic acid brazzein, or provide an
amended figure with the correct structure.

RESPONSE 4: Thank you for noticing the discrepancy. The correct 3D structure of brazzein-53 (des-
pyroglutamic acid brazzein) is available on PDB (No. 7W8E) as follows:

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

Toxicology Questions

QUESTION 1: Is the sweetening effect of brazzein transient, similar to polysaccharide-based sweeteners,

or are effects on taste receptors sustained for a significant duration? Please note that such activity has
been reported in sensory studies of other protein-based sweeteners, such as miraculin.

RESPONSE 1: The sweetening effect of brazzein was investigated in a time-intensity sensory evaluation.
Trained panelists (N=10, 2 replicates) were provided OFSP (brazzein purity 39.7%), Reb A, sucrose, and
aspartame solutions at equivalent sweetness. The results are shown in the figure below. Brazzein reached
a peak sweetness intensity that was greater than the other sugars at 20 seconds, which then progressively
declined within 60 seconds, the length of the experiment. The negative slope trend in the figure below
indicates rapid decrease of sweetness after 30 seconds from consumption. These findings suggest that
the sweetening effect of brazzein is transient and are not sustained.

Sweet Taste Time Intensity

6

5
Sweet Intensity Score

4
Brazzein
3
Stevia Reb A
2 Sucrose

1 Aspartame

0
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (Seconds)

Background information in the GRN that provides the basis for the following questions: A BLASTp of the
brazzein sequence (PDB ID: 7W8E_A) shows that it has 100% identity with defensin-like protein.

Figure 6.3.1.1.-1 shows that the OFSP band is persistent throughout the entire period of incubation,
indicating that brazzein is resistant to pepsin digestion at pH 2.0. Additionally, Figure 6.3.1.1.-2 shows that
only about OFSP is not fully digested by pancreatin at pH 7.0 after a significant duration (180 min). Since
brazzein has four disulfide bonds and disulfide bonds are difficult to hydrolyze even in acidic pH, it is
understandable why brazzein may be resistant to digestion by pepsin in acidic pH. Brazzein from
Pentadiplandra brazzeana is apparently also thermostable (PMID 7957951).

Since you state that the OFSP has >35% brazzein by weight, implying that there are significant amounts of
other proteins, such as P. pastoris proteins, it is not clear what the digestion percentage represents, that
of brazzein or other proteins in OFSP.

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

FDA notes that pepsin preferentially cleaves certain peptide bonds and not all. Hence, it is not clear that
the in silico prediction of digestibility by PeptideCutter can be accurately applied to brazzein, which is
resistant to pepsin digestion and nearly resistant to pancreatin digestion. PeptideCutter should more
accurately predict pepsin digestibility for proteins that are denatured into a linearized/nearly linearized
form following consumption with all/most of the peptide bonds exposed to pepsin action.

QUESTION 2: Since brazzein has 100% amino acid identity with defensin-like protein, does brazzein have
potential “defensin-like” function that could be cause for a safety concern to the consumers, especially
because brazzein is not digested in the GI tract following oral consumption? Please provide an
explanation.

RESPONSE 2: The amino acid sequence of brazzein-53, as provided in Section 2.1.2 of GRN 1142, was
searched against the non-redundant protein database of NCBI’s Genbank using BLASTp. The results are as
follows:

We note that a number of hits to “defensin-like proteins” sharing 98% to 100% identity with 94% to 100%
query coverage was identified, suggesting that Oobli’s OFSP containing brazzein-53 may contain similar
activity as defensins.

Plant defensins are small cationic, cysteine-rich proteins that are 45 to 54 amino acids in length. These
proteins are found in different parts of plants, such as the seeds, leaves, flowers, roots, and stems. 1Yount
and Yeaman (2004) demonstrated that brazzein from P. brazzeana shares a similar motif (γ-core) as other
known peptides, including the well-known defensin rs-AFP1 from radish (Raphanus sativus). Indeed, this
finding was confirmed through a BLASTp search of the amino acid sequence of brazzein-53 against R.
sativus, and 2 matches to defensin proteins from R. sativus (Accession No. KAJ4892244.1 and Accession
No. XP_018437331.2) were identified with approximately 42% identity and 58% query coverage. These
findings suggest that brazzein-53 may contain “defensin-like” activity, similar to other commonly
consumed agricultural products.

1 Yount NY, Yeaman MR (2004). Multidimensional signatures in antimicrobial peptides. Proc Natl Acad Sci U S A

101(19):7363-7368. DOI:10.1073/pnas.0401567101.

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

The SDS-PAGE gels within GRN 1142 are revised with the following figures. These figures have been
revised to reflect the band of interest (i.e., brazzein) at approximately 6 kDa, the molecular weight of the
protein as calculated from its amino acid sequence. The digestion percentage is reflecting that of brazzein
rather than other proteins in OFSP. The individual levels of residual K. phaffii proteins are low and
therefore cannot be detected due to detection limits of this assay.

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

It is generally recognized that dietary proteins are not absorbed intact and must undergo digestion into
amino acids, dipeptides, or tripeptides, prior to absorption within the duodenum or proximal jejunum of
the small intestine through transporters (e.g., PepT1 H+/peptide co-transporter)2 (EFSA, 2021). Within the
enterocytes, proteins are digested by peptidases into amino acids and then absorbed into the systemic
circulation where they are used in metabolic processes/function. Conversely, highly lipid-soluble peptides
may enter the enterocytes via passive diffusion3 (Miner-Williams et al., 2014). Large polar molecules (>600
Da) cannot pass through the hydrophobic enterocyte cell membrane, and instead are invaginated into
vesicles that fuse with lysosomes to form phagolysosomes, whose primary function is the enzymatic
digestion of the captured macromolecules (Miner-Williams et al., 2014). Compounds greater than 200 Da
are too large for the intercellular space between the enterocytes, and likely would not be absorbed
through paracellular transport 4 (Lennernäs, 2007). Proteins that are not fully digested or are partially
digested travel to the large intestine where they are ultimately fermented by the gut microbiota 5,6
(Portune et al., 2016; Joye, 2019). In the large intestine, where the microbiota concentration is much
higher and the transit time is longer than in the small intestine, the remaining protein is broken down to
peptides and amino acids via extracellular bacterial proteases and peptidases7 (Macfarlane et al, 1986).
As a protein that is not fully digested in the upper gastrointestinal tract, brazzein-53 would be expected
to be digested/fermented by the gut microbiota within the colon or excreted within the feces.

Taken together, the “defensin-like” activity of brazzein-53 is not expected to pose any safety concerns to
final consumers on the basis that the protein is not expected to be absorbed into the systemic circulation,
but rather digested or fermented by gut microbiota in the large intestine.

QUESTION 3: The stability of brazzein raises some questions that need to be addressed. In the literature,
there are reports that resistance of proteins to gastric digestion and thermal stability may be correlative
indicators for potential allergenic risk (e.g., PMID: 9631091, 30134536, FAO/WHO (2001)). However, other
recent publications conclude that protein stability is relevant for allergenicity of some proteins, but not
for all (e.g., PMIDs: 31063834, 33473251, 21906650). Please provide a scientific narrative that addresses
why the stability of brazzein (digestion and thermal) is not a cause for a safety concern when consumed
orally.

RESPONSE 3: We recognize that the gastric or thermal resistance of proteins may be indicative of potential
allergenicity in consumers, however, this area has been debated within the scientific community and
amongst scientific regulators (e.g., EFSA). The pepsin digestion test has been incorporated into the weight-

2 EFSA (2021). Statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of
genetically modified plants (EFSA Panel on Genetically Modified Organisms/GMO) (Question no: EFSA-Q-2020-
00314, adopted: 26 November 2020, published: 12 January 2021 by European Food Safety Authority). EFSA J
19(1):6350 [16pp]. DOI:10.2903/j.efsa.2021.6350. Available at:
https://www.efsa.europa.eu/en/efsajournal/pub/6350.
3 Miner-Williams WM, Stevens BR, Moughan PJ (2014). Are intact peptides absorbed from the healthy gut in the

adult human? Nutr Res Rev 27(2):308-329. DOI:10.1017/s0954422414000225.

4 Lennernäs H (2007). Intestinal permeability and its relevance for absorption and elimination. Xenobiotica 37(10/11):1015-

1051. DOI:10.1080/00498250701704819.
5
Portune KJ, Beaumont M, Davila A-M, Tomé D, Blachier F, Sanz Y (2016). Gut microbiota role in dietary protein metabolism
and health-related outcomes: The two sides of the coin. Trends Food Sci Technol 57(B):213-232.
DOI:10.1016/j.tifs.2016.08.011.
6
Joye I (2019). Protein digestibility of cereal products. Foods 8(6):199 [14pp]. DOI:10.3390/foods8060199.
7
Macfarlane GT, Cummings JH, Allison C (1986). Protein degradation by human intestinal bacteria. J Gen Microbiol 132(6):1647-
1656. DOI:10.1099/00221287-132-6-1647.

