A highly efficient and enzyme-recoverable method for enzymatic

Feb 11, 2019 - A novel three-liquid-phase system which contained fish oil as the nonpolar phase, was developed for the lipase-based hydrolysis of fish...
0 downloads 0 Views 2MB Size
Subscriber access provided by WEBSTER UNIV

Biotechnology and Biological Transformations

A highly efficient and enzyme-recoverable method for enzymatic concentrating omega-3 fatty acids generated by hydrolysis of fish oil in a substrate-constituted three-liquid phase system Zhigang Li, hua chen, Jinfen Su, Weifei Wang, Huayong Chen, Bo Yang, and Yonghua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06382 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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 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 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.

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 39

Journal of Agricultural and Food Chemistry

1 2

A highly efficient and enzyme-recoverable method for enzymatic concentrating

3

omega-3 fatty acids generated by hydrolysis of fish oil in a

4

substrate-constituted three-liquid phase system

5

Zhigang Li, a, c Hua Chen, a Jinfen Su, a Weifei Wang, b Huayong Chen, a, c Bo Yang, * a, c and Yonghua

6

Wang* d

7 8 9 10 11 12 13 14

a

15

510006, China.e-mail: [email protected]

16

b

17

Laboratory of Functional Food, Ministry of Agriculture, Guangdong Key Laboratory of Agricultural

18

Products Processing Guangzhou 510610, China.

19

c

20

University of Technology, Guangzhou 510006, China.

21

d

22

China. e-mail: [email protected].

23

* Corresponding author. Yonghua Wang

24

School of Biology and Biological engineering, South China University of Technology, Guangzhou

Sericultural & Agri-food Research Institute, Guangdong Academy of Agricultural Sciences, Key

Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China

School of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 39

25

Abstract: A novel three-liquid-phase system which contained fish oil as the nonpolar phase, was

26

developed for the lipase-based hydrolysis of fish oil and subsequent enrichment of the omega-3

27

polyunsaturated fatty acids (n-3 PUFA) in the glyceride fraction of the fish oil. Compared with the

28

traditional oil/water system, the enrichment factor of n-3 PUFA in this system was increased by 363.4% as

29

a result of a higher dispersity, higher selectivity of the lipase for the other fatty acids except n3-PUFA, and

30

the relief of product inhibition. The content of n-3 PUFA in the glyceride fraction could be concentrated to

31

67.97% by repeated hydrolysis after removing the free fatty acids. Furthermore, the lipase could be reused

32

for at least eight rounds. This method would be an ideal approach for enriching n-3 PUFA because it is

33

cost effective, low in toxicity, and easily scaled up.

34

Keywords: three-liquid-phase system, n-3 polyunsaturated fatty acids, Lipase, fish oil, hydrolysis

35

1 ACS Paragon Plus Environment

Page 3 of 39

Journal of Agricultural and Food Chemistry

36

Introduction

37

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially docosahexaenoic acid (DHA) and

38

eicosapentaenoic acid (EPA), have received widespread attention due to the numerous benefits that these

39

fatty acids provide for people suffering from cardiovascular diseases, diabetes, inflammation and

40

autoimmune disorders, and various neurological conditions. Since these fatty acids are not made by the

41

human body due to a lack of essential enzymes that produce n-3 PUFA, 1-3 they have to be obtained from

42

special diet, such as the oils from fish and marine microalgae. According to the suggestion put forward by

43

the American Heart Association and follow-up studies, all individual should take fish oil supplements that

44

supply 1 g n-3 PUFA per day.1-4 However, most of the natural oils from fish and marine microalgae

45

contain far more unhealthy fatty acids (e.g., monounsaturated fatty acids (MUFAs) and saturated fatty

46

acids (SFAs)) than n-3 PUFAs. Hence, the enrichment of n-3 PUFA content in natural oils has attracted a

47

great deal of interest in the fields of food and medicine. 5,6 Various techniques have been used to concentrate n-3 PUFAs present in fish oils and marine

48 49

microalgae, and these include urea complexation, vacuum or molecular distillation, supercritical fluid

50

extraction, distillation, chromatography, and enzymatic methods 6-8 Among them, selective hydrolysis of

51

n-3 PUFAs in fish oil by lipases is considered most promising because it yields the least impact on the

52

environment and requires only mild reaction conditions, while leaving no undesirable byproducts. 2,4,8

53

Most of the reported studies used lipases with high substrate specificity directed at SFAs and MUFAs to

54

release FAs while concentrating n-3 PUFA in the glyceride fraction, conferring a much higher level of

55

bioavailability to the n-3 PUFA than its corresponding free fatty acids (FFAs) and ethyl esters (EE) forms.

56

2

However, these methods have limited application because of the low enzyme-catalytic activity and/or 2 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

57 58

Page 4 of 39

high cost required by the conventional oil/water (O/W) system. In the past decades, different approaches, such as the use of reversed micelle, supercritical CO2

59

systems, and immobilized enzyme have been explored as a way to develop an efficient reaction system to

60

meet the industrial requirement.4,9-11 Unfortunately, virtually none of these approaches appear to be

61

efficient for industrial applications. In the conventional O/W phase or micro aqueous-phase reaction

62

system, production inhibition occurring in both phases and the low interfacial area can negatively affect

63

the catalytic activity of the lipases. As a result, long reaction time, high enzyme loss and high energy

64

consumption are usually difficult to avoid. Koike et al attempted to use reversed micellar systems to

65

enhance the catalytic efficiency of the lipase because of the large interfacial area in these systems, but

66

difficulties in product separation and purification cannot be avoided. 9 Recently, immobilization of lipase

67

onto solid supports has abstracted considerable attention because it can accommodate a relatively large

68

amount of lipase, which can facilitate the recovery and reuse of the lipase. However, the high cost and

69

low catalytic efficiency of this method have limited its large-scale application. In most cases, the low

70

catalytic efficiency is related to the limitation of substrate diffusion and enzyme leaking from the support.

