Study On Mycobiota In Traditionally Fermented Soybean Pastes In Northeast China And Metabonomics Of Functional Strains

Soybean paste is a traditionally Chinese fermented food with a history of nearly 3,000 years.There is a custom of making soybean paste all over the country,especially in the Northeast China,soybean paste has become a necessary food for every family.The "entire starter-making method" is the main method of making soybean paste in the Northeast China.From the lunar new year to May of the next year,soybean paste is exposed to the air,allowing the microorganisms in the air to ferment naturally,and forming a rich microbial community on the surface of soybean paste.There are many kinds of fungi in soybean paste,which produce various hydrolases to decompose the nutrient substances of soybean into small molecular substances that can be absorbed and utilized by human body.In the previous studies,there are no detailed reports about which fungi groups exist in the fermented soybean paste in Northeast China and which dominant strains participate in the fermentation process.In view of the importance of soybean paste in the dietary and living habits of Northeast China,the fermented soybean pastes were collected from the three provinces of the Northeast China for the study of fungi diversity from 2013 to 2017.388 samples of homemade soybean pastes were collected,covering a total of 72 districts in 26 cities and nearly 300 households of farmers.Traditional isolation and high-throughput sequencing methods were used to research the fungal groups in naturally fermented soybean paste,and to screen high-quality functional strains.This paper focuses on the above purposes,and summarizes and concludes with the following research contents and results:(1)A total of 25 genera of fungi were obtained by traditional isolation method,and 76 genera of fungi were detected by high-throughput sequencing.The methods of dilution separation and direct separation were used in the study.The rose bengal medium was used as the main separation medium.Through morphological and molecular biological identification,a total of 82 fungi species in 25 genera were obtained,including 14 species,3 genera of Zygomycetes,7 species,4 genera of Ascomycetes,and 61 species,18 genera of the anamorphic fungi.The isolation frequency of Penicillium is 39.2%,which is the most abundant fungal group present in the fermented soybean paste.76 fungal genera were detected by high throughput sequencing,and Ascomycetes is the most abundant phylum with 57 genera.Ophiocordyceps(14.78%),Debaryomyces(17.37%),Dipodascus(3.22%),Wickerhamomyces(1.89%),Penicillium(2.26%),Epicoccum(1.39%),Fusarium(1.15%)and some groups of which the genus are unidentified,such as Capnodiales(2.01%),Trichocomaceae(37.4%),Tremellomycetes(2.72%),Nectriaceae(1.14%),Mucoraceae(1.6%),Saccharomycetes(1.05%)and Saccharomycetales(8.78%)account for 96.76%of the total fungal sequences,distributed in all the samples.In terms of the fermented soybean paste made by the family,differences in the production process,production environment,and human disturbance factors during the production will lead to changes in the fungal groups in the soybean paste.Due to the existence of many external factors,each sample has its own unique fungal composition.(2)116 protease producing strains were obtained,among which Aspergillus oryzae HGPA20 and Penicillium glabrum GQ1-3 had the highest enzyme activities,which were 121.96 ±1.27 and 127.193±1.595 μg/mL·min,respectively.Among 116 protease producing strains,33 strains were found to have relatively high enzyme activity after removing the species with lower enzyme activity.A total of 29 strains with amylase activity were obtained through the preliminary soluble starch plate screening and the wheat bran medium rescreening.21 strains with lipase activity were obtained through the preliminary plate screening of tributyrin and the soybean solid medium rescreening.Among the active strains obtained.Penicillium and Aspergillus have an absolute advantage in the quantity and species and are important functional strains in the fermentation process of soybean pastes in the Northeast China.In addition,protease and amylase can also be produced by Mucor,Fusarium,Scopulariopsis and Cladosporium,and lipase activity can also be detected by two Mucor species.(3)Aspergillus oryzae HGPA20 and Penicillium glabrum GQ1-3 were used to ferment soybean paste with single strain,and the amino acid nitrogen content of Aspergillus oryzae HGPA20 and Penicillium glabrum GQ1-3 reached the maximum value of 0.59±0.023 g/100 mL and 0.56±0.008 g/100 mL,respectively,at 40 days of fermentation.Fermentation is a complex metabolic process.The fermentation period of the two strains was 90 days,and samples were taken every 10 days.The indicators such as water content,pH,total acid,amino acid nitrogen,reducing sugar and fat in the fermented soybean paste were measured by the national standard method.It was found that the content of amino acid nitrogen,reducing sugar and total acid in the fermentation system increased steadily with the passage of fermentation time,and the time for each substance to reach the maximum value was different,mainly concentrated in 40-60 days.The content of crude fat decreased significantly in the first 70 days of fermentation.(4)350 metabolites were screened out from the fermentation samples of the two strains GQ1-3 and HGPA20 by metabonomics.Through univariate,multivariate and KEGG analysis,72 differential metabolites were obtained,and 7 differential metabolic pathways closely related to fermentation were screened.Among them,alanine,aspartate,and glutamate metabolism had the highest correlation with differential metabolites and fermentation process.Amino acid biosynthesis and the TCA cycle are two important metabolic activities.The relative contents of metabolites involved in the process,such as a-ketoglutarate,isoleucine,ornithine,glutamine and citrulline,are higher in GQ1-3 than in HGPA20,while those of fumaric acid,L-malic acid,succinic acid,L-homoserine,aspartic acid and asparagine are higher in HGPA20.The results show that there are differences in umami substances in the fermentation system of the two fungi.This paper clarifies the diversity of fungi in the soybean paste made in Northeast China,enriches functional fungal species in fermented food,and lays a theoretical foundation and strain reserve for improving the quality of fermented soybean paste.