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

of-evidence for allergenicity assessment of novel proteins and has been adopted by the Codex
Alimentarius and EFSA. This test has its limitations in that it only evaluates gastric digestion of protein and
does not consider proceeding phases along the gastrointestinal tract where protein digestion may occur
(e.g., within the small intestine). The pepsin digestion test does not completely mimic the physiological
conditions of the human gastric digestion system 8 (EFSA, 2021). As described above and in Section 6.3.1
of GRN 1142, the digestibility of OFSP containing brazzein-53 was investigated using the standard pepsin
digestion method as well as a 2-step method which utilized both SGF and SIF to simulate the human
gastrointestinal conditions. Brazzein-53 from OFSP was demonstrated to be stable to digestion under both
conditions. Both stable and unstable (i.e., digested) proteins may elicit allergenic reactions.

As discussed within GRN 1142, brazzein-53 from OFSP and other residual proteins that may be carried
over into the ingredient from the production organism, K. phaffi, were evaluated for allergenicity potential
using a step-wise in silico approach described by the FAO/WHO, Codex Alimentarius, and EFSA. This in
silico approach has been widely employed in the allergenicity assessment of novel protein ingredients,
including those that have been the subject of a GRAS Notification that received “no questions” from the
FDA (e.g., soy leghemoglobin preparation from GRN 737 9). This in silico approach has achieved consensus
amongst the scientific community as the “best practice” in the allergenicity risk assessment of novel
proteins to predict potential cross-reactivity of novel proteins to known allergens. The in silico searches
involved searches with the full-length amino acid sequence, 80-amino acid sliding window, and an 8-
amino acid exact match. A number of curated allergen databases (e.g., AllergenOnline) were used. No
matches to any known allergens were identified that would suggest that brazzein-53 in OFSP is a known
allergen or that it would be cross-reactive to other putative allergens. Therefore, based on the totality of
evidence, despite the fact that brazzein-53 within OFSP may be resistant to digestion, it is unlikely that
this protein would pose any allergenic concern based on the findings of the bioinformatics evaluation.

In addition to the allergenic potential of brazzein-53 from OFSP, the safety of the ingredient has been
established from a toxicological perspective. The systemic toxicity of brazzein-53 was evaluated in a 90-
day repeated-dose oral toxicity study in rats. The study was conducted in accordance with OECD Test
Guideline No. 408 and OECD GLP and yielded a NOAEL of 978 mg/kg body weight/day, the highest dose
tested. Based on the proposed conditions of use of OFSP, the observed NOAEL results in a margin of
exposure of approximately 230. Considering the resistant nature of brazzein-53 to digestion as discussed
above, the intact protein would not be expected to be absorbed into the systemic circulation to elicit any
toxic effect.

Based on the available information, given that there is a large safety margin for OFSP containing brazzein-
53 under its proposed conditions of use as a sweetening agent, it is not expected that the stability profile
of brazzein would be indicative of a safety concern in final consumers.

8 EFSA (2021). Statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of
genetically modified plants (EFSA Panel on Genetically Modified Organisms/GMO) (Question no: EFSA-Q-2020-
00314, adopted: 26 November 2020, published: 12 January 2021 by European Food Safety Authority). EFSA J
19(1):6350 [16pp]. DOI:10.2903/j.efsa.2021.6350. Available at:
https://www.efsa.europa.eu/en/efsajournal/pub/6350.
9 U.S. FDA (2018). Agency Response Letter GRAS Notice No. GRN 737 [Soy leghemoglobin preparation from a

strain of Pichia pastoris, Redwood City (CA): Impossible Foods Inc.], Silver Spring (MD): U.S. Food and Drug
Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety.
Available at: https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 [Jul. 3, 2018
- FDA response - no questions].

Oobli, Inc. • 202 Cousteau Place #210 • Davis, CA 95618 • oobli.com

From: Jason Ryder
To: Kampmeyer, Christopher
Subject: Re: [EXTERNAL] Re: Questions for GRN 001142
Date: Friday, February 2, 2024 3:25:08 PM
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OFSP_Amendment to GRN 1142.pdf

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you
recognize the sender and know the content is safe.

Hi Chris,

On behalf of Oobli, Inc., I am writing to amend GRAS Notice (GRN) 001142 in order to
revise the intended use of Oubli Fruit Sweet Protein Product (OFSP) to reflect the intended
use as a general-purpose sweetener, in accordance with current good manufacturing
procedures (cGMP). The attached amendment provides updated estimates of daily intakes,
along with an updated conclusion in the narrative supporting that use of OFSP as a general-
purpose sweetener would not change the original GRAS conclusion. Oobli, Inc. confirms that
there have been no changes to the identity, method of manufacture, specifications, or technical
effect of OFSP since FDA was notified of the GRAS conclusion on April 20, 2023. The
attached amendment therefore addresses only the revised estimate of intake, and an updated
conclusion of safety based on comparison of the revised estimated daily intake with the no
observed adverse effect level (NOAEL) derived from the pivotal pre-clinical data.

Please kindly confirm receipt of this amendment and let me know if you have any questions.

Best,

Jason

--
Jason Ryder
CTO & Co-Founder

510.684.5610
[email protected]
Amendment to Notice Filed as GRN 1142
Introduction
Oobli, Inc. (“Oobli”), previously concluded that the use of Oubli Fruit Sweet Protein Product,
derived from Komagataella phaffii and containing ≥35% brazzein by weight (hereinafter “Oubli
Fruit Sweet Protein,” or “OFSP”), is Generally Recognized As Safe (GRAS) for the intended use
as a sweetener in various conventional food and beverage products. Oobli notified offices of the
Unites States (U.S.) Food and Drug Administration (FDA) of the GRAS conclusion on April 20,
2023, and the notice was filed as GRN 1142. At this time, FDA is still completing review of the
notice.

The intended use of OFSP defined in the notice filed as GRN 1142 is to provide sweetness in
select categories of foods (including beverages), with use levels of OFSP ranging from 2 to 99
mg/100 g in the different food categories (see Table 1.3-1 in GRN 1142).

Since the notice was submitted to FDA, Oobli has identified several additional food categories in
which the high-intensity OFSP sweetener can be used as a source of sweetness. To include these
recently identified uses, as well as other uses that may be feasible, Oobli has revised the intended
use of OFSP to reflect the intended use as a general-purpose sweetener, in accordance with
current good manufacturing procedures (cGMP), excluding use in infant formula and meat and
poultry products. The updated estimates of daily intakes are provided below, along with an
updated conclusion in the narrative supporting that use of OFSP as a general-purpose sweetener
would not change the original GRAS conclusion. Oobli confirms that there have been no
changes to the identity, method of manufacture, specifications, or technical effect of OFSP since
FDA was notified of the GRAS conclusion on April 20, 2023. The discussion below therefore
addresses only the revised estimate of intake, and an updated conclusion of safety based on
comparison of the revised estimate of intake with the no observed adverse effect level (NOAEL)
derived from the pivotal pre-clinical data.

Updated Intended Use and Estimated Daily Intake

OFSP is intended for use as a general-purpose sweetener in accordance with current cGMP,
excluding infant formula and meat and poultry products.

OFSP contains brazzein, which is a sweet protein. The sweetness intensity of OFSP has been
tested using a sweet potency protocol designed to measure the sweetness response of an
unknown sample compared to sucrose. Results from this testing demonstrate that brazzein is
approximately 750-fold sweeter than sucrose on a weight basis. Given that OFSP is
approximately 44% brazzein by weight, OFSP is approximately 330-fold sweeter than sucrose.

The estimated daily intake of OFSP from the intended use as a general-purpose sweetener in
foods has been derived using the replacement approach described by Renwick (2008). The
approach detailed by Renwick provided estimated intakes of rebaudioside A, a new high-
intensity sweetener at the time. Estimates of rebaudioside A intake were based on published data

2300221.000 - 9337 1
for intake of other high-intensity sweeteners among populations of children and adults with and
without diabetes and the assumption that intake of the new sweetener would fully replace intake
of existing high-intensity sweeteners. Replacement of high-intensity sweeteners was calculated
based on sweetness intensities relative to sucrose.

