Study On The Formation And Dynamic Changes Of Harmful Substances During The Broad Bean Fermentation

As an important fermented seasoning with a long history in China,broad bean paste has high edible nutritional value.Pixian broad bean paste in Sichuan Province enjoys the reputation as the soul of Sichuan cuisine due to its unique production technology,and it has a broad consumption prospect in China.The manufacturing process includes koji making,post-fermentation of broad bean,and mixed fermentation with pepper.Fermented mature broad bean,commonly known as broad bean mash,was an intermediate product,which directly affects the quality of final product.Broad bean and flour are the main raw materials for processing.Using pure Aspergillus oryzae to make koji is the preferred method for many modern bean processing companies.The koji-making process is conducive to the proliferation of Mold,while a good time to contaminate toxin-producing strains and the accumulation of aflatoxin B₁（AFB₁）.The microorganisms composition is relatively complex in the fermentation,and the decarboxylation reaction of microorganisms may promote the synthesis of biogenic amines.In recent years,researches on the harmful substances represented by biogenic amines in fermented foods have gradually attracted the attention of scholars at home and abroad,while the research on biogenic amines and AFB₁ in the fermentation process of bean paste is still relatively lacking.In this paper,the two harmful substances,quality indicators and bacterial phases during the industrial fermentation of broad bean were dynamically tracked,and the thermal insulation fermentation was simulated in the laboratory environment.Using Aspergillus oryzae 3.042 to make koji and optimize the koji-making process,and the influence of salinity and temperature on the formation of harmful substances and quality in the post-fermentation process were explored.The isolation and identification of the amines-producing strains were carried out.The aim is to provide a reference for the formation mechanism and blocking measures of harmful substances in the actual production.The main conclusions were as follows:1.The changes of the harmful substance and quality indicators of fermented broad bean during post-fermentation process in industrial production were studied.The results showed that the spermine content decreased regularly over time during traditional sealed fermentation（from 0th to 12th month）and thermal insulation fermentation（from0th to 30th day）.The histamine content continuously rise to 23.23 mg/kg in traditional fermentation,and 2-phenylethylamine and tyramine increased sharply to 84.13 mg/kg and 73.93 mg/kg respectively in December of traditional fermentation.Among them,2-phenethylamine exceeds the limit value,which has a certain safety risk.The total amines content in final product was 238.52 mg/kg in traditional fermentation and 81.62mg/kg in thermal insulation fermentation.The AFB₁ content increased continuously over time,which were 2.29μg/kg and 2.75μg/kg respectively in final product.The total amines and AFB₁ contents were both lower than the limit standards and safe to eat as a whole.The pH and water activity decreased overall,while the contents of NaCl,total acid,and amino nitrogen increased during the two fermentation processes.The moisture content were both less than 50%in final products.The NaCl,total acid,and amino acid nitrogen content in final product of traditional fermentation were 13.248 g/100g,1.471g/100g,0.725 g/100g,which were higher than those in thermal insulation fermentation.Total biogenic amines were positively correlated with the total plate count in the two fermentations,and each biogenic amines and AFB₁ had strong correlations with most physical and chemical indicators and also microorganisms.2.The changes of the harmful substance and basic quality indicators of fermented broad bean during koji making process were studied.The results showed that the optimal parameters were determined by single factor and orthogonal optimization design tests,which were Aspergillus oryzae 3.042 inoculation amount of 0.3%,koji-making time of 72 hour,temperature of 30℃and the flour addition amount of 15%,and the neutral protease activity was 646.02±7.82 U/g in mature broad bean koji.The main biogenic amines in raw broad beans were spermine,spermidine,and putrescine,while AFB₁ content was 1.16μg/kg.The dominant amines in flour was putrescine and the AFB₁ content was 0.84μg/kg.During the koji making process,the putrescine content increased to become the dominant amines.The total amine content reached a maximum values of 215.95 mg/kg at 24th hour.The highest value of AFB₁ was 2.08μg/kg at 0th hour,and the content decreased and fluctuated slightly from 12th to 72th hour.The total biogenic amines content in broad bean koji was 149.27 mg/kg,while the AFB₁ content was 1.40μg/kg,both of which were at safe levels.During the koji making process,the water content and water activity decreased regularly,and the amino nitrogen content gradually increased to remain stable.The pH and total acid content reached the minimum and maximum values at 24th hour,respectively.The amino nitrogen and total acid content in broad bean koji were 0.548 g/100g and 0.606 