Enzymatic Synthesis Of Hydrophilic β-sitosterol Glycosyl Derivatives And Their Cholesterol-lowering Properties

Phytosterols have attracted much attention due to their cholesterol-lowering activity and the effect of preventing cardiovascular disease.However,the low oil solubility,water insolubility and high melting point of phytosterols seriously affect their intestinal absorption and limit their application in food processing.Studies have shown that the hydrophilic structure modification of phytosterols is an effective way to improve their water solubility and bioavailability.At present,hydrophilic phytosterol derivatives are mostly synthesized by chemical method or chemo-enzymatic method.There are some problems such as high reaction temperature,many by-products,catalyst residue and low conversion rate.In addition,there is currently a lack of systematic research on the digestion and absorption of hydrophilic phytosterol derivatives,as well as the regulation of the physicochemical behavior of intestinal cholesterol micelles.With β-sitosterol,we plan to construct hydrophilic β-sitosterol glycosyl derivatives,to improve the physicochemical properties and bioavailability of phytosterol,explore the mechanism of β-sitosterol glycosyl derivatives on intestinal cholesterol micelles,and clarify the molecular interaction between β-sitosterol glycosyl derivatives and bile salts.The main results are as follows:(1)A two-step enzymatic transesterification reaction route for the preparation of hydrophilic β-sitosterol glycosyl derivatives was designed and the reaction conditions were optimized.In the first step,β-sitosterol and divinyl adipate were catalyzed by Candida rugosa lipase(CRL)at low temperature and in short time(35 °C,30 min).The hydrolysis rate of β-sitosterol vinyl adipate was less than 4% and the yield of β-sitosterol vinyl adipate was more than 94%.In the second step,a series of hydrophilic β-sitosterol glycosyl derivatives were prepared by using β-sitosterol vinyl adipate the intermediate and hydrophilic components(erythritol,xylitol,sorbitol,glucose,sucrose and raffinose)under the catalysis of alkaline protease from Bacillus subtilis.The hydrolysis rate of β-sitosterol vinyl adipate was less than 6%.The yield of β-sitosterol glycosyl derivatives was more than90%.The structure of β-sitosterol glycosyl derivatives was identified by FT-IR,HPLC-MS and NMR,and the key acyl sites of β-sitosterol coupling with hydrophilic components were identified.(2)Hydrophilic structure modification had positive effects on the physical and chemical properties of phytosterols.Compared with free β-sitosterol,β-sitosterol glycosyl derivatives possess good water solubility(3.65-8.02 m M at 35 °C),improved wettability(85.4-38.2 ° at static contact angle),enhanced thermal stability(310-360 °C at second stage decomposition temperature),higher emulsifying activity,lower crystallinity and good hydrogen bond networks.The change of physical and chemical properties is strongly dependent on the coupled hydrophilic groups,and changes regularly with the number of hydrophilic groups.(3)The bioaccessibility of β-sitosterol glycosyl derivatives(41.55%-63.61%)was 10 times higher than that of free β-sitosterol(< 6%)in the non-fat system during in vitro gastrointestinal simulation.In Caco-2 cell model,the uptake rate of β-sitosterol glycosyl derivatives(4.11%-6.82%)was higher than that of free β-sitosterol(< 1.8%).The results showed that the hydrophilic structure modification improved the digestion and absorption behavior of phytosterols and increased the bioavailability of phytosterols.β-Sitosterol glycosyl derivatives were partly hydrolyzed to free β-sitosterol and β-sitosterol adipate under the action of digestive enzymes in the body,and the degree of hydrolysis of β-sitosterol glycosyl derivatives was affected by the spatial structure of hydrophilic groups.(4)In the in vitro hydrolysis model,β-sitosterol and β-sitosterol glycosyl derivatives showed different cholesterol-lowering mechanisms.β-Sitosterol promoted the digestion of lipids and the release of free fatty acids,and significantly promoted the accumulation of cholesterol in the precipitated phase without affecting the distribution of oleic acid.For example,when 6 m M β-sitosterol was added,cholesterol distribution in precipitated phase was > 89% and the oleic acid distribution in precipitated phase was < 10%.β-Sitosterol might reduce the micellar solubility of cholesterol due to the effect on precipitation and competitive dissolution with cholesterol.Unlike β-sitosterol,β-sitosterol glycosyl derivatives significantly promoted the accumulation of cholesterol and oleic acid in the precipitated phase.For example,when 6 m M β-sitosterol glycosyl derivatives were added,the cholesterol distribution in the precipitated phase was 17.55%-75.57%,and the oleic acid distribution in the precipitate phase was 26.50%-68.27%.β-Sitosterol glycosyl derivatives adsorbed on the lipid droplet surface,changed the properties of the particles,reduced the electronegativity of the particles,thus interfering with cholesterol micellization.(5)The interaction between β-sitosterol and cholesterol was stronger than that betweenβ-sitosterol and cholesterol-loaded bile salt micelles,indicating that the coprecipitation mechanism was superior to the competitive dissolution mechanism.When β-sitosterol glycosyl derivatives were added into cholesterol-loaded bile salts micelles,resulting in a sharp non-linear decrease in the micellar solubility of cholesterol(from an initial micellar solubility of 1.87 ± 0.02 m M to 0.78 ± 0.07 m M).The reduced particle size,reduced surface potential electronegativity and changes in microscopic morphology of mixed micelles indicate that β-sitosterol glycosyl derivatives were indeed involved in the formation of mixed micelles.Changes in key NOE cross-peaks in the binary system of sodium taurocholate +β-sitosterol glycosyl derivatives suggested that β-sitosterol glycosyl derivatives interacted with sodium taurocholate via hydrogen bonds and altered the molecular arrangement of sodium taurocholate,thereby affecting the physical characteristics and functional properties of mixed micelles,resulting in low micellar solubility of cholesterol.In addition,the glycosyl components of β-sitosterol glycosyl derivatives obviously affected the intermolecular interaction.In conclusion,a mild and efficient enzymatic synthesis route for hydrophilic β-sitosterol glycosyl derivatives was established,and the positive effects of hydrophilic structure modification on physicochemical properties,digestion and absorption of phytosterols were systematically clarified.The key factors that hydrophilic β-sitosterol glycosyl derivatives affected cholesterol micellization were determined.This study has certain social value and practical significance to develop phytosterol dietary nutritional supplement.