Study On The Enzymatic Synthesis Of ?-sitosterol Laurate In Reverse Micelle System And Its Lowering Cholesterol Efficiency

Phytosterols are regarded as one kind of functional compounds able to reduce cholesterol in the blood.However,the low solubility in oil or water and the high melting point of free phytosterols limit their bioavailability in human body and wide applications in the fields of food and medicine.In order to solve these problems,people usually transport free phytosterols into phytosterol esters.Phytosterol esters have the significant effect on lowering cholesterol as well.What's more,due to the considerably greater solubility in oils and the lower melting point,the bioavailability of phytosterol esters is increased and the application in the industries of food and machine becomes more available.At present,the synthesis of phytosterol esters is mainly conducted in the organic solvent or free-solvent system using the lipase as the catalyst,which has the disadvantages of long reaction time and low synthetic efficiency.To solve this problem,this paper focused on establishing a new method of enzymatic synthesis of ?-sitosterol laurate(?-SLE),which adopted a reverse micelle(RM)system as the reaction system.Besides,the efficiency of lowering cholesterol by ?-SLE and the underlying mechanism were explored through animal experiments.The main research results have been listed as followed:1.The water/bis(2-ethylhexyl)succinate sodium sulfonate(AOT)/isooctane enzymatic RM carrying Candida Rugosa lipase AY30(CRL AY30)was established,which was identified by Dynamic Light Scattering technology.Through a stepwise optimization method,we found that CRL AY30 had the best activity in the enzymatic RM system which adopted 50 mmol/L phosphate buffer solution with a pH of 7.5 as the hydrophilic core to carry CRL AY30 and was prepared in a proportion of [CRL AY30](mg/mL),[water](mmol/L)and [AOT](mmol/L)= 3:375:25 in isooctane.Comparing with 25 mmol/L AOT/isooctane,375 mmol/L water/isooctane or isooctane enzymatic systems,CRL AY30 had better activity in the RM enzymatic system.It could catalyzed synthesize 55.62% ?-SLE after 24 h or 72.71% ?-SLE after 48 h when substrate mole ratio(?-sitosterol/lauric acid,25 mmol/L ?-sitosterol)was set as 1:4,enzyme load as 10%(w% total reactants)and reaction temperature as 45 �C.Thus,the above results indicated that the water/AOT/isooctane RM enzymatic system can be used for the synthesis of ?-SLE.2.The esterification reaction conditions in the water/AOT/isooctane RM enzymatic system were optimized.The identification of ?-SLE was done by thin layer chromatography,high performance liquid chromatography-triple quadrupole mass spectrometry and infrared spectroscopy methods and the quantification was detected by high performance liquid chromatography(HPLC)method.The results of singlefactor experiments and the response surface method showed that the maximum yield rate was increased to 86.82% under the conditions as follow: substrate mole ratio(?-sitosterol/lauric acid,25 mmol/L ?-sitosterol)of 1:3.5,enzyme load of 18%(w% total reactants),reaction temperature of 45 �C and reaction time of 48 h.3.0.2% cholesterol diet and peanut oil were used to establish a model of hypertriglyceridemia and hypercholesterolemia in Syrian golden hamster.At the same time,the low,medium and high dose of ?-SLE(11,22 and 44 mg per 100 g of weight,respectively)were fed to hamsters by gavage for six weeks.Through analyzing the lipids in the serum and the liver,the morphology of epididymal adipocytes and hepatic cells,and the composition of total fatty acids in the liver,it was found that ?-SLE had the significant effect on lowering cholesterol,of which the medium dose exerted the best function.In the medium dose group,the lipid level of total cholesterol in the serum and liver were significantly decreased by 30.9% and 33.6%(P < 0.05),respectively,when comparing with these in the model group.At the same time,?-SLE played a role in lowering fat and regulating the metabolism of lipids,of which the medium dose exerted the best function.In the medium dose group,the lipid level of total triglyceride in the serum and liver were decreased by 30.9%(P < 0.05)and 13.6%,respectively,as well as the sizes of epididymal adipose tissue cells and liver cells were reduced to the utmost extent when comparing with these in the model group.Besides,the proportions of total saturated fatty acid and total unsaturated fatty acid in the medium group were closest to those in the control group.In the medium group,the proportions of total saturated fatty acid and total unsaturated fatty acid were 37.62% and 60.75%,respectively,while in the control group,the proportions of total saturated fatty acid and total unsaturated fatty acid were 35.32% and 63.19%,respectively.In medium dose group,the ratio of n-6 to n-3 polyunsaturated fatty acid was the best,being 4.32,and the proportion of medium chain fatty acid was the highest,being 3.00 times that of the model group.The functional enzymes were also determined.As a result,it was found that ?