Enzymatic Synthesis of Polyglycerol Fatty Acid Esters and Their

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Food and Beverage Chemistry/Biochemistry

Study on Enzymatic Synthesis of Polyglycerol Fatty Acid Esters and its Application as an Emulsion Stabilizer Bin Peng, Chao-Yue Xiong, Yao Huang, Jiang-Ning Hu, Xue-Mei Zhu, and zeyuan deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00222 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Journal of Agricultural and Food Chemistry

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Study on Enzymatic Synthesis of Polyglycerol Fatty Acid Esters

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and its Application as an Emulsion Stabilizer

3

Bin Peng1, Chao-Yue Xiong1, Yao Huang1, Jiang-Ning Hu2*, Xue-Mei Zhu1,2,

4

Ze-Yuan Deng1*

5 6

1

7

Nanchang, Jiangxi 330047, China

8

2

9

116034, China

State Key Laboratory of Food Science and Technology, Nanchang University,

School of Food Science and Technology, Dalian Polytechnic University, Dalian

10 11

Running title:Synthesis of PGFEs and its application

12 13

* To Corresponding authors:

14

Telephone No:+86 88304449-8226,E-mail address: [email protected] (J-N Hu);

15

Telephone/ Fax No: +86 791 88304402, E-mail address: [email protected](Z-Y Deng)

16

ACS Paragon Plus Environment


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Abstract

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Polyglycerol ester is considered as a kind of excellent food emulsions. The current

19

study was to synthesize the polyglycerol fatty acid esters (PGFEs) with different

20

long-chain fatty acids, i. e. long carbon fatty acid polyglycerol esters (L-PGFEs),

21

medium carbon fatty acid polyglycerol esters (M-PGFEs) and short carbon fatty acid

22

polyglycerol esters (S-PGFEs), using Lipozyme 435 as a catalyst in a solvent-free

23

system. Thereafter, the physicochemical properties and the potential applications as a

24

food emulsifier of the newly synthesized PGFEs were investigated. The maximum

25

esterification efficiency of L-PGFEs, M-PGFEs and S-PGFEs were achieved to

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69.37%, 67.34% and 71.68%, respectively, at the optimum conditions: reaction

27

temperature 84.48℃, reaction time 6 h, the molar ratio of polyglycerol to fatty acid

28

1.35:1, enzyme usage 1.41 wt% (based on the total substrate mass). High performance

29

liquid chromatography equipped with an evaporative light scattering detector

30

(HPLC-ELSD) and electrospray ionization mass spectrum (ESI-MS) were employed

31

to identify the synthesized products. Results demonstrated that the main components

32

of these PGFEs were dimeric glycerides (68.3%), triglycerides (13.13%), and a small

33

amount of tetraglycerides (3.18%). The properties of PGFEs were characterized by

34

physical and chemical methods. Compared to M-PGFEs and S-PGFEs, L-PGFEs had

35

the best physicochemical properties without any obvious odor. Further, the emulsion

36

capabilities of these different long-chain PGFEs were evaluated via examining the

37

particle size and storage stability compared to glycerin monostearate (GMS). Results

38

showed that the emulsions prepared by L-PGFEs had the best stability and the


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smallest particle size (16.8 nm) compared to M-PGFEs, S-PGFEs and GMS, which

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were not prone to coalescence of oil droplets and separation of oil and water. From the

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current study, the newly synthesized PGFEs with long-chain fatty acids showed the

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best advantages as a food emulsifier than M-PGFEs and S-PGFEs, even glycerin

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monostearate.

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Keywords: Polyglycerol fatty acid esters, chain lengths, Lipozyme 435, esterification,

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emulsion capability



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Introduction

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Polyglycerol fatty acid esters (PGFEs) as a new type of safe and effective non-ionic

48

surfactant have been extensively used in foods, cosmetics, and pharmaceuticals due to

49

their excellent properties of emulsification, crystallization adjustment, viscosity

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modifier and antimicrobials.1-4 It is known that the lipophilic PGFEs could promote

51

the formation of crystals5 while the hydrophilic PGFEs inhibited the formation of

52

crystallization in a diacylglycerol-rich oil.6 Meanwhile, PGFEs are considered as a

53

better substitute in emulsification performance than monoglyceride emulsifier on the

54

market. It was reported that the addition of 0.2% PGFEs to the production of

55

margarine was quite equivalent to the addition of 0.4% monoglyceride probably due

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to the fact that the structure of PGFEs contained more hydrophilic hydroxyl groups

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than monoglycerides.7-8

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Currently, PGFEs can be synthesized either chemically or enzymatically. As we know,

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it is random and easy for chemical methods to produce byproducts, which make it

60

difficult to separate and purify the PGFE products from the crude products. Also the

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final products used as a food additive often have unpleasant organoleptic features.9

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Recently, the green synthesis of PGFEs catalyzed by lipase has risen attentions around

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of the world in view of that they are generally more specific, react under mild

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conditions, and produce less side-products.10-11In a previous study, Wan FL et al.

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exploited oligoglycerol with linoleic acid in a solvent-free system using Lipozyme

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435 as the catalyst to synthesis oligoglycerol fatty acid esters (OGEs).12 Under the

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optimum conditions (reaction time, 4.52 h, reaction temperature 90℃, enzyme dosage


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2 wt%), the experimental efficiency of esterification (EE) reached 95.82±0.22%.

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However, there are few reports on how the chain length of fatty acids affect the

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hydrophilic lipophilic balance (HLB), thereby affecting the emulsifying properties of

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the PGFEs.13

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Natural fats and oils are attractive ingredients for the chemical industry as they are

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renewable and more environmentally friendly compared with fossil resources. Rice

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bran oil and Cinnamomum camphora seed oil are two such types of natural fats and

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oils with specific chain lengths of fatty acids. The major fatty acids of rice bran oil

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consist of palm acid (12% to 20%), oleic acid (40% to 50%), linoleic acid (29% to

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42%),14 which is considered as a typical source oil of long-chain fatty acids. Our

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previous studies showed that Cinnamomum camphora seeds contains about 40% of

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the fat with up to 90% of capric acid and lauric acid.15However, such medium chain

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fatty acids-enriched oil is very little utilized in China.16

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Novozyme 435 (a commercially immobilized lipase B from Candida antarctica) has a

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relatively high catalytic activity in the esterification reaction and operational

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stabilities. It presents activity under a large array of conditions and can be widely used

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as a biocatalyst to catalyze esterification reactions.17-19

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In the current study, rice bran oil, Cinnamomum camphora seed oil and acetic acid

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were used as different long chain type sources of fatty acids to enzymatically

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synthesize the long carbon fatty acid polyglycerol esters (L-PGFEs), medium carbon

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fatty acid polyglycerol esters (M-PGFEs) and short carbon fatty acid polyglycerol

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ester (S-PGFEs), respectively. The reaction conditions were optimized. Thereafter, the



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physical and chemical properties of these PGFE products with different chainlength

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fatty acids were compared.

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Materials and methods

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Materials

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Glycerol was purchased from the West Long Chemical Co. (Shantou, China). Rice

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bran oil was purchased from a local grocery store. Cinnamomum camphora seeds

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were collected on campus of Nanchang University. Diglycerol (>98%) was purchased

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from ANPEL. Laboratory Technologies Inc. (Shanghai, China). Double distilled water

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(Milli-Q) was used to prepare all solutions and emulsions. All other chemicals used

99

were

of analytical grade

unless

otherwise

stated,

and

all solvents were

100

of HPLC grade.

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Preparation and purification of polyglycerols

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Glycerin (100.0 g) and sodium hydroxide (3.00 g, 3 wt% based on the glycerin) were

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added to 250 mL of three-neck flask equipped with one condenser. The temperature of

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the reaction was controlled by an oil bath (260℃). The stirring speed (300 rpm/min)

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was controlled by a magnetic stirrer, and nitrogen was continuously used to exclude

106

the air in the reaction system. Then the condensate tube was to reflux glycerol, the

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reaction process to maintain a stable temperature and stirring strength.20After reaction

108

the temperature cooled below 80℃ and the reaction product was removed. Thereafter,

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a transparent light brown viscous liquid was collected and purified through the

110

molecular distillation (MD) equipment in the distillation temperature of 120℃,

111

vacuum pressure of 4.0 Pa, and scraping speed of 250 rpm/min.


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The composition of polyglycerols was analyzed by high performance liquid

113

chromatography (HPLC) according to a modified method described by Crowther MW

114

et al.21After purification by MD, the purity of polyglycerol product was up to 86.33%,

115

including 68.37% diglycerol, 13.13% triglycerol and 3.18% teraglycerol(data not

116

shown).

117

Enzymatic synthesis of L-PGFEs, M-PGFEs and S-PGFEs in a solvent-free

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system

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Enzymatic esterification was completed in a solvent-free system. Lipozyme 435,

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polyglycerols and rice bran oil, Cinnamomum camphora seed oil, or acetic acid were

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added to a 150-mL air-tight flask and reacted at 300 rpm under nitrogen conditions to

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prevent the oxidation of the reagents, respectively. The effects of reaction temperature

123

(40~100℃), reaction time (2~24 h), enzyme dosage (0.4~2.4 wt% of the total

124

substrate mass), and different molar ratio of polyglycerol to raw oil (2:1~1:2) on the

125

efficiency of esterification (EE) were studied.

126

After each reaction, 10 mL of reaction mixture was taken and centrifuged at 4200 rpm

127

for 10 min to be separated into three layers. The middle layer of the enzyme could be

128

isolated by filtration and used repeatedly and the lower layer of polyglycerol could be

129

used as raw material. The upper layer of a mixture of synthesized polyglycerol fatty

130

acid esters were determined according to the EE calculation as follows:12

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EE(%) =

AV0 - AV ×100% AV0



132

Where AV0 is the acid value of rice bran oil/Cinnamomum camphora seed oil/acetic

133

acid, and AV is the acid value of the upper oil phase after the reaction. All

134

determinations were carried out in duplicate and the average values were reported.

