Aflatoxin B₁ Producing Aspergillus And Antagonistic Microorganisms In The Processing Of Sojae Semen Preaparatum

ObjectiveBy detecting the content of aflatoxin（AFT）,screening,identifying and quantifying AFT-producing microorganisms（referred to as toxin-producing microorganisms）and determining its toxin-producing ability at different time periods in the natural processing of Sojae Semen Preaparatum（SSP）to study the antagonistic microorganisms ability of aflatoxin B₁（aflatoxin B₁,AFB₁）and preliminarily analyze the role of toxin-producing microorganisms and antagonistic microorganisms in the growth and decline of AFB₁,to lay a foundation for exploring the growth and decline mechanism of aflatoxin in the processing of SSP.It also provides a scientific basis for the prevention and control of aflatoxins in SSP or other fermented traditional Chinese medicines and foods.MethodⅠ.Determination of aflatoxin（AFT）content in the natural processing of SSP1.Natural fermentation processing of SSP:In the early stage of this laboratory,a standardized processing technology of SSP has been established in accordance with the 2015 edition of Pharmacopoeia of the people’s Republic of China（except for 1963edition,the calendar version preparation method is the same）.In this study,SSP was prepared according to this preparation process,and the samples of SSP at different processing time periods and the finished product of SSP were obtained.2.Establish Ultra Performance Liquid Chromatography Tandem Mass Spectrometry（UPLC-MS/MS）to determine the content of AFT in the samples of SSP at different processing time periods.Ⅱ.Screening,identification,quantification and toxin-producing ability of toxin-producing microorganisms in the natural processing of SSP.1.Screening and identification of toxin-producing microorganisms in the natural processing of SSP.（1）Screening of toxin-producing microorganisms and colony count:According to the principle that AFT can produce blue-violet or green fluorescence under ultraviolet light irradiation,the microorganisms that produce blue-violet or green fluorescence are screened and isolated and colonies are counted.（2）Preliminary identification of toxin-producing microorganisms:The morphology of the colony was observed by eyes and the individual morphology of the toxin-producing microorganisms was observed using a microscope to initially identify the toxin-producing microorganisms（3）Molecular biology identification of toxigenic microorganisms:Extract the DNA of toxin-producing microorganismsand use 18Sr DNA sequence to carry out PCR amplification on microorganisms,and carry out gene sequencing analysis on the amplified products.Find the template gene sequence,construct a phylogenetic tree to identify toxin-producing microorganisms.（4）Quantitative analysis of toxigenic bacteria（The experiment is in progress）2.Comparison of AFT production capacity of toxin-producing microorganismsUPLC-MS/MS method was used to determine the AFT content in the fermentation broth of toxin-producing microorganisms and compare their AFT production ability.Ⅲ.Investigating the antagonistic ability of AFB₁antagonistic microorganisms in the processing of SSP（In the early stage of our laboratory,we isolated and identified 5beneficial microorganisms（abbreviated as antagonistic bacteria）that have been reported to can inhibit the production of AFT（such as Bacillus subtilis,Saccharomyces salmoniae,Aspergillus niger,Enterococcus faecalis and Enterococcus avium）.To investigate the antagonistic ability of these 5 strains of AFB₁antagonistic microorganisms1.The effect of antagonistic microorganisms on the growth and reproduction of AFT-producing Aspergillus flavus（1）Plate confrontation method to detect the effect of antagonistic microorganisms on the growth of AFT-producing Aspergillus flavus:The plate confrontation method was used to investigate the effects of inoculated and non-inoculated antagonistic microorganismson the growth of AFT-producing Aspergillus flavus.Inhibition rate=（diameter of Aspergillus flavus in the control group-diameter of Aspergillus flavus in the experimental group）/Aspergillus flavus in the control group×100%.（2）The effect of the supernatant of antagonistic microorganisms fermentation on the growth of Aspergillus flavus:Take the antagonistic fermentation supernatant and mix it with potato dextrose agar（PDA）medium.After solidification,inoculate the AFT-producing Aspergillus flavus standard strain in the center of the plate and measure the growth diameter.Use only PDA medium for culture Substrate,inoculate AFT-producing Aspergillus flavus standard strain in the center of the plate as a control.Inhibition rate=（diameter of Aspergillus flavus in the control group-diameter of Aspergillus flavus in the experimental group）/Aspergillus flavus in the control group×100%.3.Antagonistic microorganisms effect on the degradation of AFB₁Inoculate 5 strains of antagonistic microorganisms in the fermentation broth containing the same concentration of AFB₁standard substance for shaking culture.The fermentation broth with the same concentration of AFB₁standard without inoculation was used as a control,and the AFB₁content in the fermentation broth was detected by UPLC-MS/MS.4、Correlation analysis of the changes of AFT content,the quantity of the production of AFT microorganisms and antagonistic effect of antagonistic activity of the antagonistic microorganisms in the processing of SSPResultⅠ.Preparation and sampling of SSPSampling is taken every 3 days,which are recorded as 0 days of fermentation,3 days of fermentation,6 days of fermentation,3 days of boring,6 days of boring,9 days of boring,12 days of boring,15 days of boring,and finished product after steaming and labeled as F0,F3,F6,Z3,Z6,Z9,Z12,Z15 and Z steam respectively.