Lipase-catalyzed two-step esterification for solvent-free production of mixed lauric acid esters with antibacterial and antioxidative activities

Article abstract of DOI:10.1016/j.foodchem.2021.130650

Mixed lauric acid esters (MLE) with antibacterial and antioxidative activities were produced through lipase-catalyzed two-step esterification in solvent-free system without purification. In the first reaction, erythorbyl laurate was synthesized for 72 h. Successive reaction for 6 h at molar ratio of 1.0 (lauric acid to glycerol) produced MLE containing erythorbyl laurate and glyceryl laurate with small amounts of residual substrates, by converting 99.52% of lauric acid. MLE addition (0.5–2.0%, w/w) to Tween 20-stabilized emulsions decreased droplet size, polydispersity index, and zeta-potential, possibly enhancing the emulsion stability. In the emulsions, MLE at 0.5 and 2.0% (w/w) caused 4.4–4.6 and 5.9–6.1 log reductions of Gram-positive (Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), respectively, within 12 h. Lipid hydroperoxide concentrations decreased to 50.8–98.3% in the presence of 0.5–2.0% (w/w) MLE. These findings support a novel approach without needing purification to produce multi-functional food additives for emulsion foods.

Full text of DOI:10.1016/j.foodchem.2021.130650

Food Chemistry 366 (2022) 130650
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Lipase-catalyzed two-step esterification for solvent-free production of
mixed lauric acid esters with antibacterial and antioxidative activities
a,
b,
1
c,
1
a,b,c,d,*
Hyunjong Yu
, Yerim Byun , Pahn-Shick Chang
a
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea
Center for Agricultural Microorganism and Enzyme, Seoul National University, Seoul 08826, Republic of Korea
Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Mixed lauric acid esters (MLE) with antibacterial and antioxidative activities were produced through lipase-
catalyzed two-step esterification in solvent-free system without purification. In the first reaction, erythorbyl
laurate was synthesized for 72 h. Successive reaction for 6 h at molar ratio of 1.0 (lauric acid to glycerol)
produced MLE containing erythorbyl laurate and glyceryl laurate with small amounts of residual substrates, by
converting 99.52% of lauric acid. MLE addition (0.5–2.0%, w/w) to Tween 20-stabilized emulsions decreased
droplet size, polydispersity index, and zeta-potential, possibly enhancing the emulsion stability. In the emulsions,
MLE at 0.5 and 2.0% (w/w) caused 4.4–4.6 and 5.9–6.1 log reductions of Gram-positive (Staphylococcus aureus,
Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), respectively,
within 12 h. Lipid hydroperoxide concentrations decreased to 50.8–98.3% in the presence of 0.5–2.0% (w/w)
MLE. These findings support a novel approach without needing purification to produce multi-functional food
additives for emulsion foods.
Lipase-catalyzed esterification
Erythorbyl laurate
Glyceryl laurate
Oil-in-water emulsion
Antibacterial activity
Antioxidative activity
1
. Introduction
Emulsion foods, such as mayonnaise, yogurt, cream, and dressing,
erythorbyl laurate, with its antibacterial and antioxidative activities in a
single molecule, could be used as a multi-functional food additive to
control the microbial contamination and lipid oxidation in emulsion
foods.
have been extensively produced in food industry. In the emulsion foods,
lipid oxidation and microbial contamination are considered as major
hazards in terms of food quality and safety (Yu, Park, & Chang, 2020).
Lipid oxidation leads to formation of off-flavors and potentially toxic
compounds, and microbial contamination by foodborne pathogens re-
sults in spoilage and food poisoning (Luther et al., 2007; Prachaiyo and
McLandsborough, 2003).
The production of erythorbyl laurate in an organic solvent reaction
system has limitations, such as safety concerns and the low production
yield caused by the low solubility of the substrates in the organic solvent
(Park, Lee, Sung, Lee, & Chang, 2011). However, a solvent-free reaction
system, which is an environmentally friendly system, has several ad-
vantages derived from the absence of organic solvents, such as greater
safety and higher volumetric productivities (Freitas, Perez, Santos, &
Castro, 2007). Nevertheless, it is demanding to produce erythorbyl
laurate in a solvent-free reaction system because erythorbic acid is not
soluble in lauric acid (i.e., the reaction medium) and existed as a solid
phase at the reaction temperature, causing mass transfer limitations
(Erbeldinger, Ni, & Halling, 1998). Therefore, a solvent-free gas-solid-
liquid multiphase system (GSL-MPS), which enhances dispersion of the
solid phase by incorporation of the gaseous phase, was established in a
previous study (Yu, Lee, Shin, Park, & Chang, 2019). Although the
Erythorbyl laurate is an amphiphilic compound produced by lipase-
catalyzed esterification of erythorbic acid with lauric acid. As the
amphiphilic characteristic gives it surface-active properties and high
foaming stability, erythorbyl laurate can be used for stabilization of oil-
in-water (O/W) emulsions (Park, Lee, Jo, Choi, Lee, & Chang, 2017).
Moreover, erythorbyl laurate can act as an antioxidant to retard lipid
oxidation in O/W emulsions. It also exhibits bacteriostatic and bacteri-
cidal effects on Gram-positive foodborne pathogens through changes in
the cell membrane permeability (Park et al., 2018). Therefore,
*
Corresponding author at: Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea.
E-mail address: pschang@snu.ac.kr (P.-S. Chang).
1
These authors contributed equally to this work as first authors.
https://doi.org/10.1016/j.foodchem.2021.130650
Received 18 March 2021; Received in revised form 14 June 2021; Accepted 19 July 2021
Available online 22 July 2021
0
308-8146/© 2021 Elsevier Ltd. All rights reserved.

H. Yu et al.
Food Chemistry 366 (2022) 130650
production of erythorbyl laurate in the GSL-MPS had numerous ad-
vantages, including higher volumetric productivities in a batch reactor,
the conversion yield was still low because of the presence of the solid
phase. Accordingly, large amounts of residual substrates could not be
avoided in the synthesis of erythorbyl laurate in the GSL-MPS.
2.2. Lipase-catalyzed esterification in a gas-solid-liquid multiphase system
All lipase-catalyzed esterification was carried out in solvent-free
GSL-MPS with slight modification (Yu et al., 2019). During the reac-
tion, nitrogen gas (N
20LN200; BHL Co., Chuncheon, Republic of Korea), was distributed
through the glass filter (thickness of 0.5 cm, pore size of 27.5 m) at a
2
2
≥ 99.9%), produced by N gas generator (GN-
The purification process for the removal or recovery of the residual
substrates after synthesis is generally accomplished by solvent extrac-
tion, recrystallization, silica-gel chromatography, molecular distillation,
or a combination of these methods (Enayati, Gong, Goddard, & Abbas-
pourrad, 2018; Zhang, Wang, Xie, Zou, Jin, & Wang, 2018). However,
solvent extraction, recrystallization, and silica-gel chromatography,
which are cumbersome processes, require large amounts of organic
solvents. Molecular distillation consumes abundant energy because of its
high operating temperature. Hence, the production of a single com-
pound with a purification process generally has safety impediments and
environmental concerns with high production costs. Purification of
erythorbyl laurate, for example, has been carried out by solvent
extraction based on the differences in solubility between erythorbic
acid, lauric acid, and erythorbyl laurate, which takes a long time and
requires a complicated process to remove the solvent (Park et al., 2011).
Those intricate purification process has become an obstacle
obstructing application of enzymatically produced compounds in the
food industry. The addition of another substrate for conversion of the
residual lauric acid into new products can be considered as an alterna-
tive to the conventional purification process. Through successive two-
step esterification with the first reaction for synthesis of erythorbyl
laurate, various reaction mixture with different physicochemical prop-
erties can be produced. The reaction mixture could have better emul-
sifying properties than pure erythorbyl laurate, which enhances the
applicability of erythorbyl laurate on emulsion foods. Moreover, in
emulsion system, antibacterial and antioxidative activities of erythorbyl
laurate in the mixture formulation could be enhanced by changing
emulsion properties such as droplet size (Erdmann, Zeeb, Salminen,
Gibis, Lautenschlaeger, & Weiss, 2015; Gaysinsky, Taylor, Davidson,
Bruce, & Weiss, 2007).
μ
flow rate of 2.0 L/min. Reaction vessels were preheated, and the reac-
◦
tion temperature was kept constant at 60 C by a water circulator. Lauric
acid were added to the reaction vessel and melted for 20 min, followed
by the addition of an acyl acceptor, including erythorbic acid for the first
reaction and the second acyl acceptors (PEG 600, propylene glycol,
glycerol, and lactic acid). The reaction was initiated by addition of
Novozym® 435. After the reaction was finished, the nitrogen gas
generator was replaced with a vacuum pump, and the reaction mixture
was filtrated through the glass filter for separating the immobilized
lipase (Fig. S1). The conversion of lauric acid was calculated according
to the following equation:
C
i
ꢀ C
t
Conversion of lauric acid (%) =
× 100
C
i
where C is the initial lauric acid concentration and C is the residual
i
t
lauric acid concentration at the reaction time. The initial conversion rate
of lauric acid was obtained from the slope of time course at linear
portion. The conversion of erythorbic acid was calculated with the same
manner.
For the successive two-step esterification, erythorbyl laurate was
produced during the first reaction. The esterification was performed for
7
2 h with 24,000 PLU of Novozym® 435 at the substrate molar ratio
(
lauric acid to erythorbic acid) of 2.0, which were the previously
determined optimum reaction conditions (Yu et al., 2019). The second
reaction was performed by adding glycerol at the defined molar ratio to
residual lauric acid immediately after the first reaction. The composition
of glyceryl laurate was expressed as mole percentage of each compound
with respect to the amount of total glyceryl laurate. The pure erythorbyl
laurate (≥99.0%), used to compare with the antibacterial and anti-
oxidative properties of MLE, was produced by the same procedure as the
first reaction, followed by purification process based on solvent
extraction using n-hexane and water, as previously reported (Park et al.,
In the present study, glycerol, which had the highest reaction effi-
ciency for the conversion of lauric acid among the candidates, was
selected as a second acyl acceptor. In the solvent-free reaction system,
erythorbyl laurate was produced in the first reaction. Subsequently, in
the second reaction, the residual lauric acid was converted to glyceryl
laurate. The successive two-step esterification without purification
produced mixed lauric acid esters (MLE), mainly composed of eryth-
orbyl laurate and glyceryl laurate with small amounts of residual sub-
strates (erythorbic acid, lauric acid, and glycerol). The effects of MLE
addition on O/W emulsion properties were evaluated. Moreover, anti-
bacterial and antioxidative properties of MLE were investigated in the
O/W emulsions to assess its applicability on emulsion foods.
2
021).
2
.3. Quantitative analyses using HPLC
The esterification reaction was monitored using a high-performance
liquid chromatography (HPLC) system (LC-2002, Jasco, Tokyo, Japan)
equipped with an ultraviolet detector (UV-2075, Jasco) and a refractive
index detector (RI-2031, Jasco). Separation of the compounds was car-
ried out on a silica-based column (5.0 µm, 4.6 × 150 mm: Luna C18
,
2
. Materials and methods
Phenomenex, Torrance, CA, USA), and the column temperature was held
◦
constant at 30 C. The mobile phase for analyses of the reactants of the
2
.1. Materials
esterification reaction of lauric acid with PEG 600, propylene glycol, and
lactic acid was methanol/water/acetic acid (90:5:5, v/v/v). For analyses
of the reactants of the esterification of lauric acid with glycerol, the
mobile phase was acetonitrile/acetone (50:50, v/v), whereas the re-
actants of the esterification of lauric acid with erythorbic acid were
analyzed using a mobile phase composed of acetonitrile/water/acetic
acid (90:5:5, v/v/v). All mobile phases passed through the column with
a flow rate of 1.0 mL/min. Erythorbic acid and erythorbyl laurate were
detected by the UV detector at a wavelength of 265 nm. Other com-
pounds were detected on the RI detector. All compounds were identified
by their retention time, and the concentrations were calculated from the
Novozym® 435, immobilized lipase B from Candida antarctica, with
a catalytic activity of 10,000 PLU/g (propyl laurate unit; 1 PLU is the
amount of enzyme activity that generates 1 mol of propyl laurate per
μ
min) was purchased from Novozymes (Bagsvaerd, Denmark). Lauric
acid (≥99.0%) and soybean oil were purchased from Daejung Chemicals
&
Metals Co., Ltd. (Siheung, Republic of Korea), and Tween 20 was
purchased from Ilshinwells (Cheongju, Republic of Korea). Crystalline
erythorbic acid (≥98.0%), glycerol (≥99.5%), polyethylene glycol 600
(
PEG 600), propylene glycol, and lactic acid were purchased from Acros
Organics (Geel, Belgium), Fisher Chemical (Loughborough, Leicester-
shire, UK), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Junsei
Chemical Co., Ltd. (Tokyo, Japan), and Showa Chemical (Tokyo, Japan),
respectively. All other reagents and solvents were analytical or HPLC
grade.
2
standard curve of each compound (R > 0.994).
2

H. Yu et al.
Food Chemistry 366 (2022) 130650
2
.4. Emulsion preparation
Scientific, Inc., Gimpo, Republic of Korea). The upper organic layer
(
200 µL) was mixed with 2,800 µL methanol/butanol solution (2:1, v/v),
O/W emulsion stabilized with Tween 20 was prepared according to
followed by addition of 15 µL 3.94 M ammonium thiocyanate and 15 µL
Fe solution. The Fe solution was prepared freshly from the super-
natant of 0.132 M BaCl in 0.4 M HCl and 0.144 M FeSO . The solution
2
+
2+
the procedure previously described (Abcha et al., 2019). The water
phase consisted of Tween 20 (1.0%, w/w) and distilled water (up to
2
4
1
00.0%, w/w), whereas the oil phase consisted of the soybean oil (5.0%,
was vortexed, held for 20 min at room temperature, and the absorbance
was measured at 510 nm in a UV–vis spectrophotometer (Optizen POP-
BIO, Mecasys Co., Ltd., Daejeon, Korea). Lipid hydroperoxide concen-
trations were determined using a standard curve prepared from
w/w) and various concentrations of MLE. The concentrations of the
soybean oil and Tween 20 were designed to mimic the emulsion of a
beverage (Perugini, Cinelli, Cofelice, Ceglie, Lopez, & Cuomo, 2018).
