We performed a detailed study of the effects of electron beam irradiation on the functional characteristics of bovine blood plasma protein samples using indices of gel strength, syneresis, moisture adsorption, molecular weight distribution, viscosity, organoleptic evaluation and microbial experiments. The functional characteristics of plasma proteins subjected to five different levels of electron beam radiation were evaluated in comparison to non-irradiated samples. 1) No significant change was noted in gel strength, water holding capacity. 2) Electron beam irradiation was observed to polymerize plasma proteins. To prove this phenomenon of molecular weight, molecular alteration of plasma proteins was inferred from changes in viscosity and changes in peak components according to measured molecular weight distribution. 3) Regardless of molecular alteration, no significant changes were noted in terms of taste, flavor or the functional characteristics of plasma proteins appropriate for use in food products. It was therefore learned that such plasma proteins may be used to impart gel strength, viscosity or water retention characteristics to food items requiring sterilization treatment, regardless of the amount of electron beam radiation applied to such plasma proteins.

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We have applied an aluminum-backed reversed-phase TLC plate to the analysis of the 12 coal tar dyes permitted to use for foods in Japan. Two solvent systems were selected for the separation of the xanthene dyes (methanol-acetonitrile-5% aqueous sodium sulfate solution=5:1:3) and the others (methanol-acetonitrile-5% aqueous sodium sulfate solution=3:3:10) (Fig.1). Fifty-four commercial foods were analyzed by aluminum-backed reversed-phase TLC after clean up of the dyes using polyamide column chromatography, and chromatographic behaviors of the dyes were observed. The average Ra/Rs values, ratios of Rf values of sample and standard spots on the same TLC plate, ranged from 0.99 to 1.08 and their coefficient of variation values were 0.0-5.1% (Table 1 and 2). These results mean that the separation was not affected by coexisting substances from foods and the spots always gave the same Rf values as the standards with good reproductibility. In TLC/fast-atom bombardment mass spectrometry (FABMS) of the coal tar dyes, use of aluminum-backed reversed-phase TLC plate improved the detection limits about 2-fold in comparison with use of glass-backed reversed-phase TLC plate except for azo dyes (Table 3). Finally, TLC/FABMS with the aluminum-backed plate has been successfully applied to the identification of Azo Rubine and Wool Green S, which are not permitted to use for foods in Japan, in the imported foods (Fig. 2). Therefore, aluminum-backed reversed-phase TLC plate is considered to be useful tool for the analysis of coal tar dyes in foods.

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Shoot and callus cultures of Centaurium scilloides (Gentianaceae) were established and the secondary metabolites of the cultures were determined. The major constituents of the shoots cultured on phytohormone free 1/2 MS solid medium were bitter secoiridoid glucosides, swertiamarin (SA) and gentiopicrin (GP). The content of SA in the aerial part was much larger (ca. 6% dry weight) than that in the root portion, although GP content was not so much different in both parts. On the contrary, the major constituent in the callus cultured on 1/2 MS solid medium supplemented with 1.0 mg/l 2,4-D and 0.1 mg/l BA was 3,5,6,7,8-pentamethoxy-1-O-primeverosylxanthone (PX) and the bitter principles (SA ans GP) were produced at very low level. In the time course experiment of the callus cultures, PX showed the maximum content at week 8 of the culture, and the content decreased at the end of the culture period. Callus was also derived from the regenerants which were formed directly on the adventitious roots. The constituents of the callus were similar to those detected in the callus derived from the seedlings. The secondary metabolites as well as the morphology of the regenerants were also similar to those of the seedling plantlets in vitro. These observations showed the stability of the characters in the morphology and secondary metabolism of this plant through some tissue culture stages such as subculture, direct regeneration from the root, etc.

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Shoot and callus cultures of Osmanthus fragrans Lour. var. Thunbergii Makino (Oleaceae) were established for the first time. The production of acteoside, which is one of the strongest anti-oxidant principles in natural resource, in these cultures were determined. In addition, acteoside content in in vivo plants of three Osmanthus plants (O. fragrance Lour. var. aurantiacus Makino, O. fragrans Lour. var. Thunbergii Makino and O. x fortunei Carr.) was also analyzed by HPLC. Field-grown (in vivo) Osmanthus plants contained large amount of acteoside, particularly in stem portion (maximum: 4.72% dw, in O. fragrans Lour. var. Thunbergii Makino). On the contrary, young (about 1-year old) plants in vivo and shoot cultures of the plant contained small amount of acteoside (the content was below 1% dw). This result suggested that plant aging is an important factor for the biosynthesis and accumulation of acteoside in this species. Calli of O. fragrans Lour. var. Thunbergii Makimo, cultured on BF solid medium supplemented with 3.0 mg/l IAA and 0.1 mg/l BA in the light or dark condition, produced ca. 1% (dw) content of acteoside as the major phenolic constituent. Callus cultures of this species couls be expected to become a new valuable resource for the supply of anti-oxidant principle, acteoside.

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[in Japanese]

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Among many food additives, preservatives contribute to safe and stable supply of food. On the other hand, they may be hazardous to our health depend on the amount of use. Although acceptable daily intake (ADI) has been used for the safety assessment of food additives, this stndard is solely based on the knowledge of whole animals. Therefore, they may have some adverse effect on cellular levels even if their used level are below ADI, or commercially used level. In this paper we carried out to investigate their cellular effects on ADI and daily intake level. Five preservatives (butyl p-hydroxy benzoate, hinolitiol, sodium dehydroacetate, sodium benzoate, potassium sorbate, sodium propionate) certified by the Japanese Standard of Food Additives, and noncertified one (salicylic acid) were chosen and used. Acetyl salicylic acid was used as a referential inhibitor of platelet function. Each compound was dissolved in Ca²⁺, Mg²⁺ free Tyrode buffer pH 7.4 at concentrations corresponded to ADI, 1/5ADI, 1/10ADI, 1/20ADI, and 1/40ADI, respectively. Washed rabbit platelets were prepared by the method decribed. An aliquot (2x10⁷ platelets) was incubