Study On Hypoglycemic Activity Of Pinctada Martensii Flesh Enzymatic Hydrolysate And Product Development

Pinctada martensii(Dunker),mainly produced in Beihai,Guangxi,is the protozoan of seawater pearl.The 2020 edition of the "Chinese Pharmacopoeia" records that pearls are sweet,salty,and cold,enter the heart and liver meridians,and have the effects of soothing the nerves and calming shocks,improving eyesight and eliminating nebula,detoxifying and strengthening muscles,moisturizing skin and removing freckles.The mother-of-pearl is salty and cold.It returns to the liver and heart meridians and has the effect of calming the liver and subduing the yang,soothing the nerves and calming fear,improving eyesight and relieving nebula.Pearls and motherof-pearl originating from Pinctada martensii have been used in traditional Chinese medicine for thousands of years.Their pearls and shells are traditional Chinese medicines,and there are records of herbal medicines in all dynasties.However,in the process of producing and processing pearls and mother-of-pearl,only part of the flesh body of the mother martensii oyster that nurtures the pearls is eaten,and most of them are discarded as waste,resulting in waste of resources and pollution of the marine environment.Modern research shows that the Pinctada martensii flesh is rich in protein,polyene unsaturated fatty acids,and various inorganic salts and trace elements needed by the human body,and is rich in nutrients.Pinctada martensii flesh was taken as the research object,and the enzymatic hydrolysis technology is used to study the aspects of enzymatic hydrolysis process optimization,material basis,evaluation of hypoglycemic activity,hypoglycemic mechanism,intake and transport,and preparation process.The main contents are as follows:1.Literature researchThe current situation of utilization of marine shellfish,the research on the function and activity evaluation of marine shellfish active peptides,the preparation and identification of active peptides were reviewed,and the discovery and application of shellfish active peptides were also prospected.2.Optimization of Pinctada martensii flesh enzymatic hydrolysis processUsing DPP-Ⅳ inhibitory activity and glucose consumption of HepG-2 cells as indicators to optimize the enzymatic hydrolysis process of Pinctada martensii flesh,the optimal process was determined as follows:acid protease enzymatic hydrolysis,45℃,time 3 h,material-liquid ratio 1:3.5(w:w),1000 U/g protamex.The DPP-Ⅳ inhibition rate under this condition was 74.21%,and the glucose consumption of HepG-2 cells was 37.53%.3.Study on the substance basis of Pinctada martensii flesh glucopeptideThe total sugar content of the enzymolysis peptide was 12.99%±0.62%by phenol sulfuric acid method,and 69.74%±1.52%by polypeptide kit.263 peptides were identified by nano LCMS/MS,of which 44.11%were hydrophobic,60.08%of which had at least one hydrophobic amino acid at the N-terminus,and 85.17%of which had a molecular weight lower than 2000 Da.4.Evaluation of hypoglycemic activity of Pinctada martensii fleshThe hypoglycemic activity of enzymolysis peptide of Pinctada martensii flesh was evaluated in db/db mice with self-released diabetes.With Sitagliptin Phosphate/metformin Hydrochloride Tablets as the positive drug,the effects of high(900 mg/kg)and low dose(300 mg/kg)of flesh enzymolysis peptide on glucose index,blood lipid and key protein expression of PI3K-AKT pathway in db/db diabetic model mice were compared before and after administration.The result is that the flesh enzymolyzed peptide of Pinctada martensii can reduce the blood sugar of db/db mice,improve the glucose tolerance and insulin tolerance,and increase the concentration of IRS1,p-PI3K/PI3K and p-Akt/Akt in liver tissue.The contents of TC,TG,LDL-C,ALT and AST in serum were decreased,and the contents of HDL-C were increased.These suggest that flesh enzymolysis peptide can improve lipid metabolism,and activate PI3K-AKT pathway.5.Study on the potential pathways of flesh enzymatic peptides from Pinctada martensii in the intervention of diabetes mellitusThe method of label free quantitative proteomics was used to qualitatively and quantitatively analyze the proteins in the livers of the mice in the control group,the model group,and the flesh enzymatic peptides group.KEGG and GO analysis were performed on the screened differentially