Simultaneous rice bran oil stabilization and extraction using sub-critical water medium

Sub-critical water technique was used for simultaneous inactivation of lipase enzyme existing in rice bran and extraction of its oil in order to obtain the stabilized edible rice bran oil. Sub-critical water treatment was carried out in the temperature range between 120 and 240 °C for 10 and/or 20 min residence time in a batch reactor. The quality of the extracted oil was evaluated with respect to its total free fatty acids concentration over a 12 week period, and compared with the oil obtained by conventional extraction methods. Without sub-critical water treatment, the concentration of total free fatty acids in the rice bran significantly increased from 5.6% to 36.0%. In contrast, no increase was observed in the total free fatty acids concentration in the samples treated by sub-critical water. Experimental evidence showed that total free fatty acids concentration increased somewhat in the oils treated by conventional methods. Considering no change was observed in total free fatty acid concentration in the treated oils by sub-critical water, it was found that sub-critical water not only could efficiently extract oil from rice bran in a short residence time but also completely stabilized the extracted oil. Oil extraction yields generally increased with increases in sub-critical water treatment temperature and residence time. The highest extraction yield of oil was 249 (mg/g dry matter) obtained at 240 °C and 10 min residence time. Oil extraction by sub-critical water could be conducted in a very short residence time (10 and/or 20 min). Also, the kinetics of free fatty acids formation in untreated rice bran was investigated and developed which successfully describes the concentration of total free fatty acids in the course of rice bran storage.     

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.     

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.     

Development of an Enzyme-Linked Immunosorbent Assay for Specific Detection of Mulberroside A in Mulberry (Morus alba L.) Using Anti-mulberroside A Polyclonal Antibody

An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibody (PAb) against mulberroside A (MuA), a major active component found in the root bark of mulberry (Morus alba L.). MuA was conjugated with the carrier protein bovine serum albumin for immunization to rabbits. The results showed that the antibodies were specific only for MuA and have very low specificity for its aglycone, oxyresveratrol. The ELISA assay was suitable for quantitating MuA in the range of 0.17–15.62 μg/mL with a relative standard deviation (RSD) of less than 5 % for both intra- and inter-assay precision levels. The recovery rates of MuA in the samples were in the range of 97.6–101.4 % with a RSD of less than 5 %. The developed ELISA exhibited a good correlation value (R ²?=?0.994) with the standard high-performance liquid chromatography method in the crude extracts of plant samples. These results suggest that the developed ELISA method using PAb against MuA can be applied to determine MuA content with high specificity, rapidity, and simplicity in mulberry samples. The developed ELISA method described could prove to be useful as an analytical tool for quality control of mulberry and their products.     

CoCl(2) inhibits neural differentiation of retinoic acid-treated embryoid bodies

The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.     

Influence of the conditions of storage and cooking on growth,invasion and survival of Salmonella enteritidis in eggs

Basal studies for the confirmation of sanitary rules in the kitchen were performed, focusing on preventing an outbreak of food poisoning due to eggs contaminated with Salmonella Enteritidis (SE), using hen and quail eggs. SE did not grow at 5 degrees C but grew markedly at 25 degrees C in eggs. The invasion and growth of SE were marked under very humid conditions regardless of whether the eggshell was damaged. The invasion of SE into egg also occurred when eggs were taken in and out of the refrigerator. Moreover, SE was spread immediately to all non-contaminated eggs when SE-contaminated eggs were cracked into a bowl with non-contaminated eggs. In homemade mayonnaise containing 15% vinegar, sterilization took several hours to occur. On a stainless-steel bowl, SE survived for 2 weeks or more. These findings suggest that it is necessary to pay attention to secondary contamination.     

Characterization of the microbial community in an anaerobic ammonium-oxidizing biofilm cultured on a nonwoven biomass carrier

The enrichment and characterization of anaerobic ammonium-oxidizing biofilm cultures are ongoing in our laboratories. Biomass, with a predominately red color, demonstrating simultaneous removal of ammonium and nitrite under autotrophic and anoxic conditions, which is characteristic of anaerobic ammonium-oxidizing planctomycetes, was enriched and maintained for an extended period on a polyester nonwoven carrier. To investigate the bacterial composition of the mature biofilm community, 16S rDNA sequences were amplified by PCR and comparative analyses using DNA databases were conducted. Only one sequence had a notable similarity (92.2%) to that of the first discovered anaerobic ammonium-oxidizing planctomycete and lesser, yet significant, similarities to the 16S rDNA sequences of other recently reported anaerobic ammonium-oxidizing strains. The newly discovered strain (designated KSU-1) reported here was dominant among detectable members of the biofilm community. By fluorescence imaging, KSU-1 was shown to form spherical clusters wrapped in a thin layer of Zoogloea sp. Possible interactions and interdependencies of these two species are discussed with regard to the putative unculturability of the anaerobic ammonium-oxidizing planctomycetes.     

Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators

The present study was undertaken to examine the effects of various transition metal ion chelators, both polyaminocarboxylates (including nitrilotriacetate (NTA), ethylenediaminediacetate (EDDA), ethyleneglycolbistetraacetate (EGTA), ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA)) and non-polyaminocarboxylates (dipicolinic acid and deferoxamine), on the development in vitro of one-cell ICR strain mouse embryos to the four-cell and blastocyst stages. The order of stability constants of polyaminocarboxylates for transition metal ions such as zinc, copper and iron is as follows: NTA < or = EDDA < EGTA < EDTA < DTPA. Addition of 10 or 100 micromol polyaminocarboxylates x l(-1) to the medium significantly enhanced the development of most one-cell embryos (66-88%) beyond the two-cell stage compared with that (< 25%) in medium without polyaminocarboxylates. Although EDDA, EDTA and DTPA at 10 micromol x l(-1) induced the development of most one-cell embryos to the four-cell stage and beyond, a higher concentration (100 micromol x l(-1)) of NTA and EGTA was required to obtain a similar result. Therefore, the ability of polyaminocarboxylates to overcome the two-cell block is not correlated with their potency to chelate transition metal ions. In contrast, the non-polyaminocarboxylates dipicolinic acid and deferoxamine, at 10 and 100 micromol x l(-1), did not have the same effect. Taken together, the results indicate that the ability of polyaminocarboxylates to overcome the two-cell block in embryo development is due to some common feature or features other than the ability to chelate transition metal ions.     

Optimizing production of in vivo-matured oocytes from superstimulated Holstein cows for in vitro production of embryos using X-sorted sperm

The present study aimed to establish an efficient system for the production of female embryos from dairy cows by in vitro fertilization (IVF) using X-sorted sperm and in vivo-matured oocytes collected by ovum pick up (OPU). Nonlactating Holstein cows (n = 36) were administered a controlled intravaginal progesterone-releasing (controlled internal drug release) device (d 0), underwent dominant follicle ablation (DFA) or ovulation by administration of 100 μg of GnRH on d 5, and were superstimulated with FSH and PGF_2α, following standard procedures. Controlled internal drug release devices were removed on the evening of d 8 or on the morning of d 9, depending on the experiment. For LH surge induction, 200 μg of GnRH was administered on the morning of d 10 (0 h). In experiment 1, the peak (48.1%) of ovulating follicles was detected at 29 to 32 h after GnRH injection (0 h), and the range in the timing of the initiation of ovulation was less by timing from GnRH administration (30.0 ± 2.8 h) rather than by timing the onset of estrus (32.7 ± 4.7 h). Only 0.9% of total ovulated follicles were recorded before 26 h after GnRH injection. Therefore, OPU was carried out at 26 h and IVF occurred at 30 h after GnRH in experiments 2 and 3. In experiment 2, 83.3 ± 10.8% of oocytes with expanded cumulus cells had extruded the first polar body at 30 h after GnRH injection. The aim of experiment 3 was to compare the effect of either DFA or GnRH-induced LH surge before superstimulation on the efficiency of embryo production by IVF following superstimulation. Progesterone concentrations from d 10 to 12 in the DFA group were lower than those in the GnRH group. A greater proportion of recovered oocytes with expanded cumulus cells from ≥8-mm follicles was observed in the DFA group than in the GnRH group (95.9 and 77.4%, respectively). Blastocyst rates in the DFA and GnRH groups (58.0 and 52.8%, respectively) did not differ from those of oocytes collected from nonstimulated OPU and matured in vitro (49.9%). However, the proportion of high-quality blastocysts was higher in the DFA group compared with the GnRH group (54.9 vs. 21.5%). Our results demonstrate that high rates of good-quality blastocysts can be produced by IVF with X-sorted frozen sperm using in vivo-matured oocytes collected by OPU from cows after DFA and superstimulation combined with ovulation induction.     

Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk

We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors.