Waste and Biomass Valorization

https://doi.org/10.1007/s12649-020-01284-y

ORIGINAL PAPER

Synthesizing Precursors for Functional Food Structured Lipids

from Soybean Oil Deodorized Distillates
Ramelito Casado Agapay1 · Yi‑Hsu Ju1,2,5 · Phuong Lan Tran‑Nguyen3 · Artik Elisa Angkawijaya2 ·
Shella Permatasari Santoso4 · Alchris Woo Go2

Received: 24 May 2020 / Accepted: 26 October 2020

© Springer Nature B.V. 2020

Abstract
A “green” scheme of synthesizing diglycerides as a means of valorizing an oil processing by-product, specifically soybean
oil deodorized distillate (SODD), was established. Different glycerol dosing strategies were implemented in the solvent-free
lipase catalyzed esterification of free fatty acids with glycerol within molar ratios of 2–4 and temperatures of 40–60 °C. The
process responses were compared with non-dosed systems in this study and other similar processes reported in literature.
Better 1,3-diglyceride selectivity at comparable yields were observed with glycerol dosed-systems over non-dosed systems.
The presence of molecular sieves negatively affected 1,3-diglyceride selectivity, total diglyceride selectivity and yield.
Selectivity of 0.91 ± 0.01 g diglycerides/g (mono + triglycerides), free fatty acid conversion of 57.87 ± 3.46%, and yield of
30.59 ± 1.26 g diglyceride/100 g raw material is obtained with SODD at 40 °C, overall free fatty acid to glycerol molar ratio
of 2, 4 wt% Novozyme 435 and 48 h. The process scaled better than most solvent-based ones supported with hygroscopic
sorbents in terms of reaction mass efficiency.
Graphic Abstract

Keywords Solvent-free · Enzymatic esterification · Glycerol dosing · Selectivity · Soybean oil deodorized distillate ·
Structured lipid precursors

3
* Alchris Woo Go Department of Mechanical Engineering, Can Tho University,
[email protected] 3‑2 Street, Can Tho City, Viet Nam
4
1 Department of Chemical Engineering, Widya Mandala
Department of Chemical Engineering, National Taiwan
Surabaya Catholic University, Kalijudan 37, Surabaya 60114,
University of Science and Technology, No. 43, Keelung
Indonesia
Road, Section 4, Taipei 10607, Taiwan
5
2 Taiwan Building Technology Center, National Taiwan
Graduate Institute of Applied Science and Technology, ,
University of Science and Technology, No. 43, Keelung
National Taiwan University of Science and Technology,
Road, Section 4, Taipei 10607, Taiwan
No. 43, Keelung Road, Section 4, Taipei 10607, Taiwan

13
Vol.:(0123456789)
 Waste and Biomass Valorization

Statement of Novelty OA dosing strategies to investigate their influence on

yields. It was observed that higher diolein yield was
A green scheme of precursor synthesis from SODD that obtained if all OA was fed into the reaction in the begin-
improved selectivity and yield without the use of solvents, ning rather than adding batch-wisely during reaction.
hydroscopic sorbents & specific lipases was established. Addition of solvent minimizes mass transfer problems
as the immiscible reactants can be dissolved in it and that
product selectivity can be influenced depending on sol-
vent concentration and hydrophobicity [19, 21]. Solvent-
Introduction free esterification, however, can give better conversions
over solvent-based systems in certain cases such as in the
Diglycerides (DG) are versatile compounds used as addi- enzyme-catalyzed esterification of caprylic acid and glyc-
tives for various products and as precursors for more erol where the conversion reached more than 10% higher as
complex compounds. DG, together with monoglycerides compared to systems using cyclohexane, octane, and dioxane
(MG), form a group called partial glycerides (PG), which as solvents [22]. This difference is attributed partly on the
are nonionic but possess both hydrophilic and lipophilic inhibiting effect of solvent on lipase activity. Also, when
functional groups. Because of this, they function largely exposed to a combination of organic solvent, compounds
as emulsifiers [1] especially in food products, but are also with surfactant properties, and moderately high temperature,
important components of nutraceuticals or functional immobilized lipase can be desorbed from its matrix [23]
food [2, 3], pharmaceuticals [4], and cosmetics [5]. When making them less active in succeeding cycles of use. Fur-
reacted with certain ligands, DG can be converted to struc- thermore, solvents pose some health risks to users [24] apart
tured triglycerides (STG) with unique or important prop- from contributing to the cost of materials and operational
erties such as radical scavenging or anti-oxidative activ- cost in downstream processing.
ity with ferulic [6] and caffeic acids [7], weight control Solvents also serve to dilute glycerol in the reaction sys-
activity with D-galactopyranose and pinolenic acid [8], tem as high glycerol concentration was reported to be inhibi-
and anti-inflammatory, anti-proliferative & anti-cancer tory towards lipase activity [25, 26]. As dilution by using
effects with n-3 polyunsaturated fatty acids [9] making solvents is not an option in solvent-free esterification of alco-
them indispensable in human diet and wellness. hol and fatty acid, a means to avoid this inhibitory effect is
The synthesis of DG proceeds through several schemes to implement alcohol dosing strategies. Dosing of alcohol,
of reactions allowing for the use of reactants from varied e.g. methanol and ethanol, during enzymatic esterification
sources including waste products [10]. Among synthesis has long been applied in alkyl ester synthesis particularly in
routes that are commonly investigated are alcoholysis of biodiesel production to prevent inhibition [27], but has not
oils using either ethanol [2] or glycerol [11], and direct been exploited in fatty acid and glycerol systems.
esterification of free fatty acids (FFA) with glycerol [12, In esterification, water is a by-product that influences the
13]. Transesterification or interesterification of oils with equilibrium concentration of the desired product. When in
either fatty acids or other oils/triglycerides (TG) also pro- excess, the reaction proceeds in reverse breaking down the
duce DG as by-products [14–16]. Esterification is desir- partial glycerides and triglycerides that have been formed.
able among these routes because it produces less by-prod- However, water is required in some quantity (~ 10 wt%) as
ucts at high conversion, requiring minimal purification some lipases are not as active without them [18, 28]. Regula-
steps to attain high quality. A number of studies involving tion of water activity by removal of excess water has been per-
the esterification of oleic acid (OA) and glycerol carried formed through the addition of hygroscopic sorbents such as
out by the same group of researchers focused on the effects molecular sieves [29] and silica gel [20]. It is noted that silica
of temperature, molar ratio, catalyst type and loading [17], gel has been used as an adsorbent for glycerol and the resulting
water activity [18], and addition of solvents with vary- material utilized as a glycerol reservoir to regulate its release in
ing polarities [19] on 1,3-diolein production, and further the glycerolysis of TG [30]. It also known to adsorb different
using response surface methods to optimize 1,3-diolein classes of lipids at various degrees, more strongly with partial
yield in the process [13]. These works reported that opti- glycerides, making it useful in lipid separation [31]. These
mum diolein yield and 1,3-diolein/diolein of about 87% could compete with the adsorption of water during solvent-
are achieved at ~ 60 °C, ~ 2.5 OA to glycerol molar ratio free esterification. Preferably, silica gel is used in combination
(OA:G), 7.5 wt% Novozyme 435 and 4.8 wt% t-butanol with solvents such as n-hexane to attain better conversions of
with respect to OA. Another work involving diolein pro- the free fatty acids to acyl glycerides [32]. Certain molecu-
duction using the same reaction system but catalyzed with lar sieves also have the capacity to adsorb fatty acids [33]
lipase TL IM, which is sn-1,3-specific [20], implemented and other lipids [34]. Instead of hydroscopic sorbents, there
were also cases where reactions were carried out at vacuum