◦
2
Both the water phase and the oil phase were heated to 70 C, to melt the
hydrogen peroxide (R = 0.9978).
MLE, and then the water phase was transferred to the oil phase. The
water and oil phases were mixed using a high-speed blender (Ultra-
Turrax IKA T18 Basic, IKA Works, Inc., Wilmington, NC, USA) at 12,000
rpm for 3 min to form a coarse emulsion. The coarse emulsion was ho-
mogenized further for 10 min (1 s on, 4 s off) using an ultrasonicator
2
.8. Statistical analyses
All data were analyzed by analysis of variance (ANOVA) using SPSS
software v. 25 (SPSS, Inc., Chicago, IL, USA) and presented as means
with standard deviations of triplicate experiments. The differences be-
tween mean values were compared using Duncan’s multiple range test
(
Sonomasher, S&T Science, Seoul, Republic of Korea) to produce a fine
emulsion. The emulsion was immediately cooled to room temperature
◦
(
25 C) with tap water. The emulsions used for the time-kill assay were
(
p < 0.05).
prepared with 20 mM phosphate buffer (pH 7.4) instead of distilled
water. Each emulsion was prepared in triplicate. An emulsion prepared
without MLE was used as the control.
3
. Results and discussion
3
.1. Selection of second acyl acceptor
2
.5. Measurements of droplet size and zeta potential
To select candidates for a second acyl acceptor for solvent-free
The droplet size and zeta potential of the emulsions were analyzed at
esterification with lauric acid, the melting point of the organic com-
pounds with a hydroxyl group was considered because low conversion
can occur because of the mass transfer limitation when the acyl acceptor
exists as a solid phase in the reaction system (Erbeldinger et al., 1998).
Based on this circumstance, PEG 600, propylene glycol, glycerol, and
lactic acid were selected as candidates for the second acyl acceptor
◦
2
5 C using a Zetasizer Nano ZS90 analyzer (Malvern Instruments, Ltd.,
Worcestershire, UK), according to the method described previously
Mauriello, Ferrari, & Donsì, 2021). Each measurement was performed
in triplicate. To measure droplet size, the emulsions were diluted at
:1,000 (v/v) with distilled water to prevent multiple scattering. The
(
1
average droplet size was expressed as the intensity-weighted mean
droplet diameter (Z-average size), and the width parameter was pre-
sented as the polydispersity index. To measure zeta potential, the
emulsions were diluted at 1:100 (v/v) with distilled water.
(
Table S1). The lipase-catalyzed esterification of lauric acid with the
second acyl acceptor candidates reached equilibrium within 12 h, except
for the case of lactic acid (Fig. S2). Esterification with lactic acid did not
occur. The results could be attributed to either poor reactivity of lactic
acid in the esterification or deactivation of the immobilized lipase by
lactic acid during the reaction. It was discussed that the direct esterifi-
cation of lactic acid with fatty acid was difficult due to low nucleophi-
licity of hydroxyl group and steric hindrance by the adjacent carboxyl
group in lactic acid (Torres and Otero, 2001). Meanwhile, lactic acid
could lead to severe deactivation of the immobilized lipase during the
reaction, possible due to low pH (Pirozzi and Greco, 2004).
2
.6. Time-kill assay in oil-in-water emulsion
To evaluate the antibacterial activity of MLE in emulsion system,
time-kill plots of the Tween 20-stabilized O/W emulsions with different
concentrations of MLE were investigated (Moghimi, Aliahmadi, &
Rafati, 2017). Two Gram-positive and two Gram-negative bacteria were
used in this study: Staphylococcus aureus ATCC 12692, Listeria mono-
cytogenes ATCC 19115, Escherichia coli ATCC 35150, and Pseudomonas
aeruginosa ATCC 15692. All bacteria were cultured in Mueller–Hinton
The esterification reaction efficiency of the second acyl acceptor
candidates was determined by the initial conversion rate of lauric acid
and the conversion of lauric acid at the reaction time of 12 h (Table 1).
The initial conversion rate of lauric acid was highest in the reaction with
glycerol, which was 1.38 and 2.02-fold higher than those from the re-
action with PEG 600 and propylene glycol, respectively. In addition,
conversion of lauric acid in the esterification with glycerol was signifi-
cantly higher (p < 0.05) than those in the esterification with PEG 600
and propylene glycol.
◦
broth (MHB) at 37 C, 220 rpm, and for 12–16 h. Cultures were diluted
in MHB until the turbidity reached the 0.5 McFarland standard (1.5 ×
8
1
0 CFU/mL). The McFarland standard suspensions were diluted further
6
to a cell density of 5.0 × 10 CFU/mL. Emulsions (3.6 mL) containing
different concentrations of MLE (0–2.0%, w/w), prepared by the pro-
cedure described in Section 2.4, were mixed with bacterial suspensions
◦
(
(
0.4 mL). The mixture was incubated at 37 C and 220 rpm. Aliquots
In lipase-catalyzed esterification, the number of hydroxyl groups and
200
μ
L) were extracted at 0, 0.5, 1, 2, 4, 6, 8, 10, and 12 h and serially
L)
diluted in 20 mM phosphate buffer (pH 7.4). Diluted samples (50
μ
Table 1
were spread on tryptic soy agar (TSA), and viable cells were counted
Initial conversion rate of lauric acid and conversion of lauric acid obtained from
the esterification with the second acyl acceptor candidates.
◦
after incubation at 37 C for 24 h. All experiments were performed in
triplicate.
Second acyl
acceptor
Initial conversion rate of lauric
acid (mmol/h)
Conversion of lauric
acid (%)
2
.7. Measurement of lipid oxidation in oil-in-water emulsion
b
b
PEG 600
29.23 ± 1.50
93.59 ± 0.20
Propylene glycol
Glycerol
19.97 ± 1.19c
77.40 ± 2.16c
Emulsions (6 mL) were placed in 10 mL screw-cap glass vials and
a
a
40.28 ± 2.07
99.38 ± 0.05
◦
allowed to be oxidized with thermal acceleration at 37 C. Lipid hy-
droperoxides, as the primary lipid oxidation products, were measured
according to the method (Shantha and Decker, 1994). Every 24 h,
emulsion (300 µL) was taken out, mixed with 1,500 µL isooctane/2-
propanol solution (3:1, v/v), and vortexed (10 s, 3 times). The mixed
solution was centrifuged at 2,000 × g for 2 min (Micro-12, Hanil
Lactic acid
N.D.
