Combining Non-Targeted Metabolomics To Resolve The Formation Mechanism Of Semicarbazide (SEM) In Macrobrachium Rosenbergii

Semicarbazide(SEM)is a metabolite of furacilin,which is currently a marker for judging whether furacilin in aquatic products exceeds the standard.SEM has a certain toxic effect on the human body and will cause certain damage to the body.SEM has a wide range of sources,and the growth environment and processing process will also introduce SEM.Therefore,it is an urgent problem to study the formation mechanism of SEM and overcome the defects of traditional detection methods.In this paper,the distribution characteristics of SEM in Macrobrachium rosenbergii were quantitatively analyzed by high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS),and the formation mechanism of SEM in the process of storage,processing and breeding of Macrobrachium rosenbergii was explored.Finally,the interaction between urea and SEM was verified by stress experiment,in order to provide theoretical basis for the quality control of crustacean aquatic products.The main results are as follows.1.The distribution characteristics of SEM in Macrobrachium rosenbergiiThe samples of M.rosenbergii were collected from several farms.The existence characteristics of SEM in M.rosenbergii were studied by measuring the content of SEM in carapace,shell,tail fan,leg foot,liver and shrimp meat.The results showed that: 1)SEM was detected in all tissues of M.rosenbergii.The SEM contents in carapace,shell,tail fan,leg,liver and muscle were 26.31-37.54,10.80-50.17,16.45-30.11,17.45-24.15,4.59-7.31 and ND-2.72 μg/kg,respectively.The detection rate of SEM in shell was100 %,and that in muscle was 87.5%.2)The average contents of SEM in different tissues were 32.02,26.85,23.97,21.03,5.56 and 1.35 μg/kg,respectively.The contents of SEM in carapace and shrimp shell were higher [ average(32.02 ± 3.75)and(26.85± 13.90)μg/kg],and the contents of SEM in muscle and liver were lower [ average(1.35 ± 0.67)and(5.56 ± 0.87)μg/kg ],which exceeded the current SEM residue limit standard of 1.0 μg/kg.3)The content of SEM in different tissues from high to low was: carapace > shell > tail > leg > head.The content of SEM in carapace was the highest,and the content in muscle was the lowest.2.The change of SEM content during storage of M.rosenbergiiM.rosenbergii was refrigerated(-4 °C)and frozen(-20 °C,-40 °C,-80 °C).The SEM content in the shell and meat of M.rosenbergii was detected on days 0,7,14,21,28 and 60,and compared with the SEM content in the shell and meat of the original shrimp.The results showed that: 1)The SEM content in the shell and meat of M.rosenbergii decreased with the increase of storage time.Compared with the 0 day,the SEM content in shrimp shell and shrimp meat decreased by 65 % and 75.09 % respectively after 60 days of storage,and there were significant differences in SEM content in shrimp shell and shrimp meat with different storage days(P<0.05).3)SEM in shrimp shell was transferred and enriched into shrimp meat through the single cell epidermis connecting shell and meat.3.Analysis of SEM content and protein changes during processing of M.rosenbergii.The shell and meat of M.rosenbergii were freeze-dried,untreated(raw shrimp)and baked(50,70,100,130 and 200 °C),respectively.The SEM content and protein content in the shell and meat of M.rosenbergii under different processing methods were detected,and the correlation analysis was carried out.The results showed that: 1)there was no significant difference in SEM content and protein content between freeze-dried shrimp shell and shrimp meat and untreated shrimp shell and shrimp meat(P > 0.05);2)The SEM content in shrimp shell and shrimp meat gradually increased with the increase of temperature,and the protein content gradually decreased with the increase of temperature.There were significant differences in SEM content and protein content at different processing temperatures(P < 0.05).Compared with shrimp meat,the SEM detection base in shrimp shell was larger;3)SEM and protein were not detected in shrimp shell and shrimp meat when the heating temperature reached 200 °C.4)The content of SEM in shrimp shell and shrimp meat with high protein content was the lowest,while the content of SEM in shrimp shell and shrimp meat with relatively low protein content was relatively high.4.Effects of arginine and urea on SEM content of M.rosenbergiiArginine feeding and urea stress experiments were carried out on M.rosenbergii.The SEM content in shrimp shell and shrimp meat was detected,and the SEM changes of shrimp shell on day 0 and day 6 were analyzed by metabolomics.The results showed that: 1)the SEM content in shrimp shell and shrimp meat increased with the increase of arginine concentration,and there was a significant difference in the SEM content in shrimp shell and shrimp meat fed with different arginine concentrations(P < 0.05);2)After urea stress,with the increase of urea content in M.rosenbergii from the breeding environment,the detection amount of SEM in shrimp shell and shrimp meat increased with the increase of breeding time.3)After urea stress,there were 350 differential metabolites with variable projection importance values exceeding 1 in M.rosenbergii,of which 97 were significantly up-regulated and 48 were significantly down-regulated.These substances were mainly involved in amino acid metabolism and nucleotide metabolism.4)Pathway attribution analysis of differential metabolites showed that urea stress promoted the formation of SEM in M.rosenbergii by comprehensively regulating the anabolism of arginine,histidine,proline,L-glutamic acid and ornithine.5.Study on the change of SEM content in seawater shrimp under urea stressThrough different concentrations of urea stress on Penaeus monodon and Penaeus vannamei,the SEM content in shrimp shell,shrimp meat and viscera were detected at different times.The results showed that: 1)With the passage of culture time,when the urea concentration in the culture environment was 0 g/kg,there was no significant change in the content of SEM detected in the shell,meat and liver of Macrobrachium rosenbergii,Penaeus monodon and Penaeus vannamei.2)With the passage of culture time,when the concentration of urea in the culture environment was 50 g/kg and 80g/kg,the content of SEM in the shell,meat and liver of Macrobrachium rosenbergii showed a significant growth trend,and the content of SEM in the shell,meat and liver of Penaeus monodon and Penaeus vannamei showed a slow growth trend.