Lipase-Catalyzed Synthesis of Conjugated Linoleyl β-Sitosterol and Its Cholesterol-Lowering Properties in Mice

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Lipase-Catalyzed Synthesis of Conjugated Linoleyl β-Sitosterol and Its Cholesterol-Lowering Properties in Mice

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  pubs.acs.org/JAFC Published on Web 01/07/2010  ©  2010 American Chemical Society 1898  J. Agric. Food Chem.  2010,  58,  1898–1902 DOI:10.1021/jf902943y Lipase-Catalyzed Synthesis of Conjugated Linoleyl  β  -Sitosterol and Its Cholesterol-Lowering Properties in Mice R UI  L I , †,‡,§ C HENG -S HENG  J IA , ‡,§ L IN  Y UE , ‡ X IAO -M ING  Z HANG ,* ,‡ Q IU -Y U  X IA , † S ONG -L IN  Z HAO , † B IAO  F ENG , ‡ F ANG  Z HONG , ‡ AND  W EI -J UN  C HEN † † Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences, Wenchang 571339,Hainan, China and  ‡ State Key Laboratory of Food Science and Technology, School of Food Science andTechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China.  § These authors hadequal contributions to this work Conjugated linoleyl  β -sitosterol  ( CLS )  was prepared from  β -sitosterol and conjugated linoleic acid ( CLA )  via lipase-catalyzed synthesis in  n  -hexane in the presence of molecular sieves. Its plasmacholesterol-lowering properties were also studied. The optimal biosynthesis conditions were asfollows:  β -sitosterol concentration of 50  μ mol/mL, the molar ratio of CLA to  β -sitosterol of 1:1, thelipase concentration of 20 mg/mL, and 4 A˚ molecular sieve concentration of 60 mg/mL in  n  -hexaneat 50   C with vigorous shaking of 150 rpm for 72 h, and the highest yield of CLS reached 72.6%.The isolated CLS mixed with mice feed had good cholesterol-lowering properties. It decreasedserum total cholesterol  ( TC ) , serum triacylglycerols  ( TAGs ) , serum low-density lipoprotein choles-terol  ( LDL-C ) , atherogenic index  ( AI ) , liver weight  ( LW ) , liver index  ( LI ) , liver TC, and TAGs of mice,which was shown that CLS could prevent the formation of atherosclerosis and moderate the fatpathologic changes of liver. However, the HDL-C was not decreased, which proved the advantageof CLS over the other cholesterol-lowering products. KEYWORDS: Conjugated linoleyl  β -sitosterol  ( CLS ) ; immobilized lipase; cholesterol-lowering INTRODUCTION Plant sterols (phytosterols) are naturally occurring compo-nents of plants, which are a group of cholesterol analogues withdifferent side-chain configurations and have been used success-fully for lowering plasma cholesterol levels and shown to be safeforhalfacentury.  β -Sitosterolisthemostabundantinplantsterolanalogues ( 1 ). It is estimated that the intake of phytosterol inhumans can reach 160 - 360 mg/day, and it has been suggestedthat the daily consumption of 2 g of phytosterols can effectivelylower the cholesterol by 9 - 14% in humans ( 2 ). The mechanismfor differential absorption of cholesterol and phytosterols in theintestine is partially known. First, phytosterols are largely pre-ventedfrombeingabsorbedbyintestine.Second,phytosterolsarealso inhibitors ofintestinal cholesterol absorption and may causean effective displacement of cholesterol from micellar binding inthe intestine or affect cholesterol synthesis ( 2 ).However, practical applications of free plant sterols in foodsare limited because of their poor solubility and low bioavail-ability ( 3 ). The amount of plant and other sterols in diets iscomparable to that of dietary cholesterol, but only 5% isabsorbed ( 2 ). Therefore, it is needed to add some beneficialmoleculestoplantsterolstoenhancetheirsolubilityandbioavail-ability. Esterification or transesterification of plant sterols withfatty acids or oils and fats can increase their lipid solubility andthus facilitates the incorporation into a variety of foods ( 4 ,  5 ).Plant sterol fatty acid esters synthesized by chemical methodshave been added to margarine, and the margarine-containingplantsterolesterenteredtheUnitedStatesmarketin1999( 6 ).TheUS Food and Drug Administration had reviewed thoroughly thesafety of plant sterols and their esters and approved them to beused in foods in 2000 ( 3 ).Conjugated linoleic acid (CLA) is a naturally occurring fattyacid and has an uncommon structure containing conjugateddouble bonds in the carbon skeleton. CLA is found mainly indairy products and exhibits many physiological properties andhas many benefits for human, such as anticarcinogenic proper-ties ( 7  ), helping to normalize blood cholesterol level ( 8 ).Ithasbeenknownthat,inthepresenceoforganicsolvent,lipasecan catalyze the esterification reaction ( 4 ,  9 ,  10 ) and transesteri-fication ( 4 ,  10 - 13 ). However, lipase has a rigorous requirementfor water content in the reaction of lipase-catalyzed synthesis inorganic solvent, and the removal of the water by addition of adesiccant, i.e., molecular sieves, can shift the reaction toward thedesired product ( 13 ). But up to now, no researcher has performedthe synthesis of conjugated linoleyl  β -sitosterol (CLS) with theaddition of desiccant in lipase-catalyzed synthesis. Vu et al. usedthe lipase-catalyzed method to synthesize CLS, the molecularratio of   β -sitosterol to CLA was 1:3, the reaction temperate was55   C, and the reaction was conducted with a magnetic stirrer(175rpm)usingdifferentlipasesascatalysts.Butthedesiccantwasnot used during the reaction. The maximum yield of the ester wasonly 28.3% when the reaction was conducted for 48 h in thepresence of   n- hexane ( 6 ). *Corresponding author (e-mail, xmzhang@jiangnan.edu.cn; tele-phone, 86-510-85919106; fax, 86-510-85884496).  Article  J. Agric. Food Chem.,  Vol. 58, No. 3, 2010  1899 In the present study, we optimized the reaction conditions of CLS by condensation of   β -sitosterol and CLA in organic solventusing immobilized lipase, Chirazyme L-2 c.-f. C2 (from  Candidaantarctica ), molecular sieves were used to remove waters as thebyproduct, and a high yield of CLS was achieved. The preparedCLS was isolated by column chromatography and identified bythin-layer chromatography (TLC) and mass spectrometry (MS).Furthermore, its plasma cholesterol-lowering properties weredetected using a mouse test. MATERIALS AND METHODS Materials.  Immobilized lipase from  C. antarctica , Chirazyme L-2 c.-f.C2, was purchased from Roche Molecular Biochemicals (Mannheim,Germany).  β -Sitosterol was purchased from Fluka (Taufkirchen,Germany). Commercial conjugated linoleic acid was purchased fromDongying Dazhongnan Yellow Delta Industrial Co., Ltd. (Dongying,China). The 3 A ˚1/16 and 4 A ˚1/16 molecular sieves were purchasedfromShanghai UOP(Shanghai,China).Totalcholesterol(TC) enzymatickits, triacylglycerol (TAG) enzymatic kits, high-density lipoprotein cho-lesterol (HDL-C) kits, and low-density lipoprotein cholesterol (LDL-C)kits were purchased from Nanjing Jiancheng Bioengineering Institute(Nanjing, China). Lipase-CatalyzedSynthesisofCLS. Thereactionmixtureconsistedof   β -sitosterol (0.125 - 0.75 mmol), CLA (0.125 - 1.25 mmol), the immo-bilized lipase (100 mg), 3 or 4 A ˚activated molecular sieves (activated byheating at 180   C for 12 h in a drying oven, 100 - 500 mg), and 5 mL of solvent. The reaction mixture was put into a tightly sealed vial andincubated at 50   C with vigorous shaking (150 rpm). The solvents weredehydrated with 4 A ˚1/16 activated molecular sieves for at least 24 h. Analiquot sample (5  μ L) was removed periodically, and the product con-centration was analyzed by an HPLC system (Agilent 1100 Series, SantaClara, CA) equipped