Developing A Biogenic Amines Analysis Method And Study On The Mechanism Of Reducing Biogenic Amines By Starter Cultures In The Process Of Salted Fish

Biogenic amines(BAs)are organic nitrogenous compounds formed mainly by microbial decarboxylation of amino acids in protein-rich food and fermented products.Several BAs(especially histamine)cause safety concerns due to the risk of food intoxication.Considering the public health risks posed by the BAs,it is necessary to develop rapid and accurate methods to monitor these BAs in food products.This study focuses on the quantitative analysis and effective control of BAs in fish products.The detailed results are as follows.(1)To effectively extract BAs from complex food matrices is an important issue.In this study,a stop-and-go-extraction tip(Stage Tip)-based method for simultaneous enrichment of six BAs including histamine,tyramine,putrescine,cadaverine,tryptamine and?-phenylethylamine)in fish samples was developed and optimized.Target recovery efficiencies of BAs from spiked fish samples ranged from 84%to 110%.Linear regression models were established to determine BAs in the range of 0.4-3.0 mg/L(R~2>0.995).Good repeatability was obtained with relative standard deviation of peak areas(1.17-5.61%)and retention time(0.09-0.20%).High sensitivity was achieved for the detection of six BAs(0.064 ng/L to 1.00 ng/L for limit of detection,0.10 ng/L to 5 ng/L for limit of quantification).The proposed method had an obvious advantage in the analysis of BAs in complex food matrices such as fish tissues,since it could reflect the subtle distribution of BAs from one fish fillet.(2)Lactobacillus plantarum YC-2 and Lactobacillus plantarum YC-5,isolated from spontaneous fermented“Yucha”,were used as starter cultures in“Yucha”fermentation.Levels of the precursor FAAs(including histidine,tyrosine,lysine,ornithine,tryptophan and phenylalanine)continuously increased during the investigated period.Bacterial community analysis based on 16S r RNA gene sequences revealed that L.plantarum was the predominant bacterial strain at the end of fermentation in all the“Yucha”samples.After 35 days of fermentation,the overall BAs(including histamine,tyramine,cadaverine,putrescine,tryptamine and phenylethylamine)contents in the inoculated samples were reduced by 97%and 69%,respectively,compared to control samples.Enterococcus,Leuconostoc,Macrococcus,Morganella,Raoultella,Staphylococcus,Streptococcus,and Lactococcus were positively related to BAs formation,while Lactobacillus was negatively correlated with the production of BAs.In conclusion,two Lactobacillus starter cultures could easily colonize in fermented“Yucha”,which lead to less undesirable bacteria in inoculated samples,thereby inhibiting the accumulation of BAs.(3)The salted S.niphonius samples was used as a research system,and L.plantarum YC-5 and L.plantarum YC-6 were inoculated in the system.The traditional marinated mackerel products were used as control group.The results showed that the total content of six BAs in the inoculated sample group decreased by 18%and 54%,respectively,and the degradation rate of histamine reached 97%and 86%,compared with control group.Bacterial community analysis based on 16S r RNA gene sequences revealed that Aliivibri is the dominant genus in the early stage,Vibrio is the dominant genus in the medium term,and Fusobacterium is the dominant genus in the terminal stage.Morganella,Marinilabiaceae,Sphaerochaeta,Fusobacterium and Psychrilyobacter were positively related to BAs formation,while Aliivibrio was negatively correlated with the production of BAs.The above results indicate that inoculation of L.plantarum as starter cultures may indirectly reduce the accumulation of BAs by affecting the microbial community structure,abundance and function in the system.The research in the study provides a rapid and simple method for the determination of biogenic amines,and solves the problem of excessive accumulation of biogenic amines in fish products.It preliminarily clarifies the degradation mechanism of biogenic amines,provides theoretical basis for the follow-up study of biogenic amines,and provides technical support for the production of fish products with low biogenic amines content.