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Experiment Design for RSM Study

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In this study, polyglycerol and rice bran oil were used as substrates. According to the

137

results of single factor test and factor analysis, a three-level three-factor Box-Behnken

138

design was adopted to optimize the reaction conditions. The factors studied were the

139

reaction temperature: (Te, ℃), enzyme dosage (Ed, wt%) and the molar ratio of

140

substrate (Sr, mol/mol). The response value was the efficiency of esterification (EE%).

141

The reaction time was fixed at 6 h for all experiments. All of the experiments were

142

carried out in triplicate and the average values were recorded. Design Expert software

143

(version 8.0, Stat-ease, Inc.) was used for the analysis of variance (ANOVA) and

144

regression analysis of the experimental data.

145

Composition of the PGFEs analyzed by HPLC-ELSD and ESI-MS

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The chromatographic conditions were referred to the method of Wan F Let al.12 The

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HPLC system was composed of a Agilent HPLC system equipped with a column of

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Zorbax eclipse plus C18 (250 mm × 4.6 mm, 5 µm) and evaporative light scattering

149

detector (ELSD). The ELSD was set at 70℃ with a carrier air flow rate of 1.7 L/min,

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while column temperature was set at 30℃. The mixture of acetonitrile/isopropyl

151

alcohol (55/45, v/v) was delivered as the mobile phase at a flow rate of 0.5 mL/min.

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The samples were dissolved in isopropyl alcohol with a concentration of 0.1 mg/mL.

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About 1 mL of this sample solution was filtered through a nylon membrane filter


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(0.22 µm), and transferred to a 2 mL sample vial, then analyzed by HPLC. The

155

injection volume of each simples was 10 µL. Each sample was analyzed in duplicate.

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The MS spectrum was equipped with an ESI source. The operating parameters were

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shown in the following conditions: ions spray voltage 5500 V, capillary voltage 40 V

158

and capillary temperature 250℃.22-24 Mass spectra within a mass range of 100~2000

159

m/z were acquired in the positive ion mode. The compositions of polyglycerols were

160

identified by measuring their m/z values.

161

Determination of physical and chemical properties of the PGFEs

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The morphological and olfactory characteristics of the synthesized PGFEs were

163

investigated. The acid value (AV), saponification value (SV), iodine value (IV),

164

melting point (MP) of the PGFEs according to the National Standard of China. All

165

determinations were carried out in duplicate and the average values were reported.

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Evaluation of emulsifying properties

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Emulsions preparation

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Rice bran oil and distilled water (mass ratio of 1: 9) were mixed together, then the

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newly synthesized PGFE products and monoglyceride with different concentrations

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(0.01 wt%, 0.05 wt%, 0.1 wt%, 0.3 wt%, 0.5 wt%, 1 wt% of the total oil and water

171

mass) were added. The mixture was heated and stirred in a water bath at 50℃ for 30

172

minutes, and then a high speed homogenizer was used to produce the mixture to

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emulsions.

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Particle-size analysis of the emulsions

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The particle-size of the emulsions was analyzed according to the method reported by



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Li et al.,25 Briefly, a Malvern Mastersizer 2000 was used to determine the droplet size

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of the emulsions using static multi-angle light scattering analysis in triplicate.

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Emulsion stability test

179

Storage stability: the emulsions (10 mL) were decanted into glass test tubes and then

180

placed at room temperature. The final volume of emulsions at 1 min, 10 min, 30 min,

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1 h,1 day,1 week was recorded, respectively.

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Centrifugal stability: the emulsions (10 mL) were added in a centrifuge tube and then

183

centrifuged for 10 min at 4200 rpm/min. The final volume of emulsions in the

184

centrifuge tube was recorded.

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Thermal processing: the emulsions (10 mL) were transferred into glass test tubes, and

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incubated in water baths set at temperatures ranging from 30 to 80℃ for 30 min, and

187

then cooled to room temperature. The final volume of emulsions in the glass test tubes

188

was recorded.

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The stability of each emulsion (ES) was determined as follows:

190

ES =

191

Where V0 was the initial volume, and V was the final volume.

192

Statistical analysis

193

All measurements were performed in duplicate and reported as means and standard

194

deviations. Analyses of variance were performed, and the mean values±standard

195

deviation were evaluated by Duncan’s multiple-range test, using SPSS version 13.0

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statistical software (SPSS Inc., Chicago, IL, USA). A p-value of Te>Sr.

235

The second-order model equation for the EE (Y, %) was as follows:

236

Y=66.07+3.68A-2.13B+9.85C-0.31AB+0.43AC-0.44BC-4.45A2-4.18B2-9.59C2

237

Where Y, the EE; A, the temperature; B, molar ratio of polyglycerol/rice bran acid;

238

and C, enzyme dosage.

