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ABSTRACT: Edible brown algae are a major food material in Far East Asian countries, particularly in Korea and Japan. They contain fermentable dietary fibers, alginic acid (uronic acid polymer) and laminaran (β-1, 3-glucan), that are fermented into organic acids by intestinal bacteria. To clarify the effect of edible algae on intestinal environment, the cecal microbiota of rats fed diets containing no dietary fiber (control), 2% w/w sodium alginate or laminaran for 2 weeks was analyzed using FLX amplicon pyrosequencing with barcoded primers targeting the bacterial 16S rRNA gene. The most abundant phylum was Firmicutes in all groups. Specifically, Allobaculum was dominant in all diet groups. In addition, Bacteroides capillosus (37.1%) was abundant in the alginate group, while Clostridium ramosum (3.14%) and Parabacteroides distasonis (1.36%) were only detected in the laminaran group. Furthermore, rats fed alginate showed simplified microbiota phylotypes compared with others. With respect to cecal chemical compounds, laminaran increased cecal organic acid levels, particularly propionic acid. Alginate increased total cecal organic acids. Cecal putrefactive compounds, such as indole, H(2)S and phenol, were decreased by both alginate and laminaran. These results indicate that edible brown algae can alter the intestinal environment, with the fermentation by intestinal microbiota.
Applied and environmental microbiology 11/2012; · 3.69 Impact Factor
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ABSTRACT: Listeria monocytogenes causes listeriosis in humans, mainly through the consumption of ready-to-eat foods such as cheese. Immunocompromised persons, the elderly, and pregnant women and their fetuses or newborns are at the highest risk for the infection. We examined the effects of dietary milk-casein (MC) and soy-protein (SP), and their digested compounds tryptone (TP) and phytone peptone (PP), respectively, on L. monocytogenes invasion and infection in human enterocyte-like Caco-2 cells and A/J mice. Invasion into Caco-2 cells tended to be high with TP. In A/J mice orally infected with L. monocytogenes, viable numbers in the liver and spleen showed a tendency of decreasing with the 20% SP diet compared to the 20% MC diet. SP suppressed the inflammation marker tumour necrosis factor-α in spleen tissue. Furthermore, bacteria lipopolysaccharide (LPS)-stimulated nitric oxide (NO) secretion from murine macrophage RAW 264.7 cells was suppressed by PP more than TP. These results suggest that major dietary proteins might affect infection and inflammation by L. monocytogenes.
Food Chemistry 10/2012; 134(4):1719-23. · 3.65 Impact Factor
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ABSTRACT: Listeria monocytogenes (Lm) causes food poisoning in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons are at the highest risk for infection. We investigated effects of crude soluble polysaccharides (SPS) and ethanolic extract (EE) fractions of frond (kombu) and holdfast (ganiashi) parts of Laminaria japonica on Lm invasion into human enterocyte-like Caco-2 cells and immune and/or inflammatory reactions of murine macrophage RAW 264.7 cells. Recovery and viscosity were high in kombu SPS. Total phenolic content and antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity and Fe-reducing power) were higher in ganiashi EE. EE of ganiashi, rather than kombu, suppressed the Lm invasion into the differentiated Caco-2 cells, though the inhibitory effect of SPS was not significant. Ganiashi SPS increased the nitric oxide (NO) production of intact RAW 264.7 cells. On the other hand, the NO production from Escherichia coli O111 lipopolysaccharide-activated cells was suppressed by kombu SPS and ganiashi EE. These results suggest that L. japonica, particularly ganiashi, might suppress the invasion and infection of Lm and also the inflammation.
Applied biochemistry and biotechnology 08/2012; 168(4):928-35. · 1.94 Impact Factor
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ABSTRACT: Tandem repeats (TR), which are repetitive nucleotide sequences in DNA, are polymorphic both in repeat number and sequence. In this study, we developed a new typing method, multilocus TR sequence analysis (MLTSA), for the foodborne pathogen Listeria monocytogenes using sequence polymorphisms in three tandem repeat regions. The obtained dendrogram clustered L. monocytogenes strains of lineage I and lineage II separately, and formed three groups within the lineage I cluster, each of which included one of the three major L. monocytogenes epidemic clones (ECI, ECIa, and ECII). These results were consistent with a previously established virulence-gene-based MLST method. In comparison, our method grouped some epidemiologically related isolates together, which virulence-gene-based MLST did not. Moreover, our method, using three tandem repeat regions, showed a higher discriminatory power than the MLST method, which uses six virulence gene regions. This MLTSA approach using sequence polymorphisms in TR regions could be a useful tool in the epidemiological study of L. monocytogenes.