with a Si-60 normal phase column (4.6 mm   250mm; Hewlett-Packard, PaloAlto, CA) and an evaporative light scatteringdetector (ELSD) 200ES (Alltech, Deerfield, IL). The drift-tube tempera-ture and nitrogen carrier gas flow rate of the ELSD were set at 60   Cand 1.7 L/min, respectively. The eluent was a mixture of   n -hexane andisopropyl alcoholbya linearprogramming,andthe flowrateofthe eluentwas0.8mL/min.Theelutiongradientswere100% n -hexanefor0 - 10min, n -hexane/isopropylalcohol(90:10v/v)for10 - 20min, n -hexane/isopropylalcohol (30:70 v/v) for 20 - 25 min, and 100% isopropyl alcohol for25 - 30 min. The column temperature was 30   C. The calibration curveswerepreparedusingthepurifiedproducts.TheyieldofCLSwascalculatedby the molar ratio of CLS to  β -sitosterol at the beginning of the reaction. Isolation, Purification, and Identification of CLS.  β -Sitosterol(6 mmol), CLA (6 mmol), immobilized lipase (2.4 g), and activated 4 A ˚molecular sieves (7.2 g) were mixed with  n -hexane (120 mL), and thecondensation was carried out at 50   C with vigorous shaking (150 rpm)for 72 h.All reaction mixturewas filtered(WhatmanNo.1), and thefiltratewasthen rotary-evaporated (RE-52; Yarong Biochemical Instrument Plant,Shanghai,China).Theresiduewasdeacidifiedwith200mLof2%aqueoussodium bicarbonate solution, followed by centrifugation (Avanti J-HC;Beckman Coulter Inc., CA) at 1006  g  for 10 min. The mixture was filtered.Thefiltercakewasmixedwith200mLofwater,followedbycentrifugationat 1006  g  for 10 min to remove the unesterified acid as sodium salts. Thedeacidification process was repeated three times. The deacidified solidcontaining the desired products was dried in a vacuum at 50   C for 12 h.The crude powder of CLS obtained was applied to a silica gel column(60 - 100 mesh, 12  600 mm) and then eluted with cyclohexane/ethanol(19:1 v/v). The flow rate was 15 mL/h, and the eluent, 1 tube/10 min, wascollected and then detected by thin-layer chromatography (TLC). Theoptimum conditions for TLC were confirmed: 3  μ L samples were appliedtosilicagelGplates,developedbycyclohexane/diethylether(19:1v/v)andthen detected by spraying with 5% sulfuric acid in ethanol and heated at120   C for 15 min. The fractions containing the desired products werecollected and analyzed by the mass spectrometry.Mass spectrometry was employed to identify the isolated product. Thesamplewasdiluted1:50in n -hexane,and1  μ Lofthedilutionwasanalyzedon Platform ZMD 4000 (Waters Corp., Milford, MA). MS conditions:ionization type APCI þ , corona 3.19 kV, cone 30 V, source blocktemperature 100   C, APCI probe temperature 400   C, multiplier 700 V,desolgasflow4.2L/h,andmassscanrange200 - 800amu.Theprotonatedmolecular ion [M þ H] þ was at  m / z  677.8, and the product was identifiedto be CLS. Animal Treatments.  Eight-week-old male Kun Ming species mice(18 - 22 g) were obtained from th Medical School of Suzhou University(Suzhou, China). The mice were housed in polypropylene cages in a roommaintained at 25 ( 1   C and 60% humidity with 12 h light and 12 h darkcycle and were acclimated for 3 days before initiating studies. All of theanimal research complied with the International Guiding Principles forBiomedical Research Involving Animals (1985), and the protocols wereapprovedbytheAnimalUseandCareCommitteeofJiangnanUniversity.Formulation ofsynthetic diet is shown in Table 1 . Micewere randomlydividedintothreegroupsof20animalseach:normalgroup(NG),fedwithbasal feedfor 4 weeks consecutively;hyperlipidemicgroup(HG), fedwithhyperlipidemicfeedfor2 weeks consecutively and thenfed withbasal feedfor2weeksconsecutively;hyperlipidemicandCLSgroup(HCG),fedwithhyperlipidemicfeed for 2 weeks consecutively and thenfed with hyperlipi-demic supplemented CLS feed for 2 weeks consecutively. Every mouseintakes about 5 g of feed every day. At the end of the second and fourthweek, 10 mice from each group were kept fasting overnight and sacrificedfor biochemical analysis, respectively. Blood and tissues were collected forvarious tests. Sample Preparation.  