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Lipozyme RM IM-catalyzed acidolysis of Cinnamomum camphora seed oil with oleic acid to produce human milk fat substitutes enriched in medium-chain fatty acids Xian-Guo Zou, Jiang-Ning Hu, Man-Li Zhao, Xue-Mei Zhu, Hong-Yan Li, Xiao-Ru Liu, Rong Liu, and Ze-yuan Deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503691p • Publication Date (Web): 09 Oct 2014 Downloaded from http://pubs.acs.org on October 10, 2014

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Journal of Agricultural and Food Chemistry

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Lipozyme RM IM-catalyzed acidolysis of Cinnamomum

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camphora seed oil with oleic acid to produce human milk fat

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substitutes enriched in medium-chain fatty acids

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Xian-Guo Zou1, Jiang-Ning Hu1,2*, Man-Li Zhao1, Xue-Mei Zhu, Hong-Yan Li1,

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Xiao-Ru Liu1, Rong Liu1, Ze-Yuan Deng1*

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1

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Nanchang University, Nanchang, Jiangxi 330047, China

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2

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State Key Laboratory of Food Science and Technology, Institute for Advanced Study,

College of Food Science & Technology, Nanchang University, Nanchang, Jiangxi

330047, China

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Running title: HMFS synthesized from CCSO and oleic acid

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* To Corresponding authors:

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

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

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1

ACS Paragon Plus Environment


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ABSTRACT

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In the present study, a human milk fat substitute (HMFS) enriched in

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medium-chain fatty acids (MCFAs) were synthesized through acidolysis reaction

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from Cinnamomum camphora seed oil (CCSO) with oleic acid in a solvent-free

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system. A commercial immobilized lipase, Lipozyme RM IM, from Rhizomucor

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miehei, was facilitated as a biocatalyst. Effects of different reaction conditions,

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including substrate molar ratio, enzyme concentration, reaction temperature and

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reaction time were investigated using response surface methodology (RSM) to

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obtain the optimal oleic acid incorporation. After optimization, results showed that

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the maximal incorporation of oleic acid into HMFS was 59.68%. And compared with

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CCSO, medium-chain fatty acids at sn-2 position of HMFS possessed more than

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70%, whereas oleic acid was occupied predominantly at the sn-1,3 position (78.69%).

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Meanwhile, triacylglycerol (TAG) components of OCO (23.93%), CCO (14.94%),

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LaCO (13.58%), OLaO (12.66%) and OOO (11.13%) were determined as the major

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TAG species in HMFS. The final optimal reaction conditions were carried out as

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follows: substrate molar ratio (oleic acid: CCSO), 5:1; enzyme concentration, 12.5%

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(w/w total reactants); reaction temperature, 60℃; and reaction time, 28 h. The

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reusability of Lipozyme RM IM in the acidolysis reaction was also detected, which

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could be reused up to 9 times without significant loss of activities. Urea inclusion

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method was used to separate and purify the synthetic product. As the ratio of

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HMFS/urea increased up to 1:2, the acid value lowered to the minimum. In a

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scale-up experiment, the contents of TAG and total tocopherols in HMFS (modified 2


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CCSO) were 77.28% and 12.27 mg/100g, respectively. And all the physicochemical

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indexes of purified product were within food standard. Therefore, such

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MCFAs-enriched HMFS produced by acidolysis method might have a potential

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application in infant formula industry.

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KEYWORDS: Human milk fat substitutes (HMFS), Response surface methodology

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(RSM), Lipozyme RM IM, Cinnamomum camphora seed oil (CCSO)

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3



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INTRODUCTION

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As a naturally important subsistence source for newborn infants, human milk fat

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(HMF) provides a vital source of energy and nutrients to support infant growth.

