Study On Mechanism Of N-nitrosamines Formation And Control During Freshwater Fish Fermentation

Biological fermentation can effectively solve some common issues during Chinese traditional fermented fish products(CTFP_s)processing,such as long fermentation cycle,difficultly controllable fermentation condition,unstable product quality,and so on,which has become one of the emphasises and hot spots in the field of freshwater fish processing in recent years.In our previous researches,inoculating fermentation was used in Suan yu processing,which could significantly(P < 0.05)promote texture,flavor and nutrition properties of products.However,there are few reports focused on the N-nitrosamines(NA_s)and its precursors during Suan yu processing,which make the safety of products hard to be guaranteed.Meanwhile,the effect and mechanism of inoculating fermentation on NA_s were not clear.In this study,the safety of commercial CTFP_s was investigated first.Then,previously isolated strains(L_p-120,S_c-2018 and S_x-135)were used as starter culture.The effects of autochthonous starter cultures on NA_s and its main precursors(BAs and nitrite),main influencing factors of NA_s during Suan yu fermentation and degradation pathway of N-nitrosodimethylamine(NDMA)were studied to elaborate the degradation mechanism of NA_s by microorganisms.This study has important academic significance to reveal the effects of inoculating fermentation on NA_s during Suan yu fermentation,and also provides theoretical guidance for NA_s control during CTFP_s fermentation.The contents of BAs,residual nitrite and NA_s in commercial CTFP_s were investigated.By using traditional chemical assay methods,reversed phase high-performance liquid chromatography(RP-HPLC)and dispersive solid-phase extraction-gas chromatography-triple quadrupole mass spectrometry(d SPE-GC-MS/MS),the contents of NA_s and its precursors(BAs and nitrite)in 33 CTFP_s purchased from different provinces of China were investigated.The results showed that the nitrite contents in all samples ranged from not detcected(ND)to 1.19 mg/kg,which significantly lower than the hazardous nitrite content of food in GB 2760-2014(30 mg/kg).The contents of BAs in all samples were gennerally stayed at low levels,and only in one sample from Guangxi,the histamine(HIS)content exceeded the limit of U.S.Food and Drug Administration(50 mg/kg).A total of 6 kinds of NA_s were detected in commercial CTFP_s,including NDMA,N-nitrosoethylmethylamine(NMEA),N-nitrosopiperidine(NPIP),N-nitrosopyrrolidine(NPYR),N-nitrosomorpholine(NMOR)and N-nitrosodiphenylamine(NDPhe A).The contents of NDMA in about 36% of samples exceeded the acceptanle limint(4 μg/kg)of China national Ministry of Health,and the total NA_s contents of about 64% samples were unacceptable according to the limit suggested by the United States Dpartment of Agriculture(10 μg/kg).Primcipal component analysis indicated that the accumulation of NDMA and total NA_s in CTFP_s were closely related with the contents of HIS,tyramine(TYR)and nitrate.The effects of inoculating autochthonous starter cultures on BAs during Suan yu fermentation were studied.The changes of BAs contents and other biochemical parameters,inclluding microbiological characteristics,p H,total acid,α-amino nitrogen,TCA-soluble peptides,protease activity and free amino acids,of Suan yu inoculated with single stain(L_p-120,S_c-2018 and S_x-135)and mixed strains(L_p-120: S_c-2018: S_x-135=1: 1: 1)during fermentation were determinated.Suan yu without inoculation acted as control.The results indicated that no HIS accumulation was observed in each fermentation group.Putrescine(PUT)and TYR were the dominating BAs in Suan yu.PUT accumulation was significantly(P < 0.05)suppressed by inoculated with L_p-120 and S_x-135,and TYR was significantly(P < 0.05)inhibited by inoculated with mixed starter culures.Low levels of cadaverine(8.22-20.03 mg/kg),spermidine(6.60-8.11 mg/kg)and spermine(7.94-9.62 mg/kg)were observed in all groups at the end of the fermentation.Pearson correlation coefficient assay indicated that total BAs contents were signicantly(P < 0.05)related with protease activities and α-amino nitrogen