Study On The Anti-alcohol And Liver-protection Efficacy Of Hydrolysate From Soft Tissue Of Corbicula Fluminea

In 2016,the number of alcoholic drinkers worldwide reached 2.3 billion.The number of deaths caused by drinking worldwide is as high as 3 million.The death rate caused by drinking is higher than that of tuberculosis,AIDS and diabetes.The death toll from cirrhosis caused by alcohol in China has reached 83,341 people.At present,the control and treatment of alcoholic liver injury are manily dependent on temperance and drugs.Alcoholic liver injury drugs are mainly synthetic drugs,which all have side effects to some extent.However,food-borne anti-alcoholic peptides have a good auxiliary protective effect on alcoholic liver injury,whether in terms of effectiveness and safety.Therefore,it is urgent to find effective anti-alcoholic and liver-protecting active peptides and to study the mechanism of anti-alcoholic and liver-protecting.According to the "Compendium of Materia Medica",Corbicula fluminea has the effects of treating liver disease and anti-alcohol.However,the current research on the anti-alcohol and liver-protection of Corbicula fluminea is not systematic enough,and the active ingredients are still unknown.Moreover,Corbicula fluminea is still dominated by fresh sales,and their intensive processing technology is lacking compared with foreign countries.Therefore,Corbicula fluminea was used as raw material in this paper,controlled enzymolysis technology,ultrafiltration,separation and purification,and in vivo and in vitro activity evaluation methods.The anti-alcohol and liver-protection efficacy of hydrolysate were studied.The optimum enzymes and enzymatic process conditions for the preparation of enzymatic hydrolysates from Corbicula fluminea were optimized.The anti-alcohol and liver-protection active substances of Corbicula fluminea were tracked.To obtain peptide fragments with anti-alcohol and liver-protection activity.Designed to enrich the theory of anti-alcohol and liver-protection,and provide a theoretical basis for the high value utilization of Corbicula fluminea The results of the research are as follows:1.The basic nutrients,amino acids,fatty acid composition and inorganic elements were determined and analyzed from the perspective of food chemistry.The results show that the chemical composition of the dry base of Corbicula fluminea is as follows: 58.1% crude protein,4.7%crude fat,24.2% Total sugar,5.6% ash,1.7% non-protein nitrogen.The amino acid composition is perfect,and the essential amino acid accounts for 39.45% of the total amino acid.Lysine,histidine,proline,and alanine,leucine and other five amino acids related to the effect of sober up account for 30.04% of the total amino acid;The results of fatty acid analysis showed that Corbicula fluminea was rich in PUFA(40.01%),and the highest content was linolenic acid(14.60%).Palmitic acid was the major saturated fatty,and oleic acid was the major monounsaturated fatty acid.Their contents were determined as 25.90% and 10.00%,respectively.Also,Corbicula fluminea was an excellent source of minerals,such as Ca,Fe,Zn and Se.2.The effect of hydrolysate from Corbicula fluminea soft tissue on the activation of alcohol dehydrogenase(ADH)in vitro was determined,and its effects of anti-alcohol and liver-protection on mice were also observed by measuring the drunk time,sober-up time,concentration of ethanol in serum,the contents of malondialdehyde(MDA),triglyceride(TG)and glutathione(GSH)in liver tissue of mice.The results showed that hydrolysate from soft tissue of Corbicula fluminea could activate ADH in vitro,activation rate 52%,it could prolong drunk time and shorten the sober-up time of drunken mice significantly,by 69.42% and 13.40%,respectively,with the comparation of negative group.Which reduced the concentration of ethanol in mice serum significantly(P<0.05),reduced the contents of MDA and TG in liver tissue of mice significantly(P<0.05),and slow down the consumption of GSH in mice significantly(P<0.05).Therefore,the hydrolysate from Corbicula fluminea soft tissue can activate ADH in vitro,anti-alcohol and protect liver in drunk mice.3.The activation rate of ADH in vitro was the main evaluation index,and the protein recovery rate was the reference evaluation index.The preparation process of the hydrolyzed peptides of Corbicula fluminea was optimized by means of sieving enzyme,single factor test and response surface test,and optimized.The hydrolyzed polypeptide product of Corbicula fluminea was evaluated for its activity anti-alcoholic liver injury in vivo.The results showed that animal protease was the most suitable enzyme for the preparation of antialcoholism polypeptide from Corbicula fluminea.Compared with the other five proteases,the hydrolysate had the highest activation rate of ADH(428.40±0.66%)and the protein recovery rate reached 73.53±4.30%.The optimum conditions of enzymatic hydrolysis were as follows: time 52 min,hydrolysis enzyme amount 1530 U/g,pH 7.5.Under these conditions,the activation rate of ADH was 454.6±3.21%.Animal experiments showed that the optimized enzymatic hydrolysate of Corbicula fluminea could significantly reduce the activities of ALT and AST in serum and the contents of MDA,TG and CYP2E1,slow down the consumption of GSH,improve the activities of ADH and ALDH in liver tissues of acute alcohol intake mice.Therefore,the optimized enzymatic hydrolysate of Corbicula fluminea has an auxiliary protective effect on the liver of acute alcohol intake mice,and in the range of total nitrogen concentration of 2.5~10 mg/mL,it has a certain dose effect.4.Separation of enzymatic hydrolysate by ultrafiltration and high effect size exclusion chromatography,And determination the molecular weight distribution of each ultrafiltration component,Then,four ultrafiltration components in the <2.5 kDa group,2.5~5 kDa group,5~10 kDa group and >10 kDa group were evaluated for anti-alcoholic liver injury activity by animal test.The results showed that the hydrolyzed product of Corbicula fluminea can achieve better separation by ultrafiltration.The protective effect of 2.5~5 kDa ultrafiltration fraction on liver of acute alcohol intake mice was better than that of other fractions.5.In vitro ADH activation rate evaluation method was used for activity tracking.Sephaacryl S-100 gel chromatography and RP-HPLC were used to separate,purify and screen the 2.5~5 kDa ultrafiltration components of the hydrolysate.Finally,a peptide segment with a strong activation effect on ADH was obtained(Mw:446~476 Da).