伊春森林猪源植物乳杆菌P1M1株的鉴定及生物学特性研究

Identification and Biological Characteristics of Lactobacillus plantarum P1M1 Strain Isolated from Yichun Forest Pig

[目的]研究伊春森林猪源植物乳杆菌的生物学特性,全面评价其在益生菌制剂开发方面的应用潜力。[方法]对分离自黑龙江省伊春市健康森林猪粪便的1株乳酸菌(P1M1株)进行生化鉴定,利用细菌16S rDNA序列PCR扩增及测序技术进行分子生物学鉴定。绘制菌株生长曲线及产酸曲线,评价菌株对酸(pH值为2.0、3.0、4.0)和胆盐(浓度为0.15%和0.30%)的耐受性,考查菌株对13种抗菌药物的敏感性及其对猪源金黄色葡萄球菌的抑制作用,测定菌株的自由基清除能力及胞外多糖产量,评价菌株对小鼠的安全性。[结果]结合生化鉴定结果和分子生物学鉴定结果,将P1M1株鉴定为植物乳杆菌(Lactobacillus plantarum,L.plantarum)。L.plantarum P1M1株具有糖酵解能力;培养2~10 h为对数生长期,18 h后培养基pH值稳定在3.8~3.9;在pH值为2.0、3.0、4.0条件下的3 h存活率分别为(48.97±0.61)%、(64.19±0.94)%、(78.44±1.36)%,在0.15%和0.30%胆盐浓度下的3 h存活率分别为(53.86±1.14)%、(32.87±0.75)%;对红霉素、恩诺沙星、诺氟沙星、妥布霉素、氨苄西林、阿莫西林、头孢哌酮、头孢拉定、头孢他啶敏感;对猪源金黄色葡萄球菌表现出明显的抑制作用;无细胞上清液对DPPH自由基、羟自由基、ABTS自由基的清除率分别为(91.65±1.44)%、(87.32±0.22)%、(89.79±0.59)%,均显著(P<0.05)高于完整细胞菌悬液;胞外多糖产量为(796.73±2.43)mg/L;小鼠连续灌胃L.plantarum P1M1株2周后,未见死亡及器官病变。[结论]森林猪源L.plantarum P1M1株进入对数生长期快,具有糖酵解特性和较强的产酸能力,耐受酸和胆盐,对多种抗菌药物敏感,可抑制猪源金黄色葡萄球菌生长,抗氧化能力强,胞外多糖产量高,对小鼠具有安全性,可作为益生菌制剂开发的候选菌株。

[Objective]The aims of the present study were to assess the biological characteristics of Lactobacillus plantarum derived from Yichun forest pig and to comprehensively evaluate its application potential in the development of probiotic preparations.[Method]A lactic acid bacterial strain(P1M1)isolated from the feces of the healthy forest pig in Yichun City,Heilongjiang Province,was biochemically identified and then molecularly confirmed by using 16S rDNA PCR amplification and sequencing technology.The curves of growth and acid production of the strain were drawn,and its tolerance to acid(pH values at 2.0,3.0 and 4.0)and bile salt(concentrations at 0.15%and 0.30%)were evaluated.The sensitivity of the strain to 13 antimicrobial agents and its inhibitory effect on Staphylococcus aureus(S.aureus)of porcine origin were examined.Tests were also conducted to assess the free radical scavenging ability,exopolysaccharides yield and safety to mice of the strain.[Result]Based on the biochemical and molecular identification results,P1M1 strain was identified as Lactobacillus plantarum(L.plantarum).L.Plantarum P1M1 strain had glycolytic ability.Its logarithmic growth phase was 2-10 h of cultivation.After 18 h of cultivation,the pH value of its culture medium stabilized at 3.8-3.9.Its 3 h survival rates at pH values of 2.0,3.0 and 4.0 were(48.97±0.61)%,(64.19±0.94)%and(78.44±1.36)%,respectively,and those at bile salt concentrations of 0.15%and 0.30%were(53.86±1.14)%and(32.87±0.75)%,respectively.It was sensitive to erythromycin,enrofloxacin,norfloxacin,tobramycin,ampicillin,amoxicillin,cefoperazone,cefradine and ceftazidime,and had pronounced inhibitory effect on S.aureus of porcine origin.The scavenging rates of its cell-free supernatant against DPPH free radical,hydroxyl radical and ABTS free radical were(91.65±1.44)%,(87.32±0.22)%and(89.79±0.59)%,respectively,all significantly(P<0.05)higher than those of its intact cell suspension.Its exopolysaccharides yield was(796.73±2.43)mg/L.No death or organ damage was observed after continuous gavages of the L.plantarum P1M1 strain to mice for 2 weeks.[Conclusion]Forest pig derived L.plantarum P1M1 strain shows an early entrance into the logarithmic growth phase,has glycolytic characteristics,strong acid producing ability,and tolerance to acid and bile salt.It is sensitive to various antimicrobial agents and capable of inhibiting the growth of S.aureus of porcine origin.Furthermore,it has strong antioxidant capacity and high exopolysaccharides yield,and is safe for mice.Accordingly,it can be used as a candidate strain for the development of probiotic preparations.