奶牛源鼠李糖乳杆菌的12C6+重离子束诱变选育

本研究利用中科院重离子加速器释放的12C6+重离子束作为辐射诱变源，以酸斑值（HC）和抑菌圈值为指标，对鼠李糖乳杆菌JF12-1进行功能性诱变。通过致死率和正负突变率确定诱变的最佳辐照剂量；对最佳辐照剂量下的突变菌株利用酸斑法进行初筛，抑菌圈法复筛，而后通过连续传代之后检测乳酸含量变化来测定遗传稳定性，并对遗传稳定菌株进行16S rDNA测序，定位其突变位点。结果显示辐照剂量为300 Gy时，致死率为79.86%，正突变率为30.33%，负突变率为5.38%，确定为最佳诱变剂量；酸斑法初筛最佳诱变剂量下的突变菌株，得到20株HC值较原始野生菌株提高25%以上的突变株；抑菌法复筛得到8株体外抑菌性较原始野生菌株提高15%以上的菌株；遗传稳定性测定发现这8株突变乳酸菌株产乳酸稳定性良好；16S rDNA测序发现原始野生型菌株JF12-1的可能突变位点不在16S rRNA基因上，促使其产酸和抑菌性能增强的突变位点可能发生在其他基因区段。利用12C6+重离子束成功诱变选育出了高产乳酸及体外抑菌性优良的功能性鼠李糖乳杆菌稳定株，为下一步深入开发该菌株提供了较好的理论基础和应用依据。

Mutation Breeding of Lactobacillus rhamnosus from Dairy Cow by 12C6+ Heavy Ion Beam

In this study, 12C6+ heavy ion beam released from heavy ion accelerator of Chinese Academy of Sciences was used as the radiation mutagenesis source. With acid spot value and bacteriostatic ring value as indexes, functional mutagenesis of Lactobacillus rhamnosus JF12-1 was carried out to determine the optimal radiation dose of mutagenesis through mortality rate and positive and negative mutation rate. The mutated strains under the optimal irradiation dose were screened by acid patch method and screened by inhibition zone method. Then the genetic stability of the genetically stable strains was determined by detecting the change of lactic acid content after continuous passage. 16S rDNA sequencing was performed to locate the mutation sites of the genetically stable strains. The results showed that the mortality rate, positive mutation rate and negative mutation rate were 79.86%, 30.33% and 5.38%, respectively, when the irradiation dose was 300 Gy, which was the best mutagenesis dose. The results showed that the HC value of 20 mutant strains increased by more than 25% compared with the original wild strain. The bacteriostatic activity of 8 strains increased by 15% compared with the original wild strains. Genetic stability analysis showed that the 8 mutant Lactobacillus strains were stable in producing lactic acid. 16S rDNA sequencing revealed that the mutated sites of JF12-1 were not in 16S rRNA gene, and the mutated sites promoting acid production and bacteriostatic performance of JF12-1 were probably in other gene segments. A functional Lactobacillus rhamnosus stable strain with high lactic acid yield and good bacteriostatic activity in vitro was successfully cultured by 12C6+ heavy ion beam mutation, which provided a good theoretical basis and application basis for further development of this strain.