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Unlike the triacylglycerol (TAG) composition of vegetable oils and cow milk fat,

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HMF has a unique structure of the TAG, where the major saturated fatty acid, palmitic

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acid (16:0), locates at sn-2 position (approximately 70%), while unsaturated fatty acid,

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such as oleic acid (30-35%), and linoleic acid (7-14%) are presented at sn-1,3

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positions.1,2 Researches showed that such unique TAG profile of HMF facilitates the

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absorption of nutrients.3 The long chain saturated fatty acid palmic acid at sn-2

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position is not hydrolyzed by pancreatic lipase, and as 2-monopalmitin it forms a

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mixed micelle with bile salt, which is efficiently absorbed.4 In contrast, mostly

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traditional powdered infant formulas have a non-specific fatty acid position in their

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TAG structures, where saturated fatty acids at sn-1,3 positions are hydrolyzed by

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pancreatic lipase, producing free saturated fatty acids, then further form poorly

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absorbed calcium soap in the intestine, and result in constipation symptoms.2,5

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Considering the drawbacks of traditional infant formula and the absorption

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advantages of HMF, in recent ten years, preference for imitating the similar structure

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of HMF to produce infant formula has gained a great number of attentions.6,7

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Nowadays, “BetapolTM”, a commercial structured lipid made of vegetable oils through

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position-specific enzymatic interesterification, has been approved as HMFS by many

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European countries as well as South Korea and China. Compared to interesterification,

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many reports recently prefer to use acidolysis reaction to produce HMFS which 4


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contained palmitic, oleic, stearic, and linoleic acids,8 essential fatty acids,9

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gamma-linolenic acid,10 and omega-3 polyunsaturated fatty acids.11 Acidolysis

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reaction has the advantages of positional selectivity and specificity, low cost and less

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side products which is caused by the inevitable existence of acyl migration and can be

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minimized by the control of reaction conditions, such as water content, enzyme load,

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reaction time, substrate ratio and mainly reaction temperature.12 The acidolysis

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mechanisms include two steps:12 at initial stage, the hydrolysis of an ester bond

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between the fatty acid residue and the glycerol moiety of the triglyceride resulted in

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the release of the free fatty acid and generation of a glyceride containing at least one

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hydroxyl group, and then, a new ester bond is formed by the reaction between the

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newly created hydroxyl group and the incoming new free fatty acid.

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Besides that the fatty acids at different position of TAG affect the absorption

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efficiency, different length of fatty acids also have different absorption rates and

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different functions. Clinical experiments reveal that long-chain fatty acids (LCFAs,

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C14-C22) were more slowly absorbed than medium-chain fatty acids (MCFAs,

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C6-C12).13 An increase in change length of saturated fatty acids reduced the

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absorbability of them as well increased the loss of energy intake and calcium. MCFAs

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were absorbed more efficiently than LCFAs in gastrointestinal tract via the portal

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system, and rapidly provide energy for the body.14 It has also been reported that

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MCFAs had more antiviral and antibacterial activity than LCFAs.15 Due to the

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predominantly LCFAs in infant formula and human milk fats, the incorporation of

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MCFAs into HMFS has been suggested to provide a concentrated source of energy by 5



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many clinical nutrition experts.16 Recently, a commercial product named Neobee

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consists of MCFAs (caprylic and capric acids) has been developed as a functional

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food for patients. To our knowledge, in the literature, few data can be found regarding

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the enrichment of human milk fat substitutes with MCFAs.17

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Lipozyme RM IM, which is a immobilized lipase from Rhizomucor miehei, is

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widely used as a catalyst in pharmaceuticals and drugs production and lipid

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modifications. It has the characteristics of substrate specificity, stereospecificity, and

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regioselectivity.18 In recent study, different strategies of lipase immobilization were

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investigated to improve enzyme properties by positive alterations of structure, which

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may enhance enzyme activity, specificity or selectivity and increase its mechanical

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strength, hydrophobic or hydrophilic character and regeneration.19

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Cinnamomum camphora (lauraceae), known as an evergreen tree, is largely

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distributed in south area of the Yangtze River in China. In previous study, it has been

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reported that the oil from Cinnamomum camphora (lauraceae) seeds has a unique