The approach reported by Renwick was first used to support the exposure assessment for
rebaudioside A in the GRAS conclusion submitted to FDA (GRN 253), to which FDA responded
with a “no questions” letter. Since that time, Renwick’s replacement methodology has been used
to develop estimated daily intakes to support numerous GRAS conclusions for use of
rebaudioside A and related sweeteners providing steviol glycosides (e.g., most recently GRN
1106), as well as other high-intensity sweeteners derived from sources such as Luo Han Guo
(e.g., GRN 359). FDA issued “no questions” letters to these GRAS notices.

To provide estimates of OFSP intake assuming use as a general-purpose sweetener, the Renwick
(2008) approach was applied as follows. The estimated daily intakes of OFSP were calculated by
dividing the estimated sucrose equivalent intakes from intense sweeteners reported by Renwick
by the relative sweetness determined from the adjusted potency test score for OFSP (i.e., 330-
fold). The estimated daily intakes are reported for average and high consumers of intense
sweeteners, where “high” consumers are considered to represent intake at or above the 90th
percentile consumption level based on data compiled by Renwick (2008).

Table 1 below presents the Renwick estimates of high-intensity sweetener intake by population
group along with the estimated intake of OFSP. The estimated daily intakes of OFSP for
“average” consumers in populations of non-diabetic adults, diabetic adults, non-diabetic
children, and diabetic children are up to 0.77 mg OFSP/kg bw/day, 0.85 mg OFSP/kg bw/day,
1.29 mg OFSP/kg bw/day, and 2.04 mg OFSP/kg bw/day, respectively. The estimated daily
intakes of OFSP for “high” consumers in populations of non-diabetic adults, diabetic adults, non-
diabetic children, and diabetic children are up to 2.05 mg OFSP/kg bw/day, 2.72 mg OFSP/kg
bw/day, 3.00 mg OFSP/kg bw/day, and 2.75 mg OFSP/kg bw/day, respectively.

Table 1. Estimated daily intake of OFSP for populations in the United States using an intense
sweetener intake assessment methodology

Intake of Intense Sweeteners

(expressed as sucrose
equivalents) Estimated Intake of OFSP
(mg/kg bw/day) a (mg/kg bw/day) b
“High” “High”
Average Consumer Average Consumer
Population Group Consumer ≥90th percentile Consumer ≥90th percentile
Non-diabetic adults 255 675 0.77 2.05
Diabetic adults 280 897 0.85 2.72
Non-diabetic children 425 990 1.29 3.00
Diabetic children 672 908 2.04 2.75
a Estimates as reported in Renwick, 2008 (Table 7).
b Calculated assuming relative sweetness potency of 750-fold for brazzein and assuming 44% brazzein in OFSP.

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The estimated daily intake of OFSP by the population of “high” consumer non-diabetic children
therefore provides a conservative approach for evaluating the safety of the intended use, as
intake by this population represents the highest intake across the four subpopulations. The
estimated daily intake of OFSP for “high” consumers among populations of children and adults
with or without diabetes is up to 3.00 mg/kg bw/day. The estimates of intake developed with the
approach detailed by Renwick are based on actual estimates of high-intensity sweetener intake,
yet are conservative as they assume full replacement of the currently approved high-intensity
sweeteners with a new sweetener.

Updated Safety Conclusion

In the GRAS dossier submitted to FDA and filed as GRN 1142, the intended use of OFSP in
select categories of foods and beverages resulted in a highest estimated dietary intake of 4.25
mg/kg body weight/day (90th percentile in children 2 to 5 years of age; Table 3.1.2-2 in GRN
1142). As noted in the GRAS notice filed as GRN 1142, several assumptions included in the
assessment render exposure estimates suitably conservative, including the assumption that all
food products within a food category will contain OFSP at the maximum specified level of use.

Under the revised intended use of OFSP as a general-purpose sweetener, in accordance with
cGMP, the estimated intake of OFSP by the population of “high” consumers among populations
of children and adults with or without diabetes is up to 3.00 mg/kg bw/day. These estimates of
intake are based on an established approach for estimating intake of a high-intensity sweetener
(Renwick, 2008), and the measured 330-fold sweetness potency of OFSP relative to sucrose. The
estimated intake of up to 3.00 mg OFSP/kg bw/day corresponds to intake for children without
diabetes. Among adults, the estimated intake of OFSP is up to 2.72 mg OFSP/kg bw/day in the
population of adults with diabetes.

The notice filed as GRN 1142 presents a review of the literature available at the time of that
notice, which covered the published literature through March 2023. To ensure that the review
considers the most recent evidence, a search of PubMed was conducted on January 31, 2024,
with the term “brazzein” for literature indexed since March 1, 2023, with no limitations other
than the English language. This search identified one study with information pertinent to the
GRAS review of OFSP (Novik et al., 2023). Findings from the study reported by Novik and
colleagues are presented below.

The safety of a recombinant brazzein produced in Komagataella phaffii (referenced in the study
as Pichia pastoris, the previously recognized name for K. phaffii) was assessed in acute, sub-
chronic, and chronic toxicity tests and genotoxicity tests (Novik et al., 2023). The recombinant
brazzein test material was stated to be identical to brazzein isolated from the ripe fruits of the
Pentadiplandra brazzeana plant; purity of the test material was not reported. The reported oral
LD50 was >5000 mg/kg bw in rats and mice (species not specified). In the subacute (21-day) oral
toxicity study, recombinant brazzein was administered via gavage to guinea pigs (9/sex/dose) at
0, 2.17, or 21.7 mg/kg bw/day; no effects were reported at the highest dose tested. In the chronic
oral toxicity study in rats, recombinant brazzein was administered via gavage to outbred rats
(10/sex/dose; species not specified) at 0 (vehicle control), 2.17, or 21.7 mg/kg bw/day for a

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period of 150 days. A positive control group was administered sucrose at 4824 mg/kg bw/day.
The authors reported a statistically significant difference in relative body weight gain in male rats
at 2.17 mg/kg bw/day (-13%) and 21.7 mg/kg bw/day (-16%) when compared to the control
group. However, the dose groups administered sucrose (4824 mg/kg bw/day) also demonstrated
a decrease in mean body weight gain (-12%) compared to the control group. In a bacterial
reverse mutation assay (S. typhimurium strains TA 98, TA 97, and TA 100), recombinant
brazzein (up to 50,000 ug/mL) was reported as negative. Recombinant brazzein was reported as
negative in in vivo micronucleus and in vivo chromosome aberrations tests in mice (species not
specified) up to 5,000 mg/kg bw/day.

As reviewed in GRN 1142, findings from pivotal pre-clinical studies demonstrate OFSP to be
non-mutagenic and non-genotoxic, and the NOAEL from the 90-day study at the highest dose
tested was approximately 978 mg/kg body weight/day in males and 985 mg/kg body weight/day
in females (Lynch et al., 2023). This study remains the pivotal study for the assessment of OFSP
safety. Based on the highest estimated dietary intake of 3.00 mg/kg bw/day for the intended use
as a general-purpose sweetener (“high” consumers among populations of children without
diabetes), the margin of exposure is estimated to be 326-fold, suggesting that OFSP, under its
proposed conditions of use, does not pose any safety concerns to end consumers.

Based on the above information, Oobli has concluded that the intended use of OFSP as a
general-purpose sweetener, in accordance with cGMP, excluding use in infant formula and meat
and poultry products, would not change the GRAS conclusion as previously described in the
notice submitted on April 20, 2023, and filed as GRN 1142 and incorporated herein. Oobli
concludes that the intended use of OFSP as a general-purpose sweetener, in accordance with
cGMP, is GRAS. It is Oobli’s view that other experts qualified by scientific training and
experience in food safety evaluation would agree with Oobli’s conclusion.

References

Lynch B, Wang T, Vo T, Tafazoli S, Ryder J. Safety evaluation of oubli fruit sweet protein
(brazzein) derived from Komagataella phaffii, intended for use as a sweetener in food and
beverages. Toxicology Research and Application. 2023;7. doi:10.1177/23978473231151258.

Novik TS, Koveshnikova EI, Kotlobay AA, Sycheva LP, Kurochkina KG, Averina OA,
Belopolskaya MV, Sergiev PV, Dontsova OA, Lazarev VN, Maev IV, Kostyaeva MG, Eremeev
AV, Chukina SI, Lagarkova MA. Sweet-tasting natural proteins brazzein and monellin: safe
sugar substitutes for the food industry. Foods. 2023;12(22):4065. doi: 10.3390/foods12224065.

Renwick AG. The use of a sweetener substitution method to predict dietary exposures for the
intense sweetener rebaudioside A. Food Chem Toxicol. 2008;46 Suppl 7:S61-9. doi:
10.1016/j.fct.2008.05.009.

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