Evaluation of immunochromatographic test kits for food allergens using processed food models

It has been mandatory to label five allergenic substances (AS; egg, milk, wheat, buckwheat and peanut) in all processed foods, since April 2002 in Japan. Two kinds of ELISA kits have been provided as screening test kits for the Japanese official method. The kits have many advantages but some disadvantages, i.e., the kits are not necessarily suitable for daily monitoring in food manufacturing plants, because they require various analytical equipments and the use of complicated procedures. To overcome these drawbacks, we have developed other diagnostic kits based on immunochromatography that should enable more rapid and simple screening for food allergens. Then we examined the performance of these immunochromatographic test kits (IC kits) in terms of sensitivity, repeatability and cross-reactivity to AS proteins in 11 kinds of food models with various heating conditions and physical properties. We also examined processed food models including AS protein of constant concentration, using the IC kits and ELISA kits, and compared the results. The IC kits detected AS proteins at 5 microg/g in the extracts from processed food models, and provided highly reproducible results. Cross-reactivity among the AS proteins was not observed. The results obtained using the IC kits showed performance equivalent to that of the ELISA kits we examined in unheating processed food models including AS proteins of constant concentration. The IC kits should be more suitable for daily monitoring in food manufacturing plants.     

Measurement of vertical and horizontal vibrations of a probe for acoustic evaluation of food texture

Two‐dimensional (vertical and horizontal) vibrations of a wedge‐type probe upon food rupture were evaluated separately using two accelerometers placed perpendicular to a guide rod of a swing‐arm device for texture evaluation of the flesh of three varieties of apples and three types of potato chips. Voltage signals from the accelerometers were filtered using a half‐octave band‐pass filter. The energy texture index (ETI), based on kinetic energy of the vertical or horizontal probe vibrations, was calculated over low to high frequency bands (no. 1–21). The spectra for the flesh of the three varieties of apples included two common peaks for vertical ETI at band no. 11 (1,120–1,600 Hz) and 19 (17,920–25,600 Hz) and horizontal ETI at band no. 1 (0–10 Hz) and 15 (4,480–6,400 Hz). The spectra for the three types of potato chips had a common ETI peak at band no. 11 (1,120–1,600 Hz) for horizontal ETI and at no. 15 (4,480–6,400 Hz) for vertical ETI. The three apple varieties gave rise to different intensities of vertical and horizontal ETIs while the two peaks were maintained. The thick potato chip type had higher vertical and horizontal ETIs than the thin and soft varieties in most bands; however, the thin type had the highest vertical ETIs only in lower bands (0–200 Hz). The soft type had the lowest vertical and horizontal ETI. The above results suggest that different food textures can be distinguished by two‐dimensional vibration analyses of probe insertion into a food sample based on frequency bands.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Study of pesticide residues in agricultural products for the "Positive List" system

During a 3-year monitoring survey (April 2002-March 2005) of pesticide residues in agricultural products, 592 samples (324 domestic; 268 imported) collected in Hyogo prefecture, Japan were analyzed. The number of pesticides tested increased from 232 in FY 2002 to 323 in FY 2004. The purpose of the study was to clarify the residue status by accumulating information about pesticides detected frequently, to allow effective and efficient regulation under the new "Positive List" legislation to be implemented in FY 2006. Overall, 47% of domestic and 61% of imported samples contained detectable residues and ca. 60% of positive samples contained multiple residues. The limit of quantitation was set at 0.01 microg/g and the limit of detection was 0.001-0.003 microg/ g. Most of the residues were present at low concentrations: 80% of the detections in samples excluding imported citrus fruits were < 0.05 microg/g. More than 5 different pesticides (> 0.01 microg/g) were detected simultaneously in 13 samples. The sum of the ratios of residues to MRLs was calculated as one of the indexes to represent the risk of multiple residues, and they exceeded 100% in 3 imported frozen vegetables; baby kidney bean, spinach, Welsh onion. Samples in violation of the Food Sanitation Law were not found in our survey, but 1.9% of the samples might be in conflict with the new "Positive List" legislation.     

北京养老服务机构入住理由及位置选择的初探——关于合理布局建设养老服务机构

通过对北京市养老服务机构的入住老人进行问卷调研,解析老人的入住理由.老人在机构选择意向中重视“离子女居住地近”的要素,并在实际选择入住时,呈现选择离子女居住地较近机构的趋势.根据该倾向,研究通过整理各功能区特征,为解决北京市养老服务机构入住率分布不均衡的现状,提出各功能区养老机构的规划建设见解.     

Effects of fermentation conditions and soybean peptide supplementation on hyaluronic acid production by Streptococcus thermophilus strain YIT 2084 in milk

To increase the hyaluronic acid (HA) yield from Streptococcus thermophilus YIT 2084, fermentation conditions (pH, temperature, agitation, aeration) were optimized in milk-based medium, and the effects of supplemental soybean peptides, which have different molecular weight distributions, were determined. HA production was enhanced to approximately 100 mg/l at pH 6.8 and 33–40 °C. Agitation speed and aeration rate slightly affected HA production. Soybean peptides including those of high molecular weight (approximately 27 to 130 kDa) further increased HA production to 208 mg/l under the optimal condition (pH 6.8, 35 °C, 100 rpm), which was 20-fold greater than non-optimal condition. HA production was no longer related to the specific growth rate. The HA produced under the optimal condition included a large amount of high-molecular-weight fraction of 100 to 2000 kDa, compared with under the basal condition without optimization.     

Effect of skimmed milk and its fractions on the inactivation of Escherichia coli K12 by high hydrostatic pressure treatment

We investigated the effects of skimmed milk and its protein fractions (casein, whey, globulin, and albumin) on the injury and inactivation of Escherichia coli K-12 by high hydrostatic pressure (HHP) treatment. The protective effect of skimmed milk on HHP-mediated inactivation and injury of E. coli increased with increases in the skimmed milk concentration. However, protein fractions derived from skimmed milk did not exhibit this protective effect. Microscopy analysis by DAPI/PI staining indicated that some cells were localized in the solid portion of skimmed milk, and some of these cells were alive. The coagulated fraction derived from the autoclaved whey fraction also showed a significant protective effect. We speculate that the solid portion in skimmed milk could provide the protective effect to bacterial cells.     

Effect of light intensity and frequency of flashing light from blue light emitting diodes on astaxanthin production by Haematococcus pluvialis

Flashing light from blue light emitting diodes is an effective method for the reduction of energy consumption in the bioproduction of astaxanthin by Haematococcus pluvialis. We investigated the effects of light intensity and frequency on the final astaxanthin concentration in bioproduction by H. pluvialis grown mixotrophically. The final astaxanthin concentration under illumination with flashing light, with frequencies ranging from 25 to 200 Hz, was dependent on the light intensity and on the duty cycle and was equivalent, or higher, in comparison with that under illumination with continuous light at the same incident intensity. The light intensity determined the maximum attainable concentration of astaxanthin under continuous illumination. Under illumination with flashing light, the ratio of the final astaxanthin concentration to the maximum concentration at a specific light intensity was correlated to the duty cycle in the frequency range from 25 to 200 Hz. The effect of lower frequencies on enhanced astaxanthin production under flashing light was also studied; at levels as low as 1 Hz, higher final astaxanthin concentrations were observed under flashing light compared to concentrations attained under continuous light.     

The protein encoded by cancer/testis gene D40/AF15q14 is localized in spermatocytes, acrosomes of spermatids and ejaculated spermatozoa

We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.