Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce, using nested PCR-denaturing gradient gel electrophoresis and the plate count method.

The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.

Lactobacillus futsaii sp. nov., isolated from fu-tsai and suan-tsai, traditional Taiwanese fermented mustard products.

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 0097(T) (=JCM 17355(T)=BCRC 80278(T)).

Lactobacillus odoratitofui sp. nov., isolated from stinky tofu brine.

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 11304(T) (=JCM 15043(T) =BCRC 17810(T) =DSM 19909(T)).

Inhibitory effects of citronellol and geraniol on nitric oxide and prostaglandin E2production in macrophages.

Geranium oil has been used traditionally for diarrhea, dermatitis, and intestinal inflammation in East Asia. The aim of this study was to determine the effects of geranium oil's characteristic components, citronellol and geraniol, on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Citronellol and geraniol suppressed NO and PGE(2) production in a dose-dependent manner. The inhibitory efficacy of geraniol was concomitant with decreases in protein and mRNA expression levels of inducible nitric oxide synthase (iNOS), whereas citronellol inhibited only iNOS enzymatic activity. By adding citronellol and geraniol, the LPS-induced cyclooxygenase-2 (COX-2) protein and mRNA expression levels were significantly attenuated, whereas cytosolic degradation of I κB α and upregulation of NF-κB p65 in the nucleus were reversed. These results suggested that citronellol and geraniol exhibit anti-inflammatory activities, supporting their common use and demonstrating their therapeutic potential for inflammation-associated disorders.

Author Correction: Diversity in gut bacterial community of school-age children in Asia.

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Diversity of lactic acid bacteria in fermented brines used to make stinky tofu.

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.

Bacterial Composition and Diversity in Breast Milk Samples from Mothers Living in Taiwan and Mainland China.

Human breast milk is widely recognized as the best source of nutrients for healthy growth and development of infants; it contains a diverse microbiota. Here, we characterized the diversity of the microbiota in the breast milk of East Asian women and assessed whether delivery mode influenced the microbiota in the milk of healthy breast-feeding mothers. We profiled the microbiota in breast milk samples collected from 133 healthy mothers in Taiwan and in six regions of mainland China (Central, East, North, Northeast, South, and Southwest China) by using 16S rRNA pyrosequencing. Lactation stage (months postpartum when the milk sample was collected) and maternal body mass index did not influence the breast milk microbiota. Bacterial composition at the family level differed significantly among samples from the seven geographical regions. The five most predominant bacterial families were Streptococcaceae (mean relative abundance: 24.4%), Pseudomonadaceae (14.0%), Staphylococcaceae (12.2%), Lactobacillaceae (6.2%), and Oxalobacteraceae (4.8%). The microbial profiles were classified into three clusters, driven by Staphylococcaceae (abundance in Cluster 1: 42.1%), Streptococcaceae (Cluster 2: 48.5%), or Pseudomonadaceae (Cluster 3: 26.5%). Microbial network analysis at the genus level revealed that the abundances of the Gram-positive Staphylococcus, Streptococcus, and Rothia were negatively correlated with those of the Gram-negative Acinetobacter, Bacteroides, Halomonas, Herbaspirillum, and Pseudomonas. Milk from mothers who had undergone Caesarian section (C-section group) had a significantly higher abundance of Lactobacillus (P < 0.05) and a higher number of unique unclassified operational taxonomic units (OTUs) (P < 0.001) than that from mothers who had undergone vaginal delivery (vaginal group). These findings revealed that (i) geographic differences in the microbial profiles were found in breast milk from mothers living in Taiwan and mainland China, (ii) the predominant bacterial families Streptococcaceae, Staphylococcaceae, and Pseudomonadaceae were key components for forming three respective clusters, and (iii) a significantly greater number of unique OTUs was found in the breast milk from mothers who had undergone C-section than from those who had delivered vaginally.

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae.

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.

Diversity in gut bacterial community of school-age children in Asia.

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

Immunomodulatory Activity of Lactococcus lactis A17 from Taiwan Fermented Cabbage in OVA-Sensitized BALB/c Mice.