Journal of microbiological methods 06/2012; 90(3):285-91. · 2.43 Impact Factor
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ABSTRACT: Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan. It
is characterized by a unique cheese-like flavor and characteristic sour taste. We analyzed the changes in the microbial community
during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCR-DGGE) and by plate counts.
The plate counts showed that lactic acid bacteria reached 8.0 log10 CFU/g within 7days of fermentation initiation before decreasing slowly to 4.0 log10 CFU/g during the remainder of 1-year study period. PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and
30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L. acetotolerans. This is the first identification of L. acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive. This is the first report of PCR-DGGE being used
to assess the microbial community in funazushi. This technique was also found to be effective in profiling microbial diversity.
Keywords
Funazushi
–Bacterial community–PCR-DGGE–
Lactobacillus plantarum
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L. acetotolerans
Fisheries Science 04/2012; 77(1):151-157. · 0.94 Impact Factor
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ABSTRACT: Aji-no-susu is a Japanese fermented fish product prepared from salted horse mackerel Trachurus japonicus, and cooked rice. We studied the organic acid and free amino acid contents and microflora in 12 aji-no-susu products to clarify their features as a lactic-acid-fermented food. Salinity of the samples was approximately 7.0% (rice
portion) and 6.0% (fish portion) (w/w). Water activity was approximately 0.9, and pH was approximately 4.4 and lower. In the
rice portions, lactic acid content was very high (57mg/g sample). The predominant amino acids were alanine (2.3mg/g rice
portion) and lysine (2.1mg/g). In the case of long-fermented (4 and 12months) aji-no-susu, a high content of gamma-aminobutyric acid (GABA, 1.5 and 1.4mg/g) was detected. Total viable counts in rice and fish portions
were 7.7 and 7.4 log colony-forming units (cfu)/g, respectively. The number of lactobacilli in the rice and fish portions
was 7.3 and 7.1logcfu/g, respectively. Yeasts were detected in eight samples. Furthermore, acid-tolerant lactic acid bacteria
(LAB) (Lactobacillus plantarum), GABA-producing LAB (Lactobacillus sp.), and halophilic or halo-tolerant yeast (Debaryomyces hansenii) were isolated and identified. Results in this study indicate that aji-no-susu is a typical traditional lactic-acid-fermented fish product.
Fisheries Science 04/2012; 75(6):1499-1506. · 0.94 Impact Factor
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ABSTRACT: Listeria monocytogenes causes listeriosis in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons, the elderly, and pregnant women and their fetuses or newborns are at highest risk for the infection. To isolate probiotic lactic acid bacteria (LAB) with inhibitory effects against L. monocytogenes, we screened for acid and bile resistant LABs from narezushi, a traditional salted and long-fermented fish with cooked rice. Then, inhibitory effects of the selected LABs on L. monocytogenes invasion and infection of human enterocyte Caco-2 cells and Listeria-susceptible A/J mice were determined. From a total of 231 LAB isolates, we selected five acid and bile resistant isolates (four were Lactobacillus plantarum and one was Leuconostoc mesenteroides). Among the five isolates, Ln. mesenteroides (Lnm-1RM3) showed the highest inhibition against L. monocytogenes invasion into Caco-2 cells. In the case of L. monocytogenes orally infected A/J mice, recovery of the pathogen from the spleen was suppressed by drinking water containing 9 log CFU/ml of Lnm-1RM3 cells. The inhibitory effects were also shown by heat-killed Lnm-1RM3 cells. These results suggest that live and also heat-killed Lnm-1RM3 cell intake might prevent L. monocytogenes entero-gastric invasion and infection.
