2株乳酸菌的生长和潜在益生菌特性

摘要

通过研究和比较2株乳酸菌——植物乳杆菌D28(Lactobacillus plantarum D28)和粪肠球菌B21(Enterococcus faecium B21)的生长曲线,对NaCl和胆盐的耐受性,对人工胃液和人工肠液的耐受性,以及它们的体外抗氧化能力和胆盐水解酶活性,判断其是否具有益生菌特性,以期为后期菌株功能研究提供理论基础.生长曲线试验表明,L.plantarum D28的最大生长速度出现在18 h左右,而E.faecium B21为16 h左右.起始pH对2株菌生长的影响很大,L.plantarum D28的最适生长pH为5.5~7.0,而E.faecium B21为pH 6.0~7.0.耐受性试验表明:E.faecium B21对NaCl和胆盐的耐受性较好,高于L.plantarum D28;L.plantarum D28在人工胃液(pH 2.0,180 min)和人工肠液(pH 8.0,240 min)中的菌群存活率均显著高于E.faecium B21.采用DPPH(二苯基苦基苯肼)自由基清除率来测定2株菌的体外抗氧化活性,结果表明,2株菌均具有一定的抗氧化活性,且L.plantarum D28的抗氧化活性(34.86%)显著高于B21(25.32%).采用茚三酮法测定菌株的胆盐水解酶活性发现,E.faecium B21的胆盐水解酶活性(0.415 U/mg)显著高于L.plantarum D28(0.114 U/mg).综上表明,L.plantarum D28和E.faecium B21均具有一定的益生特性,且这2个菌株均可应用于乳酸菌降低胆固醇活性的研究,且L.plantarum D28还可以用于应用乳酸菌提高体内抗氧化活性的进一步研究中.%Probiotics are defined as"living micro-organisms which, upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition". Recent reports have confirmed that probiotics can provide a variety of beneficial health effects, which are strain dependent. The regulation effect of probiotics was based on tolerance of acid environment in the stomach, and resistance to bile salt and trypsin in the small intestine. Most of lactic acid bacteria (LAB) are considered as probiotics, and have been widely used as dietary additives in cultured dairy foods and other fermentation products. The source of LAB may be naturally fermented food or human and animal intestines.In this study, two strains were isolated from Chinese traditional food (Northeast pickle and stinky beans), and identified as Lactobacillus plantarum D28 and Enterococcus faecium B21 by 16S rRNA gene sequences, respectively. The growth and potential probiotic characteristics of the two lactic acid bacteria strains were further investigated. Growth curve and the effect of initial pH on the growth were studied by spectrophotometry method. Meanwhile, the resistance characteristics were investigated, including sodium chloride (NaCl), bile salt, artificial gastric juice and artificial intestinal juice. In addition, antioxidant activity and bile salt hydrolase activity (BSH) in vitro were determined by scavenging DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical and ninhydrin method, respectively. The results of growth curve showed that the growth rate of L. plantarum D28 was faster than E. faecium B21 under the same culture condition, and the stationary growth phase of L. plantarum D28 and E. faecium B21 occurred at 18 h and 16 h, respectively. The growth rate of the two strains was affected by different initial pH;pH ranges of 5.5-7.0 and 6.0-7.0 were most favorite initial pH for L. plantarum D28 and E. faecium B21, respectively. Moreover, L. plantarum D28 had a good tolerance against NaCl (5%) and bile salt (0.15%) in vitro, and E. faecium B21 had a better tolerance against NaCl (8%) and bile salt (0.45%). Time-dependent behavior was found in the bacterial survival amount under the artificial gastric juice (pH 2.0) and artificial intestinal juice (pH 8.0) conditions. After treatment with artificial gastric juice (pH 2.0) for 180 min, the survival rate of L. plantarum D28 was higher than E. faecium B21, remaining 41.33 lg (CFU/mL) and 8.33 lg (CFU/mL), respectively. Moreover, similar result was found with treatment of artificial intestinal juice (pH 8.0) for 240 min, 36.33 lg (CFU/mL) and 18.67 lg (CFU/mL) for L. plantarum D28 and E. faecium B21, respectively. In addition, the two strains had certain antioxidant activity in vitro, and L. plantarum D28 had a higher DPPH free radical scavenging activity (34.86%) than that of E. faecium B21 (25.32%). Besides, both of the two stains had bile salt hydrolase (BSH) activity, and E. faecium B21 (0.415 U/mg) had a higher BSH activity than that of L. plantarum D28 (0.114 U/mg). In conclusion, the growth rate of the two strains is affected by different initial pH, and L. plantarum D28 has a higher growth rate than E. faecium B21. L. plantarum D28 and E. faecium B21 both have tolerance to acid, bile salt, and NaCl, and are able to survive under the conditions of artificial gastric juice and artificial intestine juice. Therefore, both L. plantarum D28 and E. faecium B21 can be used as candidate for probiotic strains and application for lowering cholesterol research, and L. plantarum D28 can especially be applied to antioxidant-related research.