Choa An

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.

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.

Caecal environment of rats fed far East Asian-modelled diets

To clarify the effect of type of foods on the intestinal environment, Far East Asian- (FEA; rich in rice starch, soy protein and soy oil) and Far East Asian marine- (FEAM; rich in rice starch, fish meal, fish oil and brown alga) modelled diets and sucrose, casein and beef tallow-rich (SCB) diet were prepared. After the 2-week administration of diets in rats, caecal organic acids and putrefactive compounds (ammonia, indole, phenol and H2S, which are regarded as putative risk factors for tumours) were determined. The caecal microbiota was also analyzed using denaturing gradient gel electrophoresis and pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. Levels of n-butyrate, acetate, indole and phenol were high in rats fed FEA. On the other hand, H2S was clearly suppressed by both FEA and FEAM comparing with SCB. These results suggest that FEAM is preferable to FEA for optimal intestinal environment and host health. Both microbial analyses showed that the diversity of microbiota in the FEAM group was lower than in the other diet groups. Ratio of Firmicutes, Bacteroidetes and Proteobacteria in the SCB group was about 5:4:1. Firmicutes, particularly Lachnospiraceae, was promoted by FEA and FEAM.

Effects of Holdfast of Laminaria japonica on Listeria Invasion on Enterocyte-Like Caco-2 Cells and NO Production of Macrophage RAW 264.7 Cells

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.