Lipozyme RM IM-Catalyzed Acidolysis of Cinnamomum camphora

Subscriber access provided by UNIV OF SOUTHERN QUEENSLAND

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

Journal of Agricultural and Food Chemistry

1

Lipozyme RM IM-catalyzed acidolysis of Cinnamomum

2

camphora seed oil with oleic acid to produce human milk fat

3

substitutes enriched in medium-chain fatty acids

4

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

5

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

6 7

1

8

Nanchang University, Nanchang, Jiangxi 330047, China

9

2

10

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

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

330047, China

11 12

Running title: HMFS synthesized from CCSO and oleic acid

13 14 15

* To Corresponding authors:

16

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

17

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

18

1

ACS Paragon Plus Environment


19

ABSTRACT

20

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

21

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

22

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

23

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

24

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

25

including substrate molar ratio, enzyme concentration, reaction temperature and

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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


Page 2 of 44

Page 3 of 44


41

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

42

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

43

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

44

application in infant formula industry.

45

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

46

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

47

3



48

INTRODUCTION

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

Considering the drawbacks of traditional infant formula and the absorption

64

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

65

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

66

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

67

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

68

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

69

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


Page 4 of 44

Page 5 of 44


70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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



92

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

93

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

94

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

95

the enrichment of human milk fat substitutes with MCFAs.17

96

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

97

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

98

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

99

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

100

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

101

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

102

strength, hydrophobic or hydrophilic character and regeneration.19

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

plastic fats.22-24

112

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

113

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


Page 6 of 44

Page 7 of 44


114

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

115

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

116

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

117

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

118

production of HMFS and the reusability of Lipozyme RM IM.

119

MATERIALS AND METHODS

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

methyl linoleate, methyl linolenate and four positional conjugated linoleic acid

130

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

131

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

132

chemicals used were of analytical or chromatographic grade.

133

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

134

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

135

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



136

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

137

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

138

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

139

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

140

Experimental design for RSM study. Enzyme-catalyzed acidolysis was

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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


Page 8 of 44

Page 9 of 44


158

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

159

at all design points.

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

volume was 0.3 uL.

178

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

179

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



180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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


Page 10 of 44

Page 11 of 44


202

separated by suction filtration using Buchner funnel to obtain urea inclusion

203

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

204

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

205

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

206

analyzed by HPLC.

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

quantification.30

216

Physicochemical characteristics. The determination of CCSO and HMFS on

217

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

218

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

219

methods.31

220

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

221

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

222

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

223

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



224

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

225

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

226

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

227

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

228

physicochemical characteristics were analyzed by using the Statistical Analysis

229

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

230

RESULTS AND DISCUSSION

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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


Page 12 of 44

Page 13 of 44


246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

chosen for optimization in following optimization.

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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



268

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

269

further study.

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

and capric acid into borage oil.41

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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


Page 14 of 44

Page 15 of 44


290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

RM IM can therefore be written as:

303

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

304

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

305

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

306

C is temperature, and D is reaction time.

307

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

308

Table 2. The very small P values (

Lipozyme RM IM-Catalyzed Acidolysis of Cinnamomum camphora

Recommend Documents