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fatty acid profile that mainly contains MCFAs (capric acid, C10:0, 53.27%; lauric acid,

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C12:0, 39.93%).20 Liu et al.21 have also confirmed that Cinnamomum camphora seed

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oil (CCSO) is nontoxic with no side effects, which could be used as woody edible oil

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for its further development and utilization. Recently, our research group has managed

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to use CCSO for the production of medium-chain triacylglycerol (MCT) enriched

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plastic fats.22-24

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The aim of this work was to synthesize HMFS enriched with MCFAs through

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enzymatic acidolysis of CCSO and oleic acid using immobilized Lipozyme RM IM 6


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lipase as a catalyst. The effects of reaction conditions (time, temperature, enzyme

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concentration, and substrate mole ratio) on oleic acid incorporation were optimized

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by response surface methodology (RSM). Subsequently, scale-up trials were

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conducted to determine the feasibility of enzymatic modification of CCSO for the

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production of HMFS and the reusability of Lipozyme RM IM.

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MATERIALS AND METHODS

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Materials and chemicals. Oleic acid (≥85%) was purchased from local oil market

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(Nanchang, China). CCSO was extracted from Cinnamomum camphora seeds by the

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CO2 supercritical fluid method as described in Hu et al.20 Lipozyme RM IM, a

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commercial immobilized 1,3-specific lipase from Rhizomucor miehei, was purchased

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from Novozymes A/S (Bagsvaerd, Den-mark). The specific activity of Lipozyme

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RM IM was 150 IU/g, having 0.35-0.45 g/mL bulk density and 0.3-0.6 mm particle

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diameter. Carbon dioxide (99.9%) was provided by Wanli Gas Corp (Nanchang,

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China). #463 of standard fatty acid methyl esters (FAMEs) containing methyl esters

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C8-C20 saturated fatty acid, methyl myristate, methyl palmitoleate, methyl oleate,

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methyl linoleate, methyl linolenate and four positional conjugated linoleic acid

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isomers were purchased from Nu-Chek Prep Inc. (Elysian, MN). Tocopherol (α, γ,

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and δ) standards were acquired from Sigma Chemical Co. (St. Louis, MO). All other

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chemicals used were of analytical or chromatographic grade.

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Acidolysis reaction of small-scale sample. CCSO (1 g each) was blended with

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oleic acid at substrate mole ratios of 1:2, 1:4 and 1:6. Lipozyme RM IM of 6, 10,

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14% (w/w total reactants) was added to the blend in 50 ml screw-capped flask. The 7



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acidolysis was performed under 200 rpm orbital shaking water bath and temperature

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was set at 55, 60 and 65℃ for 20, 24 and 28 h incubation. After reaction, RM IM

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lipases were separated from the mixture through a 0.45-µm polytetrafluoroethylene

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(PTFE) syringe membrane filter. The filtrate was stored at -18℃ until analysis.

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Experimental design for RSM study. Enzyme-catalyzed acidolysis was

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influenced by reaction conditions such as water content, substrate mole ratio,

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enzyme concentration, temperature and reaction time.12 Before models were set up

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by RSM, single factor experiments were carried out to detect the effects of these five

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conditions on the oleic acid incorporation. Then, four of the five factors were

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selected as independent variables, and the optimal reaction conditions for the

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acidolysis reaction were established via RSM. RSM, a useful mathematical tool, has

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been widely applied to the optimization of reaction conditions for the production of

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HMFS in several studies.25,26 In our experiment, a four-factor and three-level full

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factorial design called Box-Behnken design (BBD) were employed to generate factor

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combinations by using Design Expert 7.1 software (Stat-Ease Inc., Minneapolis, MN,

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USA). The four selected factors were substrate mole ratio (A), enzyme concentration

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(B), temperature (C) and reaction time (D) and three levels were substrate mole

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ratios of 1:2, 1:4 and 1:6, Lipozyme RM IM of 6, 10, 14% (w/w total reactants),

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temperature of 55, 60 and 65℃ and reaction time of 20, 24 and 28 h. To avoid bias,

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29 runs including 16 factorial points, 8 axial points and 5 center points were

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performed in a random order. Oleic acid incorporation was acted as response value.