N.D.
The values with different superscripts in each column are significantly different
(
p < 0.05) by Duncan’s multiple range test. N.D.: Not detected. Reaction con-
ditions: lauric acid, 60.66 mmol; second acyl acceptor candidate, 30.33 mmol;
enzyme, 3,000 PLU Novozym® 435; reaction temperature, 60 ℃; reaction time,
2
12 h; N gas flow, 2.0 L/min.
3

H. Yu et al.
Food Chemistry 366 (2022) 130650
the types of alcohols affect the reaction efficiency; the primary alcohol is
more reactive than the secondary alcohol (Gustini, Noordover, Gehrels,
Dietz, & Koning, 2015). The higher reaction efficiency of PEG 600
compared to propylene glycol could be attributed to the difference in the
number of primary alcohols. PEG 600 has two primary alcohols,
whereas propylene glycol has one primary alcohol and one secondary
alcohol. The highest reaction efficiency shown by glycerol can be
explained by the fact that glycerol has two primary alcohols and one
secondary alcohol, which provides glycerol with more opportunities for
the formation of ester bonds. Thus, among the four candidates, glycerol
with its highest reaction efficiency was selected as the acyl acceptor for
the second reaction of the two-step esterification.
content (59.73%) at the molar ratio of 1.0. Meanwhile, at the molar ratio
of 1.5, 2.0, and 3.0, monolaurin content was 46.78, 25.03, and 0.46%,
respectively, indicating that the increase in the molar ratio (i.e., decrease
in glycerol) reduced the production of monolaurin. The lowest trilaurin
content (5.32%) was obtained at the molar ratio of 0.5, which was
similar to the content (5.68%) at the molar ratio of 1.0. As the molar
ratio increased from 1.0 to 3.0, significant increases (p < 0.05) in tri-
laurin content were found, and the content (72.89%) was the highest at
the molar ratio of 3.0. Increases in the molar ratio from 0.5 to 2.0
resulted in the significant increases (p < 0.05) in dilaurin content (from
34.13% to 49.62%), but the content was lowest (26.65%) at the molar
ratio of 3.0.
These results were in accordance with the results of previous studies
in which a high substrate molar ratio of fatty acid to glycerol suppressed
both monoacylglycerol production and fatty acid conversion (Freitas,
Perez, Santos, & Castro, 2007). On the other hand, no significant dif-
ference (p > 0.05) in glyceryl laurate composition was observed at
substrate molar ratio between 0.5 and 1.0. However, a higher amount of
glycerol not reactive in the esterification remained at the molar ratio of
3
.2. Effect of substrate molar ratio on esterification of lauric acid with
glycerol
Monoacylglycerols have been used widely for stabilization of O/W
emulsions with small-molecule co-emulsifiers due to their amphiphilic
characteristics (Chen et al., 2019). On the other hand, diacylglycerols
and triacylglycerols generally decrease emulsion stability because they
are highly oil soluble. During the esterification of lauric acid with
glycerol, three types of glyceryl laurates (monolaurin, dilaurin, and
trilaurin) can be produced. Among these, the content of monolaurin in
MLE was considered as the critical determinant for the emulsion prop-
erties of MLE. In lipase-catalyzed esterification, the conversion of fatty
acid and the composition are determined by the substrate molar ratio,
the specificity of the lipase, and the water activity (Chand, Adlercreutz,
0
.5, causing decreases in the concentrations of erythorbyl laurate and
glyceryl laurate in the MLE. Therefore, the optimum substrate molar
ratio (lauric acid to glycerol) for the second reaction in the two-step
esterification was determined to be 1.0.
3
.3. Two-step esterification of lauric acid
In the first reaction, erythorbyl laurate was synthesized via esterifi-
cation between erythorbic acid and lauric acid (Fig. 2A). During the
reaction for 72 h, the amount of erythorbyl laurate gradually increased
as the reaction progressed. Thereafter, additional erythorbyl laurate was
not synthesized, indicating that the reaction attained equilibrium (data
not shown). After the reaction for 72 h, 0.90 mmol of erythorbyl laurate
was produced, but low conversion of erythorbic acid (2.08%) and lauric
acid (1.17%) was obtained, in accordance with a previous study (Yu
et al., 2019). The low conversion is caused by mass transfer limitation,
which is attributed to that erythorbic acid exists as a solid phase in the
reaction system (Yadav and Devi, 2004). Accordingly, the large amounts
of substrates, 83.53 mmol of lauric acid and 42.43 mmol of erythorbic
acid, remained after the first reaction.
&
Mattiasson, 1997).
The lipase-catalyzed esterification of lauric acid with glycerol was
performed at various molar ratio of lauric acid to glycerol to investigate
the effects of the substrate molar ratio on conversion of lauric acid and
the glyceryl laurate composition (Fig. 1). As expected, the time course
showed that the molar ratio significantly affected the conversion of
lauric acid and the glyceryl laurate composition (Fig. S3). The conver-
sion of lauric acid was higher than 99.6% at substrate molar ratio be-
tween 0.5 and 2.0. However, the significantly low (p < 0.05) conversion
(
91.94%) was obtained at the molar ratio of 3.0, suggesting that the
relative decrease in glycerol led to lower conversion of lauric acid. The
results are attributed to that excessive amount of glycerol than the
stoichiometric ratio is required to shift the reaction equilibrium toward
esterification since the lipase-catalyzed reactions are reversible (Mohod
and Gogate, 2017).
In the second reaction, monolaurin, dilaurin, and trilaurin were
synthesized for 6 h after adding glycerol at a molar ratio of 1.0 with the
residual amount of lauric acid (Fig. 2B). The conversion of lauric acid in
this reaction was 99.52%, which was similar to the conversion in the
esterification between glycerol and lauric acid in the absence of eryth-
orbic acid and erythorbyl laurate (i.e., without the first reaction).
However, the first reaction for synthesis of erythorbyl laurate
affected the composition of the glyceryl laurate in the second reaction.
The composition of glyceryl laurate in the second reaction was as fol-
lows: monolaurin (60.84 ± 0.41%), dilaurin (37.59 ± 0.36%), trilaurin
(1.57 ± 0.22%). Compared to the composition in the esterification be-
tween glycerol and lauric acid without the first reaction (monolaurin,
59.73 ± 0.19%; dilaurin, 34.60 ± 0.16%; trilaurin, 5.68 ± 0.05%), the
production of monolaurin and dilaurin was higher, but the production of
trilaurin was lower. These results indicate that the esterification of
monolaurin and dilaurin with lauric acid to produce trilaurin was hin-
dered by the first reaction, possibly due to the higher viscosity of the
reaction mixture caused by insoluble (solid phase) erythorbic acid
In terms of glyceryl laurate composition, monolaurin content
(
60.56%) at the substrate molar ratio of 0.5 was similar to (p > 0.05) the
100
140
Monolaurin
Dilaurin
1
1
8
6
4
2
0
20
00
0
Trilaurin
Conversion of lauric acid
80
60
40
20
0
0
0
0
(
Hilterhaus, Thum, Liese, & Development, 2008).
