Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulase

The feasibility of using loofa sponge for immobilization of cellulase-producing microorganisms was investigated by acetylating loofa sponge. Acetylation was achieved by autoclaving process of loofa sponge immersed in acetic anhydride at various temperatures for various times. The degree of acetylation, as inferred by the weight percentage gain (WPG), was enhanced by increasing both temperature and the duration of acetylation. The acetylation of a piece of loofa sponge in an autoclave at 120 degrees C for 20 min resulted in a WPG of about 8%, which was sufficient to protect the loofa sponge against cellulose degradation. The acetylated loofa sponge prepared under this condition was not decomposed by commercial cellulase and its structure was maintained for more than 720 h during repeated-batch treatments with commercial cellulase. A flocculating yeast (Saccharomyces cerevisiae IR-2) and a fungus (Trichoderma reesei QM9414) were successfully immobilized in the acetylated loofa sponge. In each case, the percentage of immobilized cells was as high as that obtained using nonacetylated loofa sponge. Acetylation had no adverse effects on cell growth and immobilization of T. reesei QM9414, as well as on cell growth and ethanol production by S. cerevisiae IR-2. T. reesei QM9414 immobilized on an acetylated loofa sponge was successfully used for repeated-batch cellulase production from commercial cellulose powder. Although the acetylated loofa sponge showed a slight weight loss, it was not disintegrated by activated sludge. The results obtained in this study showed that acetylated loofa sponge is suitable as an immobilization carrier for bioprocesses involving cellulase.     

Safety and structural analysis of polymers produced in manufacturing process of alpha-lipoic acid

Alpha-Lipoic acid has recently been permitted for use in foodstuffs and is contained in tablets and capsules. Although alpha-lipoic acid is synthesized from adipic acid, the safety of polymers produced during the purification and drying processes has been an issue of concern. Hence, we examined the safety profiles of thermally denatured polymer (LAP-A) and ethanol-denatured polymer (LAP-B) produced in the manufacturing process of alpha-lipoic acid. Furthermore, we conducted structural analysis of these polymers by 1H-NMR and FAB-MS spectroscopy. In a consecutive ingestion test, male and female mice ingested diet containing 0.1 and 0.2% LAP-A and -B for 4 weeks. Blood uric acid, potassium and lactate dehydrogenase (LDH) tended to increase without dose-dependency. Relative liver weights were also increased. However, male dogs that were orally administered LAP-B (500 mg/kg) once did not show any abnormalities in blood parameters or general condition. These findings indicate that alpha-lipoic acid polymers are not acutely toxic; however, chronic ingestion of these polymers may affect liver and kidney functions.     

Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains

Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 microg/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.     

Inhibitory effect of zinc on beta-hexosaminidase release from RBL-2H3 cells by synthetic chemicals

The effects of foods and chemicals related to food hygiene on degranulation were evaluated using a method for assaying the enzyme activity of beta-hexosaminidase as an index of chemical mediator release from RBL-2H3 cells in vitro. Using a previously developed assay system, we had found a large number of inhibitors and promoters of degranulation of RBL-2H3 cells. In the present study, we examined the inhibitory effect of zinc chloride on the degranulation (beta-hexosaminidase release) from RBL-2H3 cells with or without antigen in the presence of the degranulation-promotive chemicals, namely, 4 food additives, 7 pesticides and 2 veterinary drugs. These promotive chemicals were classified into two types on the basis of inhibitory profile by zinc chloride: 1) those which showed marked degranulation-inhibitory action when the cells were stimulated with antigen, such as butylhydroxyanisole, dibutylhydroxytoluene, EPN, cis- and trans-permethrin, prothiofos, pyridaben, terbufos, 2) those which showed marked degranulation-inhibitory action whether the cells were stimulated with antigen or not, such as butyl p-hydroxybenzoate, o-phenylphenol, bitertanol, salinomycin. In conclusion, zinc had a dramatic inhibitory effect on enhanced degranulation induced by synthetic chemicals in vitro.     

Detection method of injured Escherichia coli O157 in noodles and vegetables

An enrichment procedure and a polymerase chain reaction (PCR) method for the detection of injured Escherichia coli O157 in foods were examined. Freeze-injured E. coli O157 inoculated in boiled spaghetti could be detected in 6-h culture within 12 h by the PCR method. Cells injured by heating in boiled spaghetti and cells injured by chlorine treatment in raw lettuce and carrot did not grow sufficiently to be detected in 6-h culture but were detected in 18-h culture using selective agar media. The injured cells could be also detected in 18-h culture within 24 h by the PCR method. Enrichment at 42 degrees C in trypticase soy broth (TSB) was more effective than that at 42 degrees C in modified EC broth with novobiocin. These results indicated that the usage of enrichment in TSB for 18 h at 42 degrees C in combination with the PCR method is suitable for screening for E. coli O157 in boiled or chlorinated foods, even if the O157 cells are injured.     