71

This has led us to consider the use of three-liquid-phase system (TLPS) to meet all the criteria.12

72

A TLPS can be considered as an integration of the aqueous two-phase system (ATPS) and O/W phase

73

system. A classic TLPS is composed of water/salt phase, nonpolar phase, and polar phase contributed by

74

a polar phase-forming component (PFC) (e.g., ionic liquid and water-soluble polymer) and water. So far,

75

TLPSs have mostly been used to purify multiple compounds, such as bioactive compounds and heavy

76

metals, because compounds with various polarities can be simultaneously extracted into the

77

corresponding phases.12,13 We have previously reported a substrate-constituted three-liquid-phase system 3 ACS Paragon Plus Environment

Page 5 of 39

Journal of Agricultural and Food Chemistry

78

(SC-TLPS) that is based on the use of a liquid substrate to constitute the nonpolar phase of TLPS. This

79

system was used to carry out complete hydrolysis of olive oil via lipase-catalyzed reaction.14 It has a

80

number of advantages over traditional systems, such as its low energy consumption, facile recovery of the

81

product and enzyme, as well as the high catalytic efficiency and low toxicity that it confers to the enzyme.

82

Therefore, the operational cost of this method can be greatly reduced because all the problems associated

83

with conventional W/O systems mentioned above could be overcome. However, to our knowledge, the

84

effect and application of the selectivity of the interfacial enzymatic reactions carried out in TLPS have not

85

yet been reported.

86

In this study, we have developed a TLPS in which the nonpolar phase was constituted by fish oil. The

87

system was used to concentrate the amount of n-3 PUFAs in fish oil. In such SC-TLPS, the catalytic

88

efficiency and selectivity of the enzyme could be significantly enhanced as a result of the relief of product

89

inhibition and an enlarged interfacial area. Furthermore, the n-3 PUFA in the system was enriched in the

90

glyceride fraction, while the glycerol and lipase were partitioned into the bottom and middle phases,

91

respectively. The products in the top phases could be readily isolated and the lipase could be recovered

92

and reused.

93

Materials and methods

94

Materials

95

All experiments were performed with deionized water. Tuna oil was supplied by Sinopharm Chemical

96

Reagent Co. (Zhejiang, China). Table S1 shows the FA profile of the tuna oil. (ESI†). Lipozyme

97

TL-100L (Thermomyces lanuginosus lipase, liquid enzyme) was purchased from Novozymes A/S was

98

obtained from Novozymes A/S (Bagsvaerd, Denmark, Beijing, China). Lipase AYS (Candida rugosa 4 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

99

lipases, spray drying powder) was obtained from Amano (Nagoya, Japan, Shanghai, China). Lipase

100

MAS1 (marine Streptomyces. sp. MAS1 lipase, bactericidal fermentation broth) was produced in our

101

laboratory by expressing the gene (UniProtKB accession number H0B8D4) in recombinant Pichia

102

pastoris X-33. Acylglycerol standards of trioleoylglycerol (TAG), dioleoylglycerol (DAG) (85% of

103

1,3-DAG and 15% of 1,2-DAG), and monooleoylglycerol (MAG) were obtained from Sigma-Aldrich

104

(Guangzhou, China). HPLC-grade isopropanol and n-hexane were purchased from Kermel Chemical

105

Reagent Co, Ltd. (Tianjin, China). BCA kit was purchased from Nanjing KeyGen Biotech. Co. Ltd. Ion

106

liquids (Nanjing, China), including 1-butyl-3-methylimidazolium bromide ([BMIM]Br),

107

1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4), were obtained from Lanzhou Institute of

108

chemical physics (Lanzhou, China). All other chemicals were of analytical grade.

109

Partition behavior of lipase in various systems

110

Lipase was dissolved in water using an enzyme: solvent ratio of 1:100 (w/v). This step was carried out

111

at room temperature (ca. 25 ºC). The partition behavior of lipase in various systems was first

112

investigated in a 2-g scale of ATPS by adding solid salt and polar solvents into the lipase solution to

113

form an aqueous two-phase system consisting of 15-20% (wt.%) salt (15% of Na2SO4, 20% of

114

(NH4)2SO4) and 20% (wt.%) polar phase-forming component based on previous studies.13, 15 After that,

115

0.4 g of tuna oil was added, and the mixture was then vortexed for 10 s followed by centrifugation at

116

2800  g. The lipase activities of in the middle and salt-enriched phases were analyzed by colorimetric

117

assay using p-nitrophenyl ester as a chromogenic substrate for the lipase. This substrate could be

118

hydrolyzed to produce p-nitrophenyl, a yellow compound that could be easily detected by a

119

spectrophotometer. 16 The partition coefficient (K) of lipase was defined as the ratio of the lipase 5 ACS Paragon Plus Environment

Page 6 of 39

Page 7 of 39

Journal of Agricultural and Food Chemistry

120

activity in the middle phase to that in the salt-enriched phase. The recovery (Y) was the ratio of lipase

121

activity partitioned in the middle phase to the total lipase activity. It was determined by the partition

122

coefficient of lipase and the volume ratio of the middle phase to the salt-enriched phase according to the

123

following equation,13 Y(%) =

124

VM CM RK = 100% (VM CM + VBCB ) (RK + 1)

(1)

125

where VM, VB are the volume of middle phase and the salt-enriched phase, respectively; CM and CB

126

are the concentration of the lipase in the middle phase and the salt-enriched phase, respectively; R is the

127

ratio of the volume of the middle phase to that of the salt-enriched phase.

128

Selective hydrolysis of tuna oil in various systems

129

The hydrolysis of tuna oil was investigated in various systems. Batch reactions were carried out in

130

10-mL conical flasks containing 2.4 g of SC-TLPS as described in the previous experiment section. A

131

control was also carried out by mixing 0.4 g of tuna oil with 2 g of enzyme solution containing 0.017 g

132

of lipase. The flasks were then placed in a shaking bath set at 200 rpm and the desired reaction

133

temperature for 2 h. The mixture from each flask was withdrawn at different time intervals and

134

centrifuged at 1000  g for 5 min (or 9500  g for 10 min in the case of the control). Samples taken

135

from the top phase were analyzed by high-performance liquid chromatography (HPLC) and gas

136

chromatography (GC).