Anaerobe 12/2011; 18(1):19-24. · 2.41 Impact Factor
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ABSTRACT: To confirm the importance of eliminating food sediments from the surfaces of food-related environments, we examined the resistance of pathogenic bacteria (Salmonella Typhimurium and Staphylococcus aureus) cells, dried and adhered on glass with 25% w/v egg albumen, 25% yolk or 50% whole egg solutions, against benzalkonium chloride and alkyldiaminoethylglycine hydrochloride. Bacterial suspensions (0.1 ml of 8 log cfu/ml) were put on 47 mmφ glass dishes and dried at room temperature (20-24 °C) for 180 min in a bio safety cabinet with ventilation. Although the viable cells in distilled water decreased 2.0 (S. aureus)-3.5 (S. Typhimurium) log fold during the drying period, the egg compounds protected the bacteria. The disinfectant treatments (2.0 mg/ml for 10 min) showed a clear bactericidal effect in the absence of egg compounds. However, the bactericidal effect disappeared in the presence of yolk and whole egg. Imaging before and after drying and the disinfectant treatments were carried out using a phase-contrast microscope and an atomic force microscope. The protective effect of egg compounds on bacterial viability disappeared with a proper washing process.
Food Microbiology 08/2011; 28(5):920-5. · 3.28 Impact Factor
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ABSTRACT: To determine the existence of p-nitrophenol (PNP)-lowering bacteria in intestine of Japanese coastal fish, the gastro-intestinal contents were incubated in Brain Heart Infusion (BHI) broth and minimal medium (MM) broths containing 1 mmol/L PNP at 30 °C for 7 days. Among 26 samples of 19 fish species, 17 samples showed a decrease in PNP of 0.5-0.8 mmol/L in BHI broth, but no decrease was shown in MM broth. Eighteen PNP-lowering bacterial strains were isolated from four fishes. All of the strains were identified as Lactococcus lactis subsp. lactis. Three L. lactis strains JS1-3 isolated from Japanese seabass Lateolabrax japonicus showed the highest PNP-lowering activity (0.44 mmol/L). Optimum temperature and pH for the growth and PNP decreasing corresponded with the marine environment. These results suggested that marine fishes have PNP decreasing bacteria in their intestine. These bacteria might protect host fish from toxicities of PNP and PNP related compounds.
Marine pollution bulletin 07/2011; 62(8):1622-7. · 2.63 Impact Factor
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ABSTRACT: Listeria monocytogenes found in minced tuna and fish roe can cause listeriosis. These products are classified in category B according to the Codex Alimentarius Commission, i.e., ready-to-eat foods in which L. monocytogenes growth can occur. We investigated the effectiveness of nisin and other commercially available antimicrobial compounds (lysozyme, ε-polylysine, and chitosan) for prevention of L. monocytogenes growth during the expected shelf life of raw minced tuna and salmon roe products. Food samples inoculated with L. monocytogenes were incubated with each antimicrobial at 10°C for 7 days or at 25°C for 12 h. Nisaplin (an antimicrobial containing nisin) effectively inhibited L. monocytogenes growth in minced tuna at 500 ppm and in salmon roe at 250 ppm within their standard shelf lives. The effective concentration of each antimicrobial was determined: 2,000 ppm for ART FRESH 50/50 (containing lysozyme) and SAN KEEPER No. 381 (containing ε-polylysine) and 10,000 ppm for SAN KEEPER K-3 (containing chitosan).
Journal of food protection 06/2011; 74(6):994-8. · 1.94 Impact Factor
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ABSTRACT: The growth kinetics of Listeria monocytogenes and natural flora (NF) in minced tuna from 2 to 30 °C were examined, and a simultaneous growth model was developed. The inhibiting effect of the NF on the growth of L. monocytogenes was examined by inoculating different levels of NF isolated from the minced tuna. The kinetic data were fitted to the Baranyi model and estimated the growth parameters such as specific growth rate (μ(max)), maximum population density (N(max)), and lag time. The temperature and inoculated NF dependency on the μ(max) of L. monocytogenes and NF were described by modified Ratkowsky's square-root model. As the initial NF level increased, the slopes of the square-root models were decreased for both L. monocytogenes and NF. The N(max) of L. monocytogenes was described as a function of temperature and inoculated NF level. Simultaneous growth prediction of L. monocytogenes and NF under constant temperature conditions was examined by using the differential equations based on the Baranyi model with the effect of interspecies competition substituted into the developed μ(max) and N(max) models. The root mean square errors between the model prediction and the observation for L. monocytogenes and NF were 0.42 and 0.34, respectively. Predictive simulation under fluctuating temperature conditions also demonstrated a high accuracy of simultaneous prediction for both L. monocytogenes and NF, representing the root mean square errors of 0.19 and 0.34, respectively. These results illustrate that the developed model permits accurate estimation of the behavior of L. monocytogenes in minced tuna under real temperature history until consumption.
