Study On Effect Of Grape Seed Proanthocyanidins On Protein Degradation Of Bighead Carp(Aristichthys Nobilis) Fillets During Cold Storage

Bighead carp(Aristichthys nobilis),which is one of the four main fish and an important commercial freshwater fish in China.Generally,freshwater fish due to neutral pH value,rich protein and high water content,and have high endogenous protease activity,and can lead to protein degradation of fish fillets in the process and storage,which result in fish quality deterioration,greatly reduce their edible quality and commodity value.The problem of fish quality degradation has attracted wide attention.How to find a natural product to slow down the degradation of fish protein or inhibit the activity of endogenous protease and its potential mechanism has been the research hotspot and bottleneck in aquaculture processing industry.Grape seed proanthocyanidin(GSP)is a kind of polyphenols extracted and isolated from grape seeds,which has various biological activities and can be used as a natural food additive to improve food quality and promote human health.However,there are few reports on whether GSP modifies fish protein and regulates the activity of endogenous protease in fish.Therefore,first,bighead carp was studied in this thesis,the quality changes of bighead carp fillets were determined by GSP at 4±1℃storage.Then,the physicochemical and structural changes of myofibrillar protein(MP)before and after GSP treatment were compared,and the changes of MP degradation induced by endogenous protease by GSP were discussed in vitro.Finally,proteomics technology were used to investigate quality changes and related key proteins during cold storage,and to reveal the mechanism of the effect of GSP on the protein changes of bighead carp fillets during cold storage from the molecular level.The main research results and conclusions as follows:(1)Firstly,the main compounds of GSP were characterized by high-resolution mass spectrometer.Results as following: catechin,epicatechin,epigafcatechin,and proanthocyanidin B series.Secondly,the quality changes of bighead carp fillets during 4±1℃ refrigeration were analyzed after the bighead carp fillets were soaked by GSP.The results showed that GSP could inhibit the decline trend of sensory indexes,color and texture of fillets.In conclusion,GSP could delay the quality decline trend of fish fillets during the period of refrigeration,and extended the shelf life for 3-4 days.The experiment results show that GSP may be related to the quality changes of fish fillets during the period of cold storage.(2)In order to research the relationship between active ingredients of GSP the physicochemical characteristics and degradation trend of MP induced by GSP.The results showed that GSP inhibited the conversion of MP amino acids to carbonyl derivatives.The decrease trend of total sulfhydryl content decreased with the increase of GSP concentration.Those could inhibit the decrease of MP surface hydrophobicity,dipoly-tyrosine and myofibrillar fracture index,and reduced the exposure of hydrophobic groups in the protein.SDS-PAGE results showed that GSP inhibited the degradation of structural proteins such as myosin heavy chain,actin and myosin.At the same time,TEM results showed that the MP microstructure of bighead carp fillets treated with GSP were relatively intact after 7 days of cold storage,and the A-band and I-band were clear.At the same time,GSP could effectively inhibit the activities of calpsin,cathepsin B and B + L in fillets.In conclusion,GSP could availability maintain the structural integrity of MP and delayed the degradation trend of myofibrin in bighead carp fillets during cold storage.GSP could be considered as a potential inhibitor to regulate the activity of endogenous enzymes.(3)The physical,chemical,structural and functional effects of GSP and MP were continuously investigated in vitro.The results showed that GSP could modify the carbonyl and sulfhydryl groups of MP amino acid side chains,interfered with the formation of protein disulphide crosslinking,slowed down the formation of MP carbonyl compounds and total sulfhydryl transformation process,reduced the secondary structure of MP protein from α-helix to β-folding,and reduced the surface hydrophobicity of MP.Those could inhibit the myosin heavy chain and actin degradation of MP.The result indicated that GSP could slow down the degradation process of MP during storage.At the same time,the scavenging ability of ABTS,DPPH and HRSA in GSP-MP suspension system were increased to a certain extent.In particular,the scavenging rate of DPPH radical were positively correlated with the concentration of GSP.The active components of GSP formed adducts with MP,which enhanced the structural stability and antioxidant capacity.(4)In vitro,those were explored between three endogenous proteases and the change trend of GSP on degradation of MP key structural proteins by SDS-PAGE and western blotting analysis.The results showed that the actin,intermuscular line protein and troponin T of GSP-MP were significantly degraded after incubation with μ-calpsin.After the incubation of GSP-MP with cathepsin B,GSP inhibited the degradation of actin and troponin-T bands with significant difference(P<0.05).After the incubation of GSP-MP with cathepsin L,GSP inhibited the degradation of actin,interomycin and troponin-T bands in a significant correlation.For troponin-T,GSP had obvious inhibitory effect at a certain low concentration.In conclusion,GSP could slow the degradation of bighead myofibrillar protein in vitro at a certain concentration,but the degree of inhibition were depended on the concentration of GSP and the type of endogenous protease.(5)Further explored the biochemical process of GSP participating in quality and protein changes of bighead carp fillets during cold storage,the differential protein changes of bighead carp fillets at 0 and 7days were analyzed by using TMT proteomics.The results showed that a total of 59 different proteins were extracted and screened as potential biomarkers for quality changes of bighead carp caused by GSP during cold storage.Combined with GO enrichment and KEGG analysis,GSP could participate in the cellular process,metabolic process,biological regulation,binding,catalytic activity,cytoskeleton and polymer complex in the muscle,accounting for the main regulatory effects.At the same time,GSP mediated transcriptional regulatory factors,ATPase activity,structural molecules,localization proteins,detoxification activities,immune system processes and stimulus responses.In conclusion,the results of this study proved that GSP could delay the texture changes of bighead carp fillets during 4 ± 1℃ storage.The regulation of major differential proteins that affect the quality indexes of fish fillets were analyzed from the molecular level.Those were described the effect of GSP on the main potential labeled differential protein sites such as triphosphate isomerase,thiolyase,heat shock protein family and actin in fish cells.Those were involved in the regulation of glycolysis,lipid metabolism,stress response and structural proteins in fish cells.Thus,those were expounded about the effect of GSP on reducing fish quality deterioration.This study could provide some scientific basis for further exploring the mechanism of how GSP improves the protein changes and delays the quality decline of bighead carp fillets during storage.