Ⅱ.The determination results of 4 kinds of AFT（aflatoxin B₁,AFB₁;aflatoxin B₂,AFB₂;aflatoxin G₁,AFG₁;aflatoxin G₂,AFG₂）at different time points during the natural processing of SSP.The UPLC-MS/MS method was used to determine the content of four kinds of AFT,and the methodological investigation was good,indicating that this method is suitable for the determination of AFT content in SSP;the measurement results show that the content of AFT changes dynamically during the natural processing of SSP.1.UPLC–MS/MS method for the determination of four AFTs contents in SSP was established（1）Linear relationship investigation:taking each concentration（ng/m L）standard product as the abscissa x and the corresponding peak area as the ordinate y,the standard curve equation of AFB₁is y=402087x+7996.2,r=0.9998;the standard curve equation of AFB₂is y=58257x+1241.8,r=0.9997;the standard curve square of AFG₁is y=331396x-6075.6,r=0.9999;the standard curve equation of AFG₂is y=29342x+92.996,r=0.9998,.It shows that the standard concentration has a good linear relationship between 0.05-20 ng/m L.（2）Repeatability investigation:the relative standard deviation（RSD）（n=6）of AFB₁,AFB₂,AFG₁and AFG₂are 0.40%,0.43%,0.93%,0.49%respectively,indicating that the repeatability is good（3）Precision inspection:the relative standard deviation（RSD）（n=6）of AFB₁,AFB₂,AFG₁and AFG₂are 0.67%,2.01%,0.77%,2.59%respectively,indicating that the precision is good.（4）Inspection of sample recovery rate:when the amount of AFB₁and AFG₁added were 5 ng/g,and the amount of AFB₂and AFG₂added were 1.25 ng/g,the average recovery rates of AFB₁,AFB₂,AFG₁and AFG₂were 97.5%,99.5%,98.2%,99.2%,RSD（n=6）were 2.32%,2.70%,0.5%,2.64%respectively;when the amount of AFB₁and AFG₁were 1.16 ng/g,AFB₂and AFG₂were 0.29 ng/g,the average recovery rates of AFB₁,and the amount of AFB₂and AFG₂were 102%,94%,92%,90.6%,RSD（n=6）were 2.24%,1.33%,1.83%,1.56%respectively,indicating that the recovery rate is good.2.Four kinds of AFTs content determination:during the natural processing of light tempeh,the content of AFT increased first and then decreased.It gradually increased from the third day of fermentation,and reached the highest value on the sixth day of re-stuffing,which was 6.95μg/kg,and then began to decrease.The AFT content dropped to 1.2μg/kg on the 9th day of re-stuffing,and the AFT content was all 0 after the 12th day of re-stuffing.Ⅲ.Screening,identification,quantification and AFT-producing ability of toxin-producing microorganisms in the natural processing of SSP15 strains of AFT-producing microorganisms were screened out,which were identified as Aspergillus flavus and Aspergillus tamarii through morphology and molecular biology.The colony count of AFT-producing microorganisms showed that the number of AFT-producing microorganisms changed dynamically during the processing of SSP,and reached the maximum on the 6th day of fermentation.The AFT-producing ability of the toxin-producing microorganisms was investigated,and the results showed that the AFT-producing microorganisms during the processing of SSP were weak（less than 5 ng/m L,much lower than the toxin-producing ability of the standard Aspergillus flavus,which was 654.90 ng/m L））,and the ability of each microorganisms to produce AFT is different.1.Screening and colony count of toxin-producing microorganisms:According to the principle that AFT can produce blue-violet or green fluorescence under ultraviolet light irradiation,a total of 15 microorganisms that produce blue-violet are screened and isolated,namely Aspergillus flavus and Aspergillus tamarii.The number of AFT-producing Aspergillus colonies increased first and then decreased.It gradually increased from the 3rd day of fermentation to the largest number of colonies on the6th day of fermentation,which was 1x10^6.30CFU/g,and then began to decrease.From the 9th day re-stuffing,no fluorescence-producing microorganisms were detected.2.Identification of toxin-producing microorganisms（1）Identification of traditional microbiological methods:the screened toxin-producing microorganisms were cultured on a plate,and the colony morphology was observed with eyes,and the colony morphology was observed to be similar to Aspergillus flavus and Aspergillus tamarii.Using a microscope to observe its individual morphology,it is found that it is similar to Aspergillus tamarii and Aspergillus flavus.