Abstract Dry aging (DA) allows for the storage of meat without packaging at 0 to 3°C for several weeks. It enhances the production of pleasant flavors, tenderness, and juiciness in meat. Due to the long storage period and roles of indigenous microbiota in the maturation of several meat products, the microbiota of DA meat is of interest in terms of microbial contributions and food hygiene but has not yet been characterized in detail. This study identified the microbiota of pork loins during DA using culturing and culture‐independent meta‐16S rRNA gene sequencing and elucidated its characteristics. The amounts of free amino acids and profiles of aroma‐active compounds were also monitored by high‐performance liquid chromatography and gas chromatography, respectively. The meta‐16S rRNA gene sequencing revealed that Pseudomonas spp. generally dominated the microbiota throughout DA; however, the culturing analysis showed marked changes in the species composition during DA. Acinetobacter spp. were the second most dominant bacteria before DA in the culture‐independent analysis but became a minor population during DA. The cell numbers of yeasts showed an increased tendency during DA, and Debaryomyces hansenii was the only microorganism isolated from all meat samples throughout DA. Well‐known foodborne pathogens were not observed in two microbiota analyses. The amounts of free amino acids were increased by DA, and the number of aroma‐active compounds and their flavor dilution values markedly changed during DA. Most microbial isolates showed positive reactions with proteolytic and lipolytic activities, suggesting their contribution to tenderness and aroma production in DA meats.

In this study, we characterized the main properties of the fermented rice bran around mackerel in the Japanese product Heshiko. We found increases in the moisture, lipids, and ash contents after fermentation, which was attributed to the effects of the Shiejiru added for filtration during the manufacturing process. However, there is no change of the general composition of the fermented rice bran of Heshiko with respect to the shipping time. We further characterized the non-starch, starch, and water-soluble polysaccharide fractions to understand better the chemical changes occurring as a result of enzyme and microbial action during fermentation. For instance, SEM photographs of non starch and starch polysaccharides indicate the breakage of cell wall and surface of the starches. Furthermore, regarding the branch chain-length distribution pattern of. amylopectin, we observed a decrease in the short chains of DP 6-12 and an increase in other chains of DP 13-20 in the fermented rice bran. Finally, we isolated amylase-producing bacteria from the fermented rice bran, which were estimated as Bacillus amyloliquefaciens ( A 1) , Paenibacillus amylolyticus ( A 3) , Bacillus subtilis ( B 1 ) , Bacillus licheniformis (B 2), and Oceanobacillus picturae (B 3).

Three Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacterial strains (IWT5T, IWT25T and IWT140), isolated from silage, were investigated by using a polyphasic taxonomic approach. Strains IWT5T and IWT25T grew at 10-37 °C and 30-37 °C, and at pH 4.0-7.5 and 4.0-7.0, respectively. The G+C contents of genomic DNA of strains IWT5T and IWT25T were 43.2 and 44.4 mol%, respectively. Strains IWT5T and IWT25T contained C16 : 0, C18 : 1 ω9c and summed feature 7 (unknown 18.846/C19 : 1 ω6c/C19 : 0cyclo ω10c) as the major fatty acids. Strain IWT5T was most closely related to the type strains of Lactobacillus mixtipabuli (99.9 % 16S rRNA gene sequence similarity) and Lactobacillus silagei (99.5 %). For IWT25T, the 16S rRNA gene sequence similarities with the closely related neighbour type strains L. mixtipabuli and L. silagei were 99.5 and 99.5 %, respectively. The 16S rRNA gene sequence similarities among the three novel isolates were 99.5-99.9 %. The average nucleotide identities of strains IWT5T and IWT25T to other neighbours of the genus Lactobacillus were less than 82 % and the genomes of IWT25T and IWT140 shared 97.3 % average nucleotide identity, demonstrating that the three strains were allocated to two different novel species of the genus Lactobacillus. Together with multilocus sequence analysis, phenotypic and chemotaxonomic characteristics, strains IWT5T (=JCM 31144T=DSM 102973T) and IWT25T (=JCM 31145T=DSM 102974T) are proposed as the type strains of novel species of the genus Lactobacillus, with the names Lactobacillus silagincola sp. nov. and Lactobacillus pentosiphilus sp. nov., respectively.

