Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.     

Effects of Phosphoryl Oligosaccharides of Calcium (POs-Ca) on Mycelial Growth and Fruiting Body Development of the Edible Mushroom,Pleurotus ostreatus

Phosphoryl oligosaccharides of calcium (POs-Ca) is a calcium salt of phosphoryl maltooligosaccharides made from potato starch. POs-Ca is highly water-soluble and can supply both the calcium ion and acidic oligosaccharides in an aqueous solution. In this study, we investigated the effects of POs-Ca on the mycelial growth and fruiting body yield of Pleurotus ostreatus , which is one of the most widely cultivated edible mushrooms in the world. We cultivated the mushroom using both potato dextrose agar (PDA) medium and sawdust-based medium, with added calcium salts. The addition of POs-Ca into the PDA medium with a calcium concentration of 10 mg increased mycelial growth significantly ( p < 0.05, vs . control). POs-Ca addition to the sawdust-based medium at concentrations of 1.0 to 3.0 g/100 g medium increased the amount of calcium in the fruiting bodies but did not affect the length of the cultivation period or the weight of the fruiting body. The calcium content in the fruiting body increased 12-fold when compared to the control. On the other hand, neither the CaHPO ₄ ･2H ₂ O group nor the CaHPO ₄ ･2H ₂ O with oligosaccharides group showed changes in the calcium content of the fruiting bodies. Our results indicate that the use of POs-Ca in mushroom cultivation allows for the possibility of developing new functional foods like calcium-enriched edible mushrooms. This is the first report describing the effects of POs-Ca on mushroom cultivation.     

Significance of the detection of staphylococcal enterotoxin A gene in low fat milk which caused a serious outbreak of food poisoning

In June 2000, there was a large-scale outbreak of food poisoning after consumption of Snow Brand low fat milk. In the evening of a day the incident made public, some cartons of low fat milk were brought to our laboratory for examination. Next day, we detected only staphylococcal enterotoxin (SE) A gene among SE (A-E) genes by PCR in left-over milk samples or samples from the same lots that patients had consumed. We presumed that the outbreak was caused by the intake of SEA. We subsequently confirmed the presence of SEA in these samples. To investigate the existence of SE (A-E) genes in milk, we examined 100 samples of commercial low fat milk and milk by PCR, but none of the genes was detected. We estimated the detection limit of SEA gene in low fat milk by PCR. Four strains of SEA-producing Staphylococcus aureus cultures were serially diluted in low fat milk. The SEA gene was detected at levels of 5.5 x 10(2) to 1.6 x 10(4) cfu/mL of S. aureus. These amounts of S. aureus are higher than the values in raw milk reported previously. Therefore we consider that SE genes in low fat milk should usually be undetectable by our PCR. This study shows that quick detection of SE genes by PCR is very helpful to analyze outbreaks, especially if no significant bacterium can be cultured.     

Determination of benzimidazole anthelmintics in livestock foods by HPLC

A simple and rapid liquid chromatographic method was developed for the simultaneous determination of twelve benzimidazole anthelmintics in livestock foods using reversed-phase high-performance liquid chromatography with photodiode array detection (PDA). A sample was homogenized with acetonitrile and n-hexane, and centrifuged. The acetonitrile phase was isolated and evaporated. The residue was dissolved in 0.1 mol/L carbonate buffer solution (pH = 9.1), sonicated, and then subjected to clean-up on a Bond Elut LRC-C18 cartridge. The benzimidazole compounds were separated isocratically on a Capcell Pak C18 UG 120 (5 microns, 150 x 4.6 mm i.d.) column and detected by PDA at 295 and 313 nm. Mixtures of acetonitrile and 0.05 mol/L ammonium acetate in mixing ratios of (20:80) and (40:60) were used as the mobile phase, and the flow-rate was 1.0 mL/min at 40 degrees C. The mean recoveries (n = 3) from 0.1-0.5 microgram/g added samples were 72.6-97.2% with coefficients of variation of 0.3-8.5%. The detection limits were 0.01-0.05 microgram/g.     

Correlation of the structures of agricultural fungicides to gene expression in Saccharomyces cerevisiae upon exposure to toxic doses

Correlations between the chemical structures of agricultural fungicides and mRNA expression levels following exposure of Saccharomyces cerevisiae to toxic doses of thiuram, zineb, maneb, TPN, and PCP were examined. Structurally, thiuram, zineb, and maneb are dithiocarbamate fungicides, whereas TPN and PCP are not. To characterize chemical toxicity, genes expression was classified according to the functional groups used by the MIPS database. However, no correlations between the classification scheme and chemical structures were found. Hierarchical clustering of gene expression profiles was performed to characterize the effects of the five chemicals. According to this analysis the similarity of gene expression profiles depended on the similarity of chemical structures. These results suggest that DNA microarray technology has potential for predicting the major chemicals which will cause environmental toxicity and will provide information on new biomonitoring methods.     

Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

Pyrolysis of sugarcane bagasse pretreated with sulfuric acid

Pyrolysis is a promising technique for the recovery of useful gas, tar, and solid products from biomass waste. However, the low tar yields obtained from lignocellulosic biomass are a significant drawback. To enhance tar yields, sugarcane bagasse, which is the most abundant agricultural waste in Fiji, was pretreated at ambient temperature and atmospheric pressure using various sulfuric acid (H₂SO₄) concentrations. Here, the ether bonds of cellulose, hemicellulose, and lignin were partially hydrolyzed. The pretreated samples were then pyrolyzed at 500 °C, and it was confirmed that H₂SO₄-pretreatment disrupted the bagasse cell structure, with the thermogravimetry and differential thermogravimetry results confirming that decomposition occurred at lower temperatures after pretreatment. In addition, tar yields were significantly enhanced from 5.6 wt% to 13.4 wt% for the untreated and 3 M H₂SO₄-pretreated samples respectively. The main components detected in this tar product were levoglucosan, andcellulose-and hemicellulose-derived products, whose proportions were increased following pretreatment. Thus, our work demonstrates that dilute acid pretreatment enhances tar production from sugarcane bagasse due to the production of shorter chain components via the partial hydrolysis of ether bonds.     

Nitric Oxide and Reactive Oxygen Species in the Pathogenesis of Preeclampsia

Preeclampsia (PE) is characterized by disturbed extravillous trophoblast migration toward uterine spiral arteries leading to increased uteroplacental vascular resistance and by vascular dysfunction resulting in reduced systemic vasodilatory properties. Its pathogenesis is mediated by an altered bioavailability of nitric oxide (NO) and tissue damage caused by increased levels of reactive oxygen species (ROS). Furthermore, superoxide (O₂⁻) rapidly inactivates NO and forms peroxynitrite (ONOO⁻). It is known that ONOO⁻ accumulates in the placental tissues and injures the placental function in PE. In addition, ROS could stimulate platelet adhesion and aggregation leading to intravascular coagulopathy. ROS-induced coagulopathy causes placental infarction and impairs the uteroplacental blood flow in PE. The disorders could lead to the reduction of oxygen and nutrients required for normal fetal development resulting in fetal growth restriction. On the other hand, several antioxidants scavenge ROS and protect tissues against oxidative damage. Placental antioxidants including catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protect the vasculature from ROS and maintain the vascular function. However, placental ischemia in PE decreases the antioxidant activity resulting in further elevated oxidative stress, which leads to the appearance of the pathological conditions of PE including hypertension and proteinuria. Oxidative stress is defined as an imbalance between ROS and antioxidant activity. This review provides new insights about roles of oxidative stress in the pathophysiology of PE.     

Thraustochytrid as a potential source of carotenoids

Thraustochytrids, marine protists whose dominant genera are Thraustochytrium and Schizochytrium, belong to the kingdom Chromista and are known as an industrial source of DHA. We describe here that thraustochytrid strain KH105, isolated as a DHA producer, also accumulates significant levels of β-carotene and xanthophylls including canthaxanthin and astaxanthin. A4-d cultivation using a medium composed of 10% glucose and less than 0.3% of nitrogen sources in a half-concentration of seawater gave an astaxanthin production up to 6.1 mg/L, and canthaxanthin content reached more than 10 mg/L under conditions where a higher concentration of nitrogen sources (6%) was employed. It might be advantageous in mass production systems for these carotenoids to be extracted readily by simply suspending the cells with organic solvents such as acetone and chloroform. Analyses on the morphological and life history features of the KH105 strain revealed that it belongs to the genus Schizochytrium. This particular species of thraustochytrids is thus considered to be a promising source of xanthophylls as well as DHA for use in the food industry.     

Catalytic activity and solid acidity of vanadium oxide thin layer loaded on TiO2, ZrO2, and SnO2

Vanadium oxide spread highly on TiO₂ (anatase, A) and SnO₂, and rather densely on TiO₂ (rutile, R) and ZrO₂ to make the monolayer in less than 4–5 V nm⁻². Profile of acid site of the monolayer was measured by temperature programmed desorption of ammonia, and its relation with the surface oxidation state was studied. The acid site density was high on the V₂O₅/TiO₂ (A) independent of the degree of oxidation. On the other hand, that of V₂O₅/TiO₂ (R) and V₂O₅/ZrO₂ depended on the oxidation state, and the high value of the concentration was observed on the oxidized one. The strength of acid site generated on the V₂O₅ monolayer on TiO₂ was as high as on the HZSM-5 zeolite. Turnover frequency (TOF) of propane conversion, and product selectivity were measured in propane oxidation. Among tested oxides, the V₂O₅/TiO₂ (A) showed the high TOF and selectivity to form propylene, while those loaded on TiO₂ (R) and ZrO₂ the small TOF and poor selectivity. Therefore, the reaction profile of activity and selectivity could be related with the extent of spreading and solid acidity. An idea of limit of the acid site density ca. 1.5 nm⁻² on the monolayer was elucidated.