Preparing Of Linoleic Acid Isomerase By Fermentation To Transform Pine Nut Oil And Its Mechanism Of Lowering Blood Lipid

Pine nut oil is a good source of Conjugated linoleic acid(CLA).Because it is rich in up to 50%linoleic acid.CLA is an essential fatty acid that can only be ingested by food.It has important physiological functions such as inhibiting fat deposition,improving immunity and treating cardiovascular diseases.CLA can be prepare by microbial fermentation and chemical isomerization from linoleic acid(LA).The most biologically active isomers are c-9,t-11 CLA and t-10,c-12 CLA.In this paper,the water-enzymatic extraction of pine nut oil(PNO)was innovated.Linoleic acid isomerase produced by Lactobacillus plantarum Lp-01 was used to transform LA in PNO into CLA,to prepare PNO containing CLA by one-step method.To explore the mechanism and correlation of conjugated linoleic acid of pine nut oil in improving lipid metabolism abnormalities induced by high fat diet and remodeling intestinal microbial homeostasis.The main results are as follows:(1)Response surface methodology was used to optimize the preparation process of linoleic acid isomerase(LAI)from Lactobacillus plantarum Lp-01.The optimal fermentation conditions were as follows:inoculation amount 2%,fermentation temperature was 36?,time 20 h,substrate concentration 11%.Under these optimal conditions,the experimental yield of CLA was 33.47 ?g/mL.Pine nut oil was fermented by Lactobacillus plantarum Lp-01 to obtain the CLA isomers mixture of c-9,t-11 CLA and t-10,c-12 CLA,with the proportions of 4.91%and 4.86%,respectively.(2)The optimal ammonium sulfate saturation of Lp-01 LAI was determined to be 40%?80%,to precipitate the crude enzyme solution.The enzyme solution was concentrated after dialysis.DEAE-Sepharose F.F.anion exchange gel and Sephadex gel G-100 were sequentially used to separate and purify the enzyme.The molecular weight of Lp-01 LAI was 41 kDa determined by SDS-PAGE electrophoresis.The enzymatic properties of LP-01 LAI were analyzed.The results showed that Lp-01 LAI was not heat-resistant to temperature.And the optimum reaction temperature was 35? and pH was 6.(3)On the basis of aqueous enzymatic extraction of PNO,the selected complex enzyme preparation was combined with Lp-01 LAI to construct complex enzyme system for synergistic isomerization to prepare PNO-CLA.The optimum extraction process was as follows:pH was 5.7,temperature was 37?,the amount of Lp-01 LAI was 1.4 ml(200 U/ml).Under the optimal conditions,the yield of pine nut oil was 56.36%,and the concentration of CLA was 49.86 ?g/mL.The chemical properties of PNO and PNO-CLA were compared,both of them met the national standard of edible oil.The acid value and peroxide value of PNO-CLA were significantly lower than PNO.The main fatty acids of PNO-CLA and PNO were comprised of oleic acid,linoleic acid and pinolenic acid.The LA content of PNO-CLA is as high as 46.22%,containing 0.6%of the CLA isomers mixture of c-9,t-11 CLA and t-10,c-12 CLA.The total content of CLA can reach 8 mg/g.The scavenging ability and reducing power of PNO-ClA on hydroxyl radical were significantly higher than those of PNO(P<0.05).(4)The mice hyperlipemia model was established,and the regulation mechanism of PNO and PNO-CLA dietary intervention on dyslipidemia induced by high fat diet was determined by in a vivo experiment.The results showed that PNO-CLA could significantly reduce the serum lipid levels(TC,LDL-C,AI index)and the risk of atherosclerosis in high-fat diet mice after 7 weeks of intervention.It also increased the antioxidant capacity(SOD,GSH-Px,MDA)and total lipase activities(LP,HPL),and decreased the levels of inflammatory factors(IL-6,TNF-?)in plasma(P<0.05).The histopathology results of liver and adipose tissue showed that PNO-CLA could ameliorate hepatocyte injury and reduce fat deposition in liver and abdomen of high-fat diet mice to a certain extent.The expression levels of HMG-CoAR and SREBP-lc in the liver of PNO-CLA group decreased,while the expression levels of AMPK?,PPARa and CYP7A1 were increased.The protein expression of SREBP-1c and HMG-CoAr decreased,and the protein expression of PPARa increased,which was consistent with the change of gene expression level.Therefore,PNO-CLA may regulate liver cholesterol metabolism and reduce liver cholesterol level by regulating the expression of biomarkers involved in cholesterol metabolism in the liver.(5)By extracting microbial DNA from feces,the 16S rDNA gene V3-V4 region of intestinal microflora of mice was amplified.The effects of PNO-CLA intervention on intestinal microflora of high-fat diet mice were detected by high-throughput sequencing.The results showed that a high-fat diet could destroy the structure of intestinal flora,reduce the Alpha diversity and richness of intestinal flora in mice.The supplementation of PNO-CLA can regulate the intestinal flora structure of the host and increase the relative abundance of Lactobacillus,Bacteroides,Akkermansia,Clostridium XlVa,Alloprevotella and Helicobacter.Restore the diversity of intestinal flora.PNO and PNO-CLA can significantly reduce the F/B value of intestinal flora,thus inhibiting obesity caused by high fat diet in mice.In the correlation analysis of lipid metabolism related indicators and intestinal microbes,the correlation results showed that SCFAs concentration was positively correlated with the relative abundance of Bacterioides,Barnesiella,Allobaculum,Alistipes and Ruminococcus.