细菌型豆豉后发酵期间菌相分析及产蛋白酶菌种的筛选
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摘要
我国细菌型豆豉生产一直采用自然发酵工艺,生产周期长,没有明确的发酵菌种,易受致病菌污染,存在产品质量不稳定和安全性问题。因此,明确豆豉发酵过程中的微生物菌相组成及变化,从中筛选蛋白酶产生菌并利用该菌种进行豆豉制作对解决目前豆豉生产中存在的问题有重要的指导意义。本文主要从细菌型豆豉后发酵期间菌相分析,筛选适于豆豉制作的高产蛋白酶菌种二方面进行研究。主要研究结果如下:
1、对豆豉后发酵期间各项理化指标进行了检测,游离态氮以及总酸含量随后发酵时间的延长呈上升趋势。
2、通过变性梯度凝胶电泳技术(DGGE)对于37℃条件下不同后发酵时间的细菌型豆豉进行研究,对DGGE条带进行切胶回收并转导大肠杆菌,通过测序、比对以及对DGGE条带进行定量分析结果表明:多种不可培养细菌(Uncultured bacterium)、枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)为豆豉后发酵期间的主要菌种。后发酵期间还会出现肠杆菌科细菌(Enterobacteriaceae bacterium)。系统进化分析表明:不可培养微生物同源性较高,可培养微生物同源性较低,说明后发酵期间可培养细菌种类较多。
3、利用末端限制性片段长度多态性(T-RFLP)技术对豆豉后发酵期间细菌多样性指数、均匀度指数和相似性指数分析表明:多样性指数先下降后升高然后逐渐降低;细菌型豆豉后发酵期间各物种丰度的变化较大。细菌序列的长度与Ribosomal Database Project II数据库进行对比表明:豆豉后发酵出现的菌群有2种不可培养细菌(Uncultured bacterium)、1种不可培养的变形菌(Uncultured proteobacterium)和8种可培养细菌,该几类菌种分别处于不同后发酵时间。枯草芽孢杆菌(Bacillus subtilis)在整个后发酵期间的各个取样时间点所占比例均大于13%,不可培养所占比例均大于1.19%。
4、利用显微镜观察及菌落形态特征对豆豉后发酵时间出现的菌种研究发现,不同时期出现的细菌种类以及各菌种所占的比例不同。同时分离出45株不同形态的菌种。对45株菌种的进行限制性片段长度多态性分析(ARDRA),共检查出6个类型,各类型代表菌株经16S rDNA扩增测序以及比对结果表明:枯草芽孢杆菌(Bacillus subtilis)占89%;解淀粉芽孢杆菌(B. amyloliquefaciens)占4%;细菌后发酵期间还会出现藤黄微萄球菌(Micrococcus luteus)和鸡葡萄球菌(Staphylococcus gallinarum)。
5、采用酪蛋白平板初筛,制曲复筛,根据曲样蛋白酶酶活以及游离态含量从45株细菌中共选出5株优良菌株用于豆豉的制作,由成品豆豉的理化指标和感官评定认为D2号菌种发酵的豆豉发风味好,滋味鲜美。结合生理生化和16S rDNA序列分析初步鉴定D2号菌种为枯草芽孢杆菌。该菌株来源于豆豉,属于安全菌种,具有研究开发价值。
Bacterial Douchi, one of the most popular traditional fermented soybean products in China,used to be made by spontaneous fermentation. It has a long production cycle. It is easily polluted by pathogenic bacteria during post-fermentation because it hasn’t explicit bacteria to ferment. The products have unstable quality and safety risks. Therefore, to reveal the bacterial flora during Douchi fermentation and screen high-yield protease producing strains has significance to solve the present problem in fermented soybean. In this thesis, there were two main aspects, which were analysising the consist of bacterial flora in Douchi post-fermentation and screening the high-yield protein producing strains. The results showed that:
1. The physical and chemical indicators were detected during the post-fermentation. The results showed that the free nitrogen and total acid content ascended gradually during the post-fermentation period.
