Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.

We examined the difference between Escherichia coli O157 and non-pathogenic E. coli in their tolerance to spices. Various spices (5 g each) were homogenized at 25 degrees C for 10 min with 5 ml of 70% ethyl alcohol, and the supernatant solutions obtained by centrifugation were used as spice extracts. When the E. coli strains were incubated with each spice extract at concentrations of 0.01% and 0.1%, a noteworthy difference was observed between the O157 and non-pathogenic strains in their tolerance to nutmeg. The populations of the non-pathogenic strains could not be reduced, but those of the O157 strains were remarkably reduced. Antibacterial activity by the nutmeg extract was also found against the enteropathogenic E. coli O111, but not against enterotoxigenic (O6 and O148) and enteroinvasive (O29 and O124) E. coli. When we examined the antibacterial effect of volatile oils in nutmeg on the O157 and non-pathogenic E. coli strains, all O157 strains tested were found to be more sensitive to beta-pinene than non-pathogenic E. coli strains.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L-to D-alo-threonine and also from D-to L-alo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the at position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.Alanine racemase (EC 5.1.1.1) catalyzes racemization of L-and D-alanine and supplies D-alanine for peptidoglycan synthesis (10). Various other amino acid racemases and epimerases have been demonstrated, but their physiological roles have not yet been clarified. Arginine racemase from Pseudomonas graveolens (11) and amino acid racemase from Aeromonas punctata (6) and Pseudomonas striata (9) show broad substrate specificity but do not act on amino acids such as threonine and valine, whose 3-methylene group is substituted. Amos (2) reported the occurrence of threonine racemase in Escherichia coli. However, it has never been purified or characterized.We 2) containing 10 mM pyridoxal 5'-phosphate was used as the buffer throughout the purification procedure unless otherwise noted. (i) Step 1. The harvested cells were washed twice with 0.85% sodium chloride solution. The washed cells (wet weight, about 1 kg) were suspended in 1 liter of the buffer containing 0.01% 2-mercaptoethanol and disrupted with a sonic oscillator (Kaijo Denki Autochaser 300, Tokyo, Japan) for 30 min. The intact cells and debris were removed by centrifugation. (ii) Step 2. The supernatant solution was brought to 30% saturation with ammonium sulfate and centrifuged at 17,000 x g for 30 min. Ammonium sulfate was added to the supernatant solution to 55% saturation. The precipitate collected by centrifugation was dissolved in the buffer. The enzyme solution was dialyzed against 100 volumes of the buffer.(iii) Step 3. The enzyme solution was applied to a QAE-ZETA prep column that had been equilibrated with the buffer. After the column was washed thoroughly with the buffer containing 20 mM NaCl, the enzyme was eluted with the buffer containing 0.2 M NaCl. The active fractions were pooled, concentrated by addition of ammonium sulfate (65% saturation), and dialyzed against 100 volumes of the b...

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Specific oligonucleotide primers for detecting Escherichia coli in various foods were designed based upon the conserved sequences of the E. coli alr gene from positions 322 to 345 and from 664 to 687. Bacteria and food samples were treated at 100°C for 10 min in 1% Tween 20 containing 5% NaCl and 1 mM EDTA, then used as templates for polymerase chain reaction (PCR). The oligonucleotide primers were specific to E. coli, except for Shigella species, when tested with 67 strains of E. coli , including such serotypes as O157:H7 and O111, and 32 strains of non-E. coli species. The oligonucleotide primers could prove useful for detecting E. coli in beef, chicken, pork, tomato, soybean, potato, cow's milk, and egg.