Blood was collected from the retroorbitalsinus at the beginning and at the end of the second and fourth weeks of the feeding trial, then placed in sterile tubes, and centrifuged at 4000  g for 20 min. The obtained serum samples were analyzed for TC, HDL-C,LDL-C, and TAGs by the commerical kits. The atherogenic index (AI,(TC-HDL-C)/HDL-C) was also calculated.The livers were excised, cleaned of adhering matter, blotted on filterpaper,weighedandrinsedwithnormalsalinesolution(1:10w/v),andthenhomogenized using a tissue homogenizer with a Teflon pestle. Thehomogenate was centrifuged at 12000  g  for 20 min, and the supernatantsamples (serum) were analyzed for TC and TAGs by the kits. The liverindex (LI, liver weight/body weight) was also calculated. Statistical Analysis.  The data were statistically analyzed usingthe SPSS 13.0 software package (SPSS, Shanghai, China) and reportedas mean  (  SD. The differences among the experimental groups wereidentified by one-way analysis of variance (ANOVA) using Duncan’smultipicrangetest,andstatisticalsignificancewasconsideredat P <0.01. RESULTS AND DISCUSSION Effect of Different Organic Solvents on the Yield of CLS.  β -Sitosterol (60.0%) containing campesterol and a little stigmas-terol was used as a starting substrate.  β -Sitosterol (0.25 mmol)was esterified with CLA (0.50 mmol) with 300 mg of 3 A ˚molecular sieves and 100 mg of lipase using either  n -hexane oracetone (5 mL) as reaction medium at 50   C with vigorous Table 1.  Formulation of Synthetic Diet basal feed  ( % )  hyperlipidemic feed  ( % )  hyperlipidemic feed supplemented with CLScorn powder 26 basal feed 57.5 0.4 g of CLS mixed with 1 kg of hyperlipidemic feedflour 34 cholesterol 2soybean residue 24.7 bile salt 0.5fish powder 5 milk powder 20soybean oil 2.3 yolk powder 10clover fodder 3 pork oil 10premix 5  1900  J. Agric. Food Chem.,  Vol. 58, No. 3, 2010 Li et al. shaking (150 rpm) for 48 h. The yields of 32.38% and 25.53%were obtained in  n -hexane and acetone, respectively. Zaks andKlibanov( 14 )reportedthatthepolarityofthesolventdeterminesthe esterifying activity of lipase in the reaction system. Generally,all immobilized lipases exhibit high yield in nonpolar solvents.When a polar organic solvent is used, significantly lower yield isdetected. In such a medium, the solvent may alter the nativeconformation of the enzyme by disrupting hydrogen-bondingand hydrophobic interactions, thereby leading to very lowyield ( 15 ).Only limited organic solvents are used in the food industry.Acetone and  n -hexane are the solvents which are approved inmany countries, and immobilized lipase, Chirazyme L-2 c.-f. C2,is active in them. The reaction in  n -hexane showed higher yieldthan that in acetone. Therefore,  n -hexane was selected as theproper reaction medium of the condensation of   β -sitosterol andCLA. Effect of   β  -Sitosterol Concentration on the Yield of CLS.  Theyield of CLS in  n -hexane at different  β -sitosterol concentrationsis shown in  Figure 1a . When  β -sitosterol concentration was50  μ mol/mL, the yield of CLS was the highest. With the increaseof the concentration of   β -sitosterol the yield of CLS decreased.Most of the  β -sitosterol remained undissolved in the solvent,especially at higher concentration, which might be the reasonfortheloweryieldathigher  β -sitosterolconcentration.Sothebest  β -sitosterol concentration was selected as 50  μ mol/mL. Effect of the Molar Ratio of CLA to  β  -Sitosterol on the Yield of CLS.  