The specific composition of MLE produced through the two-step
0.5
1.0
1.5
2.0
3.0
esterification is presented in Table S2. Compared to the amount of
erythorbic acid remained in the reaction mixture after the two-step
esterification, its content in MLE (2.84%, w/w) was substantially low-
ered. This is attributed to that most of erythorbic acid existed as solid
phase in the reaction mixture is eliminated during the filtration for
separating the immobilized lipase from the reaction mixture.
Substrate molar ratio (lauric acid/glycerol)
Fig. 1. Effect of the substrate molar ratio (lauric acid to glycerol) on the con-
version of lauric acid and glyceryl laurate composition. Reaction conditions:
lauric acid, 43.33 mmol; glycerol, 14.44–86.66 mmol; enzyme, 12,000 PLU
◦
Novozym® 435; reaction temperature, 60 C; reaction time, 6 h; N
2
gas flow,
2
.0 L/min.
In lipase-catalyzed reaction, the alkoxy group of an ester compound
4

H. Yu et al.
Food Chemistry 366 (2022) 130650
Fig. 2. Time courses of two-step esterification of lauric acid (A) with erythorbic acid at the first reaction and (B) with glycerol at the second reaction. Reaction
◦
conditions of the first reaction: erythorbic acid, 43.33 mmol; lauric acid, 86.66 mmol; enzyme, 24,000 PLU Novozym® 435; reaction temperature, 60 C; reaction
2
time, 72 h; N gas flow, 2.0 L/min. After the first reaction, the second reaction was performed for 6 h with addition of 84.70 mmol glycerol. Other reaction conditions
of the second reaction were the same as the conditions of the first reaction.
can be exchanged to another alcohol by transesterification (Demirbas,
In the two-step MLE synthesis, transesterification can be occurred be-
tween erythorbyl laurate and glycerol, which subsequently resulted in a
decrease in the amount of erythorbyl laurate during the second reaction.
2
008). Hydrolysis of the products and formation of new products may
occur depending on the reaction equilibrium in the transesterification.
Fig. 3. Effects of MLE at concentrations ranging from 0.0 to 2.0% (w/w) on (A) droplet size, (B) polydispersity index, (C) zeta potential, and (D) storage stability of
Tween 20-stabilized O/W emulsion. Different letters indicate significant differences (p < 0.05).
5

H. Yu et al.
Food Chemistry 366 (2022) 130650
However, the amount of erythorbyl laurate synthesized in the first re-
action did not change during the second reaction, indicating that
transesterification did not occur after adding glycerol as the second acyl
acceptor.
polydispersity index, which decreased gradually as the MLE concen-
tration increased from 0.0% to 1.5% (w/w); however, adding >1.5%
(w/w) MLE caused an increase in the polydispersity index. The most
monodisperse droplets formed in the emulsion containing 1.5% MLE,
which suggested that adding MLE resulted in a narrow size distribution
of Tween 20-stabilized emulsions.
3
.4. Effects of MLE on emulsion properties
The droplet size of an emulsion generally decreases as the surfactant-
to-oil ratio increases, which causes the interfacial area between the
dispersed phase (oil) and continuous phase (water) to increase (Chang
and McClements, 2014). The reduction in droplet size with increase in
MLE concentration from 0.0 to 1.0% (w/w) might be due to the
increasing concentrations of erythorbyl laurate and monolaurin, which
have amphiphilic properties. These compounds locate at the oil–water
interface, which is attributed to the splitting of oil droplets into small
pieces to minimize surface tension and maximize interfacial area
The effects of MLE on O/W emulsion stability were evaluated by
visual observation of Tween 20-stabilized emulsions with different
concentrations of MLE (Fig. S4). Emulsions with MLE at the concen-
trations higher than 2.0% (w/w) were destabilized within the first 24 h.
The separation of a lipid layer at the top and an aqueous layer at the
bottom was observed more clearly as the MLE concentration increased
from 3.0% to 5.0% (w/w). However, the emulsions containing 1.0% and
2
.0% (w/w) of MLE were stable over 24 h.
(
Saberi, Fang, & McClements, 2013). On the other hand, the increase in
The droplet size and distribution of the Tween 20-stabilized emul-
droplet size with increase in MLE concentration from 1.0% to 2.0% (w/
w) might be attributed to increasing contents of oil-soluble dilaurin and
trilaurin rather than the effects of erythorbyl laurate and monolaurin
sions with MLE at the concentrations lower than 2.0% (w/w) were
measured to evaluate the effects of MLE on the emulsion properties
(
Fig. 3A, 3B). The droplet size of the emulsions decreased as the MLE
(
Loi, Eyres, & Birch, 2019).
concentration was increased from 0.0% to 1.0% (w/w), but it increased
as the concentration was increased from 1.0% to 2.0% (w/w). The
smallest droplet size (196.0 ± 0.4 nm), which was 60 nm smaller than
the size of the emulsion stabilized with only Tween 20, was obtained
when 1.0% MLE was added to the Tween 20-stabilized emulsion. The
addition of MLE to the Tween 20-stabilized emulsions changed the
The zeta potential determines the colloidal stability of an emulsion
Masum, Chandrapala, Adhikari, Huppertz, & Zisu, 2019). Higher
(
values of absolute zeta potential indicate higher repulsive forces be-
tween oil droplets, which prevent aggregation and lead to higher
colloidal stability (Honary and Zahir, 2013). As shown in Fig. 3C, the
Fig. 4. Time-kill curves of MLE at concentrations ranging from 0.0 to 2.0% (w/w) in Tween 20-stabilized O/W emulsion against (A) Staphylococcus aureus, (B)
Listeria monocytogenes, (C) Escherichia coli, and (D) Pseudomonas aeruginosa. Dashed lines indicate the limit of detection.
6

H. Yu et al.
Food Chemistry 366 (2022) 130650
zeta potential decreased as the concentration of MLE increased, indi-
cating that MLE might enhanced the colloidal stability of the Tween 20-
stabilized O/W emulsions when used as co-stabilizer.
erythorbyl laurate and monolaurin is in emulsion foods where they exert
the best antibacterial properties.
The storage stability of the emulsions with various concentrations of
MLE was evaluated by measuring the change in droplet size during
storage (Fig. 3D). For all concentrations of MLE, no significant change (p
3
.6. Antioxidative properties of MLE in oil-in-water emulsion
The lipid oxidation inhibitory effect of MLE in the O/W emulsion
>
0.05) in droplet size was observed during the entire period of storage.
system was evaluated during thermally accelerated oxidation (Fig. 5).