137

Effects of PEG-400 and Na2SO4 concentrations on tuna oil hydrolysis in SC-TLPS

138

The effects of PEG-400 and Na2SO4 on the hydrolysis of tuna oil were investigated by adding solid

139

Na2SO4 and PEG-400 into the lipase solution (1%, wt.%) to form an ATPS consisting of 5-34% (wt.%) 6 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 39

140

PEG-400 and 6-23% (wt.%) Na2SO4 using a 2-g scale. Tuna oil (0.4 g) was then added to the ATPS to

141

form a three-liquid system. A control SC-TLPS was also carried out by adding 0.4 g of tuna oil to 2 g of

142

lipase solution containing 13 mg of lipase. To study the effect of pH, lipase solutions (1%, wt.%) with

143

different pH were first prepared by adding 1 g of lipase to 100 mL of phosphate buffer (pH: 4-9, 100

144

mM). A fixed amount (1.3 g) of each lipase solution was taken, and 0.38 g of solid Na2SO4, 0.32 g of

145

PEG-400 and 0.4 g of tuna oil were added to this lipase solution. The reaction mixtures were agitated in

146

a shaking bath at 200 rpm. Samples were withdrawn periodically and centrifuged at Samples were taken

147

periodically and subjected to centrifugation at 1000  g for 5 min. Furthermore, the effect of different

148

agitation speeds was also investigated in systems in which the concentrations of PEG-400 and Na2SO4

149

yielded optimal catalytic efficiency. In order to increase the concentrations of EPA and DHA, repeated

150

hydrolysis of the fish oil was adopted. The initial hydrolysis was carried out under the best condition for

151

2 h, and the oil in the top phase was then collected and mixed with an excess amount of 0.5 N

152

KOH-30% ethanol solution to remove the FFAs from the glyceride faction containing n3-PUFAs. The

153

upper layer was extracted with two volumes of hexane and the solvent was removed by evaporation.

154

The fatty acid–free oil was used as a substrate for another reaction under the same conditions.

155

Reuse of middle lipase-rich phase

156

A SC-TLPS was first formed by adding 3.8 g of solid Na2SO4, 3.2 g of PEG-400, and 4 g of tuna oil to

157

13 g of lipase solution (1200 U/mL). The SC-TLPS was agitated at 37 ºC for 1 h. Samples of the

158

reaction mixture were withdrawn periodically and then centrifuged at 1000  g for 5 min. The top and

159

middle phases were removed with a pipette, and the residual middle phase was then collected and

160

recycled for the next reaction. The middle phase of the reaction mixture was recycled by the addition of 7 ACS Paragon Plus Environment

Page 9 of 39

Journal of Agricultural and Food Chemistry

161

the original top and salt-enriched phase to form a SC-TLPS, which was then subjected to the same

162

process as described above

163

Analytical methods

164

Confocal microscopy

165

Fluorescent images of the SC-TLPS microstructure were obtained by Laser scanning confocal

166

microscopy (LSCM, Leica, Germany). For fluorescent imaging, the oil-phase marker (Nile Red) and the

167

middle phase marker (fluorescein) were used together; the former was excited at 553 nm and the latter at

168

460 nm line. SC-TLPS and control (no addition of lipase) were agitated for at least 5 min just before

169

observation. A drop of the stained emulsified sample was dispensed onto a 1.2-1.3 mm thick

170

microscopic slide and the microstructure in the SC-TLPS was observed under a confocal microscope.

171

Droplet size analysis

172

Droplet size was analyzed using a Mastersizer 2000 laser diffractometer equipped with a Hydrosizer

173

2000S module (Malvern Instruments, UK). The droplet sizes of fish oil in different continuous phases

174

(purified water, middle phase and bottom phase of SC-TLPS) were analyzed under 2500 rpm. The fish

175

oil was added dropwise to the continuous phase, and the mixture allowed to stand for 5 min when the

176

obscuration rate reached 10% before the distribution of the droplet (d3,2 and d4,3) within the different

177

continuous phases was determined. Each sample was measured in triplicate.

178

High-performance liquid chromatography analysis (HPLC)

179

The composition of the major compounds in the hydrolysates was analyzed by HPLC system (Waters

180

2695) coupled to a refractive index detector. 17 The components were separated on a Phenomenex Luna 8 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 39

181

silica column (250 mm × 4.6 mm i.d., 5 μm particle size) at a column temperature of 35 °C. The mobile

182

phase consisted of a mixture of n-hexane, 2-propanol and methanoic acid in a15:1:0.003 volume ratio

183

and the flow rate was set at 1.0 mL/min. 1 The volume of the injected sample was 10 μL. Peaks in the

184

HPLC profiles were evaluated by comparing their retention times with those of reference standards.

185

Each acylglycerol species and FA were expressed as weight percentage. Acquisition and processing of

186

data were carried out using the instrument integrated soft-ware (Waters 2695). The hydrolytic ratio of

187

tuna oil was defined as the ratio of the total amount of free fatty acids (FFA) generated ([FFA]t) to the

188

theoretical amount of FFA produced, 2 which was equal to three times the amount of TAG, (3×[TAG]0).

189

The conversion of oil into FFA was calculated according to the following equation.

190

191

R FFA =

[FFA ]t ×100% 3[ TAG ]0

(2)

Fatty acid composition analysis

192

Sample (200 μL) to be tested was added to a 50-mL flask and saponificated with 5 mL of 0.5 N

193

KOH-methanol for 10 min at 70 ºC. The flask was then cooled for 5 min and 3 mL of BF3-methanol was

194

then added followed by another 5 min of incubation at 70 ºC. Next, 2 mL of hexane and NaCl aq

195

mixture was added to the mixture to extract the FAME. The hexane phase was then collected and dried

196

in Na2SO4 and 1 μL of this was then injected into a GC system for analysis. The total FA composition of

197

fish oil was analyzed by FID gas chromatography (Agilent 7890 A) with a FAME capillary column

198

(0.25 mm×60 m; J&W Scientific). 18 The column temperature was raised to 150 °C and held for 5 min,

199

and then increased to 220°C at a rate of 4°C/min. It was maintained at 220 oC for 16 min. The different

200

fatty acids were evaluated by comparing their retention times with those of the reference standards. The

201

concentration yield was calculated by taking into consideration the changes in the contents of the 9 ACS Paragon Plus Environment

Page 11 of 39

202

Journal of Agricultural and Food Chemistry

different fatty acids. The enrichment ratio and recovery of the individual FA were calculated as follows:

WFA,1 - WFA,0

203

E FA =

204

R FA =

205

where EFA is the enrichment ratio of the individual FA in the glyceride fraction of the fish oil as

WFA,0

×100%

WFA,1 ×(1 - [FFA ]t ) WFA,0

(3)

(4)

×100%

206

measured by GC, WFA,0 and WFA,1 are the content of each FA in the hydrolyzed glyceride fraction before

207

and after the hydrolysis of fish oil, respectively. [FFA] t is the content of FFA in the hydrolyzed fish oil.

