Production of lepidimoide by an endophytic fungus from polysaccharide extracted from Abelmoschus sp.: identification of the product and the organism producing it

A highly potent allelopathic factor, lepidimoide, was initially extracted from mucilage of germinated cress seeds. Polysaccharide extracted from okra (Abelmoschus esculentum Moench) is considered to have a similar structure to lepidimoide as its repeating unit. We therefore initiated the screening of enzymes capable of degrading okra polysaccharide into lepidimoide from endophytes. We discovered an endophytic fungal strain AHU9748 isolated from Coleus galeatus, which produced an oligosaccharide having similar properties to lepidimoide on thin layer chromatography. The physico-chemical data from ESI-MS, NMR spectra and other analyses also showed the purified product to be identical to lepidimoide. The strain AHU9748 was identified as a fungus belonging to the coelomycetes, closely related to the genus Colletotrichum, based on morphological characteristics and sequence analysis of the 18S rDNA and ITS region.     

A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

Yoghurt fermented by Lactobacillus delbrueckii subsp. bulgaricus H+ -ATPase-defective mutants exhibits enhanced viability of Bifidobacterium breve during storage

Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+ -ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+ -ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P < or = 0.05) reduced H+ -ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (> or = 10(8) CFU/ml) of both Bifidobacteruim breve JCM 1192(T) and Bifidobacteruim breve JCM 7017 during storage at 10 degrees C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+ -ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage.     

Heat-moisture treatment of high-amylose corn starch increases dietary fiber content and lowers plasma cholesterol in ovariectomized rats

ABSTRACT:  The effect of dietary high-amylose corn starch (HACS) of varying dietary fiber (DF) content on plasma cholesterol was examined in ovariectomized (OVX) rats. Gelatinized normal corn starch (G-CS) was used as a reference. OVX rats were fed a fiber-free purified diet containing G-CS, HACS, gelatinized high-amylose corn starch (G-HACS), or heat-moisture treated HACS (HM-HACS) at 400 g starch/kg diet for 21 d. The DF content of G-CS, HACS, G-HACS, and HM-HACS measured by the AOAC method was 0.1, 19.3, 2.4, and 64.5 g/100 g, respectively. The dry weight of cecal contents, cecal wall weight, the amount of short chain fatty acids in cecal contents, the amount of bile acids in small intestinal contents, and fecal excretion of neutral sterols increased logarithmically with increasing DF, while total plasma cholesterol concentration decreased. On the other hand, hepatic CYP7A1 activity, fecal dry weight, and fecal excretion of bile acids increased linearly with increasing DF, while body weight gain decreased. The hypocholesterolemic effect of HACS in OVX-rats appeared to be more effective by heat-moisture treatment.     

A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products

A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei. For all other bacterial strains and eukaryote tested, results were negative. Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0). To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L. fructivorans was inoculated into several acidic food as an indicator. Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.     

The effects of di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) administration on human intestinal microbiota

Di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) was shown to enhance Ca absorption in rat and human intestine. The effects of DFA III administration (9 g per day for 4 weeks that corresponded to 3-fold the optimal dosage of DFA III) on human intestinal microbiota were studied using denaturing gradient gel electrophoresis (DGGE). The major groups of human intestinal microbiota reported previously: the Bacteroides, the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum group (Clostridium cluster IV), and the Bifidobacterium group were detected. The similarity of 30 DGGE profiles based on the V3 region (before and after administration to the 15 subjects) of the 16S rDNA were calculated using Pearson's correlation based on numbers, positions and intensity of bands, and then a dendrogram of DGGE profiles was constructed by the unweighted pair group method using arithmetic average (UPGMA) clustering method. By these analyses, no difference in DGGE profiles after DFA III administration was observed in healthy subjects, while two subjects with chronic constipation showed different profiles, namely on numbers, positions and the intensity of some bands. Their stools were softer and stool frequencies increased and they obtained relief from constipation.     

Moisture profiles of wheat noodles containing hydroxypropylated tapioca starch

Wheat noodles were prepared using flour to which hydroxypropylated tapioca starch was added, and the effect of this addition on the moisture distribution within the noodles during cooking was examined using a digital image processing technique. The addition of the modified starch slightly increased the moisture content and narrowed the flat distribution near the noodle surface. The distribution features reflected the changes in the water absorption behaviour caused by the properties of the modified starch and the reduction in the gluten content. Addition of the modified starch lowered Young's modulus and the energy for 99% strain of the noodles in the texture analysis to, at maximum 35% and 65%, and decreased the breakability of the noodles. These changes in the moisture distribution and textural properties have been ascribed to changes in both the state of the starch granules near the surface and the structure of the gluten network.     

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.     

Multiresidue determination of quinolones in animal and fishery products by HPLC

A simple and rapid multiresidue method was developed for the determination of twelve quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid and sarafloxacin) in muscle, liver, chicken eggs, milk, prawn and rainbow trout.The quinolones were extracted from a sample with acetonitrile-water (95 : 5). A fifth part of the filtered extract was diluted with water to keep the acetonitrile ratio at ca. 60%, and passed through a C18 mini-column. The eluate was evaporated to dryness, and the residues were dissolved in methanol-water (30 : 70) for HPLC analysis.The quinolones were separated on a Inertsil ODS-3V column (4.6 mm i.d.x250 mm) with a gradient system of 0.1% phosphoric acid-acetonitrile as the mobile phase, with fluorescence detection.No interfering peak was found on the chromatograms of animal and fishery products, except for milk. The recoveries of the quinolones were over 60% from the animal and fishery products fortified at 0.1 microg/g, and the quantification limits of the quinolones were 0.005 microg/g. This proposed method was found to be effective and suitable for the screening of the quinolones in animal and fishery products.