一株干酪乳杆菌的分离鉴定及生物学特性研究

Isolation,Identification and Biological Characteristics of a Strain of Lactobacillus casei

本研究旨在分离既能用于微生态制剂又能用于青贮饲料发酵剂的优良乳酸菌,采集规模化牛场青贮饲料,取梯度稀释液涂布MRS平板,进行厌氧培养,筛选优势菌株,观察其菌落形态,并进行生化试验和特异性PCR鉴定。研究自筛乳酸菌的生长曲线、产酸曲线、培养温度及培养基初始pH对菌株生长的影响,并研究其抑菌特性、药物敏感性和安全性。试验筛选到1株菌落形态圆形、边缘整齐、表面光滑、乳白色、镜检为革兰氏阳性的无芽孢杆菌。生化结果显示,分离菌株可厌氧生长,运动性试验、过氧化氢酶试验、硝酸盐还原试验、明胶液化试验、吲哚试验结果均为阴性,15种糖醇发酵试验结果符合干酪乳杆菌生化特征。用干酪乳酸杆菌特异性引物对菌株进行PCR鉴定,琼脂糖凝胶电泳结果显示在727 bp处出现特异性扩增条带,与生化鉴定结果相符,将分离菌株命名为RS1。RS1在0~4 h时发酵液pH变化不明显,处于生长延迟期;4~16 h时发酵液pH下降较快,进入对数生长期;16~36 h时发酵液pH下降缓慢,进入稳定期。RS1在培养温度为20~50℃范围内均能生长,适宜生长温度为25~40℃;在培养液初始pH 1.0~9.0条件下均能生长,最适pH为6.0~8.0;对金黄色葡萄球菌的抑菌圈直径为23 mm,对大肠杆菌的抑菌圈直径为17 mm;对恩诺沙星和头孢他啶耐药;经14 d灌服小鼠生长状况良好。上述结果表明,本试验筛选到的干酪乳杆菌RS1安全、无致病性,在微生态制剂及生物饲料添加剂领域具有潜在的开发价值。

The purpose of this study was to isolate the excellent lactic acid bacteria which could be used for both probiotics and silage starter.Silage was collected in large-scale cattle farm.The gradient diluent was coated on MRS plate for anaerobic culture.The dominant strains were screened and their colony morphology was observed.Biochemical test and specific PCR identification were carried out.The growth curve,acid production curve,the influence of culture temperature and initial pH of the medium on the growth of the self-screened lactic acid bacteria,and its bacteriostatic characteristics,antibiotic sensitivity and safety were studied.The results showed that a bacterial colony with round shape,neat edges,smooth surface,milky white,Gram-positive and non-bacillus bacillus was screened.The biochemical results showed that the strain could grow in the anaerobic environment.The results of exercise test,catalase test,nitrate reduction test,gelatin liquefaction test,indole test were negative.The results of 15 kinds of sugar and alcohol fermentation in keeping with the biochemical characteristics of Lactobacillus casei.The strain was identified by PCR method with specific primers of Lactobacillus casei,agarose gel electrophoresis imaging showed a specific amplified band at 727 bp,which was consistent with the biochemical identification results,and the strain was named as RS1.The pH of the fermentation broth of RS1 did not change significantly at 0 to 4 h,and was in the growth delay period.The pH of the fermentation broth decreased rapidly at 4 to 16 h and entered the logarithmic growth phase.The fermentation broth was in the logarithmic growth phase at 16 to 36 h.The pH dropped slowly and entered a stable period.RS1 could grow in the culture temperature range of 20 to 50℃,and the suitable growth temperature was 25 to 40℃.It could grow under the initial pH of the culture medium 1.0 to 9.0,and the optimum pH was 6.0 to 8.0.The diameter of the inhibition zone of Staphylococcus aureus was 23 mm,and that of Escherichia coli was 17 mm.It was resistant to enrofloxacin and ceftazidime.After 14 days of administering the mice,the growth status of mice was good.These results indicated that Lactobacillus casei RS1 was safe and non pathogenic,and had potential development value in the field of probiotics and biological feed additives.