contents.The degradation pathways of nitrite during Suan yu fermentation were explorated.In order to reveal the major influence factors of nitrite contents during Suan yu fermentation,the effects of lactic acid solution with different p H values,single stain(L_p-120,S_c-2018 and S_x-135)and soluble protein solution on nitrite degradation were investigated in model system.Additionally,comparing with spontaneous fermentation,the effects of inoculating single stain(L_p-120,S_c-2018 and S_x-135)and mixed strains(L_p-120: S_c-2018: S_x-135=1: 1: 1)on nitrite contents during Suan yu fermentation were studied.The results showed that L_p-120,S_c-2018 and S_x-135 could degrade nitrte directly in model system,and the nitrite degradation rate(NDR)were 90.70%,78.75% and 13.09%,respectively.The nitrite reductase(Ni R)activities of L_p-120 and S_c-2018 could be significantly(P < 0.05)promoted by appropriate Na Cl contents(4%-6%).Nitrite could be degraded in soluble grass carp proteins,and the NDR was positively associated with the protein concentration.Inoculation with L_p-120,S_c-2018 and S_x-135 significantly(P < 0.05)decreased the nitrite and nitrate concentrations in Sunyu.The effects of inoculating autochthonous starter cultures on NA_s contents and its degradation pathways during Suan yu fermentation were studied.The changes of NA_s and its precursors contents of Suan yu inoculated with single stain(L_p-120,S_c-2018 and S_x-135)and mixed strains(L_p-120: S_c-2018: S_x-135=1: 1: 1)during fermentation were determinated.The results indicated that L_p-120,S_c-2018 and S_x-135 could directly degrade NDMA in model system,and trimethylamine-N-oxide(TMAO)could be degraded by L_p-120 and S_c-2018 via the TMAO demethylase pathway.The accumulation of total volatile basic nitrogen and trimethylamine were significantly(P < 0.05)inhibited in inoculated Suan yu.Inoculation with L_p-120,S_c-2018 and mixed starter cultures significantly(P < 0.05)decreased the dimethylamine contents at the end of the Suan yu fermentation.A total of 6 kinds of NA_s were detected during Suan yu fermentation,including NDMA,NMEA,NPIP,NPYR,NMOR and NDPhe A,and the accumulation of NDMA,NPYR and NDPhe A were significantly(P < 0.05)suppressed by inoculating fermentation.The effects of inoculating autochthonous starter cultures on NA_s and its precursors accumulation in Suan yu during in vitro digestion were studied.The changes of microbiological characteristics,BAs,nitrite and NA_s of all fermentation samples during digestion were investigated by establishing an in vitro gastrointestinal digestive juices model system.The results showed that lactic acid bacteria,yeast,Staphylococcus and Enterobacteriaceae counts were significantly(P < 0.05)inhibited after stomach digestion,and these microbes could multiply rapidly during intestinal digestion.The contents of PUT,CAD and TYR significantly(P < 0.05)increased during intestinal digestion,while inoculating fermentation had some inhibitory effect on the accumulation of these BAs.The stomach digestion process could significantly(P < 0.05)inhibited the accumulation of nitrite in all fermentation groups,while the nitrite contents significantly(P < 0.05)increased after intestinal digestion.The increase amounts in inoculated groups were lower than than in spontaneous fermentation group,which indicated that inoculating fermentation had some positive effect on nitrite inhibiton during intestinal digestion.The NDMA contents in Suan yu significantly(P < 0.05)increased after stomach digestion,while inoculation with L_p-120,S_c-2018 and mixed starter cultures had some inhibitory effect on the accumulation of NDMA in this digestion process.This thesis defined the kinds and contents of NA_s in Suan yu,confirmed that inoculation with autochthonous starter cultures could effectively inhibite the accumulation of NA_s in Suan yu,discovered the inhibitory effect of autochthonous starter cultures on the precursors of NA_s,namely BAs and nitrite,illustrated the degradation pathway of NDMA by microorganisms,preliminarily revealed the effect and mechanism of inoculated with autochthonous starter cultures on NA_s degradation in Suan yu.