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Optimization of the reaction

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The effect of the interaction between the reaction temperature, the molar ratio of the

241

substrate, and the enzyme dosage on the esterification rate is shown in Figure 2. The


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order of influence of the factors on the esterification rate is BC>AC>AB. The optimal

243

conditions for the synthesis of L-PGFEs by Lipozyme 435 were predicted by

244

Design-Expert software. When esterification was catalyzed by Lipozyme 435 under

245

the optimal conditions (temperature, 85℃; time, 6 h; enzyme dosage, 1.4 wt%; molar

246

ratio of polyglycerol/rice bran oil fatty acid, 1.35), the predicted EE based on the

247

model was 69.82%. The small deviation between the optimal conditions and those

248

local optimal ranged was possibly due to the interaction effects of various

249

experimental factors (Figure 2). To further verify the reliability of the regression

250

model, three independent experiments were performed at the predicted optimum

251

conditions, and the actual EE was 69.37±0.12%, proving the high accuracy of the

252

RSM model used. In addition, polyglycerol esters were synthesized by using

253

Cinnamomum camphora seed oil and acetic acid under the same conditions, and

254

finally polyglycerol ester products with average esterification rate of 67.34% and

255

71.68% were obtained, respectively.

256

Composition of the PGFEs product determined by HPLC-ELSD and ESI-MS

257

The newly synthesized PGFE products were determined by HPLC-ELSD (Figure 3).

258

The retention time in the reversed-phase HPLC mode increased as longer fatty acid

259

chains were attached to polyglycerol.27 The HPLC analysis showed that the PGFE

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products could be divided into the following three groups of peaks, i.e. polyglycerol

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mono-esters, di-esters and tri-esters, which meant that the major components of

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L-PGFEs and M-PGFEs were mono-esters and di-esters with a small amount of

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triglycerides. While the products of S-PGFEs contained the main components of



264

mono-esters with a small amount of di-esters.28

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To further identify the PGFE products, the HPLC-ESI-MS in a positive ion mode was

266

employed to analyze the products. As shown in Table 3, the composition of L-PGFEs

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was mainly dimeric glycerides and triglycerides such as [oleic acid+ dimeric glycerol

268

mono Ester + Na] + and [linoleic acid + triglyceride monoester + Na] + etc. M-PGFE

269

product was similar to L-PGFE product. Also, the polyglycerol ester product also

270

detected the formation of water adducts, such as [capric acid + tetraglycerol

271

monoester + Na + 4H2O], which might be due to the presence of hydroxyl groups and

272

water molecules to form hydrogen bonds.29 However, S-PGFEs were mostly

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polyglycerol and monoesters. From the results, it could be concluded that the

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esterification reaction product of enzymatic synthesis of polyglycerol ester was

275

mainly monoester and di-ester, and only some esterification reaction occurred due to

276

the steric hindrance effects.30

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Characterization of the PGFEs

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The physicochemical properties of the PGFE products were observed. As shown in

279

Table 4, PGFEs are both light yellow and transparent, while L-PGFEs are waxy solid,

280

M-PGFEs are semi-solid, and S-PGFEs show jelly Shaped semi-solid, compared with

281

the mono-glyceride as a white powder solid.

282

Determination of particle size of PGFE emulsions

283

In the current study, these PGFE products were further tested for the capability of

284

emulsification in order to exam whether PGFEs can be as an emulsifier applied in the

285

food industry. The particle size and distribution of the same concentration of PGFE


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emulsions were prepared. As shown in Figure 4, the average particle size of the

287

L-PGFE emulsions and the M-PGFE emulsions decreased greatly to 16.8 nm and 28.7

288

nm, respectively when the dosage was at 0.3%. While particle size of the emulsions

289

prepared by S-PGFEs and GMS did not change significantly with the increase of the

290

dosage of emulsifiers, which particle sizes fluctuated at 2000 nm and 450 nm,

291

respectively. The results showed that the S-PGFE emulsions could not form the

292

interfacial film on the surface of the dispersed droplet. It should be mentioned that the

293

GMS emulsions showed poor performance in the oil-in-water emulsions at the

294

oil-to-water ratio of 1: 9, probably due to their little lipophilic contribution. In

295

addition, the emulsions prepared by L-PGFEs had a smaller particle size than the

296

other emulsions prepared at the same dosage. This might be due to the fact that the

297

HLB value of L-PGFE emulsions was larger than that of M-PGFE emulsions (data

298

not shown) due to the difference in the fatty acids of the synthetic ester and more

299

lipophilic contribution, therefore formed more stable oil-in-water emulsions. The

300

emulsion droplets in the system were not easy to aggregate and form small particle.

301

Analysis of emulsion stability

302

The storage stability of the four kinds of emulsions with different dosage of emulsifier

303

were shown in Figure 5. The storage stabilities of the emulsions with L-PGFEs and

304

M-PGFEs could be significantly improved with the increase of PGFEs concentrations.