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The independent variables (A, B, C, D) and exact experimental model are presented 8


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in Table 1. Experiments were run randomly, and duplicate reactions were performed

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at all design points.

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Fatty acid profile. The fatty acid compositions of substrates and products were

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determined. Thin-layer chromatography (TLC) analysis was carried out for the

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separation of newly-synthesized product. About fifty microliters of product was

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applied to TLC plates coated with silica gel G. The developing solvent was

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hexane/diethyl ether/acetic acid (50:50:1, v/v/v). Then, The bands were sprayed with

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0.2% 2,7- dichlorofluorescein in methanol and visualized under UV light. Finally,

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the TAG band was scraped off into a screw-capped test tube and dissolved in 2 mL

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hexane for fatty acid composition analysis. The TAG sample was converted to fatty

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acid methyl esters (FAMEs) following the procedure of Zhu et al.27 and then

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analyzed by a gas-liquid chromatograph (GLC) (model 6890N, Agilent Technologies,

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USA) equipped with an auto injector, a fused-silica capillary column (CP-Sil 88, 100

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m × 0.25 mm × 0.2 µ m i.d.) and a flame ionization detector (FID) and operated in

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splitless mode. The injector and detector temperatures were maintained at 250 and

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260℃, respectively. The column temperature was held at 45℃ for 3 min, then

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programmed to 175℃ for 27 min at the rate of 13℃/min. The temperature was then

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further increased to 215℃ at at the rate of 4℃/min and held for 35 min. The carrier

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gas was nitrogen, and the total gas flow rate was 52 mL/min. The injecting sample

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volume was 0.3 uL.

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Sn-2 positional analysis. Composition of sn-2 FAs was analyzed by the

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pancreatic hydrolysis method as previously described.28 Likewise, TLC was used to 9



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separate 2-monoacylglycerol (2-MAG) from the hydrolysate. The 2-MAG band was

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scrapped off, methylated and analysed by GLC as also described above.

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The identification of TAGs species. The different species of TAGs of CCSO and

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HMFS were separated by reversed-phase high-performance liquid chromatograph

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(HPLC) with a Nova-Pak C18 column (150 × 3.9 mm, Waters, Milford, MA) and an

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evaporative light-scattering detector (Alltech 2000ES, USA) operating at 55℃ and a

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gas flow rate of 1.5 L/min. Samples were dissolved in methanol and filtered by

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0.45-µm PTFE syringe membrane filter. Twenty microliters of filtered sample were

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injected and eluted by acetonitrile (solvent A) and 2-propanol/n-hexane (1:1, v/v)

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(solvent B) at a flow rate of 1.8 mL/min with the following profile: 0 to 20 min, 30%

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B; 21 to 36 min, 60% B; 37 to 40 min, 100% B, and then back to the initial flow rate.

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Last, the separated TAGs were identified by comparing the retention time and

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equivalent carbon number (ECN). ECN = CN-2DB, used to predict the elution order,

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where CN is the total carbon number except three carbons of glycerol in the TAG

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and DB is the total number of double bonds on the fatty acids.

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Urea inclusion for separation and purification of HMFS. The mechanism of

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urea inclusion is traditionally explained as follows: separation of mixed fatty acids

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by urea inclusion occurs in terms of different saturation degrees and carbon-chain

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lengths of the fatty acids.29 Urea/HMFS mixed with 50 mL 95% (v/v) ethanol were

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put into a round-bottomed flask. Under the nitrogen gas flow, the mixture was

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refluxed at 60℃ in a water bath till the solution became clear. Then, the product was

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cooled at room temperature for 1 h and placed at -4℃ for 24 h. The mixture was 10


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separated by suction filtration using Buchner funnel to obtain urea inclusion

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compounds and non-urea inclusion compounds. The non-urea inclusion compounds

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contained PUFA and acylglycerol were transferred to a round-bottomed flask and

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ethanol was evaporated as much as possible. Acylglycerol was separated and

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analyzed by HPLC.

