Fungal Diversity And Detection Of Mycotoxins In Naturally Fermented Soybean Paste In Some Regions Of China

Soybean paste is one of the commonest traditional condiments in China.The origin of Chinese soybean paste can be traced back to Zhou Dynasty,about 3,000 years ago.Untill now, the naturally fermented home-made soybean paste was widely consumed by Chinese people. Due to the open fermenting process,there are rich fungi in the home-made soybean paste and it is inevitable to avoid the contamination of mycotoxins and may harm the consumer's health. Many studies were conducted on bacteria and yeast in soybean paste,but its fungal studies were scarce.This study is the first systematic research on fungal diversity in naturally fermented soybean paste.The comprehensive analysis of fungal diversity was performed in aspects of paste making regions,methods and fermentation time.It is also the first detection of harmful mycotoxions in soybean paste by high-performance liquid chromatography after immunoaffinity chromatogaphy cleanup.The result shows that the presence of four mycotoxions marks the potential danger of the home-made soybean paste.The study of protease activity of some Aspergillus and Mucor isolates was also conducted,it is found that 62%of Aspergillus and 33%of Mucor possessed protease activity.The protease features of one Aspergillus strain and three Mucor strains were studied.The total results are listed as follows:The fungal species isolated from soybean pasteTotally 280 samples of soybean pastes were collected and investigated in this study, including 251 samples of home-made products in 72 regions of 11 provinces including Northeastern China,Inner Mongolia,Henan,Hebei,Sichuan,Anhui,Guizhou,Fujian and Jiangxi,26 samples of industry products from Changlong brewing factory in Changchun, Heitudi brewing factory in Dalian and Douwa brewing factory in Jinzhou,and 11 samples from markets.At last 90 fungal species belonging to 25 genera were isolated and identified. Of which 13 species of four genera are Zygomyctes,6 species of three genera Ascomyctes and 71 species of 18 genera Anamorphic fungi.Fusarium brevicatenulatum and Penicillium coprophilum are two newly recorded species for China,Aspergillus niger var.jinzhouanum and A.clavatus var.aureus are named as new taxa for their specific morphology and molecular evidence.Each fungal species was morphologically illustrated and described with microscopic photos.The species of Aspergillus and Penicillium were provided with clony features on standard media.The SEM photos of 8 main Aspergillus species were provided.The molecular identification of some suspicious fungal strainsSuch Aspergillus species as A.flavus and A.oryzae are easily misidentified due to their similar morphological characteristics.It is well-known that A.flavus is the aflatoxins producer and A.oryzae the common fungus used in food industry.In order to correctly identify the two species,the comparative studies were conducted on their cultures on standard media and rDNA ITS sequences.The comparative studies reveal that A.flavus occurred more frequently on soybean paste than A.oryzae.Theβ-tubulin sequence analyses were performed to support the new taxa of A.niger var.jinzhouanum and A.clavatus var.aureus also as P.coprophilum. A.niger var.jinzhouanum is characterized by adnate or verticillate appendages on conidiophore stalks just below the vesicles.The appedages on conidiophore stalks are new descriptive characters for Aspergillus in literature. The analysis of fungal diversity of naturally fermented soybean pastesThe research data were analyzed with the software of data processing system(2002).The results indicate that the fungal diversity was influenced by the paste making environment and methods.The predominant fungal groups in home-made pastes were Penicillium,Aspergillus, Mucor,Fusarium,Acremonium,Rhizopus,Scopulariopsis,Trichoderma and Cladosporium, while the commercial fungi of Monacsus and A.oryzae were seldom encountered.It impies that home-made soybean pastes were not good media for the two economic fungi.The continous investigation of fungi on home-made pasets shows that the fungal species population and amount had changed during the whole fermenttion course.The fungal population of soybean pastes were dominated by Mucor in early fermentation stage,and then replaced by Penicillium,Aspergillus and Fusarium with the decrease of moisture in pastes. The presence of Aspergillus from brine fermentation until mature stage of soybean pastes indicates its important role palyed in the soybean paste making process.The making methods and microenvironments influenced the quality of the soybean pastes.The method of"partial koji"in Zhuanghe which did not favor the vigorous multiplication of poisonous fungi should be recommended for the home-made soybean pastes.Detection of main mycotoxins in soybean pastesThe mycotoxin content of 21 home-made samples and two industry samples were examined by high performance liquid chromatography after immunoafinity column clean-up. The results shows that 13 samples of soybean pastes(56.5%)were detected to contain aflaxoxin,six samples(26%)contain aflatoxin B₁.The frequence of ZEA,T-2 and DON contamination was low respectively with only one sample contaminated with each one toxin. The results indicate that the potential danger exists in home-made soybean pastes.Screening of protease-producing fungal strains in soybean pastes34 strains of Aspergillus spp.and 24 Mucor spp.isolated from naturally fermented soybean paste were screened and 21 strains(62%)of Aspergillus spp.and 8 strains(33%)of Mucor spp.were detected with high protease activity,it implies that many economic fungi from home-made soybean paste could be commercially utilized in industry.Studies on the protease feature of protease-producing strainsThe protease feature of Asp-195v isolate was studied.The optimum medium is bran: bean cake=7:3 with 40%moisture content and pH 7.0.The optimum conditions for the protease production were 28℃and 48h of incubation and neutral pH condition.The favorable temperature for protease reaction was 50℃.The salt concentration influenced the protease activity.The protease activities of 3 Mucor strains were investigated.It was found that the optimum incubation conditions for protease production of the M-339,M-513 and M-808 were bran:water=1:1.1 at 26℃;bran:water=1:1.2 at 28℃and bran:water=1:1.3 at 30℃respectively.The feature of protease production for each Mucor isolate was in accordance with their fermentation environment.