305

When the dosage was less than 0.1%, only a small amount of stratification was

306

observed after standing for 10 min, and the storage stability of the emulsions

307

decreased but not significant. After 1 hour, the emulsions reached a steady stage,



308

probably due to the amount of emulsifier too small to completely reduce the surface

309

tension of the emulsion droplets in the oil droplets re-coalescence. When the dosage

310

was more than 0.1%, the emulsions stood for 1 week without any separation,

311

indicating that the emulsifiers were enough to reduce the surface tension of the

312

emulsions.31 It can be concluded that when the mass ratio of rice bran oil to distilled

313

water was 1: 9, the excellent stability of emulsions could be produced with adding the

314

minimum dosage of L-PGFEs and M-PGFEs (0.1%). In addition, it can be observed

315

that the stability of emulsions with L-PGFE was slightly better than that of emulsions

316

with M-PGFE at the same dosages. On the other hand, the stability of emulsions with

317

S-PGFE and GMS were not stable, and the separation of oil-water two phases

318

occurred within 1 min after the preparation of the emulsions. The disability of GMS

319

emulsions was much more obvious. These were consistent with the results of the

320

above formula of emulsion particles, and these two kinds of emulsifiers are poor to

321

prepare emulsions.

322

The stabilities of four kinds of emulsions with different concentrations of PGFEs

323

could be easily observed in Figure 6 storing for 1 week at room temperature. The

324

stability of the emulsions was as follows: L-PGFE emulsions> M-PGFE emulsions>

325

S-PGFE emulsions> GMS emulsions. Meanwhile, the centrifugal and thermal

326

stabilities of emulsions with PGFEs and GMS at different dosages are shown in

327

Figure 7. The centrifugal stabilities of the emulsions prepared by these PGFEs

328

increased with increasing the dosage of emulsifier. When the dosage of emulsifiers

329

was less than 0.1%, the emulsions were separated to some extent. When the emulsifier


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dosage increased gradually, the emulsions prepared by L-PGFEs and M-PGFEs did

331

not show significant oil-water separation after centrifugation, probably due to that the

332

emulsions could overcome the centrifugal force when the amount of emulsifier was

333

sufficient. When the dosage of emulsifier added to 1%, the centrifugal stabilities of

334

emulsions with S-PGFEs and GMS were far less than those of L-PGFE and M-PGFE

335

emulsions, respectively. The similar trends were also found in the thermal stabilities

336

of these emulsions (Figure 7B). Under the same conditions, the emulsions prepared by

337

L-PGFEs and M-PGFEs showed excellent thermal stability with no any oil-water

338

separation until 90℃, while the emulsions prepared by S-PGFE showed more obvious

339

separation after 70℃. The stability of the emulsions prepared by GMS was the worst,

340

and the oil and water were completely separated at 80℃.

341

Conclusively, in the current study, enzymatic synthesis of polyglycerol fatty acid

342

esters (PGFEs) with different long-chain fatty acids was studied in a solvent-free

343

reaction system. The highest EEs of L-PGFEs (69.37%), M-PGFEs (67.34%) and

344

S-PGFEs (71.68%) were achieved, respectively, under the optimal reaction conditions:

345

85℃, 6 h, enzyme dosage of 1.4 wt%, the molar ratio of polyglycerol/oilsof 1.35:1.

346

The PGFEs compositions were further identified as main three PGFEs, di-meric

347

glycerides (68.3%), tri-glycerides (13.13%), and a small amount of tetra-glycerides

348

(3.18%). Among these PGFEs, L-PGFEs showed the best physical and chemical

349

properties with no obvious odors. Meanwhile, L-PGFEs showed the best performance

350

in the preparation of oil-water emulsions whether in storage stability, centrifugal

351

stability and thermal stability, compared with M-PGFEs, S-PGFEs as well as GMS.



352

Acknowledgments

353

The authors acknowledge the National Natural Science Foundation of China (Grant

354

31571870, 31460427) and National Key Research and Development Program of

355

China (2016YFD0401404) for supporting this project.

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of

fatty

esters

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polyricinoleic fatty acids esters. UK Patent, 1981, 2, 232.

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(10) Charlemagne D.; Legoy M. D. Enzymatic synthesis of polyglycerol-fatty acid

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estersin a solvent-free system. J. Am. Oil Chem. Soc., 1995, 72(1), 61–65.

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(11) Panpipat W.; Xu X; Guo Z. Towards a commercially potential process:

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Enzymatic recovery of phytosterols from plant oil deodoriser distillates mixture.

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Process Biochem., 2012, 47(8):1256–1262.

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(12) WanF. L.; Teng Y. L.; Wang Y.Optimization of oligoglycerol fatty acid esters

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preparation catalyzed by Lipozyme 435. Grasas Y Aceites, 2015, 66(3):e088.

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(13) Pasquali R. C.; Taurozzi M. P.; Bregni C.Some considerations about the

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hydrophilic–lipophilic balance system.Int. J. pharm., 2008, 356(1): 44–51.

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(14) Liu D. N.; Li C. J. Characteristics and noval refining process of rice bran oil,

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(15) ZouX. G.; Hu J. N.; Zhao M. L.; Zhu X. M.; Li H. Y.; XR Liu; Deng Z. Y.

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Lipozyme RM IM-catalyzed acidolysis of cinnamomum camphora seed oil with oleic

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acid to produce human milk fat substitutes enriched in medium-chain fatty acids. J.

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Agric. Food Chem., 2014, 62 (43):10594–603.