2. The Douchi cultured at 37℃was studied by denaturing gradient gel electrophoresis (DGGE) at different time. Bands were excised from the DGGE Gel and re-amplified, recovered, and then ligated to pMD18-T cloning vector and transducted into E. coli DH5a. The positive clones were selected randomly for sequence analysis and quantitative analysis. The results showed that many kinds of Uncultured bacteria, Bacillus subtilis and Bacillus amyloliquefaciens were the main strains in the post-fermentation of Douchi. The Enterobacteriaceae bacterium also existed in the post-fermentation.
Phylogenetic tree demonstrating that the homoeologies of Uncultured bacteria were higher than cultured bacteria, and therefore, the composition of cultured bacteria was abundant.
3. The analysis of the diversity index, evenness and similarity indices of bacterial flora via terminal restriction fragment length polymorphism (T-RFLP) technique during post-fermentation showed that, the diversity index decreased, and then increased, the diversity decreased gradually in the end. There were greater differences in abundance of microbial flora during the post-fermentation of Douchi. The comparison of bacterial sequence length and Ribosomal Database Project II database showed that there were two kinds of uncultured bacteria, one uncultured proteobacterium and eight kinds of cultured bacteria in the different time of post-fermentation. The proportion of Bacillus subtilis was more than 13% and the proportion of uncultured bacterium was more than 1.19% in each sampling time of the whole post-fermentation period.
4. The composition of the bacteria during the post-fermentation of Douchi were analyzed by using microscopy and colony morphology. The results showed that, the different types of bacteria occured in different periods, and the proportion of each specie had significantly changed at different times. Meanwhile, forty five different bacteria were isolated and analyzed by restriction fragment length polymorphism analysis (ARDRA). The six types were checked out from those forty five strains, the 16S rDNA sequences of representative strains of the various types were amplified and aligned. The results showed that, the proportion of Bacillus subtilis was 89%, and Bacillus amyloliquefaciens was 4%. There were Micrococcus luteus and Staphylococcus gallinarum during the post-fermentation of Douchi.
5. According to the enzyme activity and the content of free nitrogen of starter of Lobster Sauce, five high-yield protease-producing strains were screened by casein plate and re-screened by starter-making from forty five strains. The five superior strains were used to ferment Lobster Sauce respectively. According to the sensory tests, physical and chemical indexes of five products, the Lobster Sauce fermented by D2 strain had a good flavor and the product was very delicious. By physiological characterization and the sequence analysis of 16S ribosomal DNA genes, the strain D2 was preliminarily identified as Bacillus subtilis. The strain was got from Lobster Sauce and it was safe, so it had further studying and developing value.
1、对豆豉后发酵期间各项理化指标进行了检测,游离态氮以及总酸含量随后发酵时间的延长呈上升趋势。
2、通过变性梯度凝胶电泳技术(DGGE)对于37℃条件下不同后发酵时间的细菌型豆豉进行研究,对DGGE条带进行切胶回收并转导大肠杆菌,通过测序、比对以及对DGGE条带进行定量分析结果表明:多种不可培养细菌(Uncultured bacterium)、枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)为豆豉后发酵期间的主要菌种。后发酵期间还会出现肠杆菌科细菌(Enterobacteriaceae bacterium)。系统进化分析表明:不可培养微生物同源性较高,可培养微生物同源性较低,说明后发酵期间可培养细菌种类较多。
3、利用末端限制性片段长度多态性(T-RFLP)技术对豆豉后发酵期间细菌多样性指数、均匀度指数和相似性指数分析表明:多样性指数先下降后升高然后逐渐降低;细菌型豆豉后发酵期间各物种丰度的变化较大。细菌序列的长度与Ribosomal Database Project II数据库进行对比表明:豆豉后发酵出现的菌群有2种不可培养细菌(Uncultured bacterium)、1种不可培养的变形菌(Uncultured proteobacterium)和8种可培养细菌,该几类菌种分别处于不同后发酵时间。枯草芽孢杆菌(Bacillus subtilis)在整个后发酵期间的各个取样时间点所占比例均大于13%,不可培养所占比例均大于1.19%。
4、利用显微镜观察及菌落形态特征对豆豉后发酵时间出现的菌种研究发现,不同时期出现的细菌种类以及各菌种所占的比例不同。同时分离出45株不同形态的菌种。对45株菌种的进行限制性片段长度多态性分析(ARDRA),共检查出6个类型,各类型代表菌株经16S rDNA扩增测序以及比对结果表明:枯草芽孢杆菌(Bacillus subtilis)占89%;解淀粉芽孢杆菌(B. amyloliquefaciens)占4%;细菌后发酵期间还会出现藤黄微萄球菌(Micrococcus luteus)和鸡葡萄球菌(Staphylococcus gallinarum)。
5、采用酪蛋白平板初筛,制曲复筛,根据曲样蛋白酶酶活以及游离态含量从45株细菌中共选出5株优良菌株用于豆豉的制作,由成品豆豉的理化指标和感官评定认为D2号菌种发酵的豆豉发风味好,滋味鲜美。结合生理生化和16S rDNA序列分析初步鉴定D2号菌种为枯草芽孢杆菌。该菌株来源于豆豉,属于安全菌种,具有研究开发价值。
