斜带石斑鱼肠道乳酸菌部分生物学特性研究

Biological properties of Lactobacillus strains isolated from grouper Epinephelus coioids

体外检测了3株斜带石斑鱼(Epinephelus coioids)肠道乳酸菌的产乳酸能力、抑菌活性、对高温、低pH值、高胆盐、人工胃液及人工肠液的耐受能力等,为海水鱼益生菌的开发提供理论依据.16S rRNA基因测序分析表明,LS61、LS64、LS77菌株分别为植物乳杆菌(Lactobacillus plantarum)、肠膜明串珠菌(Leuconostoc mesenteroides)、糊精乳杆菌(Lactobacillus dextrinicus).3株菌摇瓶培养40 h后发酵液中乳酸含量分别达到55.6、51.6、58.3 mmol/dm3且发酵上清液对致病菌有较强的拮抗活性.耐受性实验结果表明,pH值为3.5、4.5的MRS培养基对这3株菌存活影响较小;pH值为2.5的MRS培养基对LS64、LS77的影响较大,而对LS61菌株的存活基本没有影响.这3株菌对不同pH值的人工胃液的耐受性与之相似,说明LS61菌株的pH值耐受性最好,其次是LS64菌株,LS77菌株最差.3株菌对高胆盐也有不同的耐受性,含1‰和2‰胆盐的MRS培养基对3株菌的影响较小,而在3‰的胆盐处理4.5 h后,LS64菌株的存活率仍保持在30.7%,LS61菌株的存活率为2.0%左右,LS77菌株的存活率接近1.0%.在4‰的胆盐含量下,LS61、LS64菌株的存活率仍高于1‰.同样,不同胆盐含量的人工肠液对这3株菌的影响与之相似.乳酸菌LS61、LS64菌株具有较强的产乳酸和抑菌能力,具有一定的pH值耐受性和较高的胆盐耐受性,可考虑作为潜在的益生菌株应用于水产健康养殖.

The aim of this study was to evaluate the biological properties of 3 lactic acid bacterial strains （LAB） isolated from the intestine of grouper Epinephelus coioids for probiotics selection in aquaculture.Lactic acid production,the pathogen inhibition ability of strain LS61,LS64 and LS77,and the tolerance of these 3 strains to high temperature,low pH,high bile salt as well as mimic gastrointestinal environments were studied in vitro.These strains were identified as Lactobacillus plantarum （LS61）,Leuconostoc mesenteroides （LS64） and Lactobacillus dextrinicus （LS77） by 16S rRNA sequence analysis,respectively.Strain LS61,LS64 and LS77 can produce high levels of lactic acid with concentration of 55.6,51.6,and 58.3 mmol/dm3.They also showed antimicrobial activity against pathogenic bacteria and certain tolerance to low pH values.Survival rates of three strains were almost 100％ after 4.5 h incubation at pH 3.5 or pH 4.5,which suggested that pH 3.5 and pH 4.5 had little effect on the stains.However,survival rates of LS64 and LS77 declined to 36％ and 0％ while that of LS61 maintained 100％ after 4.5 h incubation at pH 2.5,which indicated that LS61 was more tolerant to low pH compared with LS64 and LS77.When treated with artificial gastric juice containing different pH values and different concentrations of pepsin,the tolerance of the three strains got similar results as mentioned above.Furthermore,all these three strains exhibited good tolerance to different concentrations of bile salt,among which both strain LS61 and LS64 could propagate rapidly after 4.5 h incubation under 0.1％ and 0.2％ bile salt,and maintained 2.0％ and 22.2％ survival rates under 0.3％ bile salt,respectively.The results were consistent with that when tested under artificial intestinal juice containing different concentrations of bile salt and trypsin.Strain LS61 and LS64 have the potential to be probiotics in aquaculture for their good lactic acid production,pathogen inhibition activity and tolerance to mimic gastrointestinal environment.