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(16) HuJ.N.;Zhang B.; ZhuX.M.; Li J.; Fan Y.W.; Liu R.; Deng Z.Y.Characterization

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of Medium-Chain Triacylglycerol (MCT)-Enriched Seed Oil from Cinnamomum

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Camphora (Lauraceae) and its Oxidative Stability. J. Agric. Food Chem., 2011,

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(17) Ortega S.; Bódalo A.; Bastida J.; Máximo M. F.; Montiel M. C.; Gómez

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M.Optimized enzymatic synthesis of the food additive polyglycerolpolyricinoleate

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(PGPR) using Novozym 435 in a solvent free system.J. Biochem. Eng., 2014,

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84:91–97.

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(18) Souza M. S.; Aguieiras ECG; Silva da MAP; Langone MAP.Biodiesel synthesis

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via esterification of feedstock with high content of free fatty acids. Appl. Biochem.

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Biotechnol. , 2009, 154:253–267.

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(19) Duan Z.Q.; Du W.; Liu Z. H. Improved synthesis of 1,3-diolein by Novozym 435.

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J. Mol. Catal. B Enzym., 2013, 89:1–5.

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(20) Zou L.; Akoh C. C. Identification of tocopherols, tocotrienols,and their fatty acid

411

esters in residues and distillates of structured lipids purified by short-path distillation.

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J. Agric. Food Chem, 2013, 61: 238–246.

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(21) Molly W. Crowther; Terrence R.O’Connell.Electrospray mass spectrometry for

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characterizing

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Am. oil Chem.Soc., 1998, 5(12):1867–1876.

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(22) Je J.; Qian Z.; Byun H.; Kim S.Purification and characterization of an antioxidant

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peptide obtained from tuna backbone protein by enzymatic hydrolysis.Process

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Biochem., 2007, 42(5):840–6.

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(23) Khemchyan L. L.; Khokhlova E. A.; Seitkalieva M. M.; Ananikov V. P.Efficient

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sustainable tool for monitoring chemical reactions and structure determination in ionic

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liquids by ESI-MS.Chemistry Open, 2013, 2:208–214.

polyglycerols and the effects of adduct ion and cone voltage. J.



L.;

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Diacylglycerol-Enriched Oil from Free Fatty Acid Using Lecitase Ultra-Catalyzed

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Esterification. J. Am. oil Chem.Soc., 2011, 88(10):1557–1565.

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(25) Li X., Fang Y.; Al-Assaf S.; Phillips G. O.; Jiang F. Complexation of bovine

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serum albumin and sugar beet pectin: Stabilising oil-in-water emulsions. J. Colloid

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Interface Sci., 2012, 388(1):103–11.

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synthesis of diacylglycerol from tuna oil and its anti-obesity effect in C57BL/6J

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mice.Process Biochem., 2010, 45:738–743.

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(27) Cassel S.; Chaimbault P.; Debaig C.; Benvegnu T.; Claude S.; Plusquellec D.;

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Lafosse M. Liquid chromatography of polyglycerol fatty esters and fatty ethers on

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porous graphitic carbon and octadecyl silica by using evaporative light scattering

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detection and mass spectrometry. J. Chromatogr. A, 2001, 919: 95–106.

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(28) Andersen T.; Holm A.; Skuland I. L.; Trones R.; Greibrokk T. Characterization of

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complex mixtures of polyglycerol fatty acid esters using temperature and solvent

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gradients in packed capillary LC. J. Sep.Sci., 2003, 26(12-13):1133–1140.

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(29) OrfanakisA.; Hatzakis

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spectroscopy, mass spectrometry and dynamic light scattering. J. Am. oil Chem.Soc.,

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of

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Hu

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polyglycerol

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Y.;

Cao

Q.

Page 22 of 38

Preparation

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polyricinoleate

formulations


using

P.

NMR

Page 23 of 38


442

(30) Liu S. C.;

443

acylglycerol synthesis

444

optimisation

445

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Limbach H. J.Foams stabilized by multi lamellar polyglycerol ester self-assemblies.

447

Langmuir, 2013, 29:38–49.

of

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C. H.; of

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glycerol

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P. Z.; Ji n-3

H. W. Lipase catalysed PUFA

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process parameters. J. Food Chem, 2007, 103: 1009–1015.

448



449

Table Captions

450

Table 1. Three-level three-factor Box-Behnken design results of L-PGFEs. Factors

Experiment No.

