Study On The Main Spoilage Bacteria And Their Source Analysis In Tofu

Tofu(soybean curd)as a kind of traditional Chinese food is very popular with comsumers in our country.Due to its high-moisture and rich-nutrition,tofu is also a highly favourable medium for the growth of microorganisms and easy to deteriorate.In this study,boxed lactone tofu(NZT)and simply packaged firm tofu(SLT)were investigated to unveil the composition of the spoilage microbiota,and the sources of these microorganisms in the production process.In addition,the spoilage potential of different bacteria isolated from spoiled tofu was also evaluated.Based on this result,simple and effective sanitary control measures were proposed to extend the shelf-life of tofu.Random samples from three companies were selected as specimens.Firstly,the strains were isolated from spoiled NZTs and SLTs that were stored at 4°C,25°C and 37°C respectively,and then sequenced and identified by 16 S rRNA gene method.The results showed that Bacillus was dominant in NZTs including Bacillus subtilis,Bacillus licheniformis,Bacillus amyloliquefaciens,et al.Meanwhile,the spesies in SLTs were complex and diverse,including Kurthia,Pseudomonas fluorescens,Leuconostoc mesenteulides,Enterobacter aerogenes,et al.Furthermore,MiSeq high-throughput sequencing technology was also used to analyze the spoilage microflora and bacterial diversity of the tofu of band C.The results indicated the microflora in NZTs was less complex than that in SLTs.The dominant bacteria in NZTs were Bacillus and their abundance were all above 90%.In addition,the microbial diversity of the spoiled SLT stored at 4°C was relatively higher compared to the other two SLT samples.Leuconostoc was the dominant bacteria in spoilage SLTs stored at 25°C and 37°C,accounting for 71% and 80% respectively.Second,the effects of several major bacteria on the spoilage potential to tofu were evaluated respectively.(1)Leuconostoc mesenteroides and Enterobacter aerogenes had strong ability to produce acid in firm tofu,while Bacillus amyloliquefaciens had strong ability to produce acid in lactone tofu,and the total acid contents in these inoculated samples were significantly increased(P<0.05)during storage.(2)Pseudomonas fluorescens and Bacillus subtilis were easier to decompose protein,resulting in higher TVB-N values in lactone tofu and firm tofu.(3)Kurthia sp and Bacillus amyloliquefaciens can cause the b* of tofu significantly increased(P < 0.05),that means,the colour of tofu become yellow.(4)Leuconostoc mesenteroides and Bacillus licheniformis made the adhesiveness of tofu increased significantly(P<0.05),while the hardness of lactone tofu sharply decreased after inoculation of Bacillus amyloliquefaciens.(5)Pseudomonas fluorescens and Enterobacter aerogenes were active producers of amine odors and Bacillus licheniformis had strongest ability to produce 1-octen-3-alcohol,which reached 12.94% relative content in lactone tofu,causing mushroom flavor.In order to trace the source of spoilage bacteria in tofu,the production process of microbial situation in the enterprise which produce the tofu of band C was monitored.With the exception of heat-resistant spores,almost all bacteria will be killed in NZTs during the insulation molding process,while the total viable counts in SLT sharply increasted after squeezing,cutting and packaging.Analysis by PCR-DGGE method indicated that Bacillus,the main spoilage bacteria in NZTs,were derived from raw soybeans and utensils which were not cleaned completely.The microbial contamination of SLT was from the subsequent such as extrusion,cutting,packaging and other processes,leading to the initial large amount of microorganism in firm tofu.Finally,corresponding improvements were provided for company C:after the end of the day's production work,the equipments,pipes and contact surfaces on the production line were cleaned by sodium hydroxide solution and then disinfected by 100 mg/kg of chlorine dioxide solution to reduce the total viable counts between 2-3 logarithmic values.The equipment needed to be rinsed with clean water before the production work on the next day.After starting of the production,the cutting knife,table,and cloth must be replaced and cleaned every two hours and workers' hands must be cleaned at least once per hour.The initial amount of microorganism in SLT decreased one logarithm by improved process and the shelf life of SLTs stored at 4°C,25°C and 37°C were extended for 24 h,5 h and 2 h,respectively.