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Tocopherol analysis. About 1g of CCSO or HMFS sample were weighted into a

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flask and fully dissolved in 10 mL of hexane. After filtering with 0.45-µm PTFE

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syringe filter, three microliters sample were injected into the Agilent 1100 series

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HPLC. Then, the contents of tocopherol in prepared sample were determined by

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reversed-phase HPLC equipped with Hypersil ODS2 column (5μm, 4.6 × 150 mm,

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Agilent Technologies, USA) and a fluorescence detector (FLD). The mobile phase

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was methanol/water (98/2, v/v) with a flow rate of 0.8 mL/min. Standards of α-,γ

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-, and δ -tocopherol dissolved in hexane were used for identification and

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quantification.30

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Physicochemical characteristics. The determination of CCSO and HMFS on

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chemical properties, such as acid value, peroxide value, saponification value and

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iodine value, were conducted following AOAC (1990) standard analytical

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methods.31

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Scale-up trial. Model verification was carried out from RSM design. To verify the

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feasibility of the scaled-up acidolysis, a batch reaction (50 times scaling-up) in 500

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-mL flask was carried out at optimum conditions. The incorporation of oleic acid and

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productivity of TAG were determined to evaluate the feasibility of scale-up trial. 11



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Statistical analysis. The effects of different substrate mole ratio, enzyme

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concentration, temperature and reaction time studied in this work and their

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interactions were analyzed by using Design Expert 7.1 software. Mean values and

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standard deviation of single-factor experiment, contents of tocopherol and

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physicochemical characteristics were analyzed by using the Statistical Analysis

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System software (SAS 2000, Cary, NC, USA).

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RESULTS AND DISCUSSION

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Effect of water content. The water content in any quantity cannot be neglected

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because lipases were activated by a small amount of water32 and it is required to a

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first breaking of the triglyceride,12 however, too much water can make the lipase

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unstable and have an adverse impact on their activity, leading to a higher amount of

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byproducts.33 Some authors reported that the optimum water contents were generally

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in the range of 0.2-3%,34-36 however, for a given enzymatic reaction, it depends on

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many factors (the selected enzyme, support, solvent and quantities of substrates).

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Thus, there is a necessity to optimize the water content in the reaction system. With

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enzyme of 10% (w/w total reactants), 4/1 substrate mole ratio (oleic aid: CCSO),

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reaction temperature of 60℃ and time of 24 h, the effects of water contents in the

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range of 1% to 20% (w/w total reactants) on the incorporation of oleic acid were

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studied (Figure 1(a)). It showed that the incorporation increased from 55.72±0.31%

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to a maximum level of 57.44±0.25% when water content increased from 1% to 5%.

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And then, a decrease till to 52.81±0.19% was observed when water content increased

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from 5% to 20%. Hence we chosed 5% water content for the ongoing study. 12


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Effect of enzyme concentration. The effect of enzyme load, by varying from 1%

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to 25% (w/w total reactants) with constant substrate mole ratio of 4:1 and 60∘C

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reaction temperature after 24 h incubation, on the oleic acid incorporation was also

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observed (Figure 1(b)). An increase in the catalyst percentage from 1% to 10% led to

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a incorporation increase from 38.41±0.54% to 57.26±0.75%. Further increase in the

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enzyme load from 10 to 25%, however, did not lead to further incorporation increase.

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This is possibly due to the saturation of the enzyme at the interface between CCSO

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and aqueous phase, in which an increase in enzyme concentration provided no

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significant changes on the incorporation.37 Our results are similar to these reported

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by Rooney et al.37 and Djéssica et al.38 Considering the incorporation of oleic acid

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and cost of Lipozyme RM IM, 10% (w/w total reactants) enzyme concentration was

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chosen for optimization in following optimization.