Te

Sr(mol/mol)

Ed

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0(80℃) -1(70℃) 0(80℃) 0(80℃) 0(80℃) -1(70℃) 1(90℃) 0(80℃) -1(70℃) 1(90℃) 1(90℃) 0(80℃) 0(80℃) 0(80℃) 1(90℃) -1(70℃) 0(80℃)

0(1.5:1) -1(1:1) -1(1:1) 0(1.5:1) 1(2:1) 1(2:1) 1(12:1) 0(1.5:1) 0(1.5:1) 0(1.5:1) -1(1:1) 0(1.5:1) -1(1:1) 0(1.5:1) 0(1.5:1) 0(1.5:1) 1(1.5:1)

0(1.2%） 0(1.2%） 1(1.6%） 0(1.2%） 1(1.6%） 0(1.2%） 0(1.2%） 0(1.2%） 1(1.6%） -1(0.8%） 0(1.2%） 0(1.2%） -1(0.6%） 0(1.2%） 1(1.6%） -1(0.8%） -1(0.8%）

451


EE（（%）） 66.82 55.93 64.21 65.37 58.76 52.6 58.31 66.07 57.92 45.28 62.89 66.94 44.95 67.14 67.14 37.77 41.27

Page 24 of 38

Page 25 of 38


452

Table 2. Analysis of variance (ANOVA) for the fitted quadratic polynomial model. Source

Squares

df

Square

Value

Prob > F

Model Ti Sr Ed AB AC BC A^2 B^2 C^2 Residual

1516.76 108.05 36.30 775.39 0.39 0.73 0.78 83.48 73.66 387.05 10.32

9 1 1 1 1 1 1 1 1 1 7

168.53 108.05 36.30 775.39 0.39 0.73 0.78 83.48 73.66 387.05 1.47

114.32 73.29 24.62 525.98 0.26 0.50 0.53 56.63 49.97 262.56

< 0.0001 significant < 0.0001 0.0016 < 0.0001 0.6226 0.5041 0.4897 0.0001 0.0002 < 0.0001

Lack of Fit Pure Error Cor Total R2 Adj.R2

5.70 4.61 1527.08 0.9932 0.9846

3 4 16

1.90 1.15

1.65

453 454


0.3132

not significant


455

Page 26 of 38

Table 3. HPLC-ESI-MS analysis of polyglycerol fatty acid esters (PGFEs) products. NO. m/z 1 189.07

L-PGFE

M-PGFE

S-PGFE

[diglycerol+H]+

[diglycerol+H]+

[diglycerol+H]+ [Acetic acid+monoesters of diglycerol+Na]+

[triglycerol+H]+

[triglycerol+H]+

[triglycerol+H]+

2 231.08 3 263.11

[Lauric acid+monoesters of glycerol+Na]+

4 296.25

[Acetic acid+monoesters of triglycerol+Na]+

5 305.12 [Capric acid + glycerol monoester+K+2H2O]

6 319.13 7 337.15 [tetraglycerol+H]+

[tetraglycerol+H]+

[tetraglycerol+H]+

[Decanoic acid+monoesters of diglycerol+Na]+ [Lauric acid+monoesters of diglycerol+Na]+

8 343.21

9 371.24

[Acetic acid+monoesters of tetraglycerol+Na]+

10 379.15 11 411.18 [pentaglycerol+H]+ 12 417.24

13 445.27

[pentaglycerol+H]+

[pentaglycerol+H]+

[Decanoic acid+monoesters of triglycerol+Na]+ [Lauric acid+monoesters of triglycerol+Na]+

[Acetic acid + dimer diglyceride diester+Na]+

[Palmitic acid+monoesters of diglycerol+Na]+ [Linoleic 15 451.16 acid+monoesters of diglycerol+Na]+

14 427.1


Page 27 of 38


[Oleic 16 453.32 acid+monoesters of diglycerol+Na]+ 17 491.28

[Capric acid + trimellate monoester+Na+4H2O] +

18 493.01 19 497.34

[Lauric acid+monoesters of tetraglycerol+H]+

[Linoleic 20 525.14 acid+monoesters of triglycerol+Na]+ [Oleic [Oleic acid+monoesters 21 527.37 acid+monoesters of of triglycerol+Na]+ triglycerol+Na]+ [Decanoic acid+diesters 22 608.38 of diglycerol+Na]+ [Lauric acid+diesters of 23 636.19 diglycerol+Na]+ [Linoleic acid+diesters 24 709.1 of diglycerol+Na]+ [Oleic acid+diesters of 25 711.24 diglycerol+Na]+ [Linoleic acid + trimer 26 787.90 diglyceride diester+Na]+ 456



457

Page 28 of 38

Table 4. Comparison of PGFEs with GMS by physical and chemical properties. Properties

L-PGFE

M-PGFE

S-PGFE

GMS

Picture

appearance

yellow、 transparent waxy solid

smell

no smell

AV (mgKOH/g) SV (mgKOH/g) IV (gI2/100g) melting point (℃)

0.28 127.0 28.1

color

56.4

light yellow、 light yellow、 transparent transparent waxy solid jellify solid cinnamomum camphora seeds little sourness fragrance 2.65 34.72 134.2 186.9 2.2 0.1 50.5

458


27.8

white、 no transparent powder solid no smell 160 - 175 56~58℃

Page 29 of 38


459

Figure Captions

460

Figure 1.Single factor experiments of lipase-catalyzed esterification of polyglycerol

461

with rice bran oil, Cinnamomum camphoraseed oil and acetic acid (produce

462

L-PGPEs,M-PGPEs,S-PGPEs respectively). The efficiency ofesterification (EE)

463

(means±SD, n=3) was affected by (A) Reaction temperature at conditions of

464

polyglycerol/fatty acid molar ratio, 1:1;enzyme dosage, 1.2 wt%;reaction time, 6 h.