The molar ratio of CLA to  β -sitosterol also influenced theyield of CLS ( Figure 1b ). At a fixed  β -sitosterol concentration of 50  μ mol/mL, the highest yield was achieved when the molar ratiowas 1:1. The yield of CLS started to decline when the molar ratiosurpassed 1:1. The reason could be that with the increase of CLAthe viscosity of reaction system increased, which led to thedecrease in the efficiency of esterification. EffectofMolecularSieveTypeandAmountontheYieldofCLS. Lipase-catalyzed esterification in organic solvents is a reactionwhere water plays a key role. A minimal amount of water isnecessary for the enzyme to ensure its optimal conformationand then to become optimally active. But, an excess of waterdecreases the enzyme catalytic activity from both kinetic andthermodynamic points of view ( 16 ). There are many methods forwater removal in the lipase-catalyzed reaction in organic sol-vent ( 16 , 17  ), and the adsorption method is most widely used. Inthis study, activated molecular sieves were used to remove thewater produced during the reaction, which has the advantage of lower cost and is easy to be separated and regenerated. Figure2a showsthe effectof3 and 4A ˚molecularsieveamounton the yield of CLS. The highest yield of CLS was achieved at60 mg/mL for both 3 and 4 A ˚molecular sieves, while the yield of CLS using 4 A ˚molecular sieves was higher than that with 3 A ˚molecular sieves at 60 mg/mL in  n -hexane. Thus, 4 A ˚molecularsieves were more fit for this condensation reaction, and itsoptimum concentration was 60 mg/mL. The effect of reactionmedium and substrate on the dehydration ability of molecularsieve is complicated ( 18 ), so the reason for the effect of molecularsieves on the yield in such a system is not very clear. The loweryield with 3 A ˚molecular sieves can be attributed to their strongwater removal ability which may reduce the lipase activity. Effect of Time Course on the Synthesis of CLS.  Figure2b showsthe time course of condensation of   β -sitosterol with CLA. Thereaction was catalyzed in  n -hexane for 96 h. The results showedthat CLS was rapidly produced within the first 24 h of the 96 hreaction. The reaction yield tended to gradually increase until72h;therefore,the optimum reaction timewas taken as72h,andthe yield was 72.6%. Effects of CLS on Plasma Cholesterol-Lowering Properties. Tables 2  and  3  show blood lipid index and liver weight (LW),LI,liverTC,andTAGsofmiceatdifferentintervals,respectively.It could be seen that at 0 week there were no differences of bloodlipid index and LW, LI, liver TC, and TAGs among the threegroups.LDL provides cholesterol to the tissues, and it is positivelyassociated with the risk of atherosclerosis (AS). HDL removesexcessive cholesterol from peripheral tissues back to the liver and Figure 1.  Effectof  β -sitosterolconcentration ( a )  andmolarratioof CLA to  β -sitosterol  ( b )  on the yield of CLS. Reaction conditions:  ( a )  molar ratioof CLA to  β -sitosterol  ( 2:1 ) , 300 mg of 3 A˚ molecular sieves, and 100 mgof lipase in 5 mL of  n  -hexane, 50   C, 150 rpm, 72 h;  ( b )  0.25 mmol of  β -sitosterol, 300 mg of 3 A˚ molecular sieves, and 100 mg of lipase in5 mLof  n  -hexane, 50   C, 150 rpm, 72 h. Figure 2.  Effect of molecular sieve type and amount  ( a )  and time course ( b )  on the yieldofCLS. Reaction conditions:  ( a )  0.25mmol of  β -sitosterol,molar ratio of CLA to  β -sitosterol  ( 1:1 ) , and 100 mg of lipase in 5 mL of n  -hexane, 50   C, 150 rpm, 72 h  [ 4 A˚  ( [ ) , 3 A˚  ( b )] ;  ( b )  0.25 mmol of  β -sitosterol, molar ratio of CLA to  β -sitosterol  ( 1:1 ) , 300 mg of 4 A˚molecular sieves, and 100 mg of lipase in 5 mL of  n  -hexane, 50   C,150 rpm.  