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Effect of reaction temperature. Temperatures from 40 to 70℃ were evaluated

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in the present study. Substrate mole ratio (oleic acid: CCSO), water content, enzyme

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load and reaction time were 4:1, 5%, 10% and 24 h, respectively. Results are shown

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in Figure 1(c). It can be noted that the increasing temperature in the range of 40 to

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60 ℃ increased oleic acid incorporation from 42.11±0.54% to 57.63±0.83%.

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However, when the temperature further increased to 70 ℃ , the incorporation

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decreased to 57.19±0.92%. This result is in good agreement with the results of

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Phuah et al.39 who investigated the effect of temperature on the hydrolysis of partial

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palm oil. And it has been stated that enzyme activity and acyl migration are both

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increased by an increase in temperature until reaching the maximal rate.40 Therefore, 13



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a higher temperature was not suggested. Temperature of 60℃ was suitable for

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further study.

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Effect of reaction time. The reaction time was set from 1 to 36 h to investigate its

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influence on the incorporation. And substrate mole ratio (oleic acid: CCSO), water

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content, enzyme load and reaction temperature were 4:1, 5%, 10% and 60℃,

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respectively. As shown in Figure 1(d), the incorporation of oleic acid increased from

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42.51±0.43% to 57.48±0.94% when reaction time increased from 1 h to 24 h. In 36 h,

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the oleic acid incorporation was 58.62±0.90%. In similar study the optimal reaction

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time was different depending on the use of enzyme and substrate.41 Fourty hours as

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the optimal reaction time was reported by Akoh et al. for the incorporation of EPA

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and capric acid into borage oil.41

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Effect of substrate mole ratio (oleic acid: CCSO). The effect of molar ratio of

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oleic acid to CCSO on oleic acid incorporation was studied from 1:1 to 8:1 at a

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constant enzyme concentration (10%, w/w total reactants) and temperature of 60℃

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for 24 h (Figure 1(e)). It showed that the incorporation increased from 32.75±0.24%

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to 58.73±0.13% as the substrate mole ratio changed from 1:1 to 4:1. And then, from

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4:1 to 8:1, the incorporation increase till to 59.84±0.26% was found. It was reported

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that the appropriate substrate molar ratio was in favor of the reaction equilibrium and

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increased the ratio of the collision between substrates and catalyst,42 but higher

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substrate ratios were not desirable on this account that a large amount of FFAs

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containing high levels of free or ionized carboxylic acid groups acidify the enzyme

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layer, leading to absorption of water from the interface which may decrease the 14


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activity of the enzyme.43 Considering the stability of enzyme and cost of materials,

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substrate mole ratio of 4:1 was chosen for further investigation.

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Experimental design for RSM. To find the optimal condition of the greatest

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incorporation and also to see whether the reaction parameters have interactions on

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each other, RSM was used. According to the results of single-fator experiment

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discussed above (Figure 1), the water content was set at constant 5% (w/w total

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reactants), the substrate mole ratio (A, oleic acid: CCSO), enzyme load (B, w/w total

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reactants), temperature (C, ℃) and reaction time (D, in hours) were chosed as the

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four optimizing factors. Experimental model is shown in Table 1.

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Model fitting and variance analysis. Quadratic models were well established by

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multiple regression and backward elimination using Design Expert 7.1 software. The

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model equation for the responses (% oleic acid incorporated) catalyzed by Lipozyme

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RM IM can therefore be written as:

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Y=57.60+3.8A+2.36B+0.81C+1.65D+1.31AB-0.82AC+0.097AD-0.012BC-0.80BD

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-0.54CD-4.80A2-1.80B2-1.54C2-0.51D2.

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Where Y is the oleic acid incorporation, A is substrate mole ratio, B is enzyme load,

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C is temperature, and D is reaction time.

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The analysis of variance (ANOVA) of models for response variables is listed in

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Table 2. The very small P values (

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