Fermented wheat starch (FWS) is produced from starch as a byproduct of gluten manufacture. FWS is used in the traditional Japanese sweet, kuzu-mochi, which originates from the Edo period. The elastic but non-sticky texture of kuzu-mochi is attributable to the properties of FWS. To elucidate the characteristic properties of FWS, the comparison morphology, and physicochemical and pasting properties of starch with/without fermentation treatment were examined. Fermentation of wheat starch (WS) led to a decrease in the abundance of small starch granules, as small starch granules formed large aggregates. The degrees of damaged starch and amylose content in FWS were lower than those in unfermented WS. The λmax of the iodine absorption spectrum of FWS was blue-shifted relative to that of WS. The maximum and final viscosities of FWS, and its consistency in the rapid visco analyzer viscogram were lower than those of WS. These results revealed that fermentation endows starch with low viscosity and the ability to retard retrogradation.

BACKGROUND:Most lactic acid bacteria are non-motile but some of them are flagellated and exhibit motility. So far, motile lactobacilli have rarely been studied, and characteristics of their flagellins are poorly understood. In this study, a highly motile strain of Lactobacillus agilis was recruited for transcriptional analysis and characterization of its flagellins.RESULTS:Unlike another motile lactic acid bacteria of intestinal isolate, Lactobacillus ruminis, flagellar filaments of the L. agilis strain probably consist of two homologous but distinct flagellins. Glycosylation of the flagellar filaments and their resistance to heat, acid and SDS were also observed. The immunological activity of the flagellins was evaluated through the stimulation of Caco-2 cells. The results show that TLR5-stimulating activity of the protein is attenuated, likely due to an incomplete TLR5-recognition site.CONCLUSIONS:The flagella filaments of L. agilis BKN88 consist of two homologous glycosylated flagellins, which likely have an incomplete TLR5-recognition site. The characteristics of the flagellin are presumably a consequence of adaptation as a commensal microbe in the gastrointestinal tract.

The purpose of this study was to examine the mechanism of pressure-induced gel (PG) formation of hen egg yolk. Egg yolk forms gels by high-pressure processing (over 450MPa) at temperatures above 15℃. PG (20℃, 650MPa) showed the highest "Gel Strength" and "Work Done values". Furthermore, these parameters increased with increasing protein concentration. N-ethylmaleimide was used to inhibit formation of PG and heat-induced gels (HG). Result of non-reducing SDS-PAGE showed that apo-LDL from plasma and granule fractions and apo-HDL from granule fraction were the main proteins that form PG and HG. In addition, disulfide bonds play an important role in gel formation; however, there were no significant differences between PG and HG in disulfide bond formation. The structure of PG and HG consisted of a network of fine strands and the surface structure was different between PG and HG as observed by scanning electron microscopy. PG showed a thick and smooth surface, whereas HG showed a thin and rough surface. These results suggested that PG formation of hen egg yolk occurred via disulfide bond formation. Therefore, the physicochemical characteristics of PG were much different from those of HG.

