Study On The Preparation Of Mussel Oligosaccharides And Its Related Hypolipidemic Mechanisms

At present,hyperlipidemia caused by lipid metabolism disorder has become a pathogenic factor that has an important impact on human life and health,especially has a close relationship with the incidence of human cardio-cerebrovascular diseases.According to the guidelines for the Prevention and treatment of Dyslipidemia in Chinese Adults(revised in 2016),the overall prevalence of dyslipidemia in China is as high as 40.4%.According to relevant data monitoring,the overall sales of lipid-regulating drugs reached 21.165 billion yuan in 2016,showing an overall upward trend.With the acceleration of the aging of the social population and the sound development of the management system of chronic diseases,it is expected that lipid-regulating drugs will still be the mainstream products of cardio-cerebrovascular drugs in the future.The popular drugs for regulating blood lipids in the market are HMG-CoA reductase inhibitors,nicotinic acids,bates,cholesterol absorption inhibitors,cholic acid chelating agents,PCSK9 inhibitors and fish oil unsaturated fatty acid preparations,among which statins are the most widely used,but statins have adverse reactions related to liver,kidney and body metabolic disorders and a variety of side effects,such as gastrointestinal reactions,rhinitis,headache,muscle toxicity and so on.Therefore,the discovery of low-toxic and efficient natural lipid-regulating drugs from natural drugs has become a new research direction.Mussel polysaccharides have many biological activities,such as immune regulation,anti-tumor,anti-oxidation and so on.Because its main chain is the same as the starch main chain,it is speculated that the branched chain connected by ?-1,2 glycosidic bond is an effective form to exert biological activity.Because MP-A is a kind of biological macromolecule that can not be absorbed by intestinal tract,it can not meet the requirements of drug declaration,so this subject will use enzymatic hydrolysis to degrade MP-A to get mussel oligosaccharides,and explore its hypolipidemic activity and its mechanism,so as to provide theoretical basis for subsequent development and clinical application of mussel oligosaccharides.The main results are as follows:(1)study on the preparation technology of MOS-1 Enzymatic hydrolysis has the characteristics of high efficiency,substrate specificity and mild conditions.in this study,the original enzymatic hydrolysis process in vitro was optimized.Under the same hydrolysis conditions,the enzymatic hydrolysis effects of mesophilic ?-amylase and Glucoamylase on MP-A were compared under the same hydrolysis conditions,and then the enzymatic hydrolysis conditions(enzyme concentration,polysaccharide concentration,enzymatic hydrolysis time),yeast monosaccharide removal conditions(yeast concentration,reaction time)and ethanol precipitation times were optimized.Finally,the optimal enzymatic hydrolysis process was obtained,which is as follows:preparation of 10 g/L MP-A solution,adjust pH to 5.2,add medium-temperature ?-amylase to make its final concentration to 5 g/L,stir evenly,boil water bath for 15 min,filter and collect filtrate,add dry yeast powder according to 4 g/L concentration,150 rpm,shaker culture at 30? for 20 h,filter and remove bacteria,add 10 times volume of ethanol to remove MP-A,and concentrate the supernatant to the original solution volume,MOS,is finally separated by series gel chromatography.Although oligosaccharides with a wide degree of polymerization can be obtained by enzymatic hydrolysis in vitro,the separation and purification process of subsequent enzymatic hydrolysis products is more complex,and the hydrolysis temperature of mesophilic ?-amylase is higher,which is easy to produce Maillard reaction with sugars,resulting in non-enzymatic browning of MP-A hydrolysate.In order to simplify the degradation of MP-A and reduce the substrate feedback inhibition in the process of enzymatic hydrolysis,the codon preference of cryogenic amylase gene Amy derived from Penicilliumsp.in P.pastoris was optimized.after whole gene synthesis,the cryogenic amylase gene Amy was integrated into P.pastoris expression system to construct recombinant P.pastoris SMD1168H-Amy,to screen and verify the positive transformants.The recombinant strain was expanded and cultured,collected and put into MP-A solution for enzymatic hydrolysis,followed by filtration removal of yeast,ethanol precipitation of undegraded MP-A and gel chromatography purification steps to isolate MOS-1 with a purity of 90.7%.At the same time,the MOS-1 was prepared by enzymatic hydrolysis in vitro and recombinant P.pastoris,and the mussel oligosaccharides were weighed respectively,and the yield of mussel oligosaccharides was calculated according to the formula(mussel oligosaccharide mass/mussel polysaccharide used × 100%).It is calculated that the yield of MOS-1,prepared by enzymatic hydrolysis is 0.59%;the yield of MOS-1,prepared by recombinant yeast is 1.87%,which is 3.17 times that of enzymatic hydrolysis.Compared with the in vitro enzymatic hydrolysis process,the high purity and one-component MOS-1,prepared by recombinant yeast can not only meet the requirements of drug declaration,but also shorten the time,reduce the cost and increase the yield.(2)study on the hypolipidemic effect and mechanism of MOS-1 the hyperlipidemic mouse model was established by high-fat diet induction,and the blood lipid regulation of MOS-1 was discussed in three aspects:blood lipid level,lipid genes and intestinal microflora.In the course of the experiment,the daily diet,activity,hair and body weight of mice were observed,the levels of blood lipids TG,TC,HDL-C and LDL-C were measured before and after administration,and the mRNA levels of lipid metabolism genes HL,LPL,FAS,ACC,CYP7A1,HMG-CoAreductase,LCAT and PPAR a in liver were measured by real-time fluorescence quantitative PCR.Finally,16s bioinformation analysis of mouse intestinal flora was carried out with the help of high-throughput sequencing,including OTU clustering,species annotation,?-diversity analysis and ?-diversity analysis.The results showed that after intragastric administration of MOS-1,the levels of serum TG and LDL-C in the MOS-1 intervention group were significantly lower than those in the model ? group(P<0.01),and the levels of HL and PPAR a transcription in the liver viscera of the MOS-1 group increased compared with the model ? group,but there was no significant difference in the transcription levels of LPL,ACC and cholesterol metabolism related CYP7A1,HMG-CoA and LCAT.MOS-1 can regulate the structure,richness and degree of freedom of intestinal flora in hyperlipidemic mice.MOS-1 can significantly reduce the proportion of pathogenic bacteria and increase the proportion of probiotics Allobaculum(P<0.001),so that the intestinal flora of hyperlipidemic mice tend to return to normal.