Study On Characteristics Of Collagen And Collagen Peptides In Cod Swim Bladder And Its Effect And Mechanism Of Intervention On Cellular Aging Process

With the continuous development of society and the increase of human longevity,the phenomenon of population aging has appeared in our country and even in the whole world since the 21st century.The population age structure chart of China calculated by the United Nations world population outlook report showed that the population distribution of all ages has changed from a triangle in the 1970s to a spindle today.The aging society will inevitably bring about changes in family structure and social structure.The lack of family material support and life care will undoubtedly increase the pressure of middle-aged people.Aging diseases and medical care costs also increased the social burden.Therefore,in order to delay or prevent aging,it is of great significance to research and develop effective anti-aging drugs to improve the quality of life of the elderly people and reduce the social and national burden.With the increasing aging of the population,the types and demands of anti-aging products are increasing.Functional food with anti-aging activity can avoid long cycle of drug development and clinical trial compared with drugs,which has a broader space for development.Swim bladder is also called flower gum or fish maw,which is known for its high collagen content.Its efficacy has been favored by consumers since a long time ago.As for the relationship between collagen and anti-aging,there were a lot of studies on the application of macromolecular collagen and small molecule collagen peptide in skin industry.The cells of skin fibroblasts and animal skin tissues were used as subjects to explore the anti-aging effect of collagen.In recent years,the relationship between collagen and anti-aging has become a focus of research.Previous studies have shown that the swim bladder hydrolysates from other species possessed good antioxidant activity.Antioxidant is one of the best ways to prevent oxidative damage of the body by scavenging oxygen free radicals.Therefore,in this paper,collagen and enzymic collagen peptides were extracted from the swim bladder of Atlantic cod using different methods,and their physicochemical properties were analyzed and compared.Single factor experiment and response surface optimization experiment were performed to determine the optimal preparation process of antioxidant peptides,and then the ability of scavenging free radicals in vitro and the stability of simulated intestinal digestion in vitro were determined.The enzymatic collagen peptides extracted from cod swim bladder by the optimal technological conditions was purified and structurally identified.2BS cells were used to establish the model of precocious senescence and replicative senescence.Two kinds of swim bladder collagen peptides were used to detect the intervention effect on aging,and the molecular mechanism was discussed through the changes of protein expression in related signaling pathways.Acid soluble collagen（ASC）was extracted by acid soluble method at low temperature;Gelatin（GEL）was extracted by hot water;Swim bladder was extracted by enzymatic hydrolysis with protamex.The products were characterized by sensory evaluation,SDS-PAGE electrophoretic analysis,ultraviolet spectral analysis,Fourier infrared spectroscopy（FT-IR）,amino acid composition analysis and Scanning electron microscopy（SEM）.The results of the basic composition showed that the water content in the cod swim bladder sample was high,reaching 76%.The content of protein is22.08%in fresh sample,and 92%in dry basis.The extraction rate of three kinds of collagen was 42.15%,72.20%and 89.11%,respectively.The content of hydroxyproline of them was 8.73%,8.97%and 7.94%.Observed in morphology,ASC and GEL were dense spongy,and SWP was powdery solid.The UV spectra showed that the three proteins had the maximum absorption at the wavelength of 230 nm,and there were absorptions at 280nm of GEL and SWP.The atlas of SDS-PAGE showed that ASC was composed of at least twoαchains and oneβchain;the GEL contains a small number ofαchains;theαandβchains were degraded in SWP.The results of scanning electron microscopy showed that ASC and GEL had a certain cross-linking structure and there was no cross-linking structure in SWP.The results above suggest that the cod swim bladder is in high protein and low fat,and the three collagens and collagen peptides prepared with different extraction had different characteristics,which has broadened the field for the development and application of collagen in cod swim bladder.Six kinds of proteases including acid proteinase,dispase,pepsin,papain,compound proteinase and trpsin were used to hydrolyze cod swim bladder in order to select optimal one by determining degree of hydrolysis and DPPH radical scavenging rate.Combined with single factor method and response surface methodology,the optimum process was ensured that the hydrolysis temperature of 58.6℃,the hydrolysis time of 6 h,the pH of7.2 and the enzyme addition of 200 U/mL.The results of confirmatory experiment showed that the scavenging rate of DPPH was 61.1%,which was in agreement with the predicted value of 62.0%and the results proved that the optimized extraction process was stable and reliable.The free radical scavenging rates of peptides were detected and the results showed that swim bladder peptides have good abilities of scavenging free radicals of DPPH·,OH·,O₂-·and chelating