465

(B)Reaction timeat conditions of polyglycerol/fatty acid molar ratio, 1:1;enzyme

466

dosage, 1.2 wt%;reaction temperature, 80℃. (C) Enzyme dosage at conditions of

467

polyglycerol/fatty acid molar ratio, 1:1;enzyme dosage, 1.2 wt%; reaction temperature,

468

80℃. (D)Mole ratio of polyglycerol to fatty acid at conditions of reaction time, 6 h;

469

Reaction temperature, 80℃; reaction time, 6 h; enzyme dosage, 1.2 wt%.

470

Figure 2. Contour plots of interactions among temperature, polyglycerol/rice bran oil

471

fatty acid molar ratio and reaction time. Plot of EE as a function of (A) temperature

472

and substrate molar ratio with the amount of enzyme fixed at 1.2 wt%, (B)

473

temperature and enzyme dosage with a molar ratio of oligoglycerol/linoleic acid at 1.5,

474

(C) oligoglycerol/rice bran oil fatty acid molar ratio and enzyme dosage at 80℃.

475

Figure 3. HPLC chromatogram of the PGFEs production catalyzed by Lipozyme 435

476

under the optimal conditions of time, 6 h;temperature, 85℃; enzyme dosage, 1.5 wt%;

477

polyglycerol/linoleic acid molar ratio, 1.5:1.

478

Figure 4. Effects of emulsifier dosage on partical size of emulsions.

479

Figure5.The storage stability of emulsions with different dosage of L-PGFE (A),

480

M-PGFE (B), S-PGFE (C) and GMS (D).



481

Figure 6.The emulsions prepared by different emulsifiers and dosage were set at

482

room temperature for 1 week.

483

Figure 7. Centrifugal stability of emulsions (A) at room temperature and thermal

484

stability of emulsions (B) prepared by different emulsifiers at 0.3% emulsifier dosage.

485


Page 30 of 38

Page 31 of 38


486

487 488 489

Figure 1.Single factor experiments of lipase-catalyzed esterification of polyglycerol

490

with rice bran oil, Cinnamomum camphoraseed oil and acetic acid (produce L-PGPEs,

491

M-PGPEs, S-PGPEs respectively). The efficiency ofesterification (EE) (means±SD,

492

n=3) was affected by (A) Reaction temperature at conditions of polyglycerol/fatty

493

acid molar ratio, 1:1;enzyme dosage,1.2 wt%;reaction time, 6 h. (B)Reaction timeat

494

conditions of polyglycerol/fatty acid molar ratio, 1:1;enzyme dosage, 1.2

495

wt%;reaction temperature,

496

polyglycerol/fatty acid molar ratio, 1:1;enzyme dosage, 1.2 wt%; reaction temperature,

497

80℃. (D) Mole ratio of polyglycerol to fatty acid at conditions of reaction time, 6 h;

498

Reaction temperature, 80℃; reaction time, 6 h; enzyme dosage, 1.2 wt%.

80 ℃ .

(C) Enzyme

499


dosage

at conditions of


500 501

Figure 2. Contour plots of interactions among temperature, polyglycerol/rice bran oil

502

fatty acid molar ratio and reaction time. Plot of EE as a function of (A) temperature

503

and substrate molar ratio with the amount of enzyme fixed at 1.2 wt%, (B)

504

temperature and enzyme dosage with a molar ratio of oligoglycerol/linoleic acid at 1.5,

505

(C) oligoglycerol/rice bran oil fatty acid molar ratio and enzyme dosage at 80℃.

506 507


Page 32 of 38

Page 33 of 38


508 509

A:L-PGFEs; B:M-PGFEs; C:S-PGFEs.

510

Figure 3. HPLC chromatogram of the PGFEs production catalyzed by Lipozyme 435

511

under the optimal conditions of time, 6 h;temperature, 85℃; enzyme dosage, 1.5 wt%;

512

polyglycerol/linoleic acid molar ratio, 1.5:1.



513 514

Figure 4. Effects of emulsifier dosage on partical size of emulsions.

515


Page 34 of 38

Page 35 of 38


516

517 518

Figure5.The storage stability of emulsions with different dosage of L-PGFEs (A),

519

M-PGFEs (B), S-PGFEs (C) and GMS (D).

520



521

522 523

Figure 6.The emulsions prepared by different emulsifiers and dosage were set at

524

room temperature for 1 week.

525


Page 36 of 38

Page 37 of 38


526 A

B

527 528

Figure 7. Centrifugal stability of emulsions (A) at room temperature and thermal

529

stability of emulsions (B) prepared by different emulsifiers at 0.3% emulsifier dosage.

530



531 532

Figure 8 TOC Gragh Study on Enzymatic Synthesis of Polyglycerol Fatty Acid

533

Esters and its Application as an Emulsion Stabilizer


Page 38 of 38

Enzymatic Synthesis of Polyglycerol Fatty Acid Esters and Their

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