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

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