Article  J. Agric. Food Chem.,  Vol. 58, No. 3, 2010  1901 plays a great role in keeping cholesterol homeostasis in theplasma ( 2 ), and hence, HDL can prevent AS. After 2 weeks thehyperlipidemia mouse model was established, and then there wasan abnormal increase in the levels of serum TC, serum TAGs,serum LDL-C, AI, LW, LI, liver TC, and liver TAGs of the HGgroup and HCG group, which were significantly higher thanthose of the NG group ( P  < 0.01).Hyperlipidemia is the primary risk factor of AS and one of the important inducing factors of cardiovascular disease. Theincrease of serum TC, serum TAGs, and serum LDL-C anddecrease of serum HDL-C can lead to the damnification of artery endothelial cells, accelerate the sedimentation of LDL-Cin the endothelium, and promote the formation of AS. Hence,the primary measure for preventing AS is to regulate lipidmetabolism. After 4 weeks, serum TC, serum TAGs, serumLDL-C, and AI of the HCG group were decreased, whichwere significantly lower than those of the HG group ( P <0.01),while HDL-C was not significantly different from that of theNG group, which is its advantage over the other cholesterol-lowering products such as HMG-CoA reductase inhibitors ( 2 ).The results showed that CLS may decrease serum AI of theHCG group, regulate the metabolism of blood lipids, andmay prevent the formation of AS. The LW, LI, liver TC, andTAGs of the HG group were higher than those of the NGgroup ( P <0.01), while those of the HCG group were signifi-cantly lower than those of the HG group ( P <0.01), and had nodifferences from those of the NG group. It showed that CLScould moderate the fat pathologic changes of liver.Inconclusion, our workprovidesa methodfor thesynthesis of CLS by lipase-catalyzed reaction in organic solvent using molec-ular sieves as a dehydrating agent. The reaction conditions wereoptimized, and high yields of CLS were achieved. Also, thecholesterol-lowering properties of CLS were investigated, whichshowed that CLS had a good effect for lowering plasma choles-terol level. ABBREVIATIONS USED AI, atherogenic index; CLA, conjugated linoleic acid; CLS,conjugated linoleyl  β -sitosterol; ELSD, evaporative light scatter-ingdetector;HDL-C,high-densitylipoproteincholesterol;HCG,hyperlipidemic and CLS group; HG, hyperlipidemic group;HPLC, high-performance liquid chromatography; LDL-C, low-density lipoproteincholesterol;LI,liverindex;MS, massspectro-metry; NG, normal group; TAGs, triacylglycerols; TC, totalcholesterol; TLC, thin-layer chromatography. LITERATURE CITED (1) Kritchevsky, D.; Chen, S. C. Phytosterols ; health benefits andpotential concerns: a review.  Nutr. Res.  ( N.Y. )  2005 ,  25 , 413–428.(2) Chen, Z. Y.; Jiao, R.; Ma, K. Y. Cholesterol-lowing nutraceuticalsand functional foods.  J. Agric. Food Chem.  2008 ,  56 , 8761–8773.(3) Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Phytosterols,phytostanols, and their conjugates in foods: structural diversity,quantitative analysis, and health-promoting uses.  Prog. 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Effect of phytosterols and phytostanols on the solubilizationof cholesterol by dietary mixed micelles: an in vitro study.  Chem.Phys. Lipids  2004 ,  127   (2), 121–141.(9) Adachi, S.; Nagae, K.; Matsuno, R. Lipase-catalyzed condensationof erythritol and medium-chain fatty acid in acetonitrile with lowwater content.  J. Mol. Catal. B: Enzym.  1999 ,  6 , 21–27.(10) Weber,N.;Weitkamp,P.;Mukherjee,K.D.Fattyacidsteryl,stanyl,and steroid esters by esterification and transesterification in vacuousing  Candida rugosa  lipase as catalyst.  J. Agric. Food Chem.  2001 , 49 , 67–71.(11) Ferrer, M.; Cruces, M. A.; Bernab  e, M.; Ballesteros, A.; Plou, F. J.Lipase-catalyzed regioselective acylation of sucrose in two-solventmixtures.  Biotechnol. Bioeng.  1999 ,  65 , 10–16.(12) Yan, Y. C.; Bornscheuer, U. T.; Cao, L. Q.; Schmid, R. D. Lipase-catalyzed solid-phase synthesis of sugar fatty acid esters: removal of byproducts by azeotropic distillation.  Enzyme Microb. Tech.  1999 , 25 , 725–728. Table 2.  Blood Lipid Index of Mice at Different Intervals  ( Mean ( SD,  n   = 10 ) a time  ( week  )  group TC  ( mmol/L )  TAGs  ( mmol/L )  HDL-C  ( mmol/L )  LDL-C  ( mmol/L )  AI  ( mmol/L ) 0 NG 2.05 ( 0.23 0.64 ( 0.06 1.13 ( 0.06 0.69 ( 0.25 0.80 ( 0.18HGHCG2 NG 2.87 ( 0.09 0.66 ( 0.10 1.48 ( 0.06 1.09 ( 0.11 0.95 ( 0.12HG 5.53 ( 0.16 b  0.86 ( 0.08 b  1.37 ( 0.08 b  3.77 ( 0.20 b  3.05 ( 0.34 b  HCG 5.55 ( 0.09 b  0.84 ( 0.07 b  1.36 ( 0.08 b  3.81 ( 0.11 b  3.10 ( 0.23 b  4 NG 2.88 ( 0.07 0.68 ( 0.08 1.46 ( 0.07 1.15 ( 0.16 0.98 ( 0.12HG 5.51 ( 0.09 b  0.85 ( 0.07 b  1.35 ( 0.06 b  3.78 ( 0.10 b  3.10 ( 0.19 b  HCG 3.41 ( 0.05 c  0.76 ( 0.09 c  1.44 ( 0.09 c  1.63 ( 0.11 c  1.38 ( 0.15 c a Resultsaremean ( SDoftriplicatemeasurement. n  :thenumberofmiceusedforthestatisticalanalysisineachdifferenttimeinterval. b  Valuesaresignificantlydifferentfromthose of NG at  P   < 0.01 within the same interval.  c  Values are significantly different from those of HG at  P   < 0.01 within the same interval. Table 3.  LW, LI, Liver TC, and TAGs of Mice at Different Intervals  ( Mean ( SD,  n   = 10 ) a time ( week  )  group LW  ( g )  LI TC  ( mg/100 g )  TAGs  ( mg/100 g ) 0 NG 1.27 ( 0.07 4.96 ( 0.41 5.28 ( 0.41 5.25 ( 0.11HGHCG2 NG 1.41 ( 0.09 4.85 ( 0.22 5.31 ( 0.33 5.29 ( 0.08HG 2.08 ( 0.14 b  5.72 ( 0.09 b  11.06 ( 1.85 b  11.67 ( 2.03 b  HCG 2.10 ( 0.15 b  5.77 ( 0.10 b  11.04 ( 1.91 b  11.75 ( 1.98 b  4 NG 1.43 ( 0.07 4.86 ( 0.09 5.33 ( 0.42 5.30 ( 0.13HG 2.07 ( 0.13 b  5.73 ( 0.08 b  11.05 ( 1.86 b  11.64 ( 2.01 b  HCG 1.46 ( 0.06 c  4.91 ( 0.09 c  6.18 ( 0.39 c  6.95 ( 0.43 c a Results are mean ( SD of triplicate measurement.  n  : the number of mice usedfor the statistical analysis in each different time interval.  b  Values are significantlydifferent from those of NG at  P   < 0.01 within the same interval.  c  Values aresignificantly different from those of HG at  P   < 0.01 within the same interval.  1902  J. Agric. Food Chem.,  Vol. 58, No. 3, 2010 Li et al. (13) Ferrer,M.;Cruces,M.A.;Plou,F.J.;Bernab  e,M.;Ballesteros,A.Asimpleprocedurefortheregioselectivesynthesisoffattyacidestersof maltose, leucrose, maltotriose and  n -dodecyl maltosides.  Tetrahe-dron  2000 ,  56 , 4053–4061.(14) Zaks, A.; Klibanov, A. M. The effect of water on enzyme action inorganic media.  J. Biotechnol.  1998 ,  14 , 157–167.(15) Soumanou, M. M.; Bornscheuer, U. T. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil. Enzyme Microb. Technol.  2003 ,  33 , 97–103.(16) Colombie, S.; Tweddell, R. J.; Condoret, J. S.; Marty, A. Wateractivity control: a way to improve the efficiency of continuous lipaseesterification.  Biotechnol. Bioeng.  1998 ,  60  (3), 362–368.(17) Gubicza, L.; Kabiri, B. A.; Keoves, E.; Belafi, B. K. Large-scaleenzymatic production of natural flavour esters in organic solventwith continuous water removal.  J. Biotechnol.  2000 ,  84 , 193–196.(18) Villeneuve, P. Lipases in lipophilization reactions.  Biotechnol. Adv. 2007 ,  25 , 515–536. Received for review August 21, 2009. Revised manuscript receivedDecember 8, 2009. Accepted December 8, 2009. This study wasfinancially supported by Free Exploring Program of State KeyLaboratory of Food Science and Technology  ( Jiangnan University,SKLF-TS20090065 ) , Self-determined Research Program of JiangnanUniversity  ( JUSRP30903 ) , Natural Science Foundation of JiangsuProvince  ( BK2008100 ) , National High-Tech Research andDevelopment Program of China  ( 2006AA10Z312 ) , Young FoodScientist Research Award of Jiangnan University  ( FS-200807 ) , andPCSIRT0627.
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