Protective Mechanism Of Three Antioxidants On Muscle Quality Deterioration Of Litopenaeus Vannamei Induced By Typical Mycotoxins

AFB₁,T-2,OTA and DON are typical mycotoxins commonly found in feeds,which are easy to accumulate in Litopenaeus vannamei and cause oxidative stress to induce its muscle quality decline.However,it is difficult to reveal the root cause of the decline of shrimp muscle quality induced by mycotoxin.Quercetin,rutin and tea polyphenols are green antioxidants with strong anti-oxidation and anti-inflammatory effects.They have been widely used to prevent and control the adverse effects such as the decrease of growth characteristics and the damage of digestive tract.However,the protective effects of quercetin,rutin and tea polyphenols on the quality of shrimp muscle have not been reported.Thus,four typical mycotoxins（AFB₁,T-2,OTA and DON）and three safety antioxidants（quercetin,rutin and tea polyphenols）were used to expose shrimp simultaneously for 20 d in this study.The effects of mycotoxins on the quality of shrimp muscle and the protective effects of antioxidants were analyzed from the aspects of muscle tissue damage,basic nutrients,amino acid and fat composition,protein denaturation,and protein composition.The differential marker proteins which are significantly related to mycotoxins and antioxidants were screened by non-labeled quantitative proteomics,and the root cause of mycotoxins inducing the decline of shrimp muscle quality and antioxidant protection was analyzed at the molecular level.The results and conclusions are showed as follows:（1）The exposure of four mycotoxins caused the damage of shrimp muscle microstructure in a dose-dependent manner,which resulted in the decrease of the appearance quality and processing quality of shrimp muscle,such as the significant enlargement of muscle fiber space,the rupture of sarcomere,inflammation and so on.The three antioxidants significantly alleviated the muscle related damage in 4-12 d,but not in16-20 d.It is suggested that 0.20-0.08%quercetin,0.30-1.20%rutin,0.04-0.16%tea polyphenols have protective effects on the damage of muscle microstructure caused by four mycotoxins,which is closely related to the protective effect of antioxidants on cell integrity.（2）The exposure of four mycotoxins caused the oxidative stress reaction of shrimp body,which resulted in the over-oxidation of various nutrients and the decrease of the nutritional quality of shrimp muscle.After exposed to four mycotoxins for 20 d,the content of basic nutrients（water,crude fat and crude protein）in shrimp muscle decreased by 7.61-9.22%,essential amino acids content decreased significantly（P<0.05）and its index decreased by 8.14-10.71%,and total polyunsaturated fatty acids decreased by16.63-18.40%.The antioxidative effects of quercetin,rutin and tea polyphenols on scavenging free radicals effectively reduced the oxidative stress response of shrimp in 4-12d,which indicated that antioxidants had an inhibitory effect on the reduction of various nutrients in shrimp muscle caused by the exposure of four mycotoxins,and the protective effect of quercetin was more significant.The content of essential amino acids and unsaturated fatty acids in quercetin group was close to that in control group.（3）The exposure of four mycotoxins induced severe denaturation of shrimp muscle protein,resulting in the decrease of nutritional quality of shrimp muscle.After exposed to four mycotoxins for 20 d,the salt solubility,Ca²⁺-ATPase activity,sulfhydryl content and ATP sensitivity of actomyosin decreased significantly（P<0.05）,and the surface hydrophobicity increased significantly（P<0.05）.Among them,Ca²⁺-ATPase activity,sulfhydryl content and surface hydrophobicity were significantly correlated with the exposed doses of mycotoxins and antioxidants（R²=0.86-0.99）,which could be used as potential evaluation indexes for mycotoxins induced denaturation of shrimp muscle protein.Quercetin and rutin had significant inhibitory effects on the biochemical characteristics of shrimp protein induced by four mycotoxins,and each index was close to the control group in 4-12 d.However,the inhibitory effect of tea polyphenols was weak,and there was no significant effect on the decrease of Ca²⁺-ATPase activity and sulfhydryl content caused by T-2 and DON.（4）The exposure of four mycotoxins caused gene and cytotoxic effects,inhibited DNA and RNA synthesis,and then affected protein composition of shrimp.The contents of sarcoplasmic protein,stroma protein and myofibrillar protein were significantly decreased（P<0.05）in 12 d,and the content of alkali-soluble protein were significantly increased（P<0.05）.Quercetin,rutin and tea polyphenols significantly inhibited the changes of protein composition of shrimp muscle caused by different mycotoxins in 12 d.Compared with the mycotoxins group,the content of sarcoplasmic protein,stroma protein and myofibrillar protein in the antioxidants groups increased by 14.04-37.16%.SDS-PAGE was used to screen different protein bands,and then LC-MS/MS was used to identify different protein spots.It was found that the number of target proteins associated with four mycotoxins（AFB₁,T-2,OTA and DON）and three antioxidants were 11,10,8 and 11respectively.Among them,the expression levels of fructose-bisphosphate aldolase（FBA）,arginine kinase（AK）and eukaryotic translation initiation factor（EIF）,which catalytic energy metabolism and regulate protein translation,are significantly related to four mycotoxins and three antioxidants.Therefore,they can be used as differential marker proteins for shrimp muscle quality decline induced by mycotoxins and the protective effects of antioxidants.In conclusion,AFB₁,T-2,OTA and DON exposure caused the damage of muscle tissue,the decrease of nutrient composition,the significant change of protein composition and biochemical characteristics,and finally led to the decrease of the appearance quality and nutritional quality of shrimp muscle.The reason may be that mycotoxins induce the abnormal expression of specific functional proteins,resulting in the disorder of energy metabolism,redox reaction and protein synthesis in shrimp.There were significant toxic differences among the four mycotoxins on the quality of shrimp muscle,which followed by AFB₁>T-2>OTA>DON.In addition,Antioxidants（Quercetin:0.20-0.08%,rutin:0.30-1.20%,tea polyphenols:0.04-0.16%）had significant protective effects on the reduction of muscle quality and nutritional value caused by four mycotoxins in 4-12 d,and the protective effects of the three antioxidants followed by quercetin>rutin>tea polyphenols.The reason may be that antioxidants can protect the integrity of cell structure,and play the role of detoxification,anti-inflammatory and tissue and organ protection through anti-oxidation and regulation of protein expression.The results of this study provide theoretical parameters and technical guidance for the control of mycotoxins and the use of antioxidants to prevent its harm during the culture and processing of Litopenaeus vannamei.