Selection of probiotic bacteria for use in permented milk product
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Isolation of probiotic lactic acid bacteria for use in fermented milk product was carried on infant faeces, pig intestine and chickens intestine. Three hundred and seventy-nine isolates were isolated from 11 of infant faeces, 13 of pig intestine, and 15 of chicken intestine. Based on their phenotypic characteristics, 216 isolates were belonged to Lactobacillus, 122 isolates were Enterococcus and the other 41 isolates were unidentified. Twenty-nine isolates were acid- and bile-resistant at pH 2.5 and 0.3% bile salt, respectively. The potentially probiotic isolates IF2-8, P2-10 and CK8-11 were selected from infant, pig and chicken, respectively. IF2-8 was the best acid-resistant isolate at survival rate 96.4% and could inhibit E. faecium, Y. enterocolitica and B. cereus. Based on their phenotypic characteristics and 16S rDNA sequence similarity, IF2-8, P2-10, and CK8-11 were closely related to L.gasseri, L. johnsonii, and L. salivarius with 99.91, 99.53and 99.91%, respectively. Furthermore the other strains P12-3, IF7-5, and IF8-1 were closely related to L. amylovorus, E. raffinosus, and E. faecalis with 99.53, 99.75 and 99.83%, respectively. Later, the three potential probiotic strains were used for fermented milk trial which divided into 3 sets, the first was inoculated with IF2-8, P2-10, and CK8-11 culture, while the second was inoculated with the mixed culture of each probiotic culture and S. thermophilus, and the last was the control that was inoculated with yoghurt starter bacteria, S. thermophilus and L. delbrueckii subsp. bulgaricus. These products were incubated at 42 C for 24 h and stored at 5 C for 3 weeks. Upon the completion of incubation, IF2-8 had greatest survival (73.0-88.4%) in both fermented milk used only probiotic and fermented milk used probiotic and S. thermophilus. Furthermore, fermented milk used IF2-8 mixed with S. thermophilus resulted increasing in viability of IF2-8 (2 log cycles) compared to fermented milk that used only IF2-8. For acid production, the control fermented milk, only IF2-8 product and IF2-8 mixed with S. thermophilus product were 1.54, 0.62 and 1.15%, respectively. Their viscosity were 488,256 and 601 cPs, respectively. Fermented milk used only IF2-8 was found that acid production and viscosity much lower than the control, whereas fermented milk mixed with S. thermophilus produced acid not different from the control. Therefore, this results indicated that IF2-8 should be applied along with S. thermophilus in fermented milk but it could not be used as starter culture alone in fermented milk.
本研究从婴儿粪便、猪肠道及鸡肠道样本中分离可用于发酵乳制品的益生性乳酸菌。共计从11份婴儿粪便、13份猪肠道及15份鸡肠道样本中分离得到379株菌株。基于表型特征，其中216株隶属于乳杆菌属（Lactobacillus），122株隶属于肠球菌属（Enterococcus），剩余41株未被鉴定。另有29株菌株可在pH 2.5及0.3%胆盐环境中分别耐受酸性与胆盐。研究分别从婴儿粪便、猪肠道及鸡肠道中筛选出三株潜在益生菌株IF2-8、P2-10与CK8-11。其中IF2-8的耐酸性最优，存活率达96.4%，且可抑制屎肠球菌（Enterococcus faecium）、小肠结肠炎耶尔森菌（Yersinia enterocolitica）及蜡样芽孢杆菌（Bacillus cereus）。结合表型特征与16S核糖体DNA（16S rDNA）序列同源性分析，IF2-8、P2-10与CK8-11分别与加氏乳杆菌（Lactobacillus gasseri）、约翰逊乳杆菌（Lactobacillus johnsonii）及唾液乳杆菌（Lactobacillus salivarius）具有99.91%、99.53%及99.91%的同源性。其余三株菌株P12-3、IF7-5与IF8-1则分别与解淀粉乳杆菌（Lactobacillus amylovorus）、棉籽糖肠球菌（Enterococcus raffinosus）及粪肠球菌（Enterococcus faecalis）具有99.53%、99.75%及99.83%的同源性。随后将上述三株潜在益生菌株应用于发酵乳试验，试验共设3组：第一组接种IF2-8、P2-10与CK8-11的纯培养物；第二组接种各益生菌株与嗜热链球菌（Streptococcus thermophilus）的混合培养物；第三组为对照组，接种酸奶发酵剂嗜热链球菌与德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）。所有发酵产物均于42℃下培养24小时，随后于5℃条件下贮藏3周。培养结束后，IF2-8在仅接种益生菌株的发酵乳以及接种益生菌株与嗜热链球菌的混合发酵乳中均表现出最高的存活率（73.0%~88.4%）。此外，与仅接种IF2-8的发酵乳相比，接种IF2-8与嗜热链球菌混合培养物的发酵乳中IF2-8的活菌数提升了2个对数级。产酸率方面，对照组发酵乳、仅接种IF2-8的发酵乳以及接种IF2-8与嗜热链球菌混合培养物的发酵乳的产酸率分别为1.54%、0.62%及1.15%；其黏度分别为488、256及601厘泊（cPs）。结果显示，仅接种IF2-8的发酵乳的产酸率与黏度均显著低于对照组，而接种IF2-8与嗜热链球菌混合培养物的发酵乳的产酸率与对照组无显著差异。综上，IF2-8需与嗜热链球菌复配应用于发酵乳生产，不可单独作为发酵乳发酵剂使用。