13
Waste and Biomass Valorization

conditions for the purpose of removing water from the reacting for esterification, with soybean oil deodorized distillate (80%
system [35]. Although this involved no additional material, FFA, TTET Union Corporation, Taiwan) as the source of
specialized equipment was necessary to perform the reac- FFA, replacing OA, in the later system or application. In all
tions. Auxiliary materials (and/or equipment) may have certain trials performed, Novozyme 435, a non-specific lipase from
advantages when used or added to the systems above but their Candida antarctica immobilized in acrylic resin (10,000
presence consequently lowers the reaction mass efficiency of U/g, Novo-Nordisk, Denmark) was used to catalyze the reac-
the process, which translates to its reduced “greenness”. tions. Ethyl acetate (99.9%, Echo, Taiwan), n-hexane (95%,
A by-product of oil processing and refining that is relevant Tedia High Purity Solvents, USA), ethyl ether (99%, Echo,
and potentially a cost-efficient raw material in DG produc- Taiwan), and formic acid (≥ 98%, Sigma-Aldrich, Germany)
tion are fatty acid distillates [36, 37]. Soybean oil deodorizer were the solvents used in the separation of products. A lipid
distillates (SODD), in particular, are in adequate supply and mix standard consisting of mono-, di- and triolein (1787-
rich in unsaturated fatty acids such as oleic and linoleic acids 1AMP, Sigma-Aldrich), and 1,3-diolein standard (≥ 99%
[38], which make them appropriate in the production of DG GC, D3627-10MG, Sigma-Aldrich) were used for identi-
or precursors for symmetrical ABA-type (unsaturated-satu- fication and calibration of components in the analysis of
rated-unsaturated) STG. Such fatty acid distillate has been samples.
utilized mainly in the synthesis of alkyl esters [38] applied as
biodiesel, and as a source of bioactive compounds including Esterification of OA or SODD with Glycerol
tocopherols [39], steroidal hydrocarbons [40] and squalene
[41]. Functional food precursors, therefore, are high-value A 15-ml mixture of OA and glycerol with an OA:G of
derivatives that can alternate with biodiesel in making use of 0.3–5.7 was prepared in a 50-ml Erlenmeyer flask. This was
its major component, the FFA. There has been a study previ- incubated without cover in an orbital shaker incubator at
ously conducted regarding DG production using SODD and 40–60 °C and 150 rpm. Lipase of 4 g per 100 g of total reac-
sn-1,3-specific lipases with Lipozyme RM IM as the preferred tants was added to start the reaction. Aliquots of 20–25 mg
enzyme resulting in a product containing 69.9 wt% DG [42]. were taken at predetermined times for analysis using gas
To achieve higher DG concentration (86.3 wt% DG) in the chromatography. After the reaction, the crude product mix-
product, a purification step was required. The ratio of 1,3-DG ture was separated from the lipase by settling and decan-
isomers over 1,2-DG in this product was about 56:44, which is tation. It was then stored at –20 °C prior to analysis. For
significantly lower than 87:13 that can be obtained in an OA- experiments with glycerol dosing, the same protocol was
glycerol system using solvents and a non-specific lipase [13]. followed except for the initial OA:G which was set around
This presents a challenge for improving 1,3-DG to 1,2-DG 4, and dosing of glycerol according to different strategies to
ratios in solvent-free systems, as higher ratios can be achieved an overall OA:G of ~ 2. Dosing of additional glycerol was
with solvents even without employing downstream processing done at 24 h, after the start of the esterification process for
or purification steps. single dose (full dose) systems; at 24 h and 36 h for two-
Considering the cases presented above, it is the objective dose (half doses) systems. A full dose is equivalent to 750 µl
of this study to explore a minimalist approach to improve of glycerol. The reaction was terminated 12–24 h after the
process responses, including conversion, product distribu- last addition of glycerol, and the whole process lasted for
tion, selectivity, and yield in the production of diglycerides as 48 h. In total, seven sets of conditions involving the model
structured triglyceride precursors from soybean oil deodorizer reaction system were investigated, each labeled with ES-T-
distillates. Employing OA and glycerol as a model reaction MRI-DS-MRF, where ES refers to the esterification system
system, glycerol dosing strategies were tested prior to imple- undergoing the reaction at temperature T, an initial molar
menting promising schemes to a system using SODD. This is ratio of MRI, added with DS number of glycerol doses, and
to verify the hypothesis that process responses, most especially resulting in an equivalent/overall molar ratio of MRF after
product distribution and (regio-)selectivity, can be improved all doses of glycerol were added. Four of these were non-
in esterification processes without using solvents, in-situ water dosed systems, namely ES-40 °C-2.0-0-2.0, ES-40 °C-4.4-
adsorbents, and sn-1,3-specific enzymes. 0-4.4, ES-60 °C-2.0-0-2.0, and ES-60 °C-4.4-0-4.4, which
served as points for comparison with three glycerol-dosed
systems, specifically ES-40 °C-4.4-1-2.0, ES-40 °C-4.4-2-
Materials and Methods 2.0, and ES-60 °C-4.4-2-2.0. The conditions which resulted
in desirable process responses were implemented in the tri-
Materials als involving SODD. The initial free fatty acid to glycerol
ratios (FFA:G) of these systems were based on the total oleic
Oleic acid (99%, Showa Chemical Co. Ltd., Japan) and glyc- and linoleic acid content of the oil distillate. As the effect
erol (≥ 99.5%, JT Baker, USA) were used as model reactants of the presence of molecular sieves was investigated with

13
 Waste and Biomass Valorization

selected SODD systems, each system with the oil distillate bands for 1,2-DG and 1,3-DG were scraped off from the
was labeled with an extended code, ES-T-MRI-DS-MRF- plate and separately placed in vials. Ethyl acetate (1 ml) was
MS. This has the same meaning as the previous one but with used to extract the diglycerides from the scraped material.
MS referring to the presence of molecular sieve, where zero The mixture was then filtered and the filtrate subjected to
(0) meant that no molecular sieve was added to the system GC analysis. The amount of each diglyceride isomer was
and one (1) meant that 0.6 g of 4 Å molecular sieves was determined using previously established calibration curves
added per g of SODD. All experiments were performed in and 1,3-DG concentration (g/100 g total DG) was calculated
duplicates. from the masses of 1,2-DG, m1,2-DG (g), and 1,3-DG, m1,3-DG
(g), respectively, as follows:
Analysis of Free Fatty Acids and Glycerides
m1,3−DG
[ ]
g
1,3DG = × 100 (3)
An aliquot of 20–25 mg of the product was dissolved in 100g m1,2−DG + m1,3−DG
1 ml of ethyl acetate, homogenized, and then filtered using
a 13-mm syringe filter with 0.20-µm PTFE membrane
(Acrodisc syringe filters, Pall Corporation, Life Sciences). Calculation of Conversion, Selectivity, Yield,
A 1.5 µl-volume of the filtrate was injected into a gas chro- and Reaction Mass Efficiency
matography unit (Shimadzu GC-2010 Plus) installed with
a ZB-5HT Inferno column (15 m × 0.32 mm × 0.1 µm), and The moles of each component present in 100 g of glycerol-
equipped with a split injector and FID. It was operated on a free product were determined using the weight percentages
program to separate different types of fatty acids and acyl- obtained from chromatograms. OA conversion, 𝜉OA (%), was
glycerides as described in Go et al. [43]. The injector and then calculated using the following equation:
detector temperatures were set at 370 °C and the initial col-
umn temperature at 80 °C. The column temperature was
[ ( ) ( ) ]
nMG + 2 × nDG + 3 × nTG
ramped to 370 °C with a total analysis time of 29 min. Nitro- 𝜉OA [%] = ( ) ( ) × 100
nOA + nMG + 2 × nDG + 3 × nTG
gen gas flow in the column was 1.4 ml/min and the injection
(4)
split ratio was 50. The peaks were identified individually
or classified in groups using fatty acid and lipid standards. where nOA, nMG, nDG, and nTG are the moles of residual OA,
The peak areas in the chromatograms were then converted monoglyceride (MG), diglyceride (DG), and triglyceride
to weight percentages using calibration curves established (TG) in the product, respectively.
with these standards. From the OA:G value, the amount of glycerol, nGlyOH
In product analysis, product distribution PDi (g/100 g), (mol), originally used in esterification was determined on
refers to the mass of a component, mi (g), e.g. residual OA the same basis as the other components. Glycerol conver-
or acylglyceride, that is contained in 100 g of glycerol-free sion, 𝜉GlyOH (%), was then calculated based on the following
product, mGFP (g). equation:
( )
[
g
]
mi nMG + nDG + nTG
PDi = × 100 (1)
𝜉GlyOH [%] = × 100 (5)
100g mGFP nGlyOH

Glyceride distribution Ci (g/100 g), is the ratio of the Diglyceride selectivity, SDG (g/g), is calculated as the
mass of a glyceride species, mi (g) to 100 g of total glycer- ratio of the mass of total diglycerides mDG (g) in the product
ides in the product, mtotG (g). to the total amount of monoglycerides mMG (g) and triglyc-
[ ] erides mTG (g).
g mi
Ci = × 100 (2) [ ] mDG
100g mtotG SDG g∕g =
mMG + mTG (6)
To determine the distribution of 1,2- and 1,3-DG, 100 mg
Yield, YDG (g/100 g), is defined as the mass of total
of crude glyceride product was dissolved in 0.5 ml of n-hex-
diglycerides mDG (g) in the product that can be obtained
ane, and 10 µl of the resulting mixture was blotted on a
from 100 g of the reactants used.
TLC Silica gel 60 F254 plate (Merck) previously dried in
an oven at 100 °C for 1 h. The separation of components
[ ]
g mDG
was then carried out in a glass chamber using a pre-mixed YDG = × 100 (7)
100g mOAi + mGlyOHi
solvent composed of 80:20:2 v/v/v of n-hexane/ethyl ether/
formic acid. The plate was air-dried and developed using where mOAi and mGlyOHi are the total amounts of OA and
iodine vapor to show the bands of separated glycerides. The glycerol used as reactants, respectively.

13
Waste and Biomass Valorization

Reaction mass efficiency, RME, is defined as the ratio of Fig. 2. Maximum product distribution in terms of total
the mass of total diglycerides mDG (g) in the product over glycerides (PDtotG) slightly increased with increasing tem-
the total mass of raw materials, ƩmRMi (g), used in the reac- perature (Fig. 2a, c, and e), reaching 54.90 ± 3.32 g/100 g
tion mixture including the catalyst mcat (g), solvents msol (g), glycerol-free product at 40 °C, 59.31 ± 2.14 g/100 g at
sorbents msor (g), and other reagents. 50 °C, and 60.97 ± 0.15 g/100 g at 60 °C. This was pos-
� � sible because a higher reaction temperature increases both
g m mDG
RME = ∑ DG = the rate of reaction and the mutual miscibility of reac-
g mRMi mOAi + mGlyOHi + mcat + msol + msor + …
tants, leading to higher conversions and more products
(8) [45]. Also, maximum selectivity for both DG (SDG) and
PG (SPG) occurred earlier but within a narrower period as
the temperature was increased (Fig. 2b, d, and f). In par-
Results and Discussion ticular, maximum SDG and SPG were attained in 18–24 h
at 40 °C, 10–15 h at 50 °C, and 4–8 h at 60 °C. A longer
Effect of Temperature and Molar Ratio period of high SDG (or SPG) implies a more flexible process
on Conversion, Distribution, and Selectivity when the intermediate components are the desired prod-
ucts. In all three temperatures, the magnitudes of the SDG
Solvent-free esterification of OA and glycerol was carried and SPG are close and are within the ranges of 1.05–1.30 g
out over a wide range of OA:G and different temperatures DG/g (MG + TG) and 3.30–3.80 g PG/g TG, respectively.
for 24 h in order to verify their effects on conversion, and Apart from these, the equivalent ξ OA and ξ GlyOH at the
more importantly, the distribution of glycerides in the prod- stated time ranges also have similar values of 37.69% and
uct (Fig. 1). Complete conversion of glycerol (ξGlyOH) can 99.98% at 40 °C, 37.88% and 93.51% at 50 °C, and 39.68%
be achieved at OA:G ≥ 3.2, but OA conversion (ξOA) can and 100% at 60 °C. Similar trends were observed in the
only reach 84% maximally even at the lowest OA:G and the esterification of OA and glycerol at initial molar ratios
highest temperature investigated. Diglycerides were predom- of 2.0–4.0, carried out between 40 and 60 °C but in the
inantly produced in all cases except at 60 °C and OA:G ≥ 3.2 presence of 15% Lipozyme TL IM (w/wOA), the solvent
where products were largely composed of TG (> 50 g/100 g toluene, and molecular sieves [20], as well as, in non-
total glycerides). Specifically, diglyceride distribution (CDG) catalyzed solvent-free systems at the same molar ratios
reached 61.27 ± 0.34 g/100 g of total glycerides at 40 °C and but at a higher temperature range of 150–200 °C [31]. The
an OA:G of 3.2. Triglyceride distribution (CTG), on the other peaks of DG selectivity also occurred faster within a nar-
hand, reached 55.99 ± 2.12 g/100 g of total glycerides at the row time frame as the reaction temperature was increased,
same OA:G but at a higher temperature of 60 °C. Monoglyc- although their occurrences were observed at much shorter
eride distribution (CMG) was low to moderate in all cases, not periods (> 4 h) alongside steep increases in TG because
exceeding 31.94 ± 2.22 g/100 g of total glycerides. Diglyc- of a higher enzyme loading in the former and higher tem-
eride selectivity (SDG) ranged from 0.42 to 1.58 g/100 g peratures in the latter compared to the current study. As
(MG + TG), peaking at the condition where CDG was also the conversion of glycerol was practically complete at
maximum. In general, DG, or more broadly PG, tend to be maximum SDG, the decrease in selectivity with time after
formed at higher concentration when the reaction is carried this point is a result of the remaining OA further reacting
out at lower temperature and high OA:G. This ostensibly with DG to form TG according to the established reaction
validates the idea that at high OA:G where the fatty acid is mechanism between OA and glycerol [46]. Because TG is
considerably in excess over glycerol, the inhibiting effect of an undesirable product in DG production, the concentra-
glycerol on lipase activity is minimized [21, 22], although tion of unreacted OA must be kept low after this stage. In
some fatty acids can also have either inhibiting or promot- biodiesel production, the stepwise addition of methanol
ing effects [27, 28]. Whichever effect is dominating in the ensured that the conversion of FFA to methyl esters was
reaction, it is desirable to perform esterification of OA and high or nearly complete such that residual acids were neg-
glycerol at 40 °C and OA:G within 2.0–4.4 considering ligible and that the product met certain standards [27, 47].
the results obtained if CDG or SDG were prioritized. This It was, therefore, hypothesized that glycerol dosing would
is supported by previously reported works on DG produc- also be a simple but effective strategy to push the reaction
tion where the optimum molar ratio was found around 3.5 towards higher FFA conversion and, possibly, minimal TG
at 40 °C [20], and between 2 and 2.5 at 60–65 °C [17, 44]. formation. Thus, at the periods when the maximum DG
The effects of temperature and time on product distri- had been achieved, dosing of glycerol was performed to
bution and glyceride selectivity were further explored at increase ξOA while still maintaining relatively high instan-
the upper end of this OA:G range and are illustrated in taneous OA:G.

13
 Waste and Biomass Valorization

100 100 100

g/100 g GlyOH-free Product

g/100 g Total Glycerides

% Conversion
80 80 80
60 60
60
40 40
40
20 20
0 0 20
0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1 0
OA:GlyOH Ratio (mol/mol) 0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1
OA:GlyOH Ratio (mol/mol)
Residual OA Total Glycerides
Conversion, OA Conversion, GlyOH MG DG TG

(a) (b)
100 100 100
g/100 g GlyOH-free Product

g/100 g Total Glycerides

% Conversion
80 80 80
60 60
60
40 40
40
20 20
0 0 20
0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1 0
OA:GlyOH Ratio (mol/mol) 0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1
OA:GlyOH Ratio (mol/mol)
Residual OA Total Glycerides
Conversion, OA Conversion, GlyOH MG DG TG

(c) (d)
100 100 100
g/100 g Total Glycerides
g/100 g GlyOH-free Product

% Conversion

80 80 80
60 60
60
40 40
40
20 20
0 0 20
0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1 0
OA:GlyOH Ratio (mol/mol) 0.3:1 0.6:1 1.2:1 1.9:1 3.2:1 4.4:1 5.7:1
OA:GlyOH Ratio (mol/mol)
Residual OA Total Glycerides
Conversion, OA Conversion, GlyOH MG DG TG

(e) (f)

Fig. 1  Product distribution and reactant conversion during esterification of OA and glycerol catalyzed by Novozyme 435 (4 g/100 g reactants) at
40 °C (a, b), 50 °C (c, d), and 60 °C (e, f) for 24 h

Effect of Glycerol Dosing on Conversion, Different glycerol dosing strategies were then carried out to
Distribution, Selectivity, and Yield determine specific effects on a number of process responses
including selectivity and yield, summarized in Table 1.
The effects of glycerol dosing that were initially assumed Without glycerol dosing, increases in conversion were
were confirmed in an experiment, with its results illustrated achieved by performing the reactions at higher tem-
in Fig. 3. The profiles showed its advantages in the following perature as can be seen with ES-40 °C-2.0-0-2.0 against
ways: (a) further reduction in residual OA from 50 g/100 g ES-60 °C-2.0-0-2.0, or ES-40 °C-4.4-0-4.4 against
glycerol-free product in the non-dosed system to approxi- ES-60 °C-4.4-0-4.4. It is already well-established that
mately 20 g/100 g glycerol-free product in a dosed one, and temperature affects rates of reaction generally in a positive
(b) maintenance of low MG and TG concentrations thereby direction, i.e. increasing the temperature increases the reac-
sustaining high DG selectivity after glycerol addition. tion rate. Also, the catalyst influences the range at which

13
Waste and Biomass Valorization

Fig. 2  Product distribution and 100 1.2 4.0

g/100 g GlyOH-free Product

glyceride selectivity during 1.0

g DG/g MG+TG
80 3.0
esterification of OA and glyc-

g PG/g TG
0.8
60
erol at an initial OA:G of 4.4, 0.6 2.0
catalyzed by Novozyme 435 40
0.4
(4 g/100 g reactants) carried out 20 1.0
0.2
under 40 °C (a, b), 50 °C (c, d),
0 0.0 0.0
and 60 °C (e, f) 0 20 40 60 80 100 0 20 40 60 80 100
Hours Hours

Residual OA Total Glycerides DG PG

(a) (b)
100 1.4 4.0
g/100 g GlyOH-free Product

1.2

g DG/g MG+TG
80 3.0
1.0

g PG/g TG
60 0.8
2.0
40 0.6
0.4 1.0
20
0.2
0 0.0 0.0
0 20 40 60 80 100 0 20 40 60 80 100
Hours Hours

Residual OA Total Glycerides DG PG

(c) (d)
100 1.2 4.0
g/100 g GlyOH-free Product

1.0

g DG/g MG+TG
80 3.0

g PG/g TG
0.8
60
0.6 2.0
40
0.4
20 1.0
0.2
0 0.0 0.0
0 20 40 60 80 100 0 20 40 60 80 100
Hours Hours

Residual OA Total Glycerides DG PG

(e) (f)

Fig. 3  Esterification of oleic 100 100

acid and glycerol at an initial
OA:G of 4.4 catalyzed by 4 g
g/100 g GlyOH-free Product

g/100 g GlyOH-free Product

80 80
Novozyme 435/100 g reac- Glycerol
tants carried out under 40 °C Dose
a without glycerol dosing, 60 60
ES-40 °C-4.4-0-4.4 and b with
750-µL glycerol dose at 24 h, 40 40
ES-40 °C-4.4-1-2.0
20 20

0 0
0 20 40 0 10 20 30 40
Hours Hours

MG DG MG DG
TG Residual OA TG Residual OA

(a) (b)

13
 Waste and Biomass Valorization

Table 1  Effect of different strategies on conversion, distribution, selectivity, and yield

Strategy Conversion (ξOA), % Glyceride Distribution, g/100 g Total Glycer- DG Selectivity (SDG), DG Yield (YDG),
(ES-T-MRI-DS-MRF)a ides g DG/g (MG + TG) g DG/100 g RM
CMG CDG CTG

ES-40 °C-2.0-0-2.0 74.47 ± 0.55 28.95 ± 0.04 56.30 ± 0.20 14.75 ± 0.24 1.29 ± 0.01 40.68 ± 0.10
ES-40 °C-4.4-0-4.4 45.98 ± 0.09 21.28 ± 0.60 53.89 ± 0.38 24.83 ± 0.98 1.17 ± 0.02 25.52 ± 0.15
ES-60 °C-2.0-0-2.0 86.82 ± 0.00 15.37 ± 0.00 54.84 ± 0.02 29.79 ± 0.02 1.21 ± 0.00 45.01 ± 0.03
ES-60 °C-4.4-0-4.4 58.08 ± 0.77 14.90 ± 0.40 29.33 ± 0.10 55.76 ± 0.50 0.42 ± 0.00 16.95 ± 0.15
ES-40 °C-4.4-1-2.0 66.44 ± 0.50 25.14 ± 0.38 56.90 ± 0.99 17.97 ± 0.61 1.32 ± 0.05 37.80 ± 0.41
ES-40 °C-4.4-2-2.0 69.54 ± 0.64 16.39 ± 0.08 65.31 ± 0.16 18.30 ± 0.08 1.88 ± 0.01 45.03 ± 0.48
ES-60 °C-4.4-2-2.0 87.62 ± 0.14 14.34 ± 0.18 54.16 ± 0.18 31.49 ± 0.36 1.18 ± 0.01 45.64 ± 0.09
a
Total process time is 48 h; Code (ES-T-MRI-DS-MRF) refers to the following: Esterification System (ES) with the reaction carried out at T—
reaction temperature, MRI—initial molar ratio, DS—number of glycerol doses implemented, and MRF—overall molar ratio after all glycerol
doses have been added; a single-dose system is added with a full dose of glycerol at 24 h, and a two-dose system is added with equal doses at
24 h and 36 h, all to an overall molar ratio of 2.0

optimum activities are observed. Novozyme 435, which were also considered such as yield, distribution, or selectiv-
is used in this study, often exhibits significant activities ity in order to determine the best conditions to be adopted.
within the range of 40–90 °C [48], and reached maximum Diglyceride distribution (C DG) or selectivity (S DG)
activities close to 60 °C in solvent-free systems [49]. At a becomes a critical factor to consider when complete sepa-
fixed reaction temperature of 40 °C, dosing with glycerol ration between the total glycerides and residual reactants
resulted in a significant increase in OA conversion for both can be achieved. Selectivity (SDG) follows the same trend
ES-40 °C-4.4-1-2.0 and ES-40 °C-4.4-2-2.0 when compared as CDG because it is essentially the same response but with
to the non-dosed system ES-40 °C-4.4-0-4.4, but were lower the effects of CMG and CTG incorporated into one value. In
when compared to ES-40 °C-2.0-0-2.0. The increase in con- this perspective, the system with the highest CDG or SDG
version of the dosed systems relative to ES-40 °C-4.4-0-4.4 will result in higher product yields in terms of the desired
was expected because, upon dosing, more glycerol became component. Dosing had a positive influence on CDG as can
available for the residual fatty acids to react, especially when be observed in all glycerol-dosed systems at 40 °C. They
at 24 h of reaction, ξGlyOH was already at 100% (Fig. 1a). The had consistently larger CDG values when compared against
difference in conversion of dosed systems when compared both non-dosed systems. It also appeared that the frequency
to ES-40 °C-2.0-0-2.0 can be explained along the same line of dosing, i.e. more doses at correspondingly lower amounts
of reasoning, although in this particular case, more glycerol (ES-40 °C-4.0-2-2.0 versus ES-40 °C-4.4-1-2.0), increases
was already available right at the beginning of the process the same response. With more glycerol introduced at first
for system ES-40 °C-2.0-0-2.0 than for the glycerol-dosed dose in ES-40 °C-4.4-1-2.0 than in ES-40 °C-4.4-2-2.0, there
systems. Implementing both dosing and increase in tem- is a tendency to form more partial glycerides particularly
perature, specifically with ES-60 °C-4.4-2-2.0, yielded the MG than TG. The second dose of glycerol in ES-40 °C-4.4-
highest conversion among the strategies, which supported 2-2.0 resulted in the same overall OA:G for both systems,
the assumption that glycerol dosing consistently improves which could have allowed glycerolysis of DG and TG to
conversion at different temperatures. Fatty acid dosing, how- occur in order for their compositions to be the same at equi-
ever, did not result in a better process response expressed in librium. But because glycerolysis is a slower process than
terms of DG yield as reported in the synthesis of 1,3-diolein esterification, as implied in a separate study comparing
catalyzed by Lipozyme TL IM with t-butanol as solvent dicapryl glycerol production via esterification of caprylic
[20]. In such case, it was observed that the yield decreased acid and glycerol against the glycerolysis of TG containing
when the dosing frequency was increased at lesser amounts tricaprylin [22], ES-40 °C-4.4-2-2.0 ended up being richer
per dose, and that the best result was obtained when all of in DG and TG when compared to ES-40 °C-4.4-1-2.0 after
the fatty acid was added at the beginning of the reaction. No 48 h. However, temperature had a negative effect as it low-
explanation was offered as to why this occurred in the said ered CDG when increased from 40 to 60 °C (ES-40 °C-4.4-
study, but apparently, OA dosing gave an opposite effect 2-2.0 versus ES-60 °C-4.4-2-2.0). This is owing to the fact
compared to glycerol dosing. As higher conversions do not that at higher temperature the formation of TG, which is an
always translate to higher concentration of the desired prod- undesirable product in DG synthesis, increased as shown
uct when pertaining to glyceride synthesis, other indicators in Fig. 1f. This occurrence has been consistently observed

