犬源乳酸片球菌GLP236的分离鉴定、体外益生功能及小鼠安全性评价

Isolation,Identification,in Vitro Probiotic Function and Mouse Safety Evaluation of Canine-Derived Pediococcus acidilactici GLP236

本研究旨在对犬源乳酸片球菌GLP236进行益生特性和安全性评价。通过革兰染色、16S rRNA测序和生理生化特性鉴定菌株分类地位,通过低pH、胆盐、胃肠道环境和高温耐受性、自凝聚性、表面疏水性、抗氧化活性、溶血活性、抑菌活性、抗生素敏感性以及小鼠灌服试验评价菌株益生特性和安全性。小鼠灌服试验中,将健康、体重相近的80只昆明小鼠随机分为4组,每组20只。对照组(CK组)小鼠灌服0.2 mL无菌生理盐水,其他3组小鼠灌服0.2 mL浓度分别为1×10^(9)(LG组)、1×10^(10)(MG组)和1×10^(11)CFU/mL(HG组)的乳酸片球菌GLP236。试验期共28 d。结果表明:1)结合16S rRNA和生理生化结果鉴定GLP236为乳酸片球菌。2)菌株生长24 h后进入稳定期,pH最终稳定在4.10。3)菌株在0.1%、0.3%和0.5%胆盐中存活率分别为96.91%、86.24%和65.73%,在人工胃液、人工肠液和人工胃肠液中的存活率分别为73.81%、94.11%和64.18%,在80、90℃条件下可耐受5 min。4)菌株8、16和24 h自凝聚率分别为57.02%、77.56%和87.22%,表面疏水率为62.56%,2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)和1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率分别为87.28%和80.70%。5)菌悬液和无细胞上清液可抑制致病菌生长,菌株对苯唑西林、氨苄西林、庆大霉素等抗生素不敏感,呈γ溶血。6)MG组小鼠平均日增重显著高于CK组和LG组(P<0.05),MG组和HG组小鼠平均日采食量显著高于CK组(P<0.05)。MG组和HG组小鼠血清超氧化物歧化酶活性显著高于CK组(P<0.05),血清丙二醛含量显著低于CK组(P<0.05);LG、MG和HG组血清天冬氨酸氨基转移酶活性显著低于CK组(P<0.05);MG组血清尿素氮含量显著低于CK组(P<0.05)。综上所述,犬源乳酸片球菌GLP236具有良好的益生菌特性,可作为犬源益生菌的候选菌。

The aim of this study was to evaluate the probiotic properties and safety of canine-derived Pediococcus acidilactici GLP236.The taxonomic status of the strain was identified by Gram staining,16S rRNA sequencing and physiological and biochemical characteristics.The probiotic properties and safety of the strain were evaluated by low pH,bile salts,gastrointestinal environment and high-temperature tolerance,strain self-coagulation,surface hydrophobicity,antioxidant activity,hemolytic activity,bacteriostatic activity,antibiotic susceptibility and mice instillation test.In the mice instillation test,eighty healthy Kunming mice with similar body weight were randomly divided into 4 groups with 20 mice in each group.Mice in the control group were orally administered 0.2 mL sterile physiological saline,and mice in other groups were orally administered 0.2 mL Pediococcus acidilactici GLP236 with concentrations of 1×10^(9)(LG group),1×10^(10)(MG group)and 1×10^(11)CFU/mL(HG group),respectively.The experiment lasted for 28 days.The results showed as follows:1)GLP236 was identified as Pediococcus acidilactici in combination with 16S rRNA and physiological and biochemical results.2)The strain entered the stabilization phase after 24 h of growth,and the pH was finally stabilized at 4.10.3)The strain survival rates were 96.91%,86.24%and 65.73%in 0.1%,0.3%and 0.5%bile salts,respectively,and the survival rates were 73.81%,94.11%and 64.18%in artificial gastric fluid,artificial intestinal fluid and artificial gastrointestinal fluid,respectively,and 80,90℃can withstand 5 min.4)The strain self-coagulation rates were 57.02%,77.56%and 87.22%at 8,16 and 24 h,respectively,the surface hydrophobicity was 62.56%,and the free radical scavenging rates of 2,2-diazo-di(3-ethylbenzothiazole-6-sulfonic acid)diamine salt(ABTS)and 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)were 87.28%and 80.70%.5)The bacterial suspensions and cell-free cell supernatant could inhibit the growth of pathogenic bacteria,and the strains were insensitive to antibiotics such as benzoxacillin,ampicillin,gentamicinand so on,and showed gamma hemolysis.6)The average daily gain of MG group was significantly higher than that of CK group and LG group(P<0.05),the average daily feed intake of MG group and HG group was significantly higher than that of CK group(P<0.05).The serum superoxide dismutase activity of MG group and HG group was significantly higher than that of CK group(P<0.05),and the serum malondialdehyde content was significantly lower than that of CK group(P<0.05);the serum aspartate aminotransferase activity of LG group,MG group and HG group was significantly lower than that of CK group(P<0.05),the serum urea nitrogen content of MG group was significantly lower than that of CK group(P<0.05).In conclusion,the canine-derived Pediococcus acidilactici GLP236 has good probiotic properties and can be used as a candidate for canine-derived probiotics.