Partial characterization of dextran-degrading enzyme obtained from blue cheese

Degradation of dextran beads was observed when the water-soluble fraction of a blue cheese extract was applied to the top of a Sephadex G-150 or G-200 column. This phenomenon suggests the presence of a specific enzyme that can hydrolyze dextran. After removal of casein components from the blue cheese fraction, ammonium sulfate treatment and gel filtration chromatography were performed to isolate the enzyme fraction. The enzymatic products were analyzed by thin-layer chromatography and gel filtration chromatography and identified as isomaltooligosaccharides. The isoelectric point of this enzyme fraction was approximately 4.9, as determined by isoelectric focusing using Rotofor, and the molecular weight of the fraction was 65 kDa, as estimated by sodium dodecyl sulfate (SDS)-PAGE. Optimum pH for enzymatic activity was 5.0 to 5.3. A partial N-terminal amino acid sequence of 20 residues was determined to be ATPDEWRSRSIYFMLTDRGA from an enzyme fraction further purified by ion-exchange chromatography and native PAGE. This sequence showed a maximum homology of 80% with alpha-amylase or Taka amylase that originated from various microorganisms.     

Isolation and characterization of poly(butylene succinate-co-butylene adipate)-degrading microorganism

Poly(butylene succinate-co-butylene adipate) (PBSA)-degrading bacterium, strain 1-A, was isolated from soil. Strain 1-A was identified as Bacillus pumilus on the basis of its physiological properties and partial 16S rRNA gene sequence. Strain 1-A also degraded poly(butylene succinate) (PBS) and poly(epsilon-caprolactone). On the other hand, poly(butylene adipate terephthalate) and poly(lactic acid) were minimally degraded by strain 1-A. The NMR spectra of degradation products from PBSA indicated that the adipate units were more rapidly degraded than 1,4-butanediol and succinate units. This seems to be one of the reasons why strain 1-A degraded PBSA faster than PBS.     

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy γ-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-κB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.     

Ethanol and lactic acid production using sap squeezed from old oil palm trunks felled for replanting

Old oil palm trunks that had been felled for replanting were found to contain large quantities of high glucose content sap. Notably, the sap in the inner part of the trunk accounted for more than 80% of the whole trunk weight. The glucose concentration of the sap from the inner part was 85.2 g/L and decreased towards the outer part. Other sugars found in relatively low concentrations were sucrose, fructose, galactose, xylose, and rhamnose. In addition, oil palm sap was found to be rich in various kinds of amino acids, organic acids, minerals and vitamins. Based on these findings, we fermented the sap to produce ethanol using the sake brewing yeast strain, Saccharomyces cerevisiae Kyokai no.7. Ethanol was produced from the sap without the addition of nutrients, at a comparable rate and yield to the reference fermentation on YPD medium with glucose as a carbon source. Likewise, we produced lactic acid, a promising material for bio-plastics, poly-lactate, from the sap using the homolactic acid bacterium Lactobacillus lactis ATCC19435. We confirmed that sugars contained in the sap were readily converted to lactic acid with almost the same efficiency as the reference fermentation on MSR medium with glucose as a substrate. These results indicate that oil palm trunks felled for replanting are a significant resource for the production of fuel ethanol and lactic acid in palm oil-producing countries such as Malaysia and Indonesia.     

Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: a collaborative study

In order to establish a rapid and sensitive method for the detection of Verotoxigenic Escherichia coli O157 and O26, a collaborative study was conducted focusing on a comparison of the efficiency of loop-mediated amplification (LAMP) assay targeting the Verocytotoxin (also called Shiga toxin) gene, utilizing a direct plating method and a plating method with immunomagnetic separation (IMS-plating method) using various agar media. In combination with enrichment with the modified EC supplemented with novobiocin, E. coli O157 was detected in most samples of ground beef and alfalfa sprouts by LAMP assay, the direct plating method and the IMS-plating method. E. coli O26 was detected in approximately 100% of the food samples by LAMP assay. However, the IMS-plating and direct plating methods recovered 80 and 50% in ground beef samples, respectively. As a result, it was demonstrated the LAMP assay is superior to the IMS-plating method. Based on these results, it appears LAMP assay is effective as a screening assay to detect E. coli O157 and O26 from positive samples.