The purpose of this study was to investigate the mechanism of β-lactoglobulin (β-lg) pressure-induced gel (PG) formation at low to medium temperatures (0～20℃). β-lactogrobulin formed gels by high-pressure processing (HPP) at 20℃. The protein concentration required for PG formation was 125 mg/mℓ or greater. The PG at 20℃ and 650MPa showed the greatest gel strength and work done values. PG and heat-induced gel (HG) formation was inhibited by N-ethylmaleimide. Free sulfhydryl (SH) groups of β-lg increased with increasing processing temperature. The number of free SH groups after HPP at a medium temperature (20℃, 600MPa) was significantly higher than after HPP at a low temperature (0～10℃, 600MPa). However, SH content after HPP at medium temperature was approximately half of the SH content after heat processing (90℃, 0.1MPa). Non-reducing SDS-PAGE showed that β-lg oligomers were formed by HPP. In this investigation, formation of oligomers and increased coomassie brilliant blue dye staining intensity were temperature-dependent. These results suggest that free SH groups of β-lg increased in a temperature-dependent manner. Therefore, formation of β-lg PG is caused by the formation of high molecular weight oligomers by intermolecular disulfide bonds.

Six strains of anaerobic bacteria, C13EG70T, C13EG118, C13EG186T, C13GAMG5, C13GAMG28 and C13GAMG40, were isolated from the caecum of a healthy chicken bred in Bogor, Indonesia. Phylogenetic analysis showed the isolates were separated into two groups. Group I (C13EG70T and C13EG118) showed nearly identical 16S rRNA gene sequences (99.9 % sequence similarity). Group II (C13EG186T, C13GAMG5, C13GAMG28 and C13GAMG40) showed nearly identical 16S rRNA gene sequences (>99.4 % sequence similarity). The isolates showed low 16S rRNA gene sequence similarities to recognized species of the genus Bacteroides. High gene sequence similarities were found between type strains (C13EG70T and C13EG186T) and Bacteroides salanitronis JCM 13657T (87.9, 91.5 %, respectively). Physiological, biochemical and genotypic characteristics demonstrated that these strains could be separated from the type strain of B. salanitronis. It is concluded that Group I and Group II represent novel species. Two novel species of the genus Bacteroides are proposed as Bacteroides caecicola sp. nov. (type strain C13EG70T = LIPI12-4-Ck732T = JSAT12-4-Ck732T = InaCC B449T = NBRC 110958T) and Bacteroides gallinaceum sp. nov. (type strain C13EG186T = LIPI12-4-Ck844T = JSAT12-4-Ck884T = InaCC B451T = NBRC 110963T).

Kuzu-mochi made from fermentation treated wheat starch is a traditional Japanese sweet from the Edo period. Kuzu-mochi has a unique texture, which is not found in wheat starch gels. In this study, to clarify the texture characteristics of kuzu-mochi, the physical properties of kuzu-mochi were compared with that of various gelatinous foods such as dango, warabi-mochi, and yokan. Kuzu-mochi was softer than yokan, but it was as hard as dango. The chewiness and the stickiness of kuzu-mochi were lower than that of dango. Kuzu-mochi had an elastic texture and not a sticky one. Many starch granules were observed within the gel matrix of kuzu-mochi. The gelatinous state of starch in kuzu-mochi is suggested to contribute to the texture and the internal structure of kuzu-mochi. The firmness of the fermented wheat starch gels was lower than that of kuzu-mochi. After storing for 72h at 4℃，the firmness of the fermented wheat starch gel underwent few changes. The results of this study suggest that the texture of kuzu-mochi is characterized by fermented wheat starch.

Three strains of anaerobic Gram-stain-negative, short to longer rod-shaped bacteria isolated from the caecum of chicken in Indonesia were studied using a polyphasic taxonomic approach. These strains belonged to the genus Bacteroides, based on sequence analysis of 16S rRNA and hsp60 (groEL) genes, with similarities of 93.2-94.1 and 89.8-90.8 %, respectively, to the closest recognized species, Bacteroides coprocola JCM 17929T. Sugar fermentation and enzyme characteristics, cellular fatty acid profiles, menaquinone profiles and metabolic end products were also investigated. Furthermore, DNA-DNA hybridization studies confirmed that the three novel strains are different from the closest related species. The strains were also found to be distinct from each other on the basis of ribotype profiles. The DNA G+C contents of the three strains were 41.1-41.8 mol%. Based on phenotypic and phylogenetic characteristics, a novel species, Bacteroides caecigallinarum sp. nov., is proposed (type strain C13EG111T = LIPI12-4-Ck773T = JSAT12-4-Ck773T = InaCC B455T = NBRC 110959T).

