Guangyong Qin

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.

Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan Plateau

A total of 140 lactic acid bacteria (LAB) strains were isolated from corn, alfalfa, clover, sainfoin, and Indian goosegrass in the Tibetan Plateau. According to phenotypic and chemotaxonomic characteristics, 16S rDNA sequence, and recA gene PCR amplification, these LAB isolates were identified as belonging to five genera and nine species. Corn contained more LAB species than other forage crops. Leuconostoc pseudomesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus brevis, and Weissella paramesenteroides were dominant members of the LAB population on alfalfa, clover, sainfoin, and Indian goosegrass, respectively. The comprehensive 16S rDNA and recA-based approach effectively described the LAB community structure of the relatively abundant LAB species distributed on different forage crops. This is the first report describing the diversity and natural populations of LAB associated with Tibetan forage crops, and most isolates grow well at or below 10°C. The results will be valuable for the future design of appropriate inoculants for silage fermentation in this very cold area.

Identification of lactic acid bacteria isolated from corn stovers

One hundred and twenty-six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram-positive, catalase-negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover-associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage.

16S ribosomal DNA analysis and characterization of lactic acid bacteria associated with traditional Tibetan Qula cheese made from yak milk

Twenty strains of lactic acid bacteria were isolated from six traditional Tibetan Qula cheese made from yak which were collected from northwest China, including Tibet, Qinghai and Gansu province. These isolates were subjected to phenotypic and genetic analyses. All isolates were Gram-positive and catalase-negative cocci that produced gas from glucose and formed D(-) isomer of lactate. Most isolates were able to grow in de Man, Rogosa and Sharpe (MRS) broth at pH values 3.0-9.0 and in 6.5% NaCl (w/v). According to analytical profile index 50 carbohydrates (API 50 CH) fermentation patterns of amygdalin and arabinose, these isolates were divided into three groups (A to C). On the basis of the phylogenetic trees of 16S ribosomal DNA (rDNA) sequence, the strains in all groups were placed in the cluster making up the genus Leuconostoc, which showed that all strains should belong to Leuconostoc species. Strains in Group A and Group B exhibited similarity of 16S rDNA sequence of over 99% to Leuconostoc mesenteroides, indicating that they each comprised a single species. Strains in group C were assigned to the Leuconostoc pseudomesenteroides and their 16S rDNA sequence showed a similarity of over 99%. This study demonstrated that Leuconostoc was the dominant member among lactic acid bacteria in Qula cheese.

Reclassification of Lactobacillus kimchii and Lactobacillus bobalius as later subjective synonyms of Lactobacillus paralimentarius

Characterization and identification of strain CW 1 ( = JCM 17161) isolated from corn silage were performed. Strain CW 1 was a Gram-positive, catalase-negative and homofermentative rod that produced the DL-form of lactic acid. This strain exhibited more than 99.6% 16S rRNA gene sequence similarity and greater than 82% DNA-DNA reassociation with type strains of Lactobacillus kimchii, L. bobalius and L. paralimentarius. To clarify the taxonomic positions of these type strains, phenotypic characterization, 16S rRNA gene sequencing, ribotyping and DNA-DNA relatedness were examined. The three type strains displayed different L-arabinose, lactose, melibiose, melezitose, raffinose and N-acetyl-β-glucosaminidase fermentation patterns. Phylogenetic analysis showed that L. paralimentarius is a closer neighbour of L. kimchii and L. bobalius, sharing 99.5-99.9% 16S rRNA gene sequence similarity, which was confirmed by the high DNA-DNA relatedness (≥82%) between L. paralimentarius JCM 10415(T), L. bobalius JCM 16180(T) and L. kimchii JCM 10707(T). Therefore, it is proposed that L. kimchii and L. bobalius should be reclassified as later synonyms of L. paralimentarius.