Fe²⁺.Besides,the half maximal inhibitory concentration(IC₅₀)of them was 8.29 mg/mL,5.03 mg/mL,5.43mg/mL and 1.35 mg/mL seperately.After being digested in vitro,the degree of hydrolysis was increased while the free radicals scavenging abilities and chelating power of with Fe²⁺were decreased.This might be occurred because the changes of reaction conditions in the simulated gastrointestinal digestion process such as the change of pH value lead to the changes of composition and structure of the peptides.Meanwhile,the changes of composition and structure lead to the changes of antioxidant activities.In conclusion,swim bladder peptides had potent antioxidant and could serve as useful ingredients in the food industry.Swim bladder peptides of Atlantic cod were obtained by enzymatic hydrolysis with the best extraction technology and two bioactive peptides（SWP-I and SWP-II）were isolated by chromatographic method with G-15.The two fractions were characterized by FT-IR,UV scanning spectrum,amino acid composition analysis and determination of molecular weight.The antioxidant and anti-aging activities of peptides were evaluated in vitro.The results showed that the main amino acids in the two components are Asp,Glu,Gly,Ala and Pro.The most abundant amino acid is glycine in SWP-I and arginine in SWP-II.The UV spectrum scanning results showed that both components had the maximum absorption at the wavelength of230 nm,which conformed to the absorption characteristics of protein.The content of aromatic amino acids in SWP-II was higher than that in SWP-I,which was consistent with the result of acid composition analysis.The results of determination of molecular weight showed that the average molecular weight was 4976 Da and 1960 Da,respectively.The results of scavenging rates of free radicals showed that SWP-I and SWP-II possessed good scavenging activities on DPPH·,HO·and O₂-·free radicals and good Fe²⁺chelating activity.Besides,the scavenging ability of SWP-II was stronger than SWP-I at the same concentration.Biological aging occurs at every level of the body,including molecules,tissues,organs and the basic unit of life—cells.Cellular aging is considered as the foundation of human aging.Various external stress conditions,such as reactive oxygen radicals and UV irradiation,will cause damage to young cells or normal cells,the damage will lead to cell aging,this phenomenon is called premature senescence of cells.Human embryonic lung diploid fibroblast（2BS）is often selected as the type cell for research on cell aging process,and it is also one of the best models internationally.The establishment of cellular aging model is of great significance for the research on the aging process and the search for anti-aging drugs.The precocious senescence cell model of 2BS cells was established by H₂O₂ treatment to study the protective effect and mechanism of collagen peptide in cod swim bladder on precocious senescence 2BS cells.Cck-8 was used to detect the effect of different concentrations of H₂O₂ on cell proliferation rate,so as to select 0.2mM H₂O₂ to establish a model of oxidation-induced precocious senescence of 2BS cells.The proliferation rate,intracellular active oxygen content,β-galactosidase content,apoptosis rate and intracellular oxidase and lipid peroxide content of 2BS cells were tested.By detecting the expression of t-Akt,p-Akt,t-GSK-3βand p-GSK-3βof the PI3K/Akt apoptosis signaling pathway,the molecular mechanism of the protective effect of cod swim bladder peptides on oxidation-induced senescence was explored.Cellular results suggested that SWP-I and SWP-II of high concentration could significantly increase the proliferation rate of precocious senescent cells（p<0.01,p<0.01）,decrease the intracellular active oxygen content（p<0.01,p<0.01）,SA-β-gal content（p<0.01,p<0.01）,apoptosis rate（p<0.01,p<0.01）.Besides,both peptides of 400 mg/mL could increase the activity of antioxidant enzyme and decrease the lipid peroxide content significantly（p<0.01,p<0.01）.The results of Western blot showed that the peptides could inhibit the increase of p-Akt and p-GSK-3βlevels to protect the precocious senescent cells,and the protecting effects of SWP-II was better than SWP-I.To study the effect and mechanism of swim bladder collagen peptides in delaying the replicative senescence of 2BS cells,the proliferation rate,intracellular active oxygen content,β-galactosidase content,apoptosis rate and intracellular oxidase and lipid peroxide content of 2BS cells of different generations were tested.Comet assay was used to assess the effect of both peptides on the repair of DNA damage in replicative senescent cells.By detecting the expression of p53 and p21 in the p53/p21 anti-apoptosis signaling pathway,the mechanism of cod swim bladder peptide delaying the process of replicative aging 2BS cells was discussed.The results showed that compared with young cells of 20PD,SWP-I and SWP-II could significantly increase the proliferation rate of precocious senescent cells（p<0.01,p<0.01）,decrease the intracellular active oxygen content（p<0.01,p<0.01）,SA-β-gal content（p<0.01,p<0.01）,apoptosis rate（p<0.05,p<0.01）of 45PD cells.Besides,both peptides could increase the activity of antioxidant enzyme and decrease the lipid peroxide content to achieve the protection of replicative aging cells.The molecular mechanism could possibly contributed to the regulation of cell replicative aging process by down-regulating the p53/p21 cell cycle and the p53 and p21 proteins in the apoptotic protein signaling pathway.