Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Temperature effects on the development of Enteromyxum spp. (Myxozoa) in experimentally infected tiger puffer, Takifugu rubripes (Temminck & Schlegel)

The effects of water temperature on the development of the enteric myxosporeans, Enteromyxum fugu and Enteromyxum leei, were investigated in experimentally infected tiger puffer, Takifugu rubripes. After naïve tiger puffer were fed gut tissue infected with both E. fugu and E. leei, they were divided into separate tanks and kept at different constant temperature regimes between 10 and 25 °C. Regardless of the water temperature tested, E. fugu was consistently detected with a high prevalence of infection (60–100%), although no sporulation occurred at 10 and 15 °C. Development of E. leei and the onset of disease were suppressed by low water temperatures (<15 °C). However, a temperature increase to 20 °C promoted the development of E. leei, followed by an increase of disease rate in the fish. The present study demonstrates that water temperatures below 15 °C have an inhibitory effect on the development of E. fugu and E. leei, resulting in suppression of enteromyxosis at low temperatures.     

Purification and some properties of alanine racemase from the marine gastropod <Emphasis Type="Italic">Cellana grata</Emphasis>

ABSTRACT:   High levels of free d -alanine were found in the muscle of a marine gastropod Cellana grata that inhabited the intertidal zone, and alanine racemase activity was detected in the muscle. The authors purified alanine racemase from the muscle of C. grata to characterize its enzymological properties. The molecular mass of the enzyme was estimated to be 40.5 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 41.4 kDa by gel filtration, suggesting that the enzyme was monomeric in nature. Kinetic experiments, performed using the purified enzyme, revealed that the Lineweaver-Burk plot for d -alanine as a substrate resulted in a K _m value of 20.4 mM, and the value for l -alanine was 43.0 mM. Of the several types of amino acids tested, alanine was found to be the specific substrate for the enzyme. In the measurement of alanine racemase activity, exogenous pyridoxal 5'-phosphate (PLP) was not required for the enzyme activity; however, aminooxyacetic acid, hydroxylamine and phenylhydrazine, which inhibit PLP-dependent enzymes, strongly inhibited the enzyme activity. These results suggest that the enzyme is PLP-dependent. This is the first report on the purification and some properties of alanine racemase in a marine gastropod.     

Regulatory effect of amino acids on the pasting behavior of potato starch is attributable to its binding to the starch chain

The binding of an amino acid, glycine (Gly), alanine (Ala), epsilon-aminocaproic acid (-AC), monosodium glutamate (GluNa), or lysine (Lys), to starch was examined by a biomolecular interaction analyzer (IAsys). A starch sample (ATS) hydrolyzed to an extent of 1% hydrolysis rate with 15% sulfuric acid was used as a model starch for the binding examination. The reducing end of ATS was oxidized by the Somogyi reagent, and the conversion of the reducing end to the carboxyl group of ATS was confirmed by a carboxylic acid fluorescence labeling reagent. The oxidized ATS was immobilized to the amino group of a sensor cuvette by using water-soluble carbodiimide and N-hydroxysuccinimide through an amide bond. The IAsys examination showed that Gly, Ala, and epsilon-AC scarcely bound to the immobilized starch chains but that GluNa and Lys favorably bound with their increasing concentrations. The relative binding index (RBI) of each amino acid was defined by the ratio of the slope of the linear regression equation between the binding response and the concentration for each amino acid to that for Gly. Because the relationships between the RBI and the pasting characteristics (pasting temperature, peak viscosity, breakdown, and swelling index) could each be expressed by a linear regression equation with a high correlation coefficient, it is concluded that the regulation of the pasting behavior of starch with an amino acid is caused by binding of the amino acid to the starch chains.     

Collembolan community dynamics during deciduous forests regeneration in Japan

The structure and feeding group composition of collembolan communities were studied in secondary deciduous forests of different ages to investigate the collembolan community response to environmental changes associated with forest cycles. The study was carried out at eight sites forming a chronosequence (1, 4, 12, 24, 51, 54, 71 and 128 years after clear cutting) of deciduous forest stands in northern Ibaraki (Japan). Total collembolan density and species richness was low at the 1-year-old site, and there was little difference in density among sites over 4 years of age. The density of sucking feeders was especially low at the 1-year-old site. Species richness of trees of a diameter at breast height (DBH)<5 cm positively correlated with the density of fungal feeders. Species richness of total Collembolans and of sucking feeders correlated positively with the water content of the organic layer. Ordination of the collembolan community with Canonical Correspondence Analysis suggested that species richness of larger trees (DBH 5 cm) contributed to the differences in species composition of fungal feeders and sucking feeders. We conclude that total abundance and species richness of collembolans recovered within 4 years after clear-cutting, but species composition of fungal feeders and sucking feeders took longer to recover.     