-SLE can increase the activity of glutathione-S-transferase(GST)in the liver.Compared with the activity of GST in the model group,the activities of GST were increased by 59.4%,68.8% and 66.2%(P < 0.05)in the low,medium and high dose of ?-SLE groups,respectively.4.HPLC method was applied to measure the content of cholesterol in the feces.It was shown that the low,medium,and high intakes of ?-SLE significantly increased the contents of cholesterol in by 13.2%,21.4% and 65.9% per gram of feces(P < 0.05),respectively,when comparing with this in the model group,suggesting that ?-SLE can inhibit the absorption of diet cholesterol.The Elisa analysis of enzymes related to the absorption of cholesterol in the small intestine showed that ?-SLE inhibited the absorption of cholesterol mainly through down-regulating the expression quantity of niemann-pick c1-like protein 1(NPC1L1),of which the medium dose exerted the best function.Comparing with what in the model group,the expression quantity of NPC1L1 significantly decreased by 22.6% in the medium group(P < 0.05).Besides,the high intake of ?-SLE significantly down-regulated the expression quantities of acyl CoA cholesterol acyltransferase 2 and microsomal triacylglycerol transport protein by 8.5% and 9.3%,respectively,when comparing with these in the model group(P < 0.05).Ultra-fast liquid chromatography-triple quadrupole mass spectrometry method was applied to measure the contents of bile acids in the feces and liver.It was shown that the contents of total bile acids(TBA)in the liver increased with the dose of ?-SLE,which were 148.018,281.303 and 830.205 ng/mg prot,respectively,in the low,medium,and high dose of ?-SLE groups.The contents of TBA in three dose groups were significantly lower than this in the model group while the contents of TBA in the medium and high dose groups were significantly higher than this in the control group(P < 0.05).The contents of TBA in the feces increased with the dose of ?-SLE as well,which were 1030.507,1430.901 and 1566.969 ng/mg prot,respectively,in the low,medium,and high dose of ?-SLE groups.The contents of TBA in three dose groups were significantly lower than this in the model group but higher than this in the control group(P < 0.05).Therefore,it was suggested that ?-SLE might directly promote the decomposition of cholesterol into bile acids in the liver,and/or promote the excretion of bile acids,thereby indirectly promoting the decomposition of cholesterol into bile acids in the liver.The further Elisa analysis of enzymes related the synthesis of bile acids in the liver showed that the low,medium,and high intakes of ?-SLE significantly up-regulated the expression quantity of cholesterol 7?-hydroxylase(CYP7A1)by 7.6%?16.9% and 12.3%,respectively,and the medium and high intakes of ?-SLE significantly up-regulated the expression quantity of sterol 27-hydroxylase(CYP27A1)by 31.% and 21.7%,respectively,when comparing with these in the model group(P < 0.05).The Elisa analysis of enzymes related the resorption of bile acids in the ileum showed that the low and medium intakes of ?-SLE significantly down-regulated the expression quantity of apical sodium-dependent bile acid by 17.7% and 14.5%,respectively,when comparing with this in the model group(P < 0.05).Therefore,it was suggested that ?-SLE might promote the excretion of bile acids,thereby indirectly promoting the decomposition of cholesterol into bile acids in the liver.In addition,the expression quantities of ileal bile acid binding protein(IBABP),farnesoid x receptor(FXR)in the ileal and FXR in the liver were reduced by ?-SLE,which also can induce the up-regulation of the expression quantities of CYP7A1 and CYP27A1.The high take of ?-SLE had biggest inhibition effect on the expression quantities of IBABP,FXR in the ileal and FXR in the liver.Comparing with what in the model group,the expression quantities of IBABP,FXR in the ileal and FXR in the liver were significantly reduced by 28.7%,39.4% and 26.4%,respectively(P < 0.05)in the high dose group.Taking this result into consideration,it was inferred that ?-SLE might directly promote the decomposition of cholesterol into bile acids in the liver.However,this hypothesis needed further verification.Consequently,the mechanism of the effect on lowering cholesterol of ?-SLE may be: 1)to lower the absorption of cholesterol in the small intestine.2)to promote the excretion of bile acids through reducing the reabsorption of bile acids in the ileum,thereby indirecetly promoting the decomposition of cholesterol into bile acids in the liver.