摘要:
以市售酸浆豆腐为样品,首先筛选样液适宜的稀释倍数,将样品稀释液接种于马铃薯葡萄糖平板和MRS平板进行纯培养,然后接种于脱脂牛奶培养基,以产透明圈为指标,再以酪蛋白为底物测定酶活,筛选分离产蛋白酶菌种,继而通过肉眼观察菌落形态、光学显微镜和电镜、显微镜观察菌体形态,进一步通过各种生理生化反应、ITS测序方法进行菌种鉴定。结果表明:25 g样品稀释至10-5浓度适宜,在脱脂牛奶培养基中,温度28 ℃、有氧培养72 h后产生透明圈,酶活为9.6 U/mL;肉眼观察菌落为乳白色,呈绒毛状,有同心圈并呈放射线状,显微镜下节孢子呈单个或链状,扫描电镜观察菌丝体宽4~5.5 μm,孢子大小多数为(3.3~5.7) μm×(7.0~17.1) μm;根据生理生化反应结果,查阅《真菌鉴定手册》,鉴定为白地霉(Geotrichum candidum);ITS测序显示与白地霉同源性达100%。根据生长曲线可知,0~8 h,菌株处于延迟期;8~40 h,菌株处于对数期;40~68 h,菌株处于稳定期;68 h以后,菌株处于衰亡期。
关键词:
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酸浆豆腐
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蛋白酶
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分离
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鉴定
Abstract:
The commercially available alkekengi tofu was used as samples. First,the appropriate dilution times of the sample solution were screened.Pure culture was carried out on potato glucose plates and MRS plates. Then the pure culture strains were inoculated on skim milk medium.Taking transparent zone as index,casein was used as substrate to determine enzyme activity to screen and isolate the strain that producing protease. Then,naked eye observation,optical microscopic observation,scanning electron microscopy,physiological and biochemical reaction,ITS sequencing method were used to identify the bacteria. The results showed that the appropriate diluted concentration for 25 g sample was 10-5.In the skim milk medium,under the condition of temperature of 28 ℃,aerobic training of 72 h,the strain produced a transparent circle and the enzyme activity was 9.6 U/mL. Macroscopic observation showed colony was ivory,villous,with concentric circle radiation,microscopic section spores single or in chain,sacnning electron microscopy observation showed the width of mycelium were 4~5.5 μm,and spore side were mostly(3.3~5.7) μm×(7.0~17.1) μm.According to the physiological and biochemical reaction results,refer to the Handbook of fungal identification,the strain belonged to Geotrichum candidum.The results of ITS sequencing showed homology of the strain on the potato glucose plates with Geotrichum candidum up to 100%.According to the growth curve,0~8 h was in the delay period,8~40 h was the logarithmic period,40~68 h was the stable period and after 68 h,the strain was in the decline period.
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