内蒙古原始森林桦树皮内生和表生乳酸菌分离鉴定及其特性

Isolation, identification, and characterization of endophytic and epiphytic lactic acid bacteria from bark of birch trees in primeval forest of Inner Mongolia

摘要:

[背景] 随着乳酸菌产业的快速发展，不同环境乳酸菌群落结构的分析和菌种资源的开发利用已经成为该领域的研究热点。从不同环境、不同来源分离、保藏乳酸菌对于优良菌种的筛选和开发利用具有重要意义。[目的] 阐明内蒙古原始森林桦树皮中乳酸菌的组成，并获得丰富的野生型乳酸菌资源，为优良菌株的筛选和工业化应用提供核心菌株，从原始森林桦树皮中分离、鉴定和筛选优良乳酸菌菌种，并研究其特性。[方法] 通过传统纯培养方法对20份桦树皮中的内生和表生乳酸菌进行分离纯化，运用16S rRNA基因序列分析以及系统发育关系研究进行种属鉴定，并对样品中的优势菌株进行筛选，得到2株具有优良特性的菌株。[结果] 16S rRNA基因序列分析结果表明，分离到的112株乳酸菌鉴定为3个属7个种，其中树皮表生分离株共53株，内生分离株为59株，包括Enterococcus mundtii （27株）、Enterococcus faecium （16株）、Enterococcus faecalis （12株）、Enterococcus durans （1株）、Lactococcus lactis （37株）、Lactococcus garvieae （18株）、Lactobacillus fermentum （1株）。通过初筛和复筛比较不同乳酸菌在同一条件下的pH和产酸量，从37株Lactococcus lactis中得到2株产酸速率较高、pH较低的乳酸菌，其编号为IMAU98457和IMAU98428。对菌株IMAU98457和IMAU98428进行生物学特性研究，发现其最适生长温度在37℃，适宜pH范围为5.5-7.5，菌株在37℃发酵18 h后菌液OD₆₀₀值分别高达2.531 0和2.518 2，pH低至4.36和4.34 （总酸）。菌株IMAU98457和IMAU98428有一定的盐耐受性，当NaCl浓度大于6%时，菌株生长受到明显抑制。[结论] 20份桦树皮中分离出的112株乳酸菌，Lactococcus lactis （37株）为内蒙古赤峰市桦树皮样品的优势菌株，占总分离株的33%。桦树皮表生样品与内生样品相比，内生样品较表生样品的菌株种类资源更为丰富，二者的优势菌属具有较大的差异，并从优势菌株Lactococcus lactis中筛得2株繁殖速度快、产酸速率较高、pH较低的菌株IMAU98457和IMAU98428。

关键词:乳酸菌;分离鉴定;16S rRNA基因序列分析;桦树皮;生物学特性

Abstract:

[Background] With the rapid development of the lactic acid bacteria (LAB) industry, it is becoming a research hotspot in the fermented field to analyse the community structure of LAB in different habitats and to develop and utilize bacterial resources. It is of great significance to isolate and preserve of LAB for the screening and utilization of excellent bacterial species. [Objective] In order to clarify the composition of LAB in the birch bark of the primitive forests of Inner Mongolia, and to obtain wild LAB strains, and to provide core strains for excellent strains screening and industrial production of LAB, we isolated and identified and screened LAB from bark of birch trees in Primeval Forest and also survey their characteristics. [Methods] Traditional pure culture method was used to isolate and purify the endogenous and epiphytic LAB from 20 samples of birch bark. The species identification were performed by 16S rRNA gene sequence analysis and the phylogenetic relationship study method. The dominant strains were screened, and 2 strains with excellent characteristics were obtained. [Results] 16S rRNA gene sequence analysis showed that the 112 isolates of LAB were identified as 3 genera and 7 species, of which 53 epiphytic isolates and 59 endophytic isolates, including Enterococcus mundtii (27 strains), Enterococcus faecium (16 strains), Enterococcus faecalis (12 strains), Enterococcus durans (1 strain), Lactococcus lactis (37 strains), Lactococcus garvieae (18 strains), Lactobacillus fermentum (1 strain). Among 37 strains of Lactococcus lactis, 2 strains IMAU98457 and IMAU98428 with higher acid production and lower pH were obtained after preliminary and re-screening by comparing the pH and lactic acid production of different LAB under the same conditions. The biological characteristics of the strains were studied and it was found that the optimum growth temperature of strains IMAU98457 and IMAU98428 was 37℃, and the suitable pH range was 5.5−7.5. After the fermentation at 37℃ for 18 hours, the OD₆₀₀ value of the bacterial solution was as high as 2.531 0 and 2.518 2 respectively. The pH was as low as 4.36 and 4.34 (total acid) respectively, and the strains IMAU98457 and IMAU98428 have certain salt tolerance. When the NaCl concentration is greater than 6%, the growth of the strain is significantly inhibited. [Conclusion] Among the 112 lactic acid bacteria isolated from 20 birch bark samples, Lactococcus lactis was the dominant strain of birch bark samples in Chifeng, Inner Mongolia, with a total of 37 strains, accounting for 33% of the total isolates. Compared with endophytic samples, endophytic samples had more abundant species resources than epigenetic samples, and there were great differences in dominant bacteria between endophytic samples and endophytic samples. Two strains IMAU98457 and IMAU98428 with fast propagation rate, high acid production rate and low pH were screened from the dominant strain Lactococcus lactis.

Key words:lactic acid bacteria;isolation and identification;16S rRNA gene sequence analysis;birch bark;biological characteristics