Effects Of HPP On The Quality Of Wine-marinade Crab And Its Regulation Mechanism On The Formation Of Crab Melanosis

Due to the seasonal growth of river crabs,it is difficult for consumers to purchase highquality fresh crabs in the market in the first half of the year,so wine-marinade crab products are more popular among consumers.Therefore,manufacturers cater to demand during crab fishing season by making wine-marinade crabs with a long-shelf life.In order to extend the shelf life of wine-marinade crab and improve the quality of products during the storage storage,the following two methods are generally adopted:(1)A lot of salt is added to the processing of wine-marinade crabs;(2)A variety of different preservatives are added to wine-marinade crabs.Although these methods can effectively extend the shelf life of wine-marinade crabs and improve the safety of products,in view of the increasing health awareness and food safety awareness of consumers,such practices will seriously affect the quality and commercial value of products.High pressure processing(HPP)technology has good sterilization,passivating enzyme,and has industrial production scale.Therefore,in order to improve the comprehensive quality of long shelf-life wine-marinade crab products,this study intends to use HPP as a treatment method to investigate its influence on the quality of raw wine-marinade crabs with long shelf-life and low salt at room temperature during storage,and to explore the mechanism of quality changes.First,the effects of HPP treatment on the sensory quality of the two most common crab products made from river crab(steamed crab and wine-marinade crab)was investigated.Analyze the differences in moisture,texture,color,flavor and sensory scores of crab meat and crab yolk treated with 100,300 and 500 MPa by comparing them with steamed crabs made by conventional steaming and wine-marinade crabs.The water analysis results showed that the water holding capacity(WHC)of steamed crab meat and wine-marinade crab meat treated with300 MPa was significantly improved,which was 1.5 and 1.9 times higher than that of conventional steamed crab and conventional wine-marinade crabz,respectively.The results of texture analysis showed that the hardness and springiness of the crab meat/yolk of winemarinade crab treated by HPP were significantly increased compared with that of the conventional wine-marinade crab,and the effect of HPP on wine-marinade crab was greater than that on the steamed crab.The color analysis results showed that the L* value of winemarinade crab meat treated with HPP was significantly increased by 35.39% compared with conventional wine-marinade crab meat,while the L* value of steamed crab was not significantly different.The GC-MS analysis results showed that the number of important volatile compounds in crab yolk(27 kinds)was less than that in crab meat(41 kinds).Compared to conventional steamed crab meat or conventional wine-marinade crab meat,the content of trimethylamine(a fishy odor)in HPP treated crab meat decreased by 54.66%(steamed crab)and 4.04%(wine-marinade crab),respectively.The sensory evaluation results showed that the overall impression score of steamed and wine-marinade crabs treated with HPP were higher than those of conventional samples.HPP-treated crab meat scored 9 points for texture and taste,up from 6 points for conventional wine-marinade crab meat.In general,the advantages of HPP technology in the processing of ready-to-eat wine-marinade crab products are greater than that of steaming crabs.Then,the effects of HPP technology on the quality changes of low salt and room temperature ready-to-eat wine-marinade crab during shelf-life and storage were studied.The wine-marinade crab with 1%,2%,3%,4% and 5% salt concentration were prepared,and then treated with the optimal pressure condition(300 MPa),and compared with the conventional non-pressurized wine-marinade crab with a high concentration of 7%.The results showed that when salt concentration was not lower than 3%,the shelf life of HPP-treated samples could reach 90 days,which was 30 days longer than that of conventional wine-marinade crab.Image J software was used to quantify the black spot area.The results showed that when the salt concentration was 3%,4% and 5%,after 60 days of storage,the black spot area of the pressurized wine-marinade crab was significantly reduced by 50.24%,50.80% and 58.30%,respectively,compared with that of the conventional wine-marinade crab.These results showed that the water holding capacity and salt content of wine-marinade crab meat with 3% salt concentration had higher water holding capacity and lower salt content of HPP-treated samples.The analysis results of endogenous enzymes,volatile alkali-nitrogen,histamine and lipid oxidation showed that 5 of the 6 endogenous enzymes(H3%)were significantly decreased during storage,while polyphenoloxidase(PPO)was continuously increased during storage,compared with conventional wine-marinade crabs.At the same time,the volatile alkali-nitrogen and histamine increased significantly during the whole storage period,but HPP-treated low salt wine-marinade crab was lower than the conventional wine-marinade crab.The histological results showed that there was no significant difference in the tissue morphology of the legs between conventional and pressurized wine-marinade crabs stored for 0 days.However,after60 days of storage,obvious cracks appeared in the tissue of the conventional group samples,and a small number of cracks also appeared in HPP-treated wine-marinade crab samples.In conclusion,HPP technology can serve as an effective salt reduction method for conventional wine-marinade crab products,while reducing the degree of quality deterioration during the storage period of low salt wine-marinade crab products.The effects of HPP technology on protein components of low salt wine-marinade crab meat and its WHC mechanism were further studied.The protein composition,myofibril protein structure,thermal properties,microstructure and water state of myofibrillar proteins of winemarinade crab meat treated with HPP at 1%,2%,3%,4% and 5% salt concentrations were analyzed.The results of protein composition analysis showed that compared with 7% salt concentration,HPP combined with low salt concentration was beneficial to increase the solubility and disulfide bond content of crab myofibrillar protein,and reduce the total sulfhydryl content.Circular dichroism spectrum and fluorescence spectrum showed that,compared with the crab meat treated with high salt concentration,the change of secondary junction of myofibrillar protein in the crab meat treated with low salt concentration was less,and the difference of tertiary structure was more significant.It mainly causes the thermal stability of myosin and actin in myofibrillar fibers,and increases the solubility of myofibrillar proteins by destroying the microstructure of these two proteins.The results of LF-NMR showed that HPP could enhance the binding of protein and difficult to entrapped water and prevent the conversion of entrapped water to bound water or free water when the strength of salt ion was not higher than 3%,which was conducive to improving the WHC of meat quality.We studied the distribution of the PPO in river crabs and the effect of HPP on PPO activity and protein structure.The analysis of the PPO activity in different parts of river crab showed that the PPO enzyme activity in crab gills was the highest,reaching 3588±55.15 U.When the HPP pressure was 300 MPa or above,HPP had a significant passivation effect on the PPO enzyme activity of river crab.The storage experiment results showed that adding hexose oxidase(HOX)into PPO of river crab treated with HPP could further inhibit enzyme activity and effectively prevent the recovery of residual enzyme activity.The results of PPO protein spectral analysis showed that the combination of HPP and HOX can cause the unfolding of the tertiary structure of PPO molecules,leading to a decrease in PPO activity.The atomic force microscope showed that the molecular height of PPO protein in natural state was 1～1.5 nm,that of HPP treatment was 6～6.5 nm,that of HOX treatment was 4.5～5 nm,and that of combined treatment was 7～8 nm,and its height increased significantly.Combined treatment caused a higher degree of aggregation of PPO protein molecules.Based on the analysis of PPO-DLDOPA reaction system in river crabs,the results showed that the combination of HPP and HOX reduced the production of 5,6-dihydroxyindoleic acid polymers,5,6-dihydroxyindolequinone polymers,dopa pigment and dobquinone polymers by inhibiting the enzyme activity of river crab PPO.The main reason for this was the reduction of the production of 5,6-dihydroxyindolequinone polymer,thereby inhibiting the development of black spots caused by enzymatic browning.Finally,proteomics technology was used to study the changes of HPP combined with HOX on PPO related proteins in crab gills and the proteome indirectly causing PPO to play a role in the storage process of wine-marinade crab,and to explore the biochemical process and mechanism of the formation and development of dark area from the molecular level.The shelf life results indicate that HPP combined with 60 ppm HOX could reduce the black spot area to less than 8.00% at the end of storage,and the shelf life was still not less than 90 days.Compared with fresh crab gills,39 down-regulated proteins were found in the gills of wine-marinade crab products treated with HPP and HOX.By protein domain analysis,GO and KEGG analysis,it was shown that β-1,3-glucan and lipopolysaccharide binding protein with hydrolase active molecular function and mitogen activated protein kinase with anionic coordination molecular function can regulate the metabolic pathway of cytochrome P450 on xenogeneic organisms and Fox O signaling pathway in vivo,respectively.The inactive prophenoloxidase was activated as active PPO;HPP combined with HOX can induce the downregulation of these two proteins,thus delaying the progression of melanosis in wine-marinade crabs.