208

Glycerol concentration was determined by an enzymatic kit.15

209

Results and discussion

210

Enrichment of n-3 PUFA by lipase-catalyzed hydrolysis in various SC-TLPSs.

211

Different salts, Na2SO4, and (NH4)2SO4, and PFCs (water-soluble polymers, and ionic liquids (IL) were

212

mixed with fish oil and water to determine whether different SC-TLPSs could be formed (Table S2, ESI†).

213

In general, the effects that the types of PFC and salt had on the formation of SC-TLPS appeared to be

214

similar to the effects they had on ATPS and SC-TLPS containing olive oil reported in our previous study.

215

Thus, the miscibility of the two polar phases was almost not affected by the fish oil containing n-3 PUFA,

216

and this could probably be caused by the large gap in polarity between the oil phase and the other phases,

217

and the small effect of n-3 PUFA on the polarity of the oil.

218

The lipase seemed to be partitioned into the middle phase in most of the tested SC-TLPSs, resulting in

219

high recovery (Table 1 and Table S3, ESI†), which was useful for the subsequent reuse of the enzyme.

220

On the other hand, FFA and glycerol tended to be extracted into the top phase and bottom phase,

221

respectively. Figure 1 shows the process of selective hydrolysis of tuna oil by lipase and the effectiveness 10 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 39

222

of the various SC-TLPSs tested. The catalytic efficiency and selectivity of the lipase were significantly

223

enhanced in SC-TLPSs that contained PEG as the PFC. For example, in PEG-400/Na2SO4 SC-TLPSs,

224

the enrichment factor of n-3 PUFA on the glycerol backbone was increased from 99.1% to 473.8%

225

relative to that achieved in the conventional O/W system. On the contrary, n-3 PUFA in the IL/salt

226

SC-TLPSs was barely enriched because the selectivity of lipase for n-3 PUFA relative to the other fatty

227

acids in the systems was too low. The reasons for these changes were not clear, but some studies have

228

shown that some ionic liquids and polymers can change the structure of the lipase as a result of altering

229

the hydrogen bond and/or interfacial electrostatic energy between the lipase and PFCs (ionic liquid or

230

PEG), thereby affecting the catalytic activity of the lipase. When SC-TLPSs that contained PEG were

231

used to replace the traditional O/W system, the release of SFA and MUFA was increased for all the three

232

lipases tested. Notably, when this system was used in the reactions catalyzed by CRL lipase, not only did

233

the catalytic rate become faster, but the release of SFA also increased. For example, the efficiency of the

234

hydrolysis yielding SFA and MUFA achieved by lipase in the SC-TLPSs comprising PEG-400/Na2SO4

235

was increased by 416.2% and 144.2%, respectively, relative to those in the O/W system. However, when

236

MAS1 lipase was used in the same system, increase in the amount of SFA released was lower than that

237

occurred in MUFA (33.5% vs 56.2%). This was an advantage since SFA might have a stronger negative

238

effect on human health compared to MUFA.

239

In the SC-TLPS described in this study, the top phase and middle phase could be economically

240

separated and recovered by gravity or low-speed centrifugal sedimentation, i.e. the middle phase

241

containing lipase could be directly reused and the product (n-3 PUFA) on the glycerol backbone and free

242

fatty acids (FFA) in the top phase could be readily isolated by extraction or molecular distillation.

243

However, the large amount of salt in the bottom phase that needs to be recovered is a key economic issue 11 ACS Paragon Plus Environment

Page 13 of 39

Journal of Agricultural and Food Chemistry

244

that needs to be resolved. From this point of view, Na2SO4 would seem to have more potential for

245

practical application because it can be easily recovered by cooling crystallization, but recovering the

246

(NH4)2SO4, usually via dilution crystallization, would need a large amount of methanol and energy.

247

Considering the enrichment factors of n-3 PUFA, salt recovery and biological compatibility of lipase, the

248

PEG-400/Na2SO4 SC-TLPS containing CRL lipase was therefore chosen for further investigation.

249

Effects of PEG-400 and Na2SO4 concentrations on the enrichment of n-3 PUFA by lipase-catalyzed

250

hydrolysis in SC-TLPS

251

To determine the possibility of reusing the enzymes, the partition behavior of lipase in

252

PEG-400/Na2SO4 SC-TLPs was studied. In most cases, the high recovery (above 89%) of the lipase

253

partitioned into the middle phase was a result of the high value of the partition coefficient of lipase.

254

With increases in Na2SO4concentration, changes in the patterns of lipase recovery and partition coefficient

255

were similar (initial increases followed by decreases) (Figure 2). However, similar trends in the changes of

256

recovery and partition coefficient of the lipase were not observed when PEG-400 concentration was

257

increased. Instead, the partition coefficient of lipase was decreased, while the recovery of lipase first

258

increased and then decreased. This was because a change in recovery was simultaneously influenced by a

259

change in K, and a phase volume ratio Rm/b (middle/bottom) Increase in PEG concentration led to a

260

decrease in the partition coefficient of lipase and an increase in the phase volume ratio Rm/b.

