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A millet alcoholic beverage that is made primarily from glutinous rice or millet is a traditional drink of indigenous Taiwanese tribes. In this study, we used both culture-dependent and culture-independent methods to analyse the diversity of the lactic acid bacteria (LAB) that are present during the process of millet alcohol fermentation, including during starter preparation and brewing. Based on random amplified polymorphic DNA (RAPD)-PCR fingerprinting profiles, 147 LAB isolates were identified and categorised into 38 groups. Phylogenetic analyses of 16S rRNA, phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) gene sequences identified representative strains from each of the 38 groups as belonging to the following ten species: Enterococcus faecium group, Lactobacillus brevis, Lactococcus garvieae, Lactococcus lactis, Leuconostoc carnosum, Leuconostoc (Leu.) mesenteroides, Pediococcus pentosaceus, Pediococcus stilesii, Weissella cibaria and Weissella soli. The results of the LAB species diversity studies were confirmed using 16S rRNA gene-based denaturing gradient gel electrophoresis (PCR-DGGE), which showed that P. pentosaceus was mainly involved in the fermentation in both the starter preparation and at the brewing stage. However, L. brevis, Leu. mesenteroides and P. stilesii were the predominant LAB strains found during the brewing stage.

Three Gram-positive-staining strains isolated from fermented stinky tofu brine were rod-shaped, non-motile, asporogenous, facultatively anaerobic, heterofermentative and did not exhibit catalase activity. Comparative analyses of 16S rRNA, rpoA and pheS gene sequences demonstrated that the novel strains were members of the genus Lactobacillus. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus collinoides (98.6 %), Lactobacillus paracollinoides (98.6 %) and Lactobacillus similis (99.6 %) were the closest neighbours. However, DNA–DNA reassociation values with these strains were less than 10 %. The phenotypic and genotypic features demonstrated that these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus odoratitofui sp. nov. is proposed. The type strain is YIT 11304T (=JCM 15043T =BCRC 17810T =DSM 19909T).

Three Gram-positive, catalase-negative, motile, rod-shaped bacteria were isolated from fermented stinky tofu brine. These strains, designated YIT 11306(T), YIT 11317 and YIT 11318, were discriminated from five isolates on the basis of randomly amplified polymorphic DNA profiles. They produced l-lactic acid as the main end product from glucose without gas formation, synthesized dextran from sucrose and hydrolysed aesculin. Ammonia was not produced from arginine. Comparative 16S rRNA gene sequence analysis demonstrated that the novel isolates were members of the genus Lactobacillus. Based on levels of 16S rRNA gene sequence similarity, the three novel strains were related most closely to the type strains of Lactobacillus mali (97.2 %) and Lactobacillus satsumensis (96.8 %). However, levels of DNA-DNA relatedness between the novel isolates and the type strains of L. mali and L. satsumensis were less than 10 %. The phenotypic and genotypic data demonstrate that the three strains represent a single novel species of the genus Lactobacillus, for which the name Lactobacillus capillatus sp. nov. is proposed. The type strain is YIT 11306(T) (=JCM 15044(T)=BCRC 17811(T)=DSM 19910(T)).

In Taiwanese alternative medicine Lu-doh-huang (also called Pracparatum mungo), mung beans are mixed with various herbal medicines and undergo a 4-stage process of anaerobic fermentation. Here we used high-throughput sequencing of the 16S rRNA gene to profile the bacterial community structure of Lu-doh-huang samples. Pyrosequencing of samples obtained at 7 points during fermentation revealed 9 phyla, 264 genera, and 586 species of bacteria. While mung beans were inside bamboo sections (stages 1 and 2 of the fermentation process), family Lactobacillaceae and genus Lactobacillus emerged in highest abundance; Lactobacillus plantarum was broadly distributed among these samples. During stage 3, the bacterial distribution shifted to family Porphyromonadaceae, and Butyricimonas virosa became the predominant microbial component. Thereafter, bacterial counts decreased dramatically, and organisms were too few to be detected during stage 4. In addition, the microbial compositions of the liquids used for soaking bamboo sections were dramatically different: Exiguobacterium mexicanum predominated in the fermented soybean solution whereas B. virosa was predominant in running spring water. Furthermore, our results from pyrosequencing paralleled those we obtained by using the traditional culture method, which targets lactic acid bacteria. In conclusion, the microbial communities during Lu-doh-huang fermentation were markedly diverse, and pyrosequencing revealed a complete picture of the microbial consortium.

Three Gram-stain-positive strains were isolated from fermented mustard and were rod-shaped, non-motile, asporogenous, facultatively anaerobic, homofermentative and did not exhibit catalase activity. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences demonstrated that the novel strains were members of the genus Lactobacillus. On the basis of 16S rRNA gene sequence analysis, the type strains of Lactobacillus crustorum (98.7 % similarity), Lactobacillus farciminis (98.9 %) and Lactobacillus mindensis (97.9 %) were the closest neighbours. However, DNA–DNA reassociation values with these strains were less than 50 %. Phenotypic and genotypic features demonstrated that these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus futsaii sp. nov. is proposed; the type strain is YM 0097T ( = JCM 17355T = BCRC 80278T).

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.