Spore Heat Resistance and Growth Ability at Refrigeration Temperatures of Bacillus spp. and Paenibacillus spp.

In this study, spore heat resistance and growth ability at refrigeration temperatures of Bacillus spp. and Paenibacillus spp. were determined. The spore D90°C of 67.6% (23 of 34 strains) of Bacillus and 73.9% (17 of 23 strains) of Paenibacillus was less than 15 min. The growth abilities of both genera were equivalent at 10°C. However, 71.1% (32 of 45 strains) of Paenibacillus and only 6.3% (3 of 48 strains) of Bacillus cereus group could grow at 4°C. Eight B. cereus strains formed spores with higher heat resistance compared to the other Bacillus strains assessed; however, they did not grow at tempreratures below 10°C. Conversely, four Paenibacillus strains formed spores with heat resistance equivalent to that of the eight B. cereus strains and grew at 6°C or lower. In particular, Paenibacillus sp. JCM13343 formed the highest heat-resistant spores (D90°C = 136.1 min) and grew well at 4°C. These results indicate that Paenibacillus can grow in processed foods during refrigerated storage and has the potential to cause spoilage as well as Bacillus. Therefore, Paenibacillus should be considered as one of the targets for microbiological control in refrigerated processed foods.

A small scale study on the effects of oral administration of the ß-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves.

BACKGROUND: The ß-(1 → 3),(1 → 6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the ß-(1 → 3),(1 → 6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves. FINDINGS: Holstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the ß-(1 → 3),(1 → 6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF. CONCLUSIONS: Our data indicated the possibility that oral administration of the ß-(1 → 3),(1 → 6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of ß-(1 → 3),(1 → 6)-D-glucans on domestic animals.

Toxicogenomics using yeast DNA microarrays.

Development of genomics and bioinformatics enable us to analyze the global gene expression profiles of cells by DNA microarray. Changes in gene expression patterns indicate changes in its physiological conditions. Following the exposure of an organism or cell to toxic chemicals or other environmental stresses, the global genetic responses can be expeditiously and easily analyzed. Baker's yeast, Saccharomyces cerevisiae, is one of the most studied and useful model eukaryotes. The biggest advantage of yeast genomics is the available functional information for each gene and a considerable number of data are accumulating in the field of toxicity assessment using yeast DNA microarray. In this review, we discuss the toxicogenomics of metal ions, alcohols and aldehydes, and other chemicals.

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE).

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 +/- 2.5 degrees C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.

DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae.

Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation.

Toxicity of methanol and formaldehyde towards Saccharomyces cerevisiae as assessed by DNA microarray analysis.

To assess the toxicity of the C1 compounds methanol and formaldehyde, gene expression profiles of treated baker's yeast were analyzed using DNA microarrays. Among approximately 6,000 open reading frames (ORFs), 314 were repressed and 375 were induced in response to methanol. The gene process category "energy" comprised the greatest number of induced genes while "protein synthesis" comprised the greatest number of repressed genes. Products of genes induced by methanol were mainly integral membrane proteins or were localized to the plasma membrane. A total of 622 and 610 ORFs were induced or repressed by formaldehyde, respectively. More than one-third of the genes found to be strongly repressed by formaldehyde belonged to the "protein synthesis" functional category. Conversely, genes in the subcategory of "nitrogen, sulfur, and selenium metabolism" within "metabolism" and in the category of "cell rescue, defense, and virulence" were up-regulated by exposure to formaldehyde. Our data suggest that membrane structure is a major target of methanol toxicity, while proteins were major targets of formaldehyde toxicity.

Pediococcus pentosaceus NB-17 for probiotic use.

The plant-derived Pediococcus pentosaceus NB-17 was isolated from Japanese traditional vegetable pickles. The production of cytokines from mouse spleen cells co-cultivated with heat-killed bacteria was investigated in vitro. The bacteria significantly induced secretion levels of interferon (IFN)-gamma and interleukin (IL)-12 p70, and suppressed IL-4 productions in ovalbumin (OVA) sensitized mouse spleen cells. Therefore, the bacteria could effectively stimulate immune activities and showed allergic inhibitory effects. Further study on acid tolerance was performed under simulated gastric conditions and NB-17 showed resistance to simulated gastric acidity at pH 3.0 and pH 2.5. Moreover, after oral administration of the intact cells to rats, bacterial colonies derived from feces were analyzed by random amplification polymorphic DNA (RAPD). It was confirmed that the administered strain NB-17 remained alive in feces. These results suggest the possibility to use the P. pentosaceus NB-17 as functional foods.

Mechanisms of copper toxicity in Saccharomyces cerevisiae determined by microarray analysis.

The effect of the heavy metal copper on the expression of a wide spectrum of genes was analyzed by using a DNA microarray. The gene expression profile of baker's yeast Saccharomyces cerevisiae grown in a medium containing a sublethal concentration of cupric sulfate was compared with that of yeast grown in a normal medium. Among approximately 6000 yeast ORFs, 143 ORFs were induced more than twofold to resist copper toxicity after exposure to copper. Copper metallothionein CUP1-1 and CUP1-2 were induced more than 20-fold. Some genes related to sulfur metabolism and oxidative stress response were also up-regulated. This DNA microarray analysis identified several molecular targets of copper toxicity.

Stimulated accumulation of lectin mRNA and stress response in Helianthus tuberosus callus by methyl jasmonate.

Callus from Helianthus tuberosus expresses a mannose-specific lectin (HTA). The level of HTA mRNA significantly increased one hour after treatment of the callus with 20 mg/l methyl jasmonate. Following this, fragmentation of the callus DNA at regular intervals was observed together with strong self-fluorescence emission in the callus cells.

Cloning, sequencing, and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii.

From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64% identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.