Production Of Alfatoxin B₁in The Chinese Fermented Soy Sauce And Its Control Research

As one of the traditional fermented condiments in China, the fermented soy saucehas high output and consumption. It is not only an indispensable condiment on thetable of Chinese residents, but also an important economic pillar of the fermented foodindustry. However, due to the growing environmental pollution and backwardproduction technology, the food safety issue of fermented food is outstandingly, thesafety of fermented soy sauce is also questioned by people. From the cultivation andstorage of raw materials to koji and the fermentation process of paste, it is inevitably tobe contaminated by aflatoxin B₁（AFB₁）. Consequently, the fermented soy sauce islikely to be contaminated with AFB₁, which results in serious harm to human healthand economic development. Therefore, in order to protect the dietary safety of Chinesepeople and the steady and rapid development of fermented food industry, it isparticularly important to survey the AFB₁comtamination in fermnented soy sauce andcontrol AFB₁contamination. In this study, the AFB₁contamination status in fermentedsoy sauce was investigated and its potential risk to people health was analyzed. Then,with the actual production process, the reasons which cause AFB₁contamination infermented soy sauce were analyzed. Based on the results, the strain that could safely,economic, efficient control AFB₁contamination was isolated. Finally, the mechanismof biocontrol AFB₁was comprehensively analyzed and the major studies are asfollows:1. The contamination of AFB₁in209fermented soy sauce samples came from8provinces of Shannxi, Shanxi, Shandong, Liaoning, Jiangsu, Zhejiang, Shanghai andGuangdong was detected by direct competitive enzyme-linked immunosorbent（ELISA）. It was found that AFB₁contamination in fermented soy sauce in China iswidespread with lower level. The concentration of all samples was below the Chineseregulation （5ng/mL） with the mean of0.40±0.42ng/mL and fits to the Normaldistribution model. Based on the data of soy sauce intake and the value of hepatitis Bsurface antigen-positive rate （HBsAg+）, the exposure and population risk models ofAFB₁through the fermented soy sauce were estimated by probabilistic approach usingthe Monte Carlo method. Results showed that the exposure is from0to0.177ng/kgbw/day with the mean of0.0417±0.0457ng/kg bw/day and fits to the Logisticdistribution model, the population risk is from3.87E-6to6.87E-3cancers/year/100,000people with the mean of1.42E-3±1.57E-3cancers/year/100,000people and fits to the Logistic distribution model. On the whole, there is high level ofAFB₁contamination, dietary intake and potential risk for cancer in south samples.2. To determine the source of AFB₁in fermented soy sauce and find the critical control point, the dynamic changes of AFB₁concentration and microbiology weremonitored during the process. Changes in AFB₁content is divided into three stages: thefirst stage is from raw materials to the4day of fermentation, AFB₁content （dry basis）decreased and got the minimum value. The second stage is from the4day to12day offermentation, toxigenic fungi began to produceAFB₁. So the AFB₁content increasedsignificantly, and reached a maximum value in the12day of fermentation. The thirdstage is from the fermentation of the12day to the end of fermentation, toxigenic fungistoped growing and AFB₁was degraded, so the content of AFB₁was decreasedgradually. Results revealed that50%of AFB₁is caused by raw materials,17%of AFB₁and33%of AFB₁is produced in early and middle fermentation period, respectively.Seven strains were isolated from fermented paste, which can inhibit AFB₁biosynthesisby Aspergillus flavus3.4408and degrade AFB₁simultaneously. For the seven strains,FS10has the strongest ability to control AFB₁contaminaton with80.33%of inhibitionand57.44%of degradation, respectively. So it was selected for further study. FS10wasidentified as Aspergillus niger by morphology and18S rDNA sequence.3. To elucidate the biocontrol mechanism, the ability of Aspergillus niger FS10andits fermentation products against growth, sporulation and AFB₁production bytoxigenic Aspergillus flavus3.4408was evaluated in vitro. Results showed that FS10fermentation products could restrain the expression of aflR and nor-1, and its ability toinhibit mycelia growth of Aspergillus flavus, sporulation and AFB₁production weresignificantly higher than FS10, with the maximal inhibition of40.97%、100%and94.48%, respectively. The MIC and MFC of FS10fermentation products againstAspergillus flavus were80μL/mL and320μL/mL, respectively. Results indicated thatthe active substance is a heat-resistant protein. In addition, the effect of FS10fermentation products to spore germination, ultrastructure monphology of spore andmycelium were also studied by optical microscopy, atomic force microscopy （AFM）and scanning electron microscopy （SEM）. Result showed that FS10fermentationproducts could significantly inhibit spore germination. Morphology of spores wasserious damaged accompanying with spore analosis and cell wall collapse, resulted ininhibition of spore germination. Forthermore, it made mycelium of Aspergillus flavusto distort, shrink; then the cell wall structure was serious damaged to prevent thegrowth of mycelium.4. The degradation of AFB₁by FS10and its fermentation products was studied.Results showed that AFB₁degradation is an enzymatic degradation by extracellularenzyme with poor thermal stability produced by FS10. When FS10fermentationproducts were salted by80%saturation of ammonium sulfate, the degradation activitywas strongest. The optimum enzymatic pH and temperature are6.0and40℃, respectively. The mutagenicity of FS10fermentation products and AFB₁degradationproducts were analyzed by Ames test. The results indicated that the fermentationproducts and degradation products did not have the ability of mutagenicity. In addition,the fluorescence spectrometry and LC-MS of AFB₁degradation product was studied.Results showed that fluorescence intensity of AFB₁was significantly reduced by thedegradation enzyme, and a substance with molecular weight of286was formed. Theprobable degradation pathway as follow: lactone ring structure of AFB₁was opened bythe degradation enzyme; then, an intermediate product with β-keto structure wasformed; finally, M was formed after decarboxylation reaction, cause greatly reductionof AFB₁toxicity.