Pharmacokinetics of Eugenol in Japanese Flounder,Paralichthys olivaceus

Eugenol, which has been used as an anesthetic for fish, was administered to Japanese flounder, Paralichthys olivaceus (127 ± 50.8 g), by bath treatment at concentrations of 0.025, 0.125, and 0.25 mL/L seawater for 10 min and intramuscular injection at 40 μL/fish to investigate the pharmacokinetic characteristics of eugenol in the plasma of Japanese flounder. In the bath treatment test, plasma eugenol concentration increased with increasing eugenol concentration up to 0.125 mL/L and reached steady state within 5 min. After a 10‐min bath treatment in 0.25 mL/L eugenol, plasma eugenol concentration was about 58.4 µg/mL. After transfer into running seawater, plasma eugenol concentration decreased biphasically with half‐lives of 0.0296 h (α‐phase) and 0.289 h (β‐phase). The AUC_0000→0800 was about 16.5 µg h/mL. In administration by intramuscular injection, plasma eugenol concentration increased rapidly after administration and decreased biphasically with half‐lives of 0.0329 h (α‐phase) and 8.08 h (β‐phase). The AUC_0000→0800 was about 52.5 µg h/mL. In both methods of administration, Japanese flounder with average weight of 127 g were effectively anesthetized when plasma eugenol concentrations were between 2.19 and 4.88 µg/mL.     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

Characteristics of phosphorus fractions in the soils derived from sedimentary and serpentinite rocks in lowland tropical rain forests,Borneo

ABSTRACT

Soil organic phosphorus (P) is an important P source for biota especially in P-limited forests. Organic P has various chemical formations which differ in bioavailability and these organic P can be degraded by phosphatase enzymes. Here, we report soil P fractions inferred from solution ³¹P-NMR spectroscopy and soil phosphatase activities of two tropical rain forests on contrasting parent materials; sedimentary and ultramafic igneous (serpentinite) rocks. Compared to the sedimentary soils　and previous studies, P fractions of the serpentinite soils have distinctly high proportions of pyrophosphate and scyllo-inositol hexakisphosphate (scyllo-IP₆). The accumulation of pyrophosphate and scyllo-IP₆ may be related to strong sorptive capacity of iron oxides present in the serpentinite soils, which implies a consequent low P availability in the serpentinite soils. Mean value of soil phosphatase activities was higher in the serpentinite soils than in the sedimentary soils, suggesting that biota in these serpentinite forests depend more on soil organic P as a P source.     

Influence of Amylose Content on Cookie and Sponge Cake Quality and Solvent Retention Capacities in Wheat Flour

Reduced amylose wheat (Triticum æstivum L.) produces better quality noodles and bread less prone to going stale, while little is known about the relationships between amylose content and the quality of soft wheat baking products such as sugar snap cookies (SSC) and Japanese sponge cakes (JSC). Near‐isogenic lines developed from wheat cultivar Norin 61, differing in their level of granule‐bound starch synthase (Wx protein) activity, were used to produce wheat grains and ultimately flours of different amylose contents. These were tested with regard to their effect on soft wheat baking quality and solvent retention capacities (SRC). Amylose content was strongly correlated to cookie diameter (r = 0.969, P < 0.001) and cake volume (r = 0.976, P < 0.001), indicating that the soft wheat baking quality associated with SSC diameter and JSC volume were improved by an incremental increases in amylose content. Among the four kinds of SRC tests (water, sodium carbonate, sucrose and lactic acid), the water SRC test showed the highest correlation with amylose content, SSC diameter, and JSC volume. When the regression analysis was conducted between the nonwaxy and partial waxy isogenic lines that are available in commercial markets, only water SRC was significantly correlated to amylose content (r = –0.982, P < 0.001) among of four SRC tests. This suggests that, unlike udon noodle quality, high‐amylose content is indispensable in improving soft wheat baking quality, a process requiring less water retention capacity.     

Effect of Growing Environment of Soft Wheats on Amylose Content and Its Relationship with Cookie and Sponge Cake Quality and Solvent Retention Capacity

The effect of growing environments of soft wheat on amylose content and its relationship with baking quality and solvent retention capacities (SRC) was investigated. Near‐isogenic soft wheat lines of Norin 61 differing in granule‐bound starch synthase (Wx protein) activity and grown in three different regions of Japan: Hokkaido (spring‐sown) for 2006 and 2007, Kanto (autumn‐sown), and Kyushu (autumn‐sown) for 2007 were evaluated. Spring‐sown samples produced grains of greater protein content (10.9–12.4%) than autumn‐sown samples (7.3–9.1%). In contrast, spring‐sown samples of 2007 with higher maturing temperature had lower amylose content (25.5% for Norin 61) compare to the autumn‐sown and spring‐sown samples of 2006 (27.6–28.4% for Norin 61). Amylose content was strongly correlated to sugar snap cookie (SSCD) diameter (r = 0.957–0.961; n = 10, all samples; P ≤ 0.001, r = 0.701–0.976; n = 7 partial waxy and nonwaxy samples; and Japanese sponge cake (JSCV) volume r = 0.971–0.993; n = 10; P≤ 0.001, r = 0.764–0.922; n = 7 partial waxy and nonwaxy samples), regardless of seeding season and growing conditions. The strength of the JSVC‐amylose relationship (slope) was similar among the three regions, whereas the strength of the SSCD‐amylose relationship was slightly weaker for spring‐sown samples and slightly stronger for partial waxy and nonwaxy autumn‐sown samples. Among of the four solvents (water, solutions of sodium carbonate, sucrose, or lactic acid), water‐SRC showed the greatest correlation to amylose content (r = –0.969 to –0.996; n = 10; P ≤ 0.001, r = –0.629 to –0.983; n = 7 partial waxy and nonwaxy samples), indicated that amylose content can be accurately estimated from the water‐SRC within the samples from the same grown environment.