Formation of lipid hydroperoxide in the emulsion was inhibited by MLE
in a concentration-dependent manner. In the control emulsion without
MLE, lipid hydroperoxides were produced without a lag phase; however,
the emulsions containing 0.5%, 1.0%, and 2.0% (w/w) MLE showed 1,
Furthermore, the polydispersity index of the emulsions also did not
change significantly (p > 0.05), which indicated that the negative
charge and the monodisperse distribution of droplets caused by Tween
2
0 with MLE maintained emulsion stability during storage (Table S3).
2
, and 3 days of lag phases, respectively. After 9 days, the lipid hydro-
3
.5. Antibacterial properties of MLE in oil-in-water emulsion
peroxide concentrations in the presence of 0.5%, 1.0%, and 2.0% (w/w)
MLE were 50.0%, 84.3%, and 98.3% lower than the concentration of the
control emulsion, respectively.
The antibacterial activity of MLE against Gram-positive and Gram-
negative foodborne pathogens was assessed in the O/W emulsion
Fig. 4). A >4.0-log reduction in the viable cells of Gram-positive
The inhibitory activities against lipid oxidation of 0.013% (w/w)
erythorbyl laurate, which corresponds to its concentration in 1.0% (w/
w) MLE, and 0.987% (w/w) MLE excluding erythorbyl laurate were
investigated to examine which compounds contributed to the anti-
oxidative activity of MLE (Fig. S6). The amounts of lipid hydroperoxide
formed in the emulsion with only erythorbyl laurate and the one with
MLE excluding erythorbyl laurate at the stated concentrations were
(
foodborne pathogens (S. aureus and L. monocytogenes) occurred within
a 10 h treatment of 0.5% (w/w) MLE. Treatment of MLE at the con-
centrations lower than 1.0% (w/w) did not affect the growth of Gram-
negative foodborne pathogens (E. coli and P. aeruginosa), while a treat-
ment of 2.0% (w/w) MLE caused >5.0-log reduction in their viable cells
within 12 h. These results indicated that MLE had antibacterial activity
against both Gram-positive and Gram-negative foodborne pathogens in
the O/W emulsion.
7
5.7% and 65.8% lower than the amount of the control emulsion at 9
days. These results indicate that the observed inhibitory effects of MLE
were primarily attributed to the antioxidative activity of erythorbyl
laurate. Additionally, erythorbic acid, which accounted for 2.84% (w/
w) of the MLE, contributed somewhat to the antioxidative activity of the
MLE.
To understand which compounds in MLE had the bactericidal effects,
the antibacterial activities of erythorbyl laurate at 0.007% (w/w), which
corresponds to its concentration in 0.5% (w/w) MLE, and 0.493% (w/w)
MLE excluding erythorbyl laurate against S. aureus were compared
The formation of lipid hydroperoxide was more effectively inhibited
by erythorbyl laurate than MLE excluding erythorbyl laurate despite the
higher content of erythorbic acid than erythorbyl laurate. This can be
explained by the locations of antioxidative molecules in the emulsion
system (Chaiyasit, Elias, McClements, & Decker, 2007). Erythorbyl
laurate can locate at oil–water interfaces, where the lipid oxidation
mainly occurs, due to its amphiphilic properties, which lead to effective
retardation of lipid oxidation (Park et al., 2017). Conversely, hydro-
philic erythorbic acid is dispersed evenly in the aqueous phase, causing
it to have a low concentration at the oil–water interface than erythorbyl
laurate.
(
Fig. S5). MLE including erythorbyl laurate at 0.5% (w/w) caused 4.4-
log reduction in the viable cells of S. aureus at 12 h. Meanwhile, the
MLE treatment excluding erythorbyl laurate and the erythorbyl laurate
treatment at the stated concentrations caused 1.4- and 0.3-log reduction
in the viable cells of S. aureus at 12 h, respectively. These results indicate
that other compounds in MLE, possibly monolaurin, had antibacterial
activity. However, the antibacterial activity of the MLE excluding
erythorbyl laurate was lower than the activity of the MLE including
erythorbyl laurate. These results suggest that the antibacterial activity of
MLE was significantly associated with erythorbyl laurate. In addition,
the antibacterial activity of MLE could be attributed to the synergistic
interaction between erythorbyl laurate and monolaurin.
4
. Conclusion
The antibacterial activities of erythorbyl laurate and monolaurin are
primarily caused by disruption of bacterial cell membranes and subse-
quent cell disorganization (Kovanda et al., 2019; Park et al., 2018). The
stronger antibacterial activity of MLE against Gram-positive bacteria
than on Gram-negative bacteria can be due to the differences in the
bacterial cell envelope (Zhao, Zhang, Hao, & Li, 2015). Gram-positive
bacteria have cell walls of peptidoglycan with meshwork structures,
which allow penetration of the compounds into the cell membrane of the
bacteria. However, the cell wall of Gram-negative bacteria, with
compact outer membranes due to the presence of lipopolysaccharide
layers, prevents insertion of the ester compounds into the cell membrane
Lipase-catalyzed two-step esterification for production of multi-
0
0
1
.0% MLE
.5% MLE
.0% MLE
60
50
2.0% MLE
40
30
20
(
Morente, Fern a´ ndez-Fuentes, Burgos, Abriouel, Pulido, & G a´ lvez,
013).
Erythorbyl laurate in aqueous solution had antibacterial activity
2
against only Gram-positive bacteria (Park et al., 2018). Meanwhile, as
erythorbyl laurate was dispersed in an O/W emulsion, it exhibited
bactericidal effects on both Gram-positive and Gram-negative bacteria
10
0
(
Kim, Yu, Park, & Chang, 2020). Similar to the antibacterial properties
of erythorbyl laurate, the antibacterial activity of monolaurin was
enhanced and its antibacterial spectrum was expanded in an emulsion
system (Fu, Zhang, Huang, Liu, Hu, & Feng, 2009). The different anti-
bacterial properties of the lauric esters depending on the dispersion
medium are not fully interpreted, but the emulsions might contribute to
enhancing permeability of the compounds into the bacterial outer
membrane. In this regard, the promising application of MLE containing
0
1
2
3
4
5
6
7
8
9
10
Incubation time (day)
Fig. 5. Effect of MLE at concentrations ranging from 0.0 to 2.0% (w/w) in
Tween 20-stabilized O/W emulsion on the formation of lipid hydroperoxides
during thermally accelerated oxidation.
7

H. Yu et al.
Food Chemistry 366 (2022) 130650
functional MLE with antibacterial and antioxidative activities was sug-
gested as an alternative to the purification process of erythorbyl laurate.
Erythorbyl laurate was produced in the first reaction, and the residual
lauric acid was converted into glyceryl laurate through the second re-
action. The addition of MLE in the O/W emulsion led to monodisperse
distribution of droplets with smaller size and higher absolute zeta po-
tential, which may enhance the colloidal stability of the emulsion.