Bacterial Douchi, one of the most popular traditional fermented soybean products in China,used to be made by spontaneous fermentation. It has a long production cycle. It is easily polluted by pathogenic bacteria during post-fermentation because it hasn’t explicit bacteria to ferment. The products have unstable quality and safety risks. Therefore, to reveal the bacterial flora during Douchi fermentation and screen high-yield protease producing strains has significance to solve the present problem in fermented soybean. In this thesis, there were two main aspects, which were analysising the consist of bacterial flora in Douchi post-fermentation and screening the high-yield protein producing strains. The results showed that:
1. The physical and chemical indicators were detected during the post-fermentation. The results showed that the free nitrogen and total acid content ascended gradually during the post-fermentation period.
2. The Douchi cultured at 37℃was studied by denaturing gradient gel electrophoresis (DGGE) at different time. Bands were excised from the DGGE Gel and re-amplified, recovered, and then ligated to pMD18-T cloning vector and transducted into E. coli DH5a. The positive clones were selected randomly for sequence analysis and quantitative analysis. The results showed that many kinds of Uncultured bacteria, Bacillus subtilis and Bacillus amyloliquefaciens were the main strains in the post-fermentation of Douchi. The Enterobacteriaceae bacterium also existed in the post-fermentation.
Phylogenetic tree demonstrating that the homoeologies of Uncultured bacteria were higher than cultured bacteria, and therefore, the composition of cultured bacteria was abundant.
3. The analysis of the diversity index, evenness and similarity indices of bacterial flora via terminal restriction fragment length polymorphism (T-RFLP) technique during post-fermentation showed that, the diversity index decreased, and then increased, the diversity decreased gradually in the end. There were greater differences in abundance of microbial flora during the post-fermentation of Douchi. The comparison of bacterial sequence length and Ribosomal Database Project II database showed that there were two kinds of uncultured bacteria, one uncultured proteobacterium and eight kinds of cultured bacteria in the different time of post-fermentation. The proportion of Bacillus subtilis was more than 13% and the proportion of uncultured bacterium was more than 1.19% in each sampling time of the whole post-fermentation period.
4. The composition of the bacteria during the post-fermentation of Douchi were analyzed by using microscopy and colony morphology. The results showed that, the different types of bacteria occured in different periods, and the proportion of each specie had significantly changed at different times. Meanwhile, forty five different bacteria were isolated and analyzed by restriction fragment length polymorphism analysis (ARDRA). The six types were checked out from those forty five strains, the 16S rDNA sequences of representative strains of the various types were amplified and aligned. The results showed that, the proportion of Bacillus subtilis was 89%, and Bacillus amyloliquefaciens was 4%. There were Micrococcus luteus and Staphylococcus gallinarum during the post-fermentation of Douchi.
5. According to the enzyme activity and the content of free nitrogen of starter of Lobster Sauce, five high-yield protease-producing strains were screened by casein plate and re-screened by starter-making from forty five strains. The five superior strains were used to ferment Lobster Sauce respectively. According to the sensory tests, physical and chemical indexes of five products, the Lobster Sauce fermented by D2 strain had a good flavor and the product was very delicious. By physiological characterization and the sequence analysis of 16S ribosomal DNA genes, the strain D2 was preliminarily identified as Bacillus subtilis. The strain was got from Lobster Sauce and it was safe, so it had further studying and developing value.
引文
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