Takashi Kuda

Comparison of PCR-DGGE and PCR-SSCP analysis for bacterial flora of Japanese traditional fermented fish products, aji-narezushi and iwashi-nukazuke

BACKGROUND 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. RESULTS 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. CONCLUSION The results suggest that the detection of some predominant lactic acid bacteria species in fermented fish by cultivation methods is difficult.

Inhibitory effects of Leuconostoc mesenteroides 1RM3 isolated from narezushi, a fermented fish with rice, on Listeria monocytogenes infection to Caco-2 cells and A/J mice.

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.

FLX Pyrosequencing Analysis of the Effects of the Brown-Algal Fermentable Polysaccharides Alginate and Laminaran on Rat Cecal Microbiotas

ABSTRACT Edible brown algae are used as major food material in Far East Asian countries, particularly in South 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 the intestinal environment, the cecal microbiotas of rats fed diets containing no dietary fiber (control) or 2% (wt/wt) sodium alginate or laminaran for 2 weeks were analyzed using FLX amplicon pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. The most abundant phylum in all groups was Firmicutes. 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, H2S, and phenol, were decreased by both alginate and laminaran. These results indicate that edible brown algae can alter the intestinal environment, with fermentation by intestinal microbiota.

Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice.

Six lactic acid bacteria strains (four Lactobacillus plantarum strains and one each of Lactococcus lactis subsp. lactis and Pediococcus pentosaceus) have been isolated and shown to possess anti-oxidant activity. In this study, we determined their acid, bile, salt resistance, and adhesion activity on human enterocyte-like HT-29-Luc and Caco-2 cells. An isolate Lc. lactis S-SU2 showed highest bile resistance and adhesion activity compared to type strains. S-SU2 could ferment both 10% skimmed milk and soy milk while the type strain could not ferment soy milk. Soy milk fermented with S-SU2 showed an increased nitric oxide (NO) secretion in the mouse macrophage RAW264.7 cells without bacterial lipopolysaccharide (LPS). Furthermore, the inhibitory effects of the fermented soy milk on Escherichia coli O111 LPS-induced NO secretion were higher than those of fresh soy milk. Inflammatory bowel disease (IBD) was induced in mice fed either 5% (w/v) dextran sodium sulfate (DSS) in drinking water or 50% soy milk in drinking water. Shortening of colon length, breaking of epithelial cells, lowering liver and thymus weights, and enlargement of spleen are some of the characteristics observed in the IBD, which were prevented by the use of soy milk fermented with Lc. lactis S-SU2.

In vitro antioxidant and anti-inflammation properties of lactic acid bacteria isolated from fish intestines and fermented fish from the Sanriku Satoumi region in Japan

The Ministry of the Environment, Government of Japan, defines a Satoumi as a coastal area where biological productivity and biodiversity has increased through human interaction. As a way to identify new starters and probiotics, we isolated and screened lactic acid bacteria (LAB) with acid, bile, and salt resistance from the intestines of 23 fish and 11 fermented fish samples obtained from the northeastern (Sanriku) Satoumi region of Japan. Of the 301 isolates, 75 strains were selected as LAB, 6 of which clearly showed increased antioxidant activities (DPPH and O2- radical scavenging and Fe-reducing power) in their cultured broth. Four isolates (S-SU1, 3-5) were identified as Lactobacillus plantarum. The two other strains (S-SU2, 6) identified were Lactococcus lactis and Pediococcus pentosaceus. In these in vitro assays, Lc. lactis S-SU2, isolated from sea pineapple kimchi showed the highest radical scavenging capacity. Moreover, the protective effect of heat-killed cells against the toxicity of 3mM H2O2 on Saccharomyces cerevisiae was the highest for Lb. plantarum S-SU1 isolated from a salted squid product, followed by P. pentosaceus S-SU6 isolated from the intestines of blue mackerel. Furthermore, Lb. plantarum S-SU5 showed inhibitory effect against the toxicity of 3mM H2O2 in human enterocyte-like HT-29-luc cells and on nitrite (NO) production in mouse RAW264.7 macrophage cells, which was induced by Escherichia coli O111 lipopolysaccharide (LPS). These results suggest that the selected LAB strains are potential starters and/or functional components with antioxidant and anti-inflammatory properties.

Use of Commercially Available Antimicrobial Compounds for Prevention of Listeria monocytogenes Growth in Ready-to-Eat Minced Tuna and Salmon Roe during Shelf Life

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).

Effect of quantity of food residues on resistance to desiccation of food-related pathogens adhered to a stainless steel surface

In order to study the effect of food residues on the survival of food-borne pathogens, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes were subjected to drying conditions in the presence of small amounts of food such as carrot juice, aqueous solution of nori, milk, and soy-milk. After drying for 2 h at room temperature in the absence of food residue, cell counts of S. Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. Five milligrams of fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm φ surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surface-adherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination.

In vitro cholesterol-lowering properties of Lactobacillus plantarum AN6 isolated from aji-narezushi.

Aji‐narezushi is a traditional lactic acid‐fermented fish. In this study, we screened for lactose‐utilizing, acidophilic, bile‐resistant and cholesterol‐lowering lactic acid bacteria (LAB) from aji‐narezushi for use as starter strains for fermented foods, as well as for use as probiotics. Of the 301 LAB isolates, 277 fermented lactose, and among these, 171 grew in de Man, Rogosa and Sharpe broth adjusted to pH 3·5. Thirty‐four of the isolates were grown in a broth containing 3% (w/v) bile. All of the isolates were lactobacilli. Seven isolates that demonstrated cholesterol‐lowering activity in ethanolic solution were selected. All of the isolates were identified as Lactobacillus plantarum. Lactobacillus plantarum AN6 showed the highest cholesterol‐lowering activity. AN6 was more resistant to acid, salt and bile than the type strain NBRC15891T. One‐half of the cholesterol‐lowering effect remained after boiling AN6 for 10 min. The Fourier transform infrared (FT‐IR) analysis indicated that the content of cell wall polysaccharides in AN6 is higher than ones in the type strain. These results indicate that Lact. plantarum AN6 can be used as a profitable starter organism and probiotic.

Effect of sodium-alginate and laminaran on Salmonella Typhimurium infection in human enterocyte-like HT-29-Luc cells and BALB/c mice

Brown algal polysaccharides such as alginate, polymers of uronic acids, and laminaran, beta-1,3 and 1,6-glucan, can be fermented by human intestinal microbiota. To evaluate the effects of these polysaccharides on infections caused by food poisoning pathogens, we investigated the adhesion and invasion of pathogens (Salmonella Typhimurium, Listeria monocytogenes and Vibrio parahaemolyticus) in human enterocyte-like HT-29-Luc cells and in infections caused in BALB/c mice. Both sodium Na-alginate and laminaran (0.1% each) inhibited the adhesion of the pathogens to HT-29-Luc cells by approximately 70-90%. The invasion of S. Typhimurium was also inhibited by approximately 70 and 80% by Na-alginate and laminaran, respectively. We observed that incubation with Na-alginate for 18 h increased the transepithelial electrical resistance of HT-29-Luc monolayer cells. Four days after inoculation with 7 log CFU/mouse of S. Typhimurium, the faecal pathogen count in mice that were not fed polysaccharides (control mice) was about 6.5 log CFU/g while the count in mice that were fed Na-alginate had decreased to 5.0 log CFU/g. The liver pathogen count, which was 4.1 log CFU/g in the control mice, was also decreased in mice that were fed Na-alginate. In contrast, the mice that were fed laminaran exhibited a more severe infection than that exhibited by control mice.

Effect of soy and milk protein-related compounds on Listeria monocytogenes infection in human enterocyte Caco-2 cells and A/J mice

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.