中国部分地区人体粪便中双歧杆菌的分离鉴定及具潜在益生特性菌株的筛选

发布时间：2018-05-08 01:28

本文选题：双歧杆菌 + 鉴定　；参考：《内蒙古农业大学》2012年硕士论文

【摘要】：本研究采用选择性培养基对河南、四川、西藏、云南、内蒙古和新疆等地区的18份人体粪便样品中的双歧杆菌进行了分离纯化,并采用16S rRNA基因序列分析法对分离到的29株双歧杆菌进行了鉴定。以B.animalis subsp.Iactis V9为参照,通过耐酸性、人工消化液和胆盐耐受试验,筛选出胃肠道转运过程中耐受性较好的4株双歧杆菌。并对筛选出的双歧杆菌进行了脱脂乳发酵特性和贮藏特性的研究。试验结果表明,从18位中国部分地区健康人体粪便中分离得到29株双歧杆菌,经16S rRNA基因序列分析法将IMAUFB064、IMAUFB065和IMAUFB091等9株菌鉴定为B. longum,其中河南6株、四川2株和新疆1株；将IMAUFB071、IMAUFB073、 IMAUFB084、IMAUFB090和IMAUFB092等20株菌鉴定为B.pseudocatenulatum,其中四川9株、西藏7株、云南3株和内蒙古1株。29株双歧杆菌中有24株在pH3.0、3.5和4.0的改良MRS培养液中生长良好,初筛出的24株双歧杆菌分别经pH3.0、2.5、2.0人工胃液消化3h后,存活率高于V9(84.97%)的菌株有2株,分别为IMAUFB084(85.98%)和MAUFB073(85.66%),占总菌数的6.90%,各菌株在不同pH值人工胃液中耐受性有显著差异(P0.05),随着pH值的降低,供试菌株对模拟人工胃液的耐受性也随之降低。4株复筛出的双歧杆菌在人工肠液消化8h后,其中MAUFB090的存活率为101.8%,高于V9(97.84%),MAUFB073(97.19%)、IMAUFB084(94.38%)和IMAUFB064(89.93%)都低于V9的存活率。 4株双歧杆菌均能耐受2.0%胆盐浓度,但各菌株的延迟时间差异显著(p0.05),IMAUFB064延迟时间为0.59h,为可耐受胆盐菌株；IMAUFB084、IMAUFB073和IMAUFB090的延迟时间表明其为轻微耐受胆盐菌株；参考菌株V9的延迟时间为0.25h,为对胆盐不敏感菌株,显而易见,各供试菌株对胆盐的耐受性均低于V9。分别以IMAUFB064、IMAUFB073、IMAUFB084、IMAUFB090和V9单菌株发酵制备酸乳,结果表明：IMAUFB073到达发酵终点的时间最短,仅用了18h,发酵结束时活菌数为6.02×108, MAUFB090、V9、IMAUFB084和IMAUFB064到达发酵终点的时间均为21h,活菌数分别为5.44×108、5.13×108、3.91×108和3.33×108。贮藏发酵乳的过程中,14d之后4株供试菌株的pH值下降速度显著低于V9；21d后V9和MAUFB084的活菌数不但没有下降反而分别增加了0.06和0.01个单位(logcfu/g),IMAUFB064的活菌数也没有明显的下降,说明V9、1MAUFB084和IMAUFB064在贮藏过程中的稳定性比IMAUFB073和IMAUFB090好,有利于益生菌制品的开发。
[Abstract]:In this study, a selective medium was used to isolate and purify Bifidobacterium in 18 human feces samples from Henan, Sichuan, Tibet, Yunnan, Inner Mongolia and Xinjiang, and 29 strains of Bifidobacterium were identified by 16S rRNA gene sequence analysis. The acid tolerance of B.animalis subsp.Iactis V9 was used as a reference. 4 strains of bifidobacteria, which were well tolerated during the transport of stomach and intestines, were screened by artificial digestive juice and bile salt tolerance test. The fermentation characteristics and storage characteristics of the screened bifidobacteria were studied.
The results showed that 29 strains of Bifidobacterium were isolated from 18 healthy human feces, and 9 strains of IMAUFB064, IMAUFB065 and IMAUFB091 were identified as B. longum by 16S rRNA gene sequence analysis, including 6 Henan, 2 Sichuan and 1 Xinjiang, 20 strains of IMAUFB071, IMAUFB073, IMAUFB084, IMAUFB090, IMAUFB092 and other strains. The bacteria were identified as B.pseudocatenulatum. Among them, 9 strains of Sichuan, 7 Tibet, 3 Yunnan and 1 strains of Bifidobacterium in Inner Mongolia were grown well in pH3.0,3.5 and 4 modified MRS culture. 24 strains of bifidobacteria were screened by pH3.0,2.5,2.0 artificial gastric juice for 3h, and the survival rate was higher than that of V9 (84.97%), which were IMA, respectively. UFB084 (85.98%) and MAUFB073 (85.66%) accounted for 6.90% of the total number of bacteria. The tolerance of each strain in the artificial gastric juice with different pH values was significantly different (P0.05). With the decrease of pH value, the tolerance of the tested strains to simulated artificial gastric juice also reduced the.4 strain of Bifidobacteria after the digestion of 8h in the artificial intestinal juice, and the survival rate of MAUFB090 was 101.8%, Higher than V9 (97.84%), MAUFB073 (97.19%), IMAUFB084 (94.38%) and IMAUFB064 (89.93%) were all lower than V9 survival rate.
4 strains of bifidobacteria could tolerate 2% bile salt concentration, but the delay time of each strain was significantly different (P0.05), the delay time of IMAUFB064 was 0.59h, and the delay time of IMAUFB084, IMAUFB073 and IMAUFB090 indicated that it was a mild resistant strain of bile salt; the delay time of the reference strain V9 was 0.25h, which was an insensitive strain to the bile salt. Obviously, the tolerance of the tested strains to bile salt was lower than that of V9..
Acid milk was prepared by single strain of IMAUFB064, IMAUFB073, IMAUFB084, IMAUFB090 and V9 respectively. The results showed that the time of IMAUFB073 to reach the end of fermentation was the shortest, only 18h was used, the number of living bacteria was 6.02 x 108 at the end of fermentation, the time of MAUFB090, V9, IMAUFB084 and IMAUFB064 reached the end of fermentation, and the number of living bacteria was 5.44 * * * * In the process of.91 x 108 and 3.33 x 108. storage fermented milk, the decrease rate of the pH value of the 4 strain strains after 14d was significantly lower than that of V9; the living bacteria number of V9 and MAUFB084 after 21d not only did not decrease, but increased 0.06 and 0.01 units respectively (logcfu/g), and the number of IMAUFB064 was not clearly decreased, indicating that V9,1MAUFB084 and IMAUFB064 were stored in storage. The stability of the process is better than that of IMAUFB073 and IMAUFB090, which is beneficial to the development of probiotic products.

【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R371

【参考文献】