g/100g,respectively.3.The reasonable post-fermentation time（0-60 days）of broad bean in laboratory conditions were studied.The results showed that the change trend of physical and chemical indicators is similar to the industrialized thermal insulation fermentation.The amino nitrogen value was the highest on the day 40 of fermentation,which was 0.646g/100g.The total content of biogenic amines decreased regularly,and the total content were all below 100 mg/kg from day 40 to day 60,while the toxic histamine,tyramine and 2-phenethylamine were far below the limit.The content of AFB₁ increased with time and changed not significantly from day 40 to day 60（P>0.05）.It was considered that the post-fermentation time of broad bean in this environment was 40 days through comprehensively considering the maturity,quality index and time efficiency.4.Effects of different salinity of 9%,12%,15%,18%and 21%in laboratory on the formation of harmful substances and quality in post-fermentation process of broad bean were studied.The results showed that highest total plate count were detected during 9%salinity fermentation.The content of 2-phenylethylamine,putrescine,tyramine and total amines in mature fermented broad bean at 9%salinity was significantly higher than that in other groups（P<0.05）,and the highest tyramine content was 103.41 mg/kg,which might has a security threat.The total biogenic amines content of the other four groups were less than 100 mg/kg.The 9%fermented mature broad bean had the highest AFB₁ content of 3.06μg/kg,while the other groups were below 3μg/kg.Low salinity accelerated the enzymatic hydrolysis to produce acid and promote sample maturation.The total acid content in the 9%salinity group was as high as 1.894 g/100g finally,which had risk of rancidity.Therefore,in order to minimize the salt content and ensure food safety,it is reasonable to select 12%salinity for broad bean fermentation.5.Effects of different fermentation temperatures of 35°C,40°C,45°C and 50°C in laboratory on the formation of harmful substances and quality in post-fermentation of broad bean were studied.The results showed that highest total plate count were detected at 35°C during fermentation,and the content of 2-phenethylamine,putrescine,histamine,tyramine,total amines and AFB₁ were highest in final product.Among them,putrescine reached 222.90 mg/kg and 2-phenethylamine reached 30.12 mg/kg,which has a certain consumption risk.At the end,the content of biogenic amines and AFB₁ were 261.75mg/kg and 3.83μg/kg respectively,which were significantly higher than those in other groups（P<0.05）.Total biogenic amines were less than 100 mg/kg,and AFB₁ was less than 3μg/kg in other three groups.The fermentation at 40℃and 45℃shortened the fermentation time effectively,and the total acid and amino nitrogen were the highest.Sensory comparison showed that the scores of 40°C and 45°C mature samples were relatively high,which were closer to products of two factory fermentation.Therefore,40℃and 45℃were selected considering edible quality and safety.6.Changes of microbial bacteria during fermentation process of broad bean were studied.The results showed that the number of Lactobacillus,Bacillus,and Enterobacteriaceae reached the maximum at 24th hour during koji-making process in laboratory,and the number decreased gradually during 24th-72th hour.The Mould increased to become the dominant bacteria at 72th hour,and the number was 6.89lg（CFU/g）.The number of Mould,Enterobacteriaceae,and Lactobacillus decreased gradually during post-fermentation in laboratory.Among them,Enterobacteriaceae,and Lactobacillus were undetectable after 5 and 10 days,respectively.The number of Yeast was only detected at low levels in the first 10 days.The number of Bacillus increased gradually and became dominant bacteria since 5th day,stabilizing around 5 lg（CFU/g）after that.During traditional fermentation and thermal insulation fermentation in factory,Lactobacillus and Enterobacteriacea only existed at the initial stage of fermentation.The number of Mould decreased over time.The number of Yeast in traditional fermentation was higher than thermal insulation fermentation.Bacillus was predominant bacteria in two processes,which were maintained at 5-6.5 lg（CFU/g）.7.Isolation and characterization of biogenic amines-producing microorganisms from broad bean fermentation process were studied.The results were as follows:Enterobacter spp including Enterobacter aerogenes and Enterobacter cloacae and E.coli showed high putrescine and cadaverine synthesis ability,and the production amounts were 83.06-208.16 mg/L and 14.03-76.20 mg/L respectively.Lactobacillus included Lactobacillus plantarum,Lactobacillus brevis,and Lactobacillus curvatus.Lactobacillus plantarum has a lower ability to produce amines,but Lactobacillus brevis produced tyramine at 36.58 mg/L.Enterococcus faecium and Enterococcus faecalis mainly synthesized 2-phenethylamine in 22.25-37.68 mg/L and tyramine in 46.04-55.63mg/L,respectively.Bacillus subtilis was the most common in amines-producing Bacillus,and the amines-producing ability of different strains were slightly different,but both showed strong ability to synthesize putrescine and cadaverine,which were25.82-71.83 mg/L and 13.15-42.88 mg/L,respectively.