Using a polyphasic taxonomic approach, we investigated three bacterial strains - IWT30T, IWT8 and IWT75 - isolated from total mixed ration silage prepared in Hachimantai, Iwate, Japan. The isolates comprised Gram-stain positive, non-motile, non-spore-forming, catalase-negative, rod-shaped bacteria. Good growth occurred at 15-45 °C and at pH 4.0-7.5. Their major cellular fatty acids were C18:1ω9c and C19:1 cyclo 9,10.The G+C content of genomic DNA of strain IWT30T was 44.6 mol%. Comparative 16S rRNA gene sequence analysis showed that these novel strains belonged to the genus Lactobacillus. These strains shared 100 % 16S rRNA gene sequence similarity and were most closely related to the type strains of Lactobacillus silagei, Lactobacillus odoratitofui, Lactobacillus similis, Lactobacillus collinoides, Lactobacillus paracollinoides and Lactobacillus kimchicus, with sequence similarity values of 99.5, 98.8, 98.7, 97.8, 97.8 and 96.8 %, respectively. The level of DNA-DNA relatedness between these strains and their closest phylogenetic neighbours was less than 30 %. On the basis of additional phylogenetic analysis of pheS and rpoA gene sequences and phenotypic and chemotaxonomic characteristics, we conclude that these three strains represent a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus mixtipabuli sp. nov. The type strain is IWT30T ( = JCM 19805T = DSM 28580T).

Synonymy of Gibbsiella dentisursi DSM 23818T ( = NUM 1720T) and Gibbsiella papilionis JCM 18389T ( = LEN33T) was suspected following multilocus sequence analysis (MLSA) of both type strains in a previous classification study, where they were found to share >99.6 % gene sequence similarity. The taxonomic relationship between these two strains was re-examined here using a polyphasic approach. A DNA-DNA hybridization value of 98 % confirmed that the two type strains belong to a single taxon, while the phenotypic profiles were found to be nearly identical. Therefore we propose Gibbsiella papilionis as a later heterotypic synonym of Gibbsiella dentisursi, with the type strain as NUM 1720T ( = DSM 23818T = JCM 17201T).

In this study, the gamma-aminobutyric acid (GABA) -producing Lactobacillus buchneri 1001 strain, isolated from kaburazushi (a regional specialty food of Japan's Toyama Prefecture consisting of pickled fish, rice, and turnip), was employed to prepare fermented butter-like food. GABA was effectively produced during fermentation by inoculating sodium glutamate-containing fresh cream with L. buchneri 1001 together with Lactobacillus bulgaricus and Streptococcus thermophilus. Furthermore, using freeze-drying instead of the churning process traditionally used after fermentation resulted in the production of fermented butter-like food with high GABA content, as well as a greater amount of protein and carbohydrates. The resulting fermented butter-like food met compositional standards, and its organoleptic characteristics were found to be as good as those of commercially available products.

Here, we report the draft genome sequence of a motile lactic acid bacterium, Lactobacillus sucicola JCM 15457(T), isolated from oak sap. Motility-related genes and their organization in the annotated genome were broadly similar to those in the sequenced genomes of related lactobacilli.

