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We constructed a self-cloning diploid baker’s yeast strain that overexpressed the transcription activator Msn2. It showed higher tolerance to freeze-thaw stress and higher intracellular trehalose level than observed in the wild-type strain. Overexpression of Msn2 also enhanced the fermentation ability of baker’s yeast cells in frozen dough. Hence, Msn2-overexpressing baker’s yeast should be useful in frozen-dough baking.

The protective effects of live Lactobacillus paracaseiNFRI 7415 on alcoholic liver disease were investigated. Male Fischer 344 rats were fed a control diet (CD), an ethanol diet (ED) (35.8% of total energy from ethanol), or an ethanol diet containing 20% live Lb. paracaseiNFRI 7415 (107cfu/g) (LD) for 10 weeks. The results indicated that live Lb. paracaseiNFRI 7415 reduced the total cholesterol concentration of the plasma and liver in the rats fed the LD. The level of docosahexaenoic acid (DHA; 22:6n-3) in the plasma and liver of the LD group was higher than in the ED group. Chronic alcohol consumption decreased the level of n-3 fatty acid in the plasma and liver of the ED group. These results indicated that live Lb. paracaseiNFRI 7415 can adjust the fatty acid composition of the plasma and liver, and that it is possible to decrease liver damage due to chronic alcohol intake.

γ-Aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracaseiNFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS–PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 °C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 μmol min−1mg−1. The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracaseito produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.

A thermophilic bacterium, strain TAT105, was isolated from compost made of animal wastes. TAT105 had high tolerance to ammonium nitrogen up to 1200 mM, and highly assimilated nitrogen during the growth on swine feces. The strain was classified into Bacillus, close to Bacillus pallidus. To evaluate the effect of adding TAT105 to ammonia (NH3) emission during the composting process of animal wastes, laboratory scale composting was done. NH3emission tended to be lower and nitrogen loss was smaller in the TAT105-added material than in the control material to which TAT105 was not added. Thermophilic ammonium-tolerant bacteria in the TAT105-added material increased to about 8×109CFU/g of dry matter on the average during the tests, and most of them were judged to be TAT105 from morphological colony discrimination. These results suggested the possibility of reducing NH3emission from composting of animal wastes by adding TAT105.

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker’s yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker’s yeast for frozen dough and high-sugar dough.

Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δand ccz1Δstrains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δandmon1Δare the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.

Enterococcus sp. K-4, with a bacteriocin-like activity against E. faecium, was isolated from grass silage in Thailand. Morphological, physiological, and phylogenetic studies clearly identified strain K-4 as a strain of E. faecalis. Strain K-4 produced a maximal amount of bacteriocin at 43-45°C. We purified, for the first time, the bacteriocin produced at high temperature by E. faecalis to homogeneity, using adsorption on cells of the producer strain and reversed-phase liquid chromatography. The bacteriocin, designated enterocin SE-K4, is a peptide of about 5 kDa as measured by SDS-PAGE, and Mass spectrometry analysis found the molecular mass of 5356.2, which is in good agreement. The amino acid sequencing of the N-terminal end of enterocin SE-K4 showed apparent sequence similarity to class IIa bacteriocins. Enterocin SE-K4 was active against E. faecium, E. faecalis, Bacillus subtilis, Clostridium beijerinckii, and Listeria monocytogenes. Enterocin SE-K4 is very heat stable.

Natto is a traditional Japanese food made from soybeans fermented by Bacillus subtilis natto. Because natto is high in protein and contains healthful bacteria, dieticians and producers have campaigned for higher consumption of natto in areas such as Kansai, where our university is located. One of the reasons for the low consumption of natto is its distinctive flavor. As a problem-solving exercise, undergraduate students attempted to make natto more marketable. Students set a goal of modifying natto flavor by adding spices to achieve flavors deemed palatable by organoleptic tests. During the exercise, they noticed that lemongrass, Cymbopogon citratus, had a flavor-masking ability that reduced the intensity of natto flavor, and they finally determined that it was a useful food additive to make natto marketable. The exercise was an active learning process that effectively induced voluntary student effort of to solve problems originating from microorganisms.