expressed proteins(p<0.05),and the proteins related to T2DM and the changes and regulatory pathways of related proteins after the intervention of Pinctada martensii flesh were screened.Western blot and qRT-PCR were used to verify the differences in the expression of differential proteins at the protein level and mRNA level between different groups.As a result,a total of 231 potential differential proteins were obtained for the regulation of T2DM by flesh enzymolysis peptides of Pinctada martensii.The protein biomarkers involved in T2DM were screened(p<0.05,|log2FC|>1.2),mainly Cyp1a2,Mup20,Cyp2a5,Lumican,Col6a1,Mrpl3,Etnppl,Dynlrb1,Gsdmdc1,Slc10a1,Bclf1,H2-D1,Magohb,Cyp2a4,Magoh,involved in retinol metabolic pathways,oxidative phosphorylation,glycolysis/gluconeogenesis and other processes,cytoplasmic and mitochondrial components,small molecule metabolism,REDOX,drug metabolism,nucleotide metabolism,and other biological processes,cytoplasm,mitochondria and other cellular components,focusing on the molecular functions of REDOX enzyme activity,catalytic activity,small molecule binding,cofactor binding and so on.The high-dose group of flesh enzymolyzed peptides from Pinctada martensii can significantly up-regulate the expressions of Cyp1a2,Adh4,and Rdh11 in the liver of mice,and significantly down-regulate the expressions of Aldh1a1 and Retsat mRNAs in the livers of mice,which suggests that the flesh enzymolyzed peptide of Pinctada martensii may improve T2DM by regulating the retinol metabolic pathway.The other differential proteins screened were verified,and the results showed that the highdose group of flesh enzymolyzed peptides from Pinctada martensii could significantly up-regulate the expressions of Col6a1,Lumican,Magohb,Mup 20 mRNA,and H2-D1 mRNA in mouse liver,suggesting that Pinctada martensii flesh enzymatic peptides may improve diabetic retinopathy by increasing cell adhesion and regulating retinal clarity.6.Study on uptake and transport of Pinctada martensii fleshThe flesh enzymolysis peptides of Pinctada martensii were labeled by Pseudo-isobaric dimethyl method,and the uptake and transport of enzymolysis peptides were studied based on Caco-2 cells.After the labeled peptide was given to Caco-2 cells for 2 h,90 peptides were identified in the control group,99 peptides were identified in the administration group.After removing the peptides in the control group,23 peptides were identified to coincide with labeled peptides in the administration group,suggesting that these 23 peptides may be taken up by Caco2 cells.Caco-2 cells were inoculated into a transwell plate and cultured for 21 d.The labeled peptide was given to Caco-2 cells,and samples were taken from the lower chamber of the transwell plate at 0,0.5,1,1.5,2,2.5,and 3 h for nano LC-MS/MS detection and PEAKS search,respectively.It was found that Caco-2 cells could transport more than 95%of the peptide after 0.5 h of labeled peptide administration,the transport reached saturation after 1 h of administration,and the transport rate almost did not change with time.7.Research on preparation technology of Pinctada martensii fleshPreliminary studies have found that compound protease and acid protease obtained enzymolysis peptides have comparable hypoglycemic activity,but acid protease enzymolysis conditions are cumbersome,so compound protease was selected for enzymolysis in pilot test.In this paper,complex protease was used for enzymatic hydrolysis of Pinctada martensii flesh,and the spray-drying process of the enzymatic hydrolysis solution was optimized according to the powder yield and moisture absorption rate.The drying temperature was 130℃,and the injection flow rate was 6.0 mL/min for spray drying,and the obtained intermediate had the best hygroscopicity.The flesh fishy smell of Pinctada martensii is relatively heavy,and the enzymatic hydrolysis solution prepared from this raw material still retains its fishy smell is very easy to absorb moisture and is intended to be made into tablets.The wetting agent was determined to be 95%ethanol,and 40%sorbitol and 0.5%magnesium stearate were added during wet granulation,and tableting was performed,each 0.5 g,to obtain "Pinctada martensii peptide tablets".The quality of intermediates and preparation on the "Pinctada martensii tablets" was studied..Finally,we worked out a draft on "Pinctada martensii tablets".