261

The effect of PEG-400 and Na2SO4 concentrations on the enrichment of n-3 PUFA by lipase-catalyzed

262

hydrolysis is shown in Figure 3. Increases in PEG-400 and Na2SO4 concentrations in the SC-TLPS

263

appeared to result in similar patterns of changes for the enrichment ratios of n-3 PUFA and the recoveries

264

of lipase (i.e. initial increase followed by subsequent decrease). This may be because the hydrolytic

265

ratio of fish oil was positively correlated with the amount of lipase in the middle phase. In most cases, 12 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 39

266

the amount of lipase in the middle phase increased with increasing concentrations of PEG-400 and

267

Na2SO4. However, the change in the enrichment ratios of n-3 PUFA was more complicated than the

268

change in the hydrolytic ratio of fish oil because the enrichment ratio of n-3 PUFA was not only affected

269

by the hydrolytic ratio of fish oil, but also by some other factors, such as the selectivity of the lipase for

270

different fatty acids. The selectivity of the lipase for different FAs in the SC-TLPS was dramatically

271

affected by the salt and PFC. For example, the selectivity of the lipase for the double unsaturated fatty

272

acids (DUFA) decreased with increasing Na2SO4 concentrations. It has been reported that salt or ionic

273

liquid can change the selectivity of an enzyme toward a substrate, depending on the extent to which the

274

size of the catalytic cavity in the enzyme is reduced, a phenomenon originating from hydrogen bonding

275

interaction and/or interfacial electrostatic energy between the salt/IL and lipase. 19,20 This may be the

276

main reason for the negative impact of the salt on the lipase-mediated hydrolysis of the long chains of

277

larger FAs (DUFAs and n-3 PUFA) as these molecules might present a high degree of steric hindrance

278

effect. In contrast to the effect of salt on enzyme selectivity, PEG could increase the efficiency of the

279

hydrolysis of FAs (except n-3 PUFA) in SC-TLPS, probably because some PFCs could enhance the

280

conformational flexibility of the enzyme and orientate its active site more toward the interface. 21,22

281

However, in the case of n-3 PUFA, PEG hardly promoted its hydrolysis in the SC-TLPS because of the

282

larger steric hindrance, which could be caused by the presence of more double bonds. Increased lipase

283

conformational flexibility induced by PEG was not sufficient to effectively bind and hydrolyze n-3

284

PUFA. Interestingly, with increased PEG concentration in SC-TLPS, n-3 PUFA hydrolysis was inhibited

285

by other FFAs present in this system.

286 287

At 19% (w/w) Na2SO4 and 16% (w/w) PEG-400, the enrichment ratio of n-3 PUFA reached 90.5%, while the partition coefficient and recovery of lipase reached 41.9% and 96.4%, respectively. This 13 ACS Paragon Plus Environment

Page 15 of 39

Journal of Agricultural and Food Chemistry

288

indicated that SC-TLPS could be used to improve the hydrolytic activity and selectivity of lipase, as well

289

as allowing the lipase to be reused because almost all of it was enriched in the middle phase. Therefore,

290

this condition was chosen as the optimum system for further investigation.

291

Effects of pH on enrichment of n-3 PUFA by lipase-catalyzed hydrolysis in SC-TLPS

292

The catalytic ability of a lipase is highly affected by the pH of the reaction, and each lipase seems to have

293

a specific optimum pH.23 Thus, it is important to investigate the effect of pH on the partition behavior of

294

lipase and the enrichment of n-3 PUFA facilitated by lipase-catalyzed hydrolysis in SC-TLPS. When the

295

pH of the system reached 6, the partition coefficient and recovery of the lipase reached their peak values,

296

83.3% and 97.9%, respectively (Figure 4). Overall, both the enrichment ratio of n-3 PUFA and the

297

recovery of lipase displayed similar trends in the changes associated with the response to increasing pH

298

values. This was probably due to the positive effect that the high proportion of lipase in the middle phase

299

had on the hydrolytic ratios of fish oil. Furthermore, change in pH had a more significant effect on the

300

release SFA than MUFA through lipase-catalyzed hydrolysis in the SC-TLPS. For example, stearic acid

301

could not be effectively released in most of the tested cases, but when the pH was set at either 6 or 7, this

302

fatty acid could be preferentially released. By considering the high recovery of lipase, enrichment factors

303

of n-3 PUFA and easy removal of SFA, pH 6.0 was chosen for further investigation.

304

The effect of agitation speed on the enrichment of n-3 PUFA achieved by lipase-catalyzed hydrolysis

305

is shown in Figure 5 (A). The enrichment dramatically increased with increasing agitation speeds

306

(200-750 rpm), then gradually decreased when the agitation speed increased above 750 rpm (the

307

concentration of n3-PUFA increased by less than 2.5%.) A previous study has reported that high stirring

308

speed can increase the catalytic efficiency of the enzyme in the bi-phase system because it can improve

309

the emulsifying properties and mass transfer of substrates and products in the reaction system.25 In the 14 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 39

310

TLPSs, similar trends are also found. Indeed, temperature is another important factor that can affect the

311

diffusion and emulsifying properties of the reaction system. However, it not only affects the phase

312

dispersion, but also greatly changes the activity and stability of the enzyme. As a result, the catalytic

313

efficiency of enzymes decreases when the temperature was too high, because the enzymes were

314

destroyed and inactivated by high temperatures. (Figure S1,ESI†). The trend of n-3 PUFA enrichment

315

versus reaction time is shown in Figure 5 (B). In the first 1.5 h, the content of n-3 PUFA dramatically

316

increased up to 50.4%, but after 2.5 h, the rate of increase started to slow down with prolonged reaction.

317

Our previous study on the enzymatic hydrolysis of olive oil in SC-TLPS showed that almost all of the

318

olive oil can be hydrolyzed in a similar system because the by-product, glycerol, can be simultaneously

319

removed. However, in this case, the hydrolysis ratio of fish oil was only 36.6%, although glycerol had

320

been also removed by partitioning into the salt-enriched phase. This phenomenon could suggest that the

321

accumulation of FFA (another by-product) might be the main reason for the low hydrolysis of fish oil

322

rich in n-3 PUFA. Therefore, the n-3 PUFA could be concentrated by carrying out a second round of

323

hydrolysis on the product in the glyceride obtained from the first round of hydrolysis.

324

The n3-PUFA in the glyceride fraction of the oil obtained from the first round of hydrolysis was

325

subjected to alkali extraction to remove the FFAs. The n3-PUFA was then recovered after the extractant

326

was removed by distillation. This material was subjected to four additional rounds of the same hydrolysis.

327

The content of n-3 PUFA in the glyceride could be further concentrated to 67.97% after four rounds of

328

hydrolysis, and this was possible mainly because the content of DHA was enriched, from 23.16% to

329

52.52% (Figure 7). One previous study investigating the repeated hydrolysis of fish oil by CRL lipase

330

managed to achieve a n-3 PUFA content of up to 52.4%, but a long hydrolysis time (96 h) was

331

necessary.24 Furthermore, the recovery of n3-PUFA in the glyceride fraction was only 25.78%. Another 15 ACS Paragon Plus Environment

Page 17 of 39

Journal of Agricultural and Food Chemistry

332

work based on CRL lipase-catalyzed hydrolysis of fish oil 2 managed to concentrate n-3 PUFA in the

333

glycerol backbone after the removal of FFA, achieving a product containing 50.58% of the n-3 PUFA in

334

the system.2 However, its industrial application is limited because of the long reaction time and the

335

potential risk associated with high temperature molecular distillation process (above 100℃), which may

336

be related to the high omega-3 oxidation and the formation of some by-products harmful to health. In our

337

case, not only high temperature molecular distillation process was avoided, but higher n-3 PUFA recovery

338

was also obtained in the glyceride fraction, 63% (Figure 7) versus 26%, as previously reported.2

339

Furthermore, it is worth noting that the concentration of n-3 PUFA in the FFA fraction was similar to that

340

found in the raw fish oil in the second to fourth rounds of the fish oil hydrolysis, with the total recovery of

341

n3-PUFA reaching 25% (Table S4, ESI†). This would result in some health hazardous by-products

342

because the n-3 PUFA fraction of the FFA could be easily recovered by extraction with n-hexane after

343

lowering the pH value to between 1 and 3. This form of PUFA can also be efficiently enriched by

344

enzymatic esterification with glycerol.24 If this part of the n-3 PUFA was also included, the total recovery

345

of n-3 PUFA would have reached 90%, which is particularly attractive for industrial application.

346

Microstructure of the SC-TLPS and its effect on enzyme catalytic efficiency

347

In order to understand the mechanism that led to an increase in the efficiency of lipase-catalyzed reaction

348

in the PEG/salt SC-TLPSs, the size distributions of the fish oil droplets in the various phases of the

349

SC-TLPS was measured by a laser particle size analyzer (Figure 8 and Table S5, ESI†). When water was

350

replaced by the middle phase and bottom phase of SC-TLPS, the surface area of the fish oil droplet

351

enhanced from 0.105 m2g-1 to 0.190 m2g-1 and 0.291 m2g-1, respectively, indicating that a larger

352

interfacial area in the oil/middle (O/M) phase and oil/bottom (O/B) phase obtained in the SC-TLPS was

353

the main reason for the increased catalytic efficiency of the lipase. Furthermore, the high increase in the 16 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 39

354

interfacial area in the O/M phase may be more beneficial to the enzymatic catalytic efficiency, because

355

the lipase was mainly partitioned into the middle phase and the hydrolysis of fish oil may occur mainly at

356

the interface formed by the oil and the middle phase.

357

There are only two phases in an oil/water phase system, and therefore, the interfacial area of the oil

358

droplets can be used to define the interfacial reaction area. However, this would not represent the true

359

area of the reaction surface in the case of SC-TLPS, because the salt-enriched (bottom) phase can also be

360

distributed around the oil droplets. Therefore, the dispersion of SC-TLPS was examined by confocal

361

microscopy. The PEG-enriched (middle) phase was labeled with fluorescein (green) and the oil (top)

362

phase with Nile-red (red). As shown in Figure 9 (a), (b), and (c), in the part of the field in SC-TLPS, a

363

W1/O/W1/W2 multiple emulsion was observed (W1 and W2 represent the middle and bottom phase,

364

respectively), i.e. dispersed droplets of the oil phase were surrounded by the PEG-enriched phase, which

365

was suspended in the salt-enriched phase. Furthermore, in the inner part of some fish oil droplets, a small

366

amount of PEG-enriched phase droplets was also present. Similar to the O/M phase system and the TLPS

367

containing olive oil reported in our previous study 14, SC-TLPS exhibited smaller fish oil droplets

368

compared with O/W system (Figure 9(c) vs. Figure 9(d)). The increased catalytic efficiency of SC-TLPS

369

could be explained by its larger interfacial area. Recently, the double emulsion has attracted a lot of

370

attention as an enzymatic reaction system because of the larger area of reaction surface. 26 The

371

W1/O/W1/W2 multi-emulsion did not only have this advantage, but it could also be used to further

372

improve the catalytic efficiency of the enzyme through relieving the inhibition by polar product.

373

Reuse of the middle phase contained lipase in SC-TLPS

374

Although repeated hydrolysis of the product could dramatically enhance the enrichment efficiency of n-3

375

PUFA, the number of repetitions that could be carried out for this method was limited because of the 17 ACS Paragon Plus Environment

Page 19 of 39

Journal of Agricultural and Food Chemistry

376

high consumption of enzyme in each repetition. When SC-TLPS was used, this problem could be

377

resolved because the PEG-enriched phase containing the lipase could be reused as a liquid

378

immobilization support since almost no lipase was lost to other phases. Compared to the traditional

379

enzyme immobilization system, the novel immobilization support demonstrated by SC-TLPS had a series

380

of advantages such as no leakage of the enzyme from the support and high interfacial area under

381

high-speed stirring. Furthermore, most of the available solid immobilization supports might pose a

382

potential health hazard, and therefore, PEG could represent a safer and greener option since it is

383

non-toxic and has low vapour pressure.

384

To investigate the ability of SC-TLPS to maintain its functional efficiency after repeated process, the

385

PEG-enriched phase was subjected to several batches of fish oil hydrolysis. The results of eight repeated

386

batches are shown in Figure 10. By considering the activity of the lipase and the enrichment ratio of n-3

387

PUFA in the first batch as 100%, the lipase could be reused for at least eight batches without significant

388

loss of activity and catalytic efficiency. The addition of PEG might have contributed to the good

389

reusability of the lipase in the PEG-enriched phase, since PEG has been reported to help protect the

390

enzyme again harmful substances during the n-3 PUFA enrichment process. 27 However, in the previous

391

study, the lipase was immobilized on a solid support, which required longer reaction time and higher

392

enzyme dosage than those used in this study (Table S6, ESI†). Recycling the immobilized lipase also

393

requires a more complex procedure, and this can limit its application in industry, since washing the lipase

394

with a solvent such as cyclohexane is usually needed. Furthermore, under high-speed stirring, the

395

enzyme may be irreversibly inactivated because the high-speed rotating impeller might break down the

396

solid support. In this system, this problem could be overcome because the scattered droplets formed upon

397

strong agitation could automatically reassemble and reform after the stirring process was ended. In 18 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 39

398

summary, the SC-TLPS described in this study not only could increase the catalytic efficiency and

399

selectivity of the lipase, but also offered other important features, such as facile recovery of the product,

400

low toxicity and energy consumption, and high reusability of the lipase. Therefore, we believe this

401

system could be an ideal method for interfacial enzymatic selective catalysis.

402

Corresponding Author

403

[email protected]

404

ORCID

405

Yonghua Wang:0000-0002-3255-752X

406

Funding

407

This work was supported by the National Natural Science Foundation of China (Grant No. 2017MS078),

408

Chinese National Natural Science Foundation (Grant No. 21776103), the Science and Technology

409

Program of Guangzhou, China (Grant No. 201707010391) and the Characteristic Innovation Project of

410

Department of Education of Guangdong Province (Grant No. 2016KTSCX006).

411

Notes

412

The authors declare no competing financial interest.

413

Abbreviations

414

SC-TLPS, substrate-constituted three-liquid-phase system; PUFA, omega-3 polyunsaturated fatty acids;

415

FFAs, free fatty acids; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acid;

416

MUFA, monounsaturated fatty acids; EE, ethyl esters; O/W, oil/water; TLPS, three-liquid-phase systems;

417

ATPS, aqueous two-phase system; PFC, phase-forming component; K, The partition coefficient; LSCM,

418

Laser scanning confocal microscopy; HPLC-RID, HPLC (Waters 2695) with refractive index detector; 19 ACS Paragon Plus Environment

Page 21 of 39

Journal of Agricultural and Food Chemistry

419

FFA, free fatty acids; TAG, the amount of triglyceride; Lipozyme TL-100L, Thermomyces lanuginosus

420

lipase; Lipase AYS, Candida rugosa lipases; Lipase MAS1, marine Streptomyces. Sp.MAS1 lipase;

421

[BMIM]Br, 1-butyl-3-methylimidazolium bromide; [BMIM]BF4, 1-butyl-3-methylimidazolium

422

tetrafluoroborate.

423

Supporting Information

424

Main fatty acid composition of tuna oil; information on the establishment of SC-TLPS; effect of

425

phase-forming component on the lipases; changes in fatty acid composition and recoveries of the products

426

obtained by multi-repeated hydrolysis; diameters and specific surfaces of oil in different systems.

427

20 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

428

Page 22 of 39

References

429

(1) Qi, B.; Fraser, T.; Mugford, S.; Dobson, G.; Sayanova, O.; Butler, J.; Napier, J. A.; Stobart, A. K.;

430

Lazarus, C. M. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in

431

plants. J. Nat Biotechnol. 2004, 22 (6), 739-745.

432

(2) Kahveci, D.; Xu, X. Repeated hydrolysis process is effective for enrichment of omega 3

433

polyunsaturated fatty acids in salmon oil by Candida rugosa lipase. J. Food Chem. 2011, 129,

434

1552-1558.

435

(3) Ashjari, M.; Mohammadi, M.; Badri, R. Selective concentration of eicosapentaenoic acid and

436

docosahexaenoic acid from fish oil with immobilized/stabilized preparations of Rhizopus oryzae

437

lipase. J. Mol. Catal. B: Enzym. 2015, 122, 147-155.

438

(4) Mohammadi, M.; Habibi, Z.; Dezvarei, S.; Yousefi, M.; Ashjari, M. Selective enrichment of

439

polyunsaturated fatty acids by hydrolysis of fish oil using immobilized and stabilized Rhizomucor

440

miehei lipase preparations. J. Food. Bioprod. Process. 2015, 94, 414-421.

441

(5) Lei, Q.; Ba, S.; Zhang, H.; Wei, Y.; Lee, J. Y.; Li, T. Enrichment of omega-3 fatty acids in cod liver

442

oil via alternate solvent winterization and enzymatic interesterification. J. Food Chem. 2016, 199,

443

364-371.

444

(6) Valverde, L. M.; Moreno, P. A. G.; Cerdán, L. E.; López, E. N.; López, B. C.; Medina, A. R.

445

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with

446

different acyl-acceptors. J. Biochem. Eng. 2014, 91, 163-173.

447 448

(7) Shahidi, F.; Wanasundara, U. N. Omega-3 fatty acid concentrates: nutritional aspects and production technologies. J. Trends. Food. Sci. Tech. 1998, 9, 230-240. 21 ACS Paragon Plus Environment

Page 23 of 39

Journal of Agricultural and Food Chemistry

449

(8) Rubio-Rodrí guez, N.; Beltrán, S.; Jaime, I.; Sara, M.; Sanz, M. T.; Rovira, J. Supercritical fluid

450

extraction of fish oil from fish by-products: A comparison with other extraction methods. J. Food

451

Eng. 2010, 11, 1-12.

452

(9) Koike, H.; Imai, M.; Suzuki, I. Enrichment of triglyceride docosahexanoic acid by lipase used as a

453

hydrolysis medium in lecithin-based nano-scale molecular assemblage. J. Biochem. Eng. 2007, 36,

454

38-42.

455

(10) Lin, T. J.; Chen, S. W.; Chang, A. C. Enrichment of n-3 PUFA contents on triglycerides of fish oil

456

by lipase-catalyzed trans-esterification under supercritical conditions. J. Biochem. Eng. 2006, 29,

457

27-34.