Journal of food protection 02/2011; 74(2):176-87. · 1.94 Impact Factor
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ABSTRACT: In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated.
Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons.
This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.
PLoS ONE 01/2011; 6(8):e21951. · 4.09 Impact Factor
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ABSTRACT: The bacterial flora of two Japanese traditional fermented fish products, aji-narezushi (salted and long-fermented horse mackerel (Trachurus japonicas) with rice) and iwashi-nukazuke (salted and long-fermented sardine (Sardinops melanostica) with rice bran), was analysed using non-culture-based polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) and culture-based PCR single-strand conformation polymorphism (SSCP) methods.
Viable plate counts in aji-narezushi and iwashi-nukazuke were about 6.3-6.6 and 5.7-6.9 log colony-forming units g(-1) respectively. In the PCR-DGGE analysis, Lactobacillus acidipiscis was detected as the predominant bacterium in two of three aji-narezushi samples, while Lactobacillus versmoldensis was predominant in the third sample. By the PCR-SSCP method, Lb. acidipiscis and Lactobacillus plantarum were isolated as the predominant bacteria, while Lb. versmoldensis was not detected. The predominant bacterium in two of three iwashi-nukazuke samples was Tetragenococcus muriaticus, while Tetragenococcus halophilus was predominant in the third sample.
The results suggest that the detection of some predominant lactic acid bacteria species in fermented fish by cultivation methods is difficult.
Journal of the Science of Food and Agriculture 08/2010; 90(11):1796-801. · 1.44 Impact Factor
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ABSTRACT: A novel universal real-time PCR, consisting of newly designed oligonucleotide subsets, was designed for a bacterial housekeeping gene encoding the peptide elongation factor Tu. Specificity and universality were confirmed in 66 bacterial strains, including 51 genera and 63 species. The amplification kinetics of tuf gene-targeted real-time quantitative PCR were consistent in a wide range of bacterial species tested. A calibration curve (r(2) = 0.97) was produced for the estimation of bacterial counts, based on measurements of representative inoculations with 10-fold serial dilutions of the cells of representative bacterial species. Linear regression analysis of the real-time PCR-derived bacterial counts and aerobic plate counts, in a total 149 samples consisting of 25 minced meat, 34 fresh-cut vegetables, and 90 fish, exhibited a high correlation (r(2) = 0.84, 0.87, and 0.95, respectively) over the range of 3.0 to 9.0 log CFU/g. In total, the difference between the two methods was less than 0.5 log in 75 of these samples, and in the remaining 74 samples, the difference was 0.5 to 1.0 log. Presently, our tuf gene-targeted real-time quantitative PCR assay achieves a rapid (within 2 h) estimation of bacterial counts of 3.0 to 9.0 log CFU/g, in a practical manner.
Journal of food protection 04/2010; 73(4):670-9. · 1.94 Impact Factor
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ABSTRACT: A quantitative real-time PCR using SYBR Green dye was developed to target the neurotoxin type A (boNT/A) gene of Clostridium botulinum type A. Primer specificity was confirmed by analyzing 63 strains including 5 strains of C. botulinum type A and 11 of non-type A C. botulinum. The highly similar amplification efficiencies of the real-time PCR assay were observed for 5 strains of C. botulinum type A. The DNA extraction with NucliSENS miniMAG provided sufficient performance to obtain the purified DNA from steamed rice samples and to develop the standard curve for the enumeration of C. botulinum in steamed rice samples. The real-time PCR assay could detect 10 cells per milliliter of 10 x rice homogenate, thus indicating that more than 100 C. botulinum cells per g of rice sample was quantifiable by the real-time PCR assay. The inoculation of aseptic rice samples with low numbers of C. botulinum type A cells revealed that the fate of inoculated C. botulinum type A cells in rice samples could be monitored accurately by the real-time PCR assay. These results indicate that the real-time PCR assay developed in this study provides rapid, effective, and quantitative monitoring of C. botulinum in steamed rice samples.