Moreover, MLE exhibited the bactericidal effects and inhibitory activity
on lipid oxidation in the emulsion system, which was the same proper-
ties as those of erythorbyl laurate. The two-step esterification to convert
residual lauric acid with high reaction efficiency can reduce both pro-
duction cost and time, compared to the production of pure erythorbyl
laurate, due to no need for purification process. Moreover, the solvent-
free production with high substrate concentration allows to obtain more
product with the same reaction volume, rendering the process amenable
to large-scale production. These findings suggest that MLE, produced
through the two-step esterification, can be utilized as a multi-functional
food additive to control both microbial contamination and lipid oxida-
tion, particularly for emulsion foods, as similarly as erythorbyl laurate
does.
Demirbas, A. (2008). Comparison of transesterification methods for production of
biodiesel from vegetable oils and fats. Energy Conversion and Management, 49(1),
1
25–130. https://doi.org/10.1016/j.enconman.2007.05.002.
Enayati, M., Gong, Y., Goddard, J. M., & Abbaspourrad, A. (2018). Synthesis and
characterization of lactose fatty acid ester biosurfactants using free and immobilized
lipases in organic solvents. Food Chemistry, 266, 508-513. 10.1016/ j.
foodchem.2018.06.051.
Erbeldinger, M., Ni, X., & Halling, P. J. (1998). Enzymatic synthesis with mainly
undissolved substrates at very high concentrations. Enzyme and Microbial Technology,
2
3(1–2), 141–148. https://doi.org/10.1016/S0141-0229(98)00039-8.
Erdmann, M. E., Zeeb, B., Salminen, H., Gibis, M., Lautenschlaeger, R., & Weiss, J.
2015). Influence of droplet size on the antioxidant activity of rosemary extract
(
loaded oil-in-water emulsions in mixed systems. Food & Function, 6(3), 793–804.
https://doi.org/10.1039/C4FO00878B.
Freitas, L., Perez, V. H., Santos, J. C., & Castro, H. F.d. (2007). Enzymatic synthesis of
glyceride esters in solvent-free system: Influence of the molar ratio, lipase source and
functional activating agent of the support. Journal of the Brazilian Chemical Society,
1
8(7), 1360–1366. https://doi.org/10.1590/s0103-50532007000700011.
Fu, X., Zhang, M., Huang, B., Liu, J., Hu, H., & Feng, F. (2009). Enhancement of
antimicrobial activities by the food-grade monolaurin microemulsion system.
Journal of Food Process Engineering, 32(1), 104–111. https://doi.org/10.1111/j.1745-
4530.2007. 00209.x.
Gaysinsky, S., Taylor, T. M., Davidson, P. M., Bruce, B. D., & Weiss, J. (2007).
Antimicrobial efficacy of eugenol microemulsions in milk against Listeria
monocytogenes and Escherichia coli O157:H7. Journal of Food Protection, 70(11),
2631–2637. https://doi.org/10.4315/0362-028x-70.11.2631.
Gustini, L., Noordover, B. A. J., Gehrels, C., Dietz, C., & Koning, C. E. (2015). Enzymatic
synthesis and preliminary evaluation as coating of sorbitol-based, hydroxy-
functional polyesters with controlled molecular weights. European Polymer Journal,
CRediT authorship contribution statement
6
7, 459–475. https://doi.org/10.1016/j.eurpolymj.2014.12.025.
Hilterhaus, L., Thum, O., & Liese, A. (2008). Reactor concept for lipase-catalyzed solvent-
free conversion of highly viscous reactants forming two-phase systems. Organic
Process Research & Development, 12(4), 618–625. https://doi.org/10.1021/
op800070q.
Hyunjong Yu: Conceptualization, Data curation, Validation, Visu-
alization, Writing - original draft, Writing - review & editing. Yerim
Byun: Investigation, Validation, Formal analysis, Visualization, Writing
Honary, S., & Zahir, F. (2013). Effect of zeta potential on the properties of nano-drug
delivery systems - a review (Part 2). Tropical Journal of Pharmaceutical Research, 12
-
original draft. Pahn-Shick Chang: Supervision, Project administra-
tion, Funding acquisition, Writing - review & editing.
(
2). https://doi.org/10.4314/tjpr.v12i2.20.
Kim, J.-W., Yu, H., Park, K.-M., & Chang, P.-S. (2020). Antimicrobial characterization of
erythorbyl laurate for practical applications in food and cosmetics. Journal of
Chemistry, 2020, 1–8. https://doi.org/10.1155/2020/5073508.
Declaration of Competing Interest
Kovanda, L., Zhang, W., Wei, X., Luo, J., Wu, X., Atwill, E. R., … Liu, Y. (2019). In vitro
antimicrobial activities of organic acids and their derivatives on several species of
Gram-negative and Gram-positive bacteria. Molecules, 24(20), 3770. https://doi.org/
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
1
0.3390/molecules24203770.
Loi, C. C., Eyres, G. T., & Birch, E. J. (2019). Effect of mono- and diglycerides on physical
properties and stability of a protein-stabilised oil-in-water emulsion. Journal of Food
Engineering, 240, 56–64. https://doi.org/10.1016/j.jfoodeng.2018.07.016.
Acknowledgments
Luther, M., Parry, J., Moore, J., Meng, J., Zhang, Y., Cheng, Z., & Yu, L. (2007).
Inhibitory effect of Chardonnay and black raspberry seed extracts on lipid oxidation
in fish oil and their radical scavenging and antimicrobial properties. Food Chemistry,
This work was carried out with the supports of “R&D Program for
Forest Science Technology (2019146C10-2121-AB02)” provided by
Korea Forest Service (Korea Forestry Promotion Institute) and “Coop-
1
04(3), 1065–1073. https://doi.org/10.1016/j.foodchem.2007.01.034.
Masum, A., Chandrapala, J., Adhikari, B., Huppertz, T., & Zisu, B. (2019). Effect of
lactose-to-maltodextrin ratio on emulsion stability and physicochemical properties
of spray-dried infant milk formula powders. Journal of Food Engineering, 254, 34–41.
https://doi.org/10.1016/j.jfoodeng.2019.02.023.
erative Research Program for Agriculture Science
& Technology
Development (Project No. PJ01602001)” provided by Rural Develop-
ment Administration, Republic of Korea.
Mauriello, E., Ferrari, G., & Donsì, F. (2021). Effect of formulation on properties,
stability, carvacrol release and antimicrobial activity of carvacrol emulsions. Colloids
and Surfaces B: Biointerfaces, 197, Article 111424. https://doi.org/10.1016/j.
colsurfb.2020.111424.
Appendix A. Supplementary data
Moghimi, R., Aliahmadi, A., & Rafati, H. (2017). Ultrasonic nanoemulsification of food
grade trans-cinnamaldehyde: 1,8-Cineol and investigation of the mechanism of
antibacterial activity. Ultrasonics Sonochemistry, 35, 415–421. https://doi.org/
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130650.
1
0.1016/j.ultsonch. 2016.10.020.
Mohod, A. V., & Gogate, P. R. (2017). Intensified synthesis of medium chain triglycerides
using novel approaches based on ultrasonic and microwave irradiations. Chemical
Engineering Journal, 317, 687–698. https://doi.org/10.1016/j.cej.2017.02.102.
Ortega Morente, E., Fern a´ ndez-Fuentes, M. A., Grande Burgos, M. J., Abriouel, H., P ´e rez
Pulido, R., & G a´ lvez, A. (2013). Biocide tolerance in bacteria. International Journal of
Food Microbiology, 162(1), 13–25. https://doi.org/10.1016/j.
References
Abcha, I., Souilem, S., Neves, M. A., Wang, Z., Nefatti, M., Isoda, H., & Nakajima, M.
(
2019). Ethyl oleate food-grade O/W emulsions loaded with apigenin: Insights to
ijfoodmicro.2012.12.028.
their formulation characteristics and physico-chemical stability. Food Research
International, 116, 953–962. https://doi.org/10.1016/j.foodres.2018.09.032.
Chaiyasit, W., Elias, R. J., McClements, D. J., & Decker, E. A. (2007). Role of physical
structures in bulk oils on lipid oxidation. Critical Reviews in Food Science and
Nutrition, 47(3), 299–317. https://doi.org/10.1080/10408390600754248.
Chand, S., Adlercreutz, P., & Mattiasson, B. (1997). Lipase-catalyzed esterification of
ethylene glycol to mono-and diesters. The effect of process parameters on reaction
rate and product distribution. Enzyme and Microbial Technology, 20(2), 102–106.
https://doi.org/10.1016/S0141-0229(96)00090-7.
Chang, Y., & McClements, D. J. (2014). Optimization of orange oil nanoemulsion
formation by isothermal low-energy methods: Influence of the oil phase, surfactant,
and temperature. Journal of Agricultural and Food Chemistry, 62(10), 2306–2312.
https://doi.org/10.1021/jf500160y.
Park, J.-Y., Myeong, J., Choi, Y., Yu, H., Kwon, C. W., Park, K.-M., & Chang, P.-S. (2021).
Erythorbyl fatty acid ester as a multi-functional food emulsifier: Enzymatic
synthesis, chemical identification, and functional characterization of erythorbyl
myristate. Food Chemistry, 353, 129459. https://doi.org/10.1016/j.
foodchem.2021.129459.
Park, K. M., Jo, S. K., Yu, H., Park, J. Y., Choi, S. J., Lee, C. J., & Chang, P. S. (2018).
Erythorbyl laurate as a potential food additive with multi-functionalities:
Antibacterial activity and mode of action. Food Control, 86, 138-145. 10.1016/ j.
foodcont.2017.11.008.
Park, K. M., Lee, D. E., Sung, H., Lee, J., & Chang, P. S. (2011). Lipase-catalysed synthesis
of erythorbyl laurate in acetonitrile. Food Chemistry, 129(1), 59-63. 10.1016/ j.
foodchem.2011.04.019.
Park, K. M., Lee, M. J., Jo, S. K., Choi, S. J., Lee, J., & Chang, P. S. (2017). Erythorbyl
laurate as a potential food additive with multi-functionalities: Interfacial
characteristics and antioxidant activity. Food Chemistry, 215, 101-107. 10.1016/ j.
foodchem.2016.07.174.
Chen, W., Liang, G., Li, X., He, Z., Zeng, M., Gao, D., … Chen, J. (2019). Impact of soy
proteins, hydrolysates and monoglycerides at the oil/water interface in emulsions on
interfacial properties and emulsion stability. Colloids and Surfaces B: Biointerfaces,
1
77, 550–558. https://doi.org/10.1016/j.colsurfb.2019.02.020.
8

H. Yu et al.
Food Chemistry 366 (2022) 130650
Perugini, L., Cinelli, G., Cofelice, M., Ceglie, A., Lopez, F., & Cuomo, F. (2018). Effect of
the coexistence of sodium caseinate and Tween 20 as stabilizers of food emulsions at
acidic pH. Colloids and Surfaces B: Biointerfaces, 168, 163-168. 10.1016/ j.
colsurfb.2018.02.003.
Pirozzi, D., & Greco, G. (2004). Activity and stability of lipases in the synthesis of butyl
lactate. Enzyme and Microbial Technology, 34(2), 94–100. https://doi.org/10.1016/j.
enzmictec.2003.01.002.
Prachaiyo, P., & Mclandsborough, L. A. (2003). Oil-in-water emulsion as a model system
to study the growth of Escherichia coli O157:H7 in a heterogeneous food system.
Journal of Food Science, 68(3), 1018–1024. https://doi.org/10.1111/jfds.2003.68.
issue-310.1111/j.1365-2621.2003.tb08281.x.
Torres, C., & Otero, C. (2001). Part III. Direct enzymatic esterification of lactic acid with
fatty acids. Enzyme and Microbial Technology, 29(1), 3–12. https://doi.org/10.1016/
s0141-0229(01)00344-1.
Yadav, G. D., & Devi, K. M. (2004). Immobilized lipase-catalysed esterification and
transesterification reactions in non-aqueous media for the synthesis of
tetrahydrofurfuryl butyrate: Comparison and kinetic modeling. Chemical Engineering
Science, 59(2), 373–383. https://doi.org/10.1016/j.ces.2003.09.034.
Yu, H., Lee, M. W., Shin, H., Park, K. M., & Chang, P. S. (2019). Lipase-catalyzed solvent-
free synthesis of erythorbyl laurate in a gas-solid-liquid multiphase system. Food
Chemistry, 271, 445–449. https://doi.org/10.1016/j.foodchem.2018.07.134.
Yu, H., Park, K.-M., & Chang, P.-S. (2020). Lipase-catalyzed synthesis of lauroyl
tripeptide-KHA with multi-functionalities: Its surface-active, antibacterial, and
antioxidant properties. Food Chemistry, 319, 126533. https://doi.org/10.1016/j.
foodchem.2020.126533.
Zhang, Y., Wang, X., Xie, D., Zou, S., Jin, Q., & Wang, X. (2018). Synthesis and
concentration of 2-monoacylglycerols rich in polyunsaturated fatty acids. Food
Chemistry, 250, 60–66. https://doi.org/10.1016/j.foodchem.2018.01.027.
Zhao, L., Zhang, H., Hao, T., & Li, S. (2015). In vitro antibacterial activities and
mechanism of sugar fatty acid esters against five food-related bacteria. Food
Chemistry, 187, 370–377. https://doi.org/10.1016/j.foodchem.2015.04.108.
Saberi, A. H., Fang, Y., & McClements, D. J. (2013). Fabrication of vitamin E-enriched
nanoemulsions: Factors affecting particle size using spontaneous emulsification.
Journal of Colloid and Interface Science, 391, 95–102. https://doi.org/10.1016/j.jcis.
2
012.08.069.
Shantha, N. C., & Decker, E. A. (1994). Rapid, sensitive, iron-based spectrophotometric
methods for determination of peroxide values of food lipids. Journal of AOAC
International, 77(2), 421–424. https://doi.org/10.1093/jaoac/77.2.421.
9