Two strains of lactic acid bacteria, Nu27(T) and Nu29, were isolated from Nukadoko, rice bran paste for Japanese pickles. The isolates were Gram-stain-positive, rod-shaped, catalase-negative, non-motile and facultatively anaerobic lactic acid bacteria. The isolates showed identical 16S rRNA gene sequences. The closest relatives to strain Nu27(T) based on 16S rRNA gene sequence similarities were Lactobacillus versmoldensis KU-3(T) (98.9% 16S rRNA gene sequence similarity), Lactobacillus nodensis iz4b(T) (96.3%) and Lactobacillus tucceti CECT 5290(T) (97.2%). DNA-DNA relatedness values revealed genotype separation of the two isolates from the above three species. Based on the physiological, biochemical and genotypic characteristics provided, the isolates represent a novel species of the genus Lactobacillus, for which name is Lactobacillus furfuricola proposed. The type strain is Nu 27(T) ( = JCM 18764(T) = NRIC 0900(T) = DSM 27174(T)).

Kuzu-mochi, which is a traditional Japanese sweet, is made from fermented wheat starch. In this study, we examined changes in wheat starch granules and the microflora during the fermentation of wheat starch. Large, broken granules of starch and gram-positive bacilli were observed during the fermentation process. The pH ranged from 3 to 4 throughout the process. Lactic acid and acetic acid were detected in each fermentation period, with lactic acid being the predominant acid. This result indicates that lactic acid bacteria (LAB) are heterolactic fermenters. Viable cell count was determined at the same pH as that of the fermentation medium, and microorganisms that can grow in acidic conditions were isolated. The microflora present during the fermentation process included 2—3 species of aerobic bacteria, fungi and yeast, and approximately 10 species of LAB. PCR-denaturing-gradient-gel-electrophoresis profiles indicated that the microflora remained stable from the pre-fermentation period to about 6 months post-fermentation. Furthermore, the supernatant collected during the pre-fermentation period showed starch hydrolysis activity, and wheat starch was broken by its enzyme. These results suggest that the bacterial community present at the pre-fermentation period can serve as a starter culture for the traditional processing of fermented wheat starch.

Spoilage of cooked rice occurred as a result of heat insulation. The cooked rice had an off-flavor that was caused by spoilage. This phenomenon is thought to be caused by microorganisms. Therefore, in this study, we attempted to isolate the contaminating microorganisms from cooked rice and identify them by using polymerase chain reaction (PCR) and PCR-denaturing gradient gel electrophoresis (DGGE). No microorganisms were detected in normal cooked rice; however, microorganisms were detected at 4.6×106 cfu/g in rice spoiled by 72h of heat insulation. The isolates were able to grow at 60～65℃ on the cooked rice, and they produced an off-flavor. Based on phylogenetic analysis, the isolates were identified as members of the genus Geobacillus, and were closely related to Geobacillus thermoleovorans. Contaminating microorganisms in the cooked rice were identified by PCR using bacterial universal primers and G. thermoleovorans was only detected using PCR-DGGE. These results suggest that G. thermoleovorans was the contaminating microorganism in the spoiled cooked rice and produced the off-flavor.

We constructed a gramineous plant-specific primer set-matK 792F1GC and matK 979R1, targeting the maturase K (matK) gene, for detection of gramineous raw materials in processed food. For this, we used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The primer set was evaluated using agarose gel electrophoresis and DGGE. Our results confirmed that the primer set was specific for gramineous plants. From our analysis, we confirmed that up to 6 types of gramineous plants can be simultaneously detected using PCR-DGGE. Results of a model test showed that by using this method, it was possible to detect wheat from a sample, if present more than 0.1% (w/w). Thus, our method of PCR-DGGE with matK 792F1GC and matK 979R1 can be used to detect the presence of gramineous raw materials in processed food.

ABSTRACT Lactobacillus is the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13 Lactobacillus species, all belonging to the Lactobacillus salivarius clade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin–Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside the L. salivarius clade, Lactobacillus curvatus (strain NRIC 0822). The motility of L. curvatus NRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15°C and 37°C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced the L. curvatus NRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon of Lactobacillus acidipiscis KCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon of L. curvatus suggests recent horizontal transfer between members of two distinct Lactobacillus clades: L. acidipiscis in the L. salivarius clade and L. curvatus in the L. sakei clade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli.