Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.     

Shiga toxin 2eB‐transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin‐producing Escherichia coli infection

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB‐transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB‐lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB‐lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB‐lettuce is a promising first oral vaccine candidate against ED.     

Field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows

A field study of a vaccine; prepared by solubilizing cells infected with bovine coronavirus, Triton X-100, and mixing with an oil adjuvant, was performed at 9 farms over 4 prefectures. The cattle tested were Holstein dairy cows aged 2 to 10 years. A vaccination group consisted of 157 animals (including 132 pregnant cows) and a non-vaccinated control group consisted of 50 animals. The cows received 2 intramuscular injections of vaccine (2 mL) at 3-week intervals. Vaccinated cows did not develop abnormalities, such as a decrease in milk production volume, and all pregnant animals calved normally. The geometric mean of the hemagglutination inhibition antibody titer was 34.2 before vaccination in test cows. The titer had increased to 105.6, 3 weeks after the 1st injection and peaked at 755.6, 1 month after the 2nd injection. A high antibody titer persisted at 396.0; 241.0; and 201.5, at 3, 6, and 9 months after the 2nd injection, respectively.

This confirms the safety and high antibody-response induced by this prototype vaccine. Therefore, this vaccine may be useful for the prevention of winter dysentery caused by bovine coronavirus infection.

     

Microscopic structure of the large intestinal mucosa in piglets during an antibiotic-associated diarrhea

Antibiotic-associated diarrhea (AAD) is caused by the treatments of broad-spectrum antimicrobials that seriously affect the activity and composition of the large intestinal microflora. The pathogenic bacteria or low concentration of short-chain fatty acids (SCFAs) has been repeatedly discussed in relation to AAD. Recently, we reported the detection of a large amount of succinate and lactate in the diarrheal feces in AAD-induced piglets. In this study, we investigated histologically the large intestinal mucosa in AAD-induced piglets, in which succinate and lactate were accumulated. AAD was induced in the piglets by an oral dose of polymyxin B sulfate (PL) or by an intra-muscular injection of enrofloxacin (ERFX). When the piglets were defecating diarrheal feces with a high concentration of succinate and/or lactate, the large intestine was removed and separated into four segments (cecum, gyri centripetales, gyri centrifugales, and rectum). Healthy piglets were used as the control. In the AAD-induced piglets, the lamina propria was edematous in the gyri centripetales. Piglets treated with ERFX were also edematous in gyri centrifugales. These edematous lamina propria contained larger amounts of inflammatory cells than observed in control tissues. ERFX-treated piglets had a more shallow crypt than PL-treated and control piglets. The mucosal tissue of the large intestine was more seriously damaged in the ERFX- than in the PL-treated piglets, which might have been caused by the high succinate and low SCFAs concentration in the digesta.     

Effects of bacterial strains on the development of the ripening flavor of cured pork loins

To determine the effect of bacteria on the development of the ripening flavor of cured meat, pork loins were cured with pickles containing 8 or 16% sodium chloride at 0 and 8°C. The bacterial flora, total free amino acid content and total free fatty acid content in the cured loins, sensory properties of cooked pork loins and the relationships between the proteolytic and lipolytic activities of isolates and curing conditions were investigated. Desirable bacteria, including salt‐tolerant bacteria and lactic acid bacteria, were predominant under the condition of 8% sodium chloride in the pickles and a curing temperature of 8°C. The total free amino acid content and total free fatty acid content at a curing temperature of 8°C were higher than those at 0°C. Cooked pork loins cured in pickles containing 8% sodium chloride at a temperature of 8°C for more than 7 days were preferred in terms of color, flavor and taste. Before the curing procedure, gram‐negative bacteria (Vibrio, Acinetobacter, Pseudomonas and Enterobacteriaceae) were predominant in the pork loins. During the curing period, the numbers of viable gram‐positive bacteria (Micrococcus, Staphylococcus and Pediococcus) increased and the numbers of viable gram‐negative bacteria decreased. Strains of Micrococcus and Staphylococcus with proteolytic and/or lipolytic activities in cured meat also increased during the curing period and were more predominant in the pork loins cured in pickles containing 8% sodium chloride and at a curing temperature of 8°C than in pork loins cured in pickles containing 16% sodium chloride and at a curing temperature of 0°C. The actions of these bacteria were thought to be important factors affecting the flavor of the cured pork. Micrococcus and Staphylococcus with proteolytic activity might contribute to the development of the ripening flavor of ham. The results of the present study together with the results of further investigations on the relationships of the enzyme activities of Micrococcus and Staphylococcus in cured meat with a preferable flavor would be useful for establishing a novel effective method for using bacteria to produce ham of a high quality.     

Application of genomic in situ hybridization for phylogenetic study between Mangifera indica L. and eight wild species of Mangifera

The phylogenetic relationship between mango (Mangifera indica L.) and eight wild species of Mangifera were analyzed by comparing signal intensity of genomic in situ hybridization (GISH) on somatic metaphase chromosomes of M. indica, using labeled DNA of eight wild Mangifera species. The eight wild species were divided into four groups based on intensity and number of hybridization signals on chromosomes of M. indica in GISH analyses. The probe of Mangifera sylvatica Roxb. gave the highest intensities on the chromosome of M. indica, indicating a close relationship between M. indica and M. sylvatica. For the other species, classification of GISH was comparable to that of the phylogenetic analysis using AFLP markers, as previously reported ( Eiadthong et al., 2000). This suggested a possibility that GISH analysis can be effectively used in the classification of Mangifera species.     

The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets

Porcine edema disease (ED) is caused by Shiga toxin 2e‐producing Escherichia coli (STEC). Post‐weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial‐resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21‐day‐old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.     

Long-term changes in the chemical properties of Japanese cedar (Cryptomeria japonica) forest soils under high precipitation in southwest Japan

There is insufficient direct evidence of long-term changes in the chemical properties of forest soils to provide information for forestry management plans for sustainable site productivity. To understand changes in the chemical properties of forest soils in a specific high-precipitation climate in Japan, we re-investigated, in 1997, soil pits within a Japanese cedar (Cryptomeria japonica) forest in the Yanase region of Kochi Prefecture which had previously been surveyed in 1976. Comparison of the 1976 and 1997 results revealed significantly lower pH of surface and subsurface soils, and fewer exchangeable cations over time. In contrast, subsoil pH, concentrations of total carbon and nitrogen, and cation-exchange capacity (CEC) had not changed over the 21-year period. Estimation of proton and cation budgets in the forest ecosystem over 21 years, using data for the amounts in soil, biomass accumulation, and input through precipitation, revealed that biomass accumulation did not explain the cation depletion in soils. This suggests that cations were leached from the ecosystem. In contrast with previous reports, which showed Ca accumulation in Japanese cedar forest soils, our results indicated that high precipitation of more than 4,000 mm combined with acid deposition resulted in soil acidification and leaching of cations from soils, following the decrease in base saturation.     

柿(Diospyros kaki L.)果实发育及成熟过程中内源GAs活性和ABA含量的变化

利用生物学方法和气相色谱法分别对柿有代表性的早、中、晚熟品种果实中内源赤霉素类物质(GAs)活性及脱落酸(ABA)含量的变化进行了测定。GAs 活性和 ABA 含量在幼果初期都很高,以后随果实的发育而迅速降低,在果实生长速度缓慢的第二期变化不大。但自果实进入成熟期开始,GAs 活性再次迅速降低,而 ABA 含量却呈增加趋势。发生这种变化的时期,与果实的成熟期一致。讨论了内源值物生长调节物质的平衡与果实成熟的关系。