（2）Molecular biology method identification:the toxin-producing strains were identified,and the identification results:F6–1,F6–2,F6–3,F6–4,F6–5,F6–6（the fluorescent reaction strains screened on the sixth day of fermentation）,F3Fluorescence-producing strains screened on the third day of fermentation),Z6（for the fluorescence-producing strains screened on the 6th day of the fermentation）,Z3–5,Z3-2（for the fluorescence-producing reaction screened on the 3rd day of the fermentation）Strains)are Aspergillus flavus,Z3–1,Z3–3,Z3–4,Z3–6,and Z3–7（the fluorescent reaction strains screened on the third day of repetition）are Aspergillus tamarii.3.Fluorescence quantitative PCR method to determine the number of toxin-producing microorganisms（experiment in progress）4.Toxin-producing ability of each toxin-producing strain（1）UPLC–MS/MS method for the determination of AFT in fermentation broth was establishedLinear relationship investigation:by taking the standard of each concentration（ng/ml）as the abscissa x,the corresponding peak area is the vertical coordinate y,the standard curve equation of AFB₁is y=35783x+12495,r=09996;the standard curve equation of AFB₂is y=38106x+3381.7,r=0.9996;the standard curve equation of AFG₁is y=22839x+7820.1,r=0.9995;the standard curve equation of AFG₂is y=9212.8x+1372.9,r=0.9996.Repeatability investigation:the relative standard deviation（RSD）（n=6）of AFB₁,AFB₂,AFG₁and AFG₂were 2.08%,3.50%,1.18%and 1.66%respectively,indicating that the repeatability was good.Sample recovery rate investigation:when the amount of AFB₁and AFG₁is 5 ng/m L,and when the amount of AFB₂and AFG₂is 1.5 ng/m L,the average recoveries of AFB₁,AFB₂,AFG₁,and AFG₂are 94.53%,92.53%,94.87%and 91.47%respectively,RSD（n=3）are 1.01%,1.80%,1.60%,2.20%,respectively.When AFB₁and AFG₁were added at 2 ng/m L and AFB₂and AFG₂were added at 0.6 ng/m L,the average recoveries of AFB₁,AFB₂,AFG₁,and AFG₂were 92.83%,93.33%,92.83%,92.0%,RSD（n=3）1.12%,2.47%,1.89%,2.17%respectively,indicating that the sample recovery rate was good.（2）Determination of AFT content in fermentation broth of toxigenic microorganisms The UPLC-MS/MS method was used to detect the AFT content of the fluorescence-producing microorganisms in the fermentation broth.The results showed that out of the 15 fluorescence-producing microorganisms,5 strains did not produce AFT,10 strains produced AFT,and the AFT content was 2.0-4.10 ng./m L between.Ⅳ.The antagonistic ability of AFB₁antagonistic microorganisms in the processing of SSPIn the early stage of our laboratory,we isolated and identified 5 strains（abbreviated as antagonistic microorganisms）that have been reported to inhibit the production of AFT,such as Bacillus subtilis（JX2）,Saccharomyces salmoniae（EJ1）,Aspergillus niger（JC2）,Enterococcus faecium（BR5）,Enterococcus avium（9R2）,to investigate the antagonistic ability of these 5 strains of AFB₁.The results showed that these five strains had a certain inhibitory effect on the growth of AFT-producing Aspergillus flavus standard strains.Among them,S.salmoniae had the strongest inhibitory effect and the strongest degradation effect on AFB₁.1.The effect of the seed liquid of 5 strains to be tested on the growth of the standard strain of Aspergillus flavus:the results showed that the seed culture solution of 5strains had a certain inhibitory effect on the growth of toxigenic Aspergillus flavus.Among them,S.salmoniae has the strongest inhibitory effect on the growth of Aspergillus flavus,with an inhibition rate of 64.29%.The inhibition rates of Enterococcus faecium,Enterococcus avium,Bacillus subtilis,and Aspergillus niger on the growth of Aspergillus flavus are all about 30%.2.The effect of the fermentation supernatant of 5 strains to be tested on the growth of toxin-producing Aspergillus flavus:the results showed that the fermentation supernatant of the 5 strains to be tested had a certain inhibitory effect on the growth of toxin-producing Aspergillus flavus.Among them,Enterococcus faecium had the strongest inhibitory effect,with an inhibitory rate of 29.63%.3.Degradation of AFB₁by 5 strains of microorganisms to be tested:the results showed that the fermentation broth of the 5 strains all had a certain degradation effect on AFB₁.Among them,S.salmoniae had the strongest degradation effect on AFB₁,reaching 43.65%,and the Aspergillus niger had the weakest degradation effect.Conclusion1.The UPLC-MS/MS method was established for the determination of AFT content in SSP.This method is simple,rapid and highly sensitive.The content of AFT changes dynamically during the processing of SSP,showing a trend of first increasing and then decreasing.which further confirms the importance of"re-stuffy"and the rationality of the time of“re-stuffy”.2.There are Aspergillus flavus and Aspergillus tamarii that produce AFT during the processing of SSP.Aspergillus flavus accounts for the majority.3.There are antagonistic microorganisms that degrade AFT and inhibit the growth and reproduction of AFT-producing strains such as Aspergillus flavus during the processing of SSP.4.The AFT-producing microorganisms and the antagonistic microorganisms interact during the processing of SSP,and the AFT during the processing of SSP is dynamically changed.The inhibitory effect of antagonistic microorganisms on the AFT-producing microorganisms and the degradation of AFT make the finished product of SSP not Containing AFT.To lay a foundation for revealing the growth and decline mechanism of aflatoxin in the processing of SSP,and to provide a scientific basis for the prevention and control measures of aflatoxin in SSP.