| Debaryomyces hansenii,as a microbial starter,can metabolize sugars and proteins,and produce aromas at the same time.It is often used in meat product fermentation,cheese fermentation,pickle fermentation,fruit and vegetable disease antagonism.There are still many safety issues in the application of microbial starters in the fermentation process of food production,such as biogenic amines,nitrosamines and toxins,among which histamine is the most toxic and the most harmful.This project is based on a strain of Debaryomyces hansenii isolated and screened from sausages.Through transcriptomics and proteomics sequencing,the analysis of the changes in the expression of genes and regulatory proteins in the process of histamine synthesis and metabolism is used to clarify histamine metabolism.The regulation mechanism lays the foundation.The main research contents of this paper are as following:1.Research on Debaryomyces hansenii strain characteristics and histamine production ability: It was found that the logarithmic growth period of Debaryomyces hansenii was between 24 h and 40 h,the optimum growth p H was around 5.5,and the optimum growth temperature at about 30?;Histamine began to accumulate after 8-24 h of L-histidine stress,and there was no big difference between the groups.After 24 h,there was a difference in each group and the rate of production increased,and the rate of histamine production decreased after 40 h.The accumulation of histamine after stimulation with 0.1% L-histidine and 0.3%L-histidine was slightly higher than that of the blank group,compared with the accumulation of histamine after stimulation with 0.5% L-histidine and 0.8% L-histidine The blank group is higher.2.Study on the expression of histamine synthesis regulation genes in Debaryomyces hansenii: After adding histamine,the precursor of histamine synthesis,the Illumina high-throughput sequencing platform was used to sequence the transcriptome of the samples,and the sequencing results were differentiated Analysis and enrichment analysis.The analysis results showed that 42 M clean reads were detected in the control group and 41 M clean reads in the experimental group.The difference analysis showed that there were 133 differential genes,among which 78 were differentially up-regulated and 50 were differentially down-regulated.GO enrichment analysis showed that the cell membrane There are many differentially expressed genes such as components,cellular processes,intracellular localization,generation process,membrane differential catalytic activity differences,and binding proteins.KEGG enrichment analysis shows that most of the differential genes are distributed in metabolic pathways,thiamine metabolism,The biosynthesis of secondary metabolites,peroxisomes and other pathways;analysis of metabolic pathways revealed that the key coenzyme factor of histamine synthesis and metabolism,enzymes related to the metabolism of pyridoxal phosphate have differential metabolism.3.Study on the expression level of histamine synthesis regulatory proteins in Debaryomyces hansenii: The results showed that there were 261 differentially up-regulated proteins and 235 differentially down-regulated proteins;GO enrichment analysis showed the process of heme metabolism,redox process,and amino acid decomposition process There are many different genes in the process of catabolism of cellular nitrogen compounds,lipid biosynthesis,metal ion transport,pyridoxal phosphate binding,etc.;KEGG enrichment analysis shows histidine metabolism,sulfur metabolism,arginine and proline Acid metabolism,tryptophan metabolism,thiamine metabolism,lysine degradation,folic acid biosynthesis,?-alanine metabolism,valine,leucine and isoleucine degradation,porphyrin and chlorophyll metabolism,etc.There are many different genes in the process;enrichment analysis shows that the differential protein is involved in the global regulation of protein metabolism in TOR related to histamine synthesis and metabolism.4.Knockout of related genes(RAPTOR)in the TOR complex of Debaryomyces hansenii histamine synthesis and metabolism and its engineering bacteria application: successfully knocked out the regulatory related genes(RAPTOR)in the yeast TOR complex,and set the blank as In contrast,a comparative analysis of its histamine production ability was carried out.The results showed that histamine synthesis decreased after RAPTOR was knocked out and had no major influence on the growth of the strain,indicating that the yeast TOR complex had a regulatory effect on histamine synthesis. |
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