Study On The Preparation Of Chicken Protein Source ADH Activating Peptides And The Protective Mechanisms Against Alcohol-induced Liver Injury In Mice

In recent years,alcoholic liver disease caused by excessive alcohol ingestion has become a worldwide problem that threatens human health.The pathogenesis mechanism of alcoholic liver disease is very complicated,while no clear therapy has been found so far.Hence,ameliorating alcohol-induced liver injury through daily nutritional supplement its subsequent metabolic regulation in human has gradually attracted people's attention.Bioactive peptides can exert various bioactivities according to the difference in amino acid composition and sequence.Bioactive peptides,as a result,are considered natural alternatives for regulating metabolism and promoting human health.In this study,alcohol dehydrogenase?ADH?activating peptides were prepared through the controlled hydrolysis of chicken breast.Peptide stability during food processing and gastrointestinal digestion was studied before the evaluation of their bioactivity and potential mechanism using a rodent model.The key peptides that exerted ADH activation activity were identified and synthesized for further understanding the molecular mechanism how peptides can activate ADH.Results from present study can provide theoretical guidance for the production and application of active peptides in food industry.The research contents and results are listed as follows:?1?The effects of 5 commercial enzymes?Alcalase,Neutrase,Bromelian,Flavorzyme and Papain?and 5 hydrolysis time?1,2,4,8 and 12 h?on the ADH activation activity of the prepared ADH activating peptides were studied.Research showed that ADH activating peptides prepared by 8 h hydrolysis?0.5%Alcalase,55°C?exerted significantly ADH activation activity?90%activation,2 mg/m L?.Physicochemical study indicated that ADH activating peptides showed strong stability against heat treatment?25,40,60,80,100 and 121°C,60 min?,acid and alkali treatment?p H 2,4,6,8,10 and 12?,various ionic strength?0.01,0.03,0.05,0.1,0.2,0.5,1 and2M Na Cl?and low concentrations of metal ions(<0.1 m M Zn²⁺,Ca²⁺,Fe²⁺and Fe³⁺).Results from in vitro digeston indicated that ADH activating peptides remained stable after gastric digestion,and retained up to 70%ADH activity after intestinal tract indicating the high applicability of ADH activating peptides.?2?A mouse model was set up and used to evaluate the activity in vivo of ADH activating peptide.Results indicated that the ingestion of ADH activating peptide could significantly facilitate alcohol metabolism and ameliorate liver injury in mice.Behavioral analysis showed that the loss of righting reflex?LORR?rate decrease by 50%under high dose of peptide ingestion?600 mg/kg?,the latency of LORR doubled,and the duration shortened by half,indicating enhanced tolerance against alcohol in mice.Biochemical and histopathological analysis revealed that ADH activating peptides significantly alleviated the symptoms of liver injury in mice.On one hand,the catalytic activity of major alcohol metabolism enzymes?ADH and ALDH?in mouse liver were markedly increased,which subsequently accelerate the scavenging of alcohol and its metabolites from the blood and reduces the direct damages to liver tissue.On the other hand,the catalytic activity of antioxidant enzyme SOD increased 28.6%in mouse liver,while the degree of lipid peroxidation decreased by 54.3%?MDA?.As a result,the blood transaminase?AST and ALT?dropped to normal levels,the liver triglyceride content significantly decreased,and the lesions of the liver tissue disappeared,indicating that the ingestion of ADH activating peptides could significantly protect mice against alcohol-induced liver injury.?3?The ADH activation activity-oriented separation and identification of key ADH activating peptide sequences were conducted using size-exclusion chromatography?SEC?,high-performance liquid chromatography?HPLC?,liquid chromatography tandem mass spectrometry and in silico analysis.Through peptide synthesis and bioactivity validation,3peptides?DPQYPPGPPAF,KPC and APGH?were confirmed as novel peptides that can significantly improve the catalytic activity of ADH.The most active KPC?5 m M?could retain almost 100%ADH activity after 120 min incubation at 37°C,whereas the control ADH was completely inactivated under the same conditions,indicating that KPC can significantly improve the stability of ADH.Further fluorescence quenching and molecular docking study showed that KPC could bind to the active site of ADH through 2 hydrogen bonds and hydrophobic interaction,preventing the oxidative modification of Cys-43 and Cys-153 residues,and thus stabilizing the molecular structure of ADH.?4?Since antioxidant ADH activating peptides may also protect mice agaist alcohol-induced liver injury through oxidative stress reduction,ADH activating peptides were further subjected to separation and identificatino oriented by antioxidant activity.After SEC and HPLC separation,a total of 22 peptides were identified from the fraction exerting the strongest antioxidant activity through liquid chromatography tandem mass spectrometry.Among the identified peptides,MH,YR,HY,YQ,YE and IE have been reported to exert strong free radical scavenging ability.In addition,RWGG,YYCQ,KAPKL,EVH and HED were first reported,and they contain antioxidant peptide fragments?RW,WG,YY,CQ,KA,VH and HE?.The presence of such antioxidant peptides may contribute to the ROS scavenging potential of ADH activating peptides,which might be a key factor to the enhanced the hepatic SOD activity in mice,inhibiting liver lipid peroxidation,and subsequently alleviating oxidative damage in liver tissue.?5?The effects of cooking and in vitro digestion on the peptide profile and bioactivity of endogenous peptides in chicken were studied.Results indicated that endogenous peptides exerted ADH activation and antioxidant activity,though their ADH activation activity and stability were not as strong as those of Alcalase hydrolyzed ADH activating peptides,indicating the vital role of Alcalase digestion on the release of effective ADH activating peptides.Cooking did not significantly influence the bioactivity of endogenous peptides,however,further in vitro digestion resulted in the decrease of ADH activation,reducing power and DPPH radical scavenging activity,whereas increase ORAC and ABTS radical scavenging activity.The whole fraction identification and quantification of endogenous peptides were conducted using nano-LC-MS/MS,a total of 777 peptides were detected.Peptidomic analysis showed that cooking and in vitro digestion could markedly change the constitution and content of endogenous peptides.Peptides originated from titin and collagen are major contributors to the difference in peptide profile and activity.Among these peptides,the oxidation of Pro residue in collagen peptides during cooking and in vitro digestion may be responsible for the loss of ADH activation,reducing power and DPPH radical scavenging activity.So it is speculated that dietary supplementation of chicken breast may exert potential bioactivity to facilitate alcohol metabolism and ameliorate alcohol-induced liver injury,however,enzymatic hydrolysis could improve the release of effective peptides,and the prepared ADH activating peptides were more stable,indicating the vital role of enzymatic hydrolysis on the development of ADH activating peptides.