From fermented Taiwan foods, we have isolated numerous lactic acid bacteria (LAB) of plant origin and investigated their biological activities. This study aimed to investigate the immunomodulatory effect and mechanism of Lactococcus lactis A17 (A17), isolated from Taiwan fermented cabbage, on ovalbumin (OVA)-sensitized mice. Human peripheral blood mononuclear cells were used to verify immune responses of A17 by IFN-γ production. Live (A17-A) and heat-killed A17 (A17-H) were orally administered to OVA-sensitized BALB/c mice to investigate their effects on immunoglobulin (Ig) and cytokine production. The mRNA expression of Toll-like receptors (TLR) and nucleotide binding oligomerization domain (NOD)-like protein receptors in spleen cells was analyzed by real-time RT-PCR. Both live and heat-killed A17 modulate OVA-induced allergic effects. B-cell response was modulated by diminishing IgE production and raising OVA-specific IgG2a production, while T-cell response was modulated by increasing IFN-γ production and decreasing IL-4 production. The mRNA expression of NOD-1, NOD-2, and TLR-4 was down-regulated by A17 as well. This is the first report to describe a naïve Lactococcus lactis A17 strain as a promising candidate for prophylactic and therapeutic treatments of allergic diseases via oral administration. Our results suggest the ameliorative effects of A17 may be caused by modulating NOD-1 NOD-2, and TLR-4 expression.

Microbial diversity analysis of fermented mung beans (Lu-Doh-Huang) by using pyrosequencing and culture methods.

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.

Evaluation of the antioxidant and melanogenesis inhibitory properties of pracparatum mungo (lu-do huang).

Pracparatum mungo (Lu-Do Huang) is a traditional Chinese functional medicine made from the natural fermentation of mung bean (Lǜ Dòu) mixed with other Chinese medicines. It has been recognized as having liver protecting and detoxifying effects. As mung beans have been verified to possess anti-inflammatory, antioxidant, antipyretic, and whitening actions, the present research utilized the in vitro, ex vivo, and in vivo experimental models to investigate the antioxidant and melanin inhibiting effects of P. mungo on the skin. The in vitro experiment revealed that P. mungo methanol extract (PMME) and P. mungo ethanol extract (PMEE) possess the capacity to clear α,α-diphenyl-2-picrylhydrazyl (DPPH) radicals and inhibit tyrosinase activity. The ex vivo experiment indicated that PMEE can promote the growth of MDCK cells and increase the enzymatic activities of superoxide dismutase (SOD) and catalase in MDCK cells. On the other hand, PMME and PMEE can suppress the proliferation of A375 cells, and PMEE can reduce the enzymatic activities of SOD and catalase in A375 cells. The in vivo results showed that P. mungo can enhance the enzymatic performance of SOD, Catalase, and glutathione peroxidase (GPx) in the liver. The results also showed that P. mungo has antioxidant characteristics and can inhibit tyrosinase activity, thereby promoting the growth of skin tissues and suppressing the proliferation of A375 cells, and thus enhancing the effects that the antioxidant enzymatic performance has on the liver. These results can be applied in the development of tyrosinase inhibitors or antioxidants used for the inhibition of melanin biosynthesis or for auto-oxidation in further industrial applications, particularly those relating to functional food or cosmetic compositions.

Ligustilide prevents LPS-induced iNOS expression in RAW 264.7 macrophages by preventing ROS production and down-regulating the MAPK, NF-κB and AP-1 signaling pathways.

Angelica sinensis (AS), an herb used in traditional Chinese medicine, is thought to have anti-inflammatory activities. Ligustilide is its most abundant ingredient. This study sought to determine ligustilide's effects on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. Ligustilide significantly suppressed the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor-α (TNF-α). The inhibition of NO was concomitant with a decrease in the protein and mRNA levels of LPS-induced nitric oxide synthase (iNOS). Furthermore, activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) in the nucleus and the cytosolic degradation of IκBα were abrogated by ligustilide. Ligustilide also inhibited the phosphorylation of IκB kinase (IKK) and mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). The intracellular reactive oxygen species (iROS) level was also significantly decreased. These results suggest that ligustilide exhibits anti-inflammatory activities by blocking the activation of MAPKs/IKK and the downstream transcription factors AP-1 and NF-κB, which may result from ligustilide's down-regulation of iROS production.

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan.

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.

Lactobacillus capillatus sp. nov., a motile bacterium isolated from stinky tofu brine.

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)).

Characterization of a novel PepF-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A.

An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepFBa oligopeptidase is a secreted protein and may facilitate the process of sporulation.