458

(11) Kralovec, J. A.; Zhang, S.; Zhang, W.; Barrow, C. J. A review of the progress in enzymatic

459

concentration and microencapsulation of omega-3 rich oil from fish and microbial sources. J. Food

460

Chem. 2012, 131, 639-644.

461 462

(12) Liu, L.; Dong, Y. S.; Xiu, Z. L. Three-liquid-phase extraction of diosgenin and steroidal saponins from fermentation of Dioscorea zingibernsis C. H. Wright. J. Process. Biochem. 2010, 45, 752-756.

463

(13) Zhang, C.; Huang, K.; Yu, P.; Liu, H. Salting-out induced three-liquid-phase separation of Pt(IV),

464

Pd(II) and Rh(III) in system of S201−acetonitrile−NaCl−water. J. Sep. Purif. Technol. 2011, 80,

465

81-89.

466

(14) Li, Z.; Chen, H.; Wang, W.; Qu, M.; Tang, Q.; Yang, B.; Wang, Y. Substrate-constituted

467

three-liquid-phase system: a green, highly efficient and recoverable platform for interfacial

468

enzymatic reactions. J. Chemi. Commun. 2015, 51 (65), 12943-12946.

469 470

(15) Li. Z.; Jiang, B.; Zhang, D.; Xiu, Z. Aqueous two-phase extraction of 1,3-propanediol from glycerol-based fermentation broths. J. Sep. Purif. Technol. 2009, 66 (3), 472-478. 22 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

471 472

Page 24 of 39

(16) Shome, A.; Roy, S.; Das, P. K. Nonionic Surfactants:  A Key to Enhance the Enzyme Activity at Cationic Reverse Micellar Interface. J. Langmuir. 2007, 23 (8), 4130-4136

473

(17) Li, D.; Wang, W.; Durrani, R.; Li, X.; Yang, B.; Wang, Y. Simplified Enzymatic Upgrading of

474

High-Acid Rice Bran Oil Using Ethanol as a Novel Acyl Acceptor. J. Agric. Food Chem. 2016, 64

475

(35), 6730-6737.

476 477

(18) Wang, X.; Qin, X.; Li, D.; Yang, B.; Wang, Y. An Innovative Deacidification Approach for Producing Partial Glycerides-Free Rice Bran Oil. J. Bioresour Technol. 2017, 235, 18-24.

478

(19) Kim, H. S.; Ha, S. H.; Sethaphong, L.; Koo, Y. M.; Yingling; Y. G. The relationship between

479

enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and

480

experimental study. J. Phys. Chem. Chem. Phys. 2014, 16 (7), 2944-2953.

481 482 483 484

(20) Klibanov, A. M. Improving enzymes by using them in organic solvents. J. Nature. 2001, 409 (6817), 241-246. (21) Liang, Y. R.; Wu, Q.; Lin, X. F. Effect of Additives on the Selectivity and Reactivity of Enzymes. J. Chem Rec. 2017, 17 (1), 90-121.

485

(22) Talukder, M. M. R.; Hayashi, Y.; Takeyama, T.; Zamam, M. M.; Wu, J. C.; Kawanishi, T.; Shimizu,

486

N. Activity and stability of Chromobacterium viscosum lipase in modified AOT reverse

487

micelles. Mol. Catal. B: Enzym. 2003, 22 (3), 203-209.

488 489 490 491 492

(23) Cain, F. W.; Harris; J. B.; Moore, S. R.; McNeill, G. P. Sterol concentrates its application and preparation. US. US6399138. 2002. (24) Kim, I.W.; Lee, S. Synthesis of Diacylglycerols Containing CLA by Lipase catalyzed Esterification. J. Food. Sci. 2006, 71(7), 378-382. (25) Wang, X.; Li, D.; Qu, M.; Durrani, R.; Yang, B.; Wang, Y. Immobilized MAS1 lipase showed high 23 ACS Paragon Plus Environment

Page 25 of 39

Journal of Agricultural and Food Chemistry

493

esterification activity in the production of triacylglycerols with n-3 polyunsaturated fatty acids. J.

494

Food Chem. 2017, 216, 260-267.

495

(26) Blin, J.L.; Jacoby, J.; Kim, S.; Stébé, M.J; Canilho, N.; Pasc, A. A meso-macro compartmentalized

496

bioreactor obtained through silicalization of "green" double emulsions: W/O/W and W/SLNs/W. J.

497

Chemi. Commun. 2014, 50 (80), 11871-11874.

498

(27) Cipolatti, E. P.; Valério, A.; Ninow, J. L.; Oliveira, D. D.; Pessela, B. C. Stabilization of lipase

499

from Thermomyces lanuginosus by crosslinking in PEGylated polyurethane particles by

500

polymerization: Application on fish oil ethanolysis. J. Biochem Eng. 2016, 112, 54-60.

501

24 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

502

Page 26 of 39

Figure Captions

503

Figure 1. Concentration of n-3 PUFA produced from fish oil hydrolysis by different lipases in

504

SC-TLPSs. *I: Release of FFAs; *II: Concentration of FFAs; AYS: candida rugosa lipase; MAS1;

505

marine Streptomyces. sp. MAS1 lipase; TL-100L: Thermomyces lanuginosus lipase. Histogram: Red,

506

[BMIM]BF4/Na2SO4 SC-TLPS; Blue, PEG400/(NH4)2SO4 SC-TLPS; Orange, PEG600/Na2SO4

507

SC-TLPS; Orange, PEG600/(NH4)2SO4 SC-TLPS; Gray, control (O/W system); Black words: SFAs;

508

Violet words: MUFA; Red words: DUFA; Wathet words: n-6 PUFA. Green words: n-3 PUFA.

509

Figure 2. Effects of PEG-400 and sodium sulfate concentrations on the partition coefficients and

510

recoveries of lipases AYS in the PEG-400/Na2SO4 SC-TLPS. A: Concentration of Na2SO4 (wt.%) is

511

15%; B: concentration of PEG-400 (wt.%) is 16%. Symbol: ●: Klipase, ■:Ylipase; Different lowercase

512

letters indicate significant differences (one-way analysis of variance followed by Duncan's test,

513

P