Bacterial compositions of indigenous Lanna (Northern Thai) fermented foods and their potential functional properties.

Many indigenous fermented foods of Northern Thailand and neighbouring regions have traditionally been known for their health benefits. In this study, we explored the communities of bacteria in selected fermented foods which are commonly consumed among ethnic groups around Northern Thailand, for which information on their microbial compositions or their functional properties is still limited. The selected food groups included Thua Nao (alkaline fermented soybean product), Nham (fermented pork sausage/loaf), Nam phak (fermented Chinese cabbage) and Miang (fermented leaves from Miang Tea trees). Bacteria in these fermented foods were isolated and enumerated. Bacterial communities were determined using a culture-independent (pyrosequencing) approach. Lactic acid bacteria were recovered from all of these fermented food samples, with levels ranging from 3.1 to 7.5 log CFU/g throughout the fermentation processes. Analysis of the 16S rRNA gene from the fermented food samples using 454-pyrosequencing resulted in 113,844 sequences after quality evaluation. Lactic acid bacteria were found in high proportions in Nham, Nam phak and Miang. Bacillus was predominant in Thua nao, in which significant proportions of Lactic acid bacteria of the family Leuconostocaceae were also found. Groups of lactic acid bacteria found varied among different food samples, but three genera were predominant: Lactococcus, Lactobacillus and Leuconostoc, of which many members are recognised as probiotics. The results showed that these traditional Thai fermented food products are rich sources of beneficial bacteria and can potentially be functional/probiotic foods.

Microbial niches in raw ingredients determine microbial community assembly during kimchi fermentation.

Fermented foods constitute hubs of microbial consortia differentially affecting nutritional and organoleptic properties, quality, and safety. Here we show the origin source of fermentative microbes and fermentation dynamics of kimchi. We partitioned microbiota by raw ingredient (kimchi cabbage, garlic, ginger, and red pepper) to render kimchi fermented by each source-originated microbe pool and applied multi-omics (metataxonomics and metabolomics), bacterial viability, and physiochemical analyses to longitudinally collected samples. Only kimchi cabbage- and garlic-derived microbial inoculums yielded successful kimchi fermentations. The dominant fermentative microbial taxa and subsequent metabolic outputs differed by raw ingredient type: the genus Leuconostoc, Weissella, and Lactobacillus for all non-sterilized ingredients, garlic, and kimchi cabbage, respectively. Gnotobiotic kimchi inoculated by mono-, di-, and tri- isolated fermentative microbe combinations further revealed W. koreensis-mediated reversible microbial metabolic outputs. The results suggest that the raw ingredient microbial habitat niches selectively affect microbial community assembly patterns and processes during kimchi fermentation.

Leuconostoc spp. Associated with Root Rot in Sugar Beet and Their Interaction with Rhizoctonia solani.

Rhizoctonia root and crown rot is an important disease problem in sugar beet caused by Rhizoctonia solani and also shown to be associated with Leuconostoc spp. Initial Leuconostoc studies were conducted with only a few isolates and the relationship of Leuconostoc with R. solani is poorly understood; therefore, a more thorough investigation was conducted. In total, 203 Leuconostoc isolates were collected from recently harvested sugar beet roots in southern Idaho and southeastern Oregon during 2010 and 2012: 88 and 85% Leuconostoc mesenteroides, 6 and 15% L. pseudomesenteroides, 2 and 0% L. kimchi, and 4 and 0% unrecognized Leuconostoc spp., respectively. Based on 16S ribosomal RNA sequencing, haplotype 11 (L. mesenteroides isolates) comprised 68 to 70% of the isolates in both years. In pathogenicity field studies with commercial sugar beet 'B-7', all Leuconostoc isolates caused more rot (P < 0.0001; α = 0.05) when combined with R. solani than when inoculated alone in both years. Also, 46 of the 52 combination treatments over the 2 years had significantly more rot (P < 0.0001; α = 0.05) than the fungal check. The data support the conclusion that a synergistic interaction leads to more rot when both Leuconostoc spp. and R. solani are present in sugar beet roots.

Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases.

The ginsenoside Rg3(S), which is one of the exceptional components of Korean red ginseng extract, has been known to have anti-cancer, anti-metastatic, and anti-obesity effects. An enzymatic bioconversion method was developed to obtain the ginsenoside Rg3(S) with a high specificity, yield, and purity. Two glycoside hydrolases (BglBX10 and Abf22-3) were employed to produce Rg3(S) as a 100g unit. The conversion reaction transformed ginsenoside Rc to Rd using Abf22-3, followed by Rb1 and Rd to Rg3(S), using BglBX10. It was performed in a 10L jar fermenter at pH 6.0 and 37°C for 24h, with a high concentration of 50mg/ml of purified ginsenoside mixture obtained from ginseng roots. Finally, 144g of Rg3(S) was produced from 250g of root extract with 78±1.2% chromatographic purity. These results suggest that this enzymatic method would be useful in the preparation of ginsenoside Rg3(S) for the functional food and pharmaceutical industries.

Leuconostoc holzapfelii sp. nov., isolated from Ethiopian coffee fermentation and assessment of sequence analysis of housekeeping genes for delineation of Leuconostoc species.

A Gram-positive, ovoid lactic acid bacterium, strain LMG 23990(T), was isolated from Ethiopian coffee fermentation. 16S rRNA gene sequence analysis indicated that the novel strain belongs to the genus Leuconostoc, with Leuconostoc citreum and Leuconostoc lactis as the closest neighbours (99.6 and 99.0 % 16S rRNA gene sequence similarity, respectively). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism, whole-cell protein electrophoresis, DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA, atpA, and physiological and biochemical tests allowed us to differentiate strain LMG 23990(T) from all established Leuconostoc species. Strain LMG 23990(T) (=CCUG 54536(T)) therefore represents a novel species, for which the name Leuconostoc holzapfelii sp. nov. is proposed.

In Vivo PCR-DGGE analysis of Lactobacillus plantarum and Oenococcus oeni populations in red wine.

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety "Primitivo di Puglia"), a polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30-60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.

Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation.

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.

The arcABC gene cluster encoding the arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene.

Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.

Multiplex PCR-based detection and identification of Leuconostoc species.

A multiplex polymerase chain reaction (PCR) assay has been developed for rapid and reliable identification of Leuconostoc species, by using species-specific primers targeted to the genes encoding 16S rRNA. This assay can detect and differentiate Leuconostoc species from mixed populations in natural sources as well as from pure cultures, within 3 h. This assay system consists of a total of 10 primers, two primers from each target species, and comprises two multiplex PCR reactions: one reaction for Leuconostoc carnosum, Leuconostoc citreum and Leuconostoc mesenteroides, and another reaction for Leuconostoc gelidum and Leuconostoc lactis. This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA-DNA hybridization. In addition, this assay enables simultaneous detection of the above-mentioned Leuconostoc species when chromosomal DNA from these Leuconostoc species was mixed. Thus, these results suggest that this multiplex PCR is a rapid and reliable method for identification of Leuconostoc species in pure cultures or in mixed populations.