Journal of food protection 04/2010; 73(4):688-94. · 1.94 Impact Factor
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ABSTRACT: Examination of Listeria monocytogenes prevalence among ready-to-eat foods in Japan revealed frequent (5.7 to 12.1%) contamination of minced tuna and fish roe products, and the isolates had the same virulence levels as clinical isolates in terms of invasion efficiency and infectivity in cell cultures and a murine infection model, respectively. Premature stop codons in inlA were infrequent (1 out of 39 isolates). Cell numbers of L. monocytogenes in minced tuna and salmon roe increased rapidly under inappropriate storage temperatures (from a most probable number [MPN] of 10(0) to 10(1)/g to an MPN of 10(3) to 10(4)/g over the course of 2 days at 10 degrees C). Thus, regulatory guidelines are needed for acceptable levels of L. monocytogenes in these foods.
Applied and environmental microbiology 03/2010; 76(10):3383-6. · 3.69 Impact Factor
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ABSTRACT: A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This T-RFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.
Journal of food protection 01/2010; 73(1):104-13. · 1.94 Impact Factor
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ABSTRACT: Rapid enumeration of Escherichia coli strains by quantitative real-time PCR targeting the uidA gene was developed and confirmed for minced beef, tuna and raw oyster. Higher sensitivity (1 CFU/g of E. coli in all three food samples) was obtained by incubating for 7 h in TSB. Colony-directed E. coli specific TaqMan PCR assay could effectively distinguish colonies grown on various selective media within 1.5-h. Inspection of E. coli in food testing laboratories is important, and our rapid E. coli detection strategy will contribute to quality control in food industries.
Journal of microbiological methods 09/2009; 79(1):124-7. · 2.43 Impact Factor
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ABSTRACT: Listeria monocytogenes is of great concern as a foodborne pathogen. Many ready-to-eat foods are widely contaminated with this organism and have caused listeriosis outbreaks and sporadic cases in many countries. In Japan, there is a high incidence of L. monocytogenes contamination, specifically in raw ready-to-eat seafood. Identical L. monocytogenes subtypes have been isolated repeatedly from samples of food manufactured at a given store or processing plant, and researchers suspected that certain L. monocytogenes isolates have formed biofilms at these sites. A microtiter plate biofilm formation assay was conducted, and all raw ready-to-eat seafood isolates tested were able to form biofilms to various degrees. Biofilm formation by L. monocytogenes isolates of lineage I was significantly greater (P = 0.000) than that by isolates of lineage II. However, isolates of clonal lineages formed different levels of biofilms, indicating that the ability to form a biofilm is affected positively or negatively by environmental factors.
Journal of food protection 08/2009; 72(7):1476-80. · 1.94 Impact Factor
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ABSTRACT: Photobacterium damselae subsp. damselae has been known as an opportunistic pathogen in fish and mammals. Human infectious cases are often very serious and occasionally fatal. We previously reportedtwo fatal cases caused by this subspecies where the patients developed multiple organ failure within 20-36 h after the onset of initial symptoms. Despite its ability to cause serious infections in humans, this subspecies has not been well studied because human infectious cases caused by this subspecies are very rare. However, this subspecies has been reported to be present in a wide range with high incidence rate in aquatic environments. Thus, we investigated the genotypic and phenotypic differences between clinical and environmental strains of Photobacterium damselae subsp. damselae. Using molecular typing methods, such as ribotyping, AFLP (Amplified Fragment Length Polymorphism), and PFGE (Pulsed-Field Gel Electrophoresis) and sequencing analysis, we determined that thetwo clinical strains were genetically similar yet distinguishable from environmental strains, but not significantly so. On the other hand, phenotypic differences were clear; moreover, mouse assay and hemolytic assay indicated strong pathogenicity of only clinical isolates. Based on these data, we concluded that there are differences in pathogenicity potential among isolates of this subspecies, and some environmental isolates have the potential to become highly pathogenic.
Microbial Pathogenesis 09/2008; 45(2):150-8. · 1.94 Impact Factor