13
Waste and Biomass Valorization

in several studies on DG production based on esterification ES-40 °C-2.0-0-2.0, esterification was performed between
processes [31, 49]. The DG yield (YDG) is another response SODD and glycerol, labelled ES-40 °C-4.4-2-2.0-0 and
that is important to consider as it gives a more practical basis ES-40 °C-2.0-0-2.0-0 respectively, and the results listed in
of process efficiency. Because it is influenced by conversion Table 2, together with one response added, which is the 1,3-
and selectivity, the observed trends with dosing can differ DG concentration with respect to the total DG.
from the trends of both responses when taken separately. In The values obtained for all process responses were lower
this case, dosing generally improved yields at both tempera- with SODD than those achieved with the correspond-
tures investigated, with selectivity contributing largely for ing systems involving OA and glycerol. This is possibly
the increase at 40 °C whereas conversion played a larger role influenced by the presence of other components already
at 60 °C (Table 1). The implementation of dosing at high in SODD prior to esterification, most notably TG, which
temperature resulted in the highest YDG which suggests that altered the effective concentrations of the reactants and
this combination is best for DG production when the type consequently, the distribution of glycerides as well as the
and amounts of other glycerides is not a critical matter. selectivity at the end of the process. In a previous study
Overall, glycerol dosing is an effective means of improv- [52], pretreatment by enzymatic hydrolysis of mixed deo-
ing process responses, with effects on the selectivity for dorized distillates prior to lipase-catalyzed esterification
diglycerides that are the most consistent. Among all schemes of the product with glycerol eliminated the interference of
implemented, ES-40 °C-4.4-2-2.0 is evidently the most acyl glycerides and improved DG concentration from 46.0
appropriate one to explore further or apply to other systems to 66.8 wt%. The trend in conversion remained the same
as it had collectively the best results upon considering dif- as with the model system, slightly decreasing with glyc-
ferent process responses. erol dosing, but apparently there is no significant difference
between ES-40 °C-4.4-2-2.0-0 and ES-40 °C-2.0-0-2.0-0
Esterification of SODD with Glycerol when looking at other process output variables. However,
glycerol dosing has drawn out an increase in the selectiv-
Soybean oil deodorizer distillates (SODD) contain FFA ity for 1,3-diglycerides. This constituted an improvement in
in substantial concentrations ranging from 28% [50] to as the process even though other product characteristics such
much as 85.5% [51], with > 75% unsaturated fatty acids with as glyceride distribution and yield remained practically the
respect to the total FFA [38, 51]. The higher the FFA con- same. To verify further whether this observation was not
centration the more suited it is for conversion to DG [52]. In an artifact but a result of dosing, a lower initial FFA:G was
this study, the SODD collected comprised of 80.07 ± 0.34 implemented, still within 2.0–4.0, plus dosing with glycerol
wt% FFA mainly oleic and linoleic acids, 14.89 ± 0.41 wt% to a final FFA:G of 2.0 (ES-40 °C-3.2-1-2.0-0). The con-
TG, 5.04 ± 0.06 wt% unsaponifiable matter mostly toco- version increased when compared to both ES-40 °C-4.4-2-
pherols, and traces of MG and DG. Following the con- 2.0-0 and ES-40 °C-2.0-0-2.0-0, but DG yield and selectivity
ditions of ES-40 °C-4.4-2-2.0 and the reference system decreased as CMG increased at the expense of both DG and

Table 2  DG production using SODD and glycerol with modified dosing strategies
Strategy FFA Conversion (%) Glyceride Distribution Selectivity, SDG (g 1,3-DG DG Yield (g/100 g
(ES-T-MRI-DS- (g/100 g Total Glycerides) DG/g MG + TG) (g/100 g total RM)
MRF-MS)a DG)
CMG CDG CTG

ES-40 °C-2.0-0- 62.44 ± 2.82 23.67 ± 2.91 47.86 ± 0.09 28.47 ± 3.00 0.92 ± 0.00 60.97 32.41 ± 1.14
2.0-0
ES-40 °C-4.4-2- 57.87 ± 3.46 23.70 ± 3.75 47.67 ± 0.24 28.63 ± 3.51 0.91 ± 0.01 63.96 30.59 ± 1.26
2.0-0
Effect of the presence of molecular sieves
ES-40 °C-3.2-1- 64.89 ± 2.07 29.05 ± 0.20 44.18 ± 0.21 26.78 ± 0.02 0.79 ± 0.01 63.35 30.02 ± 0.83
2.0-0
ES-40 °C-3.2-1- 65.03 ± 0.83 21.32 ± 0.85 39.00 ± 0.35 39.69 ± 0.50 0.64 ± 0.01 59.26 26.11 ± 0.46
2.0-1
a
Total process time is 48 h; Code (ER-T-MRI-DS-MRF-MS) refers to the following: Esterification System (ES) with the reaction carried out
at T—reaction temperature, MRI—initial molar ratio, DS—number of glycerol doses implemented, MRF—final/overall molar ratio, and MS—
presence of molecular sieves where 0 has no molecular sieves (activated at 175 °C under vacuum for 24 h prior to their use) & 1 has 0.6 g of 4 Å
molecular sieves per 1 g of SODD; a single-dose system is added with a full dose of glycerol at 24 h, and a two-dose system is added with equal
doses at 24 h and 36 h to an overall molar ratio of 2.0

13
 Waste and Biomass Valorization

TG (Table 2), which are consistent with the result between also have detrimental effects in solvent-free DG production
ES-40 °C-4.4-1-2.0 and ES-40 °C-4.4-2-2.0 (Table 1) especially when using SODD. In fact, the use of molecular
involving the model reactants. More importantly, the amount sieves may not be necessary as reported in a study involving
of 1,3-DG relative to the total DG remained higher than the production of high-purity DG via solvent-free esterifi-
the non-dosed system but closer and incrementally lower cation of OA and glycerol catalyzed by a Rhizopus oryzae
than that of ES-40 °C-4.4-2-2.0-0 having had closer initial lipase immobilized in magnetic nanoparticles [53], as there
and overall molar ratios. With higher conversion of the fatty was no substantial difference in the concentration of 1,3-DG
acids in ES-40 °C-3.2-1-2.0-0, more water is expected to be between the systems with and without this water-adsorbing
produced which could have increased the rate of hydrolysis material.
that resulted in higher MG concentration as compared to
ES-40 °C-4.4-2-2.0-0. To validate this assumption, trials Comparison with Related Esterification‑Based DG
based on ES-40 °C-3.2-1-2.0-0 conditions were run with Production Systems
molecular sieves (ES-40 °C-3.2-1-2.0-1) as a means of in-
situ water removal. From the values obtained for CDG, SDG, The production of DG based on the esterification of OA
and YDG, the presence of these hygroscopic sorbents had a and glycerol had been studied in several occasions (Table 3),
negative impact as the reaction pushed towards higher TG but employing different reaction strategies. Simply compar-
formation. More importantly, the 1,3-DG decreased rela- ing the product quality and typical process performance as
tive to the total DG formed. Apart from these, the product obtained from the strategy employed in this study, either
obtained had a darker hue when compared to the system with OA or SODD, the values fall within the ranges previ-
without molecular sieves. From the GC chromatograms, it ously reported in literature. Among the works presented,
was seen that some peaks of the unsaponifiable matter, spe- the conversion, distribution, and selectivity of the product
cifically tocopherols, disappeared, which implied that they from ES-40 °C-4.4-2-2.0 resemble that of the reaction sys-
may have been oxidized in the presence of molecular sieves. tem involving Candida sp. 99–125 lipase as catalyst and
In any case, these showed that employing molecular sieves cyclodextrin as a stabilizer [28]. The two systems were

Table 3  Comparison with lipase-catalyzed systems for DG production reported in literature

Temp (°C)/ Reactants FFA:G, Catalyst Auxiliary Materi- ξFFA SDG PDDG/CDG YDG (g/100 g) RME References
Time (h) (mol/mol) (wt%, specificity) als/Special Condi- (%) (g/g) (g/100 g) (g/g)
tions
(wt%)

40/4 OA & GlyOH Candida sp. cyclodextrin, 65.9 1.80 44.2/64.3 18.2 0.14 Table 1 [28]
1:5 99–125 lipase 1.5:1a (54.3)b
10, non-sp H2O, 10
30/24 OA & GlyOH Candida rugosa n-hexane, 80 76.9 1.01 39.6/50.1 8.7 0.05 Figure 3 [29]
1:10 lipase H2O, 6
4.0, non-sp
60/7 OA & GlyOH Novozyme 435
t-butanol, 53 88.0 3.54 69.6/78.0 68.4 0.31 Figure 5 [17]
2.5:1 9, non-sp molecular
sieves, 57
40/3 OA & GlyOH Lipozyme TL IM toluene, 80 40.4 2.12 29.6/68.2 29.1 0.11 Figure 9 [20]
3.5:1 18, sp H2O, 0.33c
molecular sieves,
61
65/2 OA & GlyOH Lipozyme RM 0.01 Mpa vacuum 79.6 (87.7) c 2.64 59.1/72.6 55.9 0.53 Figure 5 [44]
2:1 IM
6, sp
40/48 OA & GlyOH Novozyme 435 – 69.5 1.88 46.9/65.3 45.0 0.43 This study
2:1d 4, non-sp
40/48 OA + LA (SODD) Novozyme 435 – 57.9 0.91 30.6/47.7 30.6 0.29 This study
& GlyOH 4, non-sp
2:1d

Data were calculated based on available information in figures/tables from respective references; all wt% are relative to the initial mass of reac-
tants, SDG is in g DG/g (MG + TG), PDDG in g DG/100 g glycerol-free product, CDG in g DG/100 g total glycerides, YDG in g DG/100 g total
weight of reactants, and RME in g DG/g total raw materials; acyclodextrin/lipase mass ratio, breported as is in reference, cwater activity, doverall
FFA:G

13
Waste and Biomass Valorization

both catalyzed by a non-specific lipase and carried out at vacuum at 65 °C [33]. By itself, manipulation of operating
40 °C. There is a large difference in terms of YDG, however, conditions, in this case pressure, instead of changing reac-
mainly because the latter used excess glycerol (OA:G of 1:5) tant mixture composition is a generally a green means of
resulting in a low yield despite of the higher enzyme loading manipulating process output. However, reactors designed
involved. This is consistent with another study which used a to operate under vacuum conditions are cost-intensive as it
much lower OA:G of 1:10, also catalyzed with a non-specific would require accessories for pressure control apart from
lipase from Candida rugosa at 30 °C, that brought in YDG of the fact that materials of construction should also with-
a much lower value [29]. On the other hand, better conver- stand such extreme condition.
sion, distribution, selectivity, and DG yield were achieved The weakest aspect of the process investigated in this
with a system using higher OA:G (2.5:1), t-butanol as sol- study is the process time. This is due to the time intervals
vent, molecular sieves for water removal, and Novozyme of glycerol dosing involved. This can be addressed through
435, which contains a non-specific lipase from Candida ant- a reactor design with features compatible with the imple-
arctica [17]. All these supported the previous results pre- mentation of this strategy, such as in a plug flow reactor,
sented in Fig. 1, that led to the decision of performing the where after achieving steady state, this will become a minor
esterification reactions at higher OA:G. The product from issue. It is also possible to shorten process time through an
ES-40 °C-4.4-2-2.0-0, which utilized SODD as the FFA increase in enzyme loading as can be noted with other pro-
source, had a more unique set of response values, because cesses reported, except for the work of Yesiloglu and Kilic
unlike the others, SODD contained mixed fatty acids and [29], where employed enzyme loadings are 1.5–4.5 times
other components. higher than the one implemented in the present study. This
The use of sn-1,3-specific lipases generally yielded better made it possible for them to reach maximum SDG within
DG selectivity as those obtained with lipozyme TL IM [20] shorter reaction times.
and lipozyme RM IM [44]. The former, which worked with Overall, the process currently investigated had gener-
high OA:G (3.5:1), molecular sieves, and toluene as solvent ated products with qualities that are within those reported
resulted in an SDG of 2.12, whereas the latter with an OA:G in literature but excel in terms of its simple raw material
of 2:1 but carried out under vacuum produced an SDG of 2.64. composition and flexible strategy for manipulating conver-
The experiments performed at the conditions implemented sion, distribution, and selectivity. Without solvents, there
in this study with the use of lipase RM IM supported these is no need for a solvent recovery step and the health issues
results as higher SDG of 1.49 g DG/g (MG + TG) against related to the consumption of products contaminated with
1.17 g DG/g (MG + TG) with Novozyme 435 was achieved these substances are avoided. Furthermore, special acces-
while practically having similar total glycerides formed at sories required in reactors to regulate pressure such as those
40 °C (54.90 ± 3.32 g total glycerides/100 g glycerol-free employing vacuum conditions could potentially be avoided.
product for Novozyme 435 against 50.58 ± 1.18 g/100 g for
Lipase RM IM). Unfortunately, lipase RM IM was poor in
stability and lost its catalytic activity after the first cycle.
Thus, the robustness of Novozyme 435 over lipase RM IM, Conclusion
and more importantly, many other lipases [54] makes Novo-
zyme 435 attractive to use despite its non-specific property. Solvent-free lipase-catalyzed esterification of OA and glyc-
In fact, its non-specific character allows for further esterifi- erol performed at high OA:G with glycerol dosing led to
cation of the DG with another fatty acid to produce designer improved conversion, selectivity, distribution and yield for
lipids without the addition of another type of catalyst. More- diglycerides. The best strategy at a fixed lipase loading of
over, the specificity of Novozyme 435 can be manipulated 4 wt% was by using an OA:G of 4.4, 40 °C, 50/50 glyc-
through the use of certain solvents, as was reported when erol dosing in a total reaction time of 48 h, which resulted
various types of solvents were investigated in the synthesis in ξOA of 69.54%, SDG of 1.88 g DG/(g MG + TG), CDG of
of 1,3-diolein by esterification [19], and also to a significant 65.31 g DG/g total glycerides, and YDG of 45.03 g PG/100 g
extent through glycerol dosing as shown in this study. RM. When applied with SODD, the corresponding process
The best characteristic of the process presented in this response values were obtained: ξOA of 57.87%, SDG of 0.91 g
study is its “greenness” as it is solvent-free and the strat- DG/(g MG + TG), CDG of 47.67 g DG/g total glycerides, and
egy involved in improving process responses is based on YDG of 30.59 g PG/100 g RM. Additionally, dosing increased
a reactant feeding scheme instead of the use of solvents or the 1,3-DG concentration to 63.96 g/100 g DG. Glycerol
hygroscopic sorbents. This can be seen from its reactant dosing is a “green” strategy of manipulating output vari-
mass efficiency value (RME) of 0.43. The other system ables, as it does so without using other raw materials apart
which gave a better RME value is also solvent-free but from those necessary for the reaction to occur.
used Lipozyme RM IM and carried out the operation under

13
 Waste and Biomass Valorization

Author Contributions Methodology, investigation, formal analysis, 11. Ferretti, C.A., Spotti, M.L., Di Cosimo, J.I.: Diglyceride-
visualization, writing—original draft, writing—review and editing, rich oils from glycerolysis of edible vegetable oils. Catal.
data curation [RCA]; supervision, project administration, funding Today. 302, 233–241 (2018). https​: //doi.org/10.1016/j.catto​
acquisition, writing—review and editing [Y-HJ]; writing—review and d.2017.04.008
editing [PLT-N]; writing—review and editing [AEA]; writing—review 12. Singh, D., Patidar, P., Ganesh, A., Mahajani, S.: Esterification of
and editing [SPS]; conceptualization, formal analysis, writing—review oleic acid with glycerol in the presence of supported zinc oxide
and editing, project administration and funding acquisition [AWG]. as catalyst. Ind. Eng. Chem. Res. 52, 14776–14786 (2013). https​
://doi.org/10.1021/ie401​636v
Funding This work was financially supported by the Taiwan Build- 13. Duan, Z.-Q., Du, W., Liu, D.-H.: Rational synthesis of
ing Technology Center (Grant/Project Number—108P011) from the 1,3-diolein by enzymatic esterification. J. Biotechnol. 159,
Featured Areas Research Center Program within the framework of 44–49 (2012). https​://doi.org/10.1016/j.jbiot​ec.2012.02.006
the Higher Education Sprout Project by the Ministry of Education 14. Devi, B.L.A.P., Zhang, H., Damstrup, M.L., Guo, Z., Zhang,
in Taiwan, as well as the National Taiwan University of Science and L., Lue, B.-M., Xu, X.: Enzymatic synthesis of designer lipids.
Technology for the teaching and research start-up grant (Grant/Project Oléagineux, Corps gras, Lipides. 15, 189–195 (2008). https​://
Number—1090410305) provided for 2019–2021. doi.org/10.1051/ocl.2008.0194
15. Oliveira, P.D., Rodrigues, A.M.C., Bezerra, C.V., Silva, L.H.M.:
Chemical interesterification of blends with palm stearin and
Compliance with Ethical Standards patawa oil. Food Chem. 215, 369–376 (2017). https​: //doi.
org/10.1016/j.foodc​hem.2016.07.165
Conflict of interest The authors declare that they have no known com- 16. Jiménez, M.J., Esteban, L., Robles, A., Hita, E., González, P.A.,
peting financial interests or personal relationships that could have ap- Muñío, M.M., Molina, E.: Production of triacylglycerols rich
peared to influence the work reported in this paper. in palmitic acid at sn-2 position by lipase-catalyzed acidolysis.
Biochem. Eng. J. 51, 172–179 (2010). https​://doi.org/10.1016/j.
bej.2010.06.015
17. Duan, Z.-Q., Du, W., Liu, D.-H.: Novozym 435-catalyzed
References 1,3-diacylglycerol preparation via esterification in t-butanol
system. Process Biochem. 45, 1923–1927 (2010a). https​://doi.
1. Lauridsen, J.B.: Food emulsifiers: surface activity, edibility, man- org/10.1016/j.procb​io.2010.03.007
ufacture, composition, and application. J. Am. Oil Chem. Soc. 53, 18. Duan, Z.-Q., Du, W., Liu, D.-H.: The pronounced effect of water
400–407 (1976). https​://doi.org/10.1007/BF026​05731​ activity on the positional selectivity of Novozym 435 during
2. Zhang, Y., Wang, X., Zou, S., Xie, D., Jin, Q., Wang, X.: Synthe- 1,3-diolein synthesis by esterification. Catal. Commun. 11,
sis of 2-docosahexaenoylglycerol by enzymatic ethanolysis. Biore- 356–358 (2010b). https​://doi.org/10.1016/j.catco​m.2009.10.030
sour. Technol. 251, 334–340 (2018). https​://doi.org/10.1016/j. 19. Duan, Z.-Q., Du, W., Liu, D.-H.: The solvent influence on the
biort​ech.2017.12.025 positional selectivity of Novozym 435 during 1,3-diolein syn-
3. Zheng, M.-M., Huang, Q., Huang, F., Guo, P., Xiang, X., Deng, thesis by esterification. Bioresour. Technol. 101, 2568–2571
Q., Li, W., Wan, C., Zheng, C.: Production of novel “functional (2010c). https​://doi.org/10.1016/j.biort​ech.2009.11.087
oil” rich in diglycerides and phytosterol esters with “one-pot” 20. Wang, Z., Du, W., Dai, L., Liu, D.: Study on Lipozyme TL IM-
enzymatic transesterification. J. Agric. Food Chem. 62, 5142– catalyzed esterification of oleic acid and glycerol for 1,3-diolein
5148 (2014). https​://doi.org/10.1021/jf500​744n preparation. J. Mol. Catal. B Enzym. 127, 11–17 (2016). https​
4. Khaddaj-Mallat, R., Morin, C., Rousseau, É.: Novel n-3 PUFA ://doi.org/10.1016/j.molca​tb.2016.01.010
monoacylglycerides of pharmacological and medicinal interest: 21. Duan, Z.-Q., Du, W., Liu, D.-H.: The mechanism of solvent
anti-inflammatory and anti-proliferative effects. Eur. J. Pharmacol. effect on the positional selectivity of Candida antarctica lipase
792, 70–77 (2016). https​://doi.org/10.1016/j.ejpha​r.2016.10.038 B during 1,3-diolein synthesis by esterification. Bioresour.
5. Sun, S., Hou, X., Hu, B.: Synthesis of glyceryl monocaffeate using Technol. 102, 11048–11050 (2011). https​://doi.org/10.1016/j.
ionic liquids as catalysts. J. Mol. Liq. 248, 643–650 (2017). https​ biort​ech.2011.09.003
://doi.org/10.1016/j.molli​q.2017.10.102 22. Hu, D., Chen, J., Xia, Y.: A comparative study on production
6. Sun, S., Song, F., Bi, Y., Yang, G., Liu, W.: Solvent-free enzy- of middle chain diacylglycerol through enzymatic esterification
matic transesterification of ethyl ferulate and monostearin: opti- and glycerolysis. J. Ind. Eng. Chem. 19, 1457–1463 (2013).
mized by response surface methodology. J. Biotechnol. 164, https​://doi.org/10.1016/j.jiec.2013.01.009
340–345 (2013). https​://doi.org/10.1016/j.jbiot​ec.2013.01.013 23. Ortiz, C., Ferreira, M.L., Barbosa, O., dos Santos, J.C.S., Rod-
7. Sun, S., Hu, B.: Enzymatic preparation of novel caffeoyl struc- rigues, R.C., Berenguer-Murcia, Á., Briand, L.E., Fernandez-
tured lipids using monoacylglycerols as caffeoyl acceptor and Lafuente, R.: Novozym 435: the “perfect” lipase immobilized
transesterification mechanism. Biochem. Eng. J. 124, 78–87 biocatalyst? Catal. Sci. Technol. 9, 2380–2420 (2019). https​://
(2017). https​://doi.org/10.1016/j.bej.2017.05.002 doi.org/10.1039/C9CY0​0415G​
8. Kim, J., Chung, M., Choi, H.-D., Choi, I., Kim, B.H.: Enzymatic 24. Brautbar, N., Williams, J., II.: Industrial solvents and liver toxic-
synthesis of structured monogalactosyldiacylglycerols enriched in ity: risk assessment, risk factors and mechanisms. Int. J. Hyg.
pinolenic acid. J. Agric. Food Chem. 66, 8079–8085 (2018). https​ Environ. Heal. 205, 479–491 (2002)
://doi.org/10.1021/acs.jafc.8b025​99 25. Bélafi-Bakó, K., Kovács, F., Gubicza, L., Hancsók, J.: Enzy-
9. Morin, C., Rousseau, É., Fortin, S.: Anti-proliferative effects of matic biodiesel production from sunflower oil by Candida ant-
a new docosapentaenoic acid monoacylglyceride in colorectal arctica lipase in a solvent-free system. Biocatal. Biotransforma-
carcinoma cells. Prostaglandins Leukot. Essent. Fat. Acids. 89, tion. 20, 437–439 (2002). https​://doi.org/10.1080/10242​42021​
203–213 (2013). https​://doi.org/10.1016/j.plefa​.2013.07.004 00004​0855
10. Mostafa, N.A., Maher, A., Abdelmoez, W.: Production of mono-, 26. Lu, J., Deng, L., Nie, K., Wang, F., Tan, T.: Stability of immobi-
di-, and triglycerides from waste fatty acids through esterification lized Candida sp. 99–125 lipase for biodiesel production. Chem.
with glycerol. Adv. Biosci. Biotechnol. 04, 900–907 (2013). https​ Eng. Technol. 35, 2120–2124 (2012). https​://doi.org/10.1002/
://doi.org/10.4236/abb.2013.49118​ ceat.20120​0254

13
Waste and Biomass Valorization

27. Lai, C.C., Zullaikah, S., Vali, S.R., Ju, Y.H.: Lipase-catalyzed 43. Go, A.W., Sutanto, S., Ismadji, S., Ju, Y.H.: Catalyst free pro-
production of biodiesel from rice bran oil. J. Chem. Technol. Bio- duction of partial glycerides: acetone as solvent. RSC Adv. 5,
technol. 80, 331–337 (2005). https​://doi.org/10.1002/jctb.1208 30833–30840 (2015). https​://doi.org/10.1039/c5ra0​3249k​
28. Zhao, Y., Liu, J., Deng, L., Wang, F., Tan, T.: Optimization of 44. Meng, Z., Lu, S., Geng, W., Huang, J., Wang, X., Liu, Y.: Prelimi-
Candida sp. 99–125 lipase catalyzed esterification for synthesis nary study on acyl incorporation and migration in the production
of monoglyceride and diglyceride in solvent-free system. J. Mol. of 1,3-diacylglycerol by immobilized lipozyme RM IM-catalyzed
Catal. B Enzym. 72, 157–162 (2011). https​://doi.org/10.1016/j. esterification. Food Sci. Technol. Res. 20, 175–182 (2014). https​
molca​tb.2011.05.014 ://doi.org/10.3136/fstr.20.175
29. Yesiloglu, Y., Kilic, I.: Lipase-catalyzed esterification of glycerol 45. de Gomes, S.M., Santos, M.R.D., Salviano, A.B., Mendonça,
and oleic acid. J. Am. Oil Chem. Soc. 81, 281–284 (2004). https​ F.G., Menezes, I.R.S., Jurisch, M., Rodrigues, G.D., Augusti, R.,
://doi.org/10.1007/s1174​6-004-0896-5 Martins, P.S., Lago, R.M.: Biphasic reaction of glycerol and oleic
30. Yeoh, C.M., Choong, T.S.Y., Abdullah, L.C., Yunus, R., Siew, acid: Byproducts formation and phase transfer autocatalytic effect.
W.L.: Influence of silica gel in production of diacylglycerol via Catal. Today. 344, 227–233 (2020). https:​ //doi.org/10.1016/j.catto​
enzymatic glycerolysis of palm olein. Eur. J. Lipid Sci. Technol. d.2019.02.011
111, 599–606 (2009). https​://doi.org/10.1002/ejlt.20080​0265 46. Kong, P.S., Pérès, Y., Wan Daud, W.M.A., Cognet, P., Aroua,
31. Ting, R.R., Agapay, R., Angkawijaya, A.E., Tran Nguyen, P.L., M.K.: Esterification of glycerol with oleic acid over hydrophobic
Truong, C.T., Ju, Y.: Diglyceride production via noncatalyzed zirconia-silica acid catalyst and commercial acid catalyst: opti-
esterification of glycerol and oleic acid. Asia-Pacific J. Chem. mization and influence of catalyst acidity. Front. Chem. 7, 1–11
Eng. 14, 1–11 (2019). https​://doi.org/10.1002/apj.2383 (2019). https​://doi.org/10.3389/fchem​.2019.00205​
32. Castillo, E., Dossat, V., Marty, A., Stéphane Condoret, J., Combes, 47. Andrade-Tacca, C.A., Chang, C.C., Chen, Y.H., Ji, D.R., Wang,
D.: The role of silica gel in lipase-catalyzed esterification reac- Y.Y., Yen, Y.Q., Chang, C.Y.: Reduction of FFA in jatropha cur-
tions of high-polar substrates. JAOCS J. Am. Oil Chem. Soc. 74, cas oil via sequential direct-ultrasonic irradiation and dosage of
77–85 (1997). https​://doi.org/10.1007/s1174​6-997-0148-3 methanol/sulfuric acid catalyst mixture on esterification process.
33. Üstün, G.: Separation of fatty acid methyl esters from tall oil by Energy Convers. Manag. 88, 1078–1085 (2014). https​://doi.
selective adsorption. JAOCS J. Am. Oil Chem. Soc. 73, 203–210 org/10.1016/j.encon​man.2014.09.020
(1996). https​://doi.org/10.1007/BF025​23896​ 48. Huang, Z., Cao, Z., Guo, Z., Chen, L., Wang, Z., Sui, X., Jiang, L.:
34. Chandrasekar, G., Vinu, A., Murugesan, V., Hartmann, M.: Lipase catalysis of α-linolenic acid-rich medium- and long-chain
Adsorption of vitamin E on mesoporous silica molecular sieves. triacylglycerols from perilla oil and medium-chain triacylglycer-
In: Raj, S. (ed.) Materials research bulletin, pp. 1169–1176. Else- ols with reduced by-products. J. Sci. Food Agric. 100, 4565–4574
vier, Amsterdam (2005) (2020). https​://doi.org/10.1002/jsfa.10515​
35. Rosu, R., Yasui, M., Iwasaki, Y., Yamane, T.: Enzymatic synthe- 49. Abd Razak, N.N., Abd Razak, N.N., Pérès, Y., Gew, L.T., Cog-
sis of symmetrical 1,3-diacylglycerols by direct esterification of net, P., Aroua, M.K., Aroua, M.K.: Effect of reaction medium
glycerol in solvent-free system. JAOCS J. Am. Oil Chem. Soc. 76, mixture on the lipase catalyzed synthesis of diacylglycerol. Ind.
839–843 (1999). https​://doi.org/10.1007/s1174​6-999-0074-7 Eng. Chem. Res. 59, 9869–9881 (2020). https​://doi.org/10.1021/
36. Chongkhong, S., Tongurai, C., Chetpattananondh, P., Bunya- acs.iecr.0c002​98
kan, C.: Biodiesel production by esterification of palm fatty acid 50. Wang, L., Du, W., Liu, D., Li, L., Dai, N.: Lipase-catalyzed bio-
distillate. Biomass Bioenergy 31, 563–568 (2007). https​://doi. diesel production from soybean oil deodorizer distillate with
org/10.1016/j.biomb​ioe.2007.03.001 absorbent present in tert-butanol system. J. Mol. Catal. B Enzym.
37. Chongkhong, S., Tongurai, C., Chetpattananondh, P.: Continuous 43, 29–32 (2006). https​://doi.org/10.1016/j.molca​tb.2006.03.005
esterification for biodiesel production from palm fatty acid distil- 51. Gangopadhyay, S., Nandi, S., Ghosh, S.: Biooxidation of fatty acid
late using economical process. Renew. Energy 34, 1059–1063 distillates to dibasic acids by a mutant of Candida tropicalis. J.
(2009). https​://doi.org/10.1016/j.renen​e.2008.07.008 Oleo Sci. 56, 13–17 (2007). https​://doi.org/10.5650/jos.56.13
38. D’Amato Villardi, H.G., Leal, M.F., De Azevedo Andrade, P.H., 52. Tangkam, K., Weber, N., Wiege, B.: Solvent-free lipase-cata-
Pessoa, F.L.P., Salgado, A.M., De Oliveira, A.R.G.: Catalytic and lyzed preparation of diglycerides from co-products of vegeta-
non-catalytic esterification of soybean oil deodorizer distillate by ble oil refining. Grasas Aceites 59, 245–253 (2008). https​://doi.
ethanol: kinetic modelling. Chem. Eng. Trans. 57, 1999–2004 org/10.3989/gya.2008.v59.i3.515
(2017). https​://doi.org/10.3303/CET17​57334​ 53. Zhao, J.F., Tao-Wang, Lin, J.P., Yang, L.R., Wu, M.B.: Preparation
39. Shimada, Y., Nakai, S., Suenaga, M., Sugihara, A., Kitano, M., of high-purity 1,3-diacylglycerol using performance-enhanced
Tominaga, Y.: Facile purification of tocopherols from soybean oil lipase immobilized on nanosized magnetite particles. Biotech-
deodorizer distillate in high yield using lipase. JAOCS J. Am. Oil nol. Bioprocess Eng. 24, 326–336 (2019). https:​ //doi.org/10.1007/
Chem. Soc. 77, 1009–1013 (2000). https​://doi.org/10.1007/s1174​ s1225​7-018-0458-3
6-000-0160-z 54. Encinar, J.M., González, J.F., Sánchez, N., Nogales-Delgado, S.:
40. Kasim, N.S., Gunawan, S., Ju, Y.H.: Isolation and identification Sunflower oil transesterification with methanol using immobilized
of steroidal hydrocarbons in soybean oil deodorizer distillate. lipase enzymes. Bioprocess Biosyst. Eng. 42, 157–166 (2019).
Food Chem. 117, 15–19 (2009). https​://doi.org/10.1016/j.foodc​ https​://doi.org/10.1007/s0044​9-018-2023-z
hem.2009.03.066
41. Gunawan, S., Kasim, N.S., Ju, Y.H.: Separation and purification Publisher’s Note Springer Nature remains neutral with regard to
of squalene from soybean oil deodorizer distillate. Sep. Purif. jurisdictional claims in published maps and institutional affiliations.
Technol. 60, 128–135 (2008). https​://doi.org/10.1016/j.seppu​
r.2007.08.001
42. Lo, S.K., Baharin, B.S., Tan, C.P., Lai, O.M.: Lipase-catalysed
production and chemical composition of diacylglycerols from
soybean oil deodoriser distillate. Eur. J. Lipid Sci. Technol. 106,
218–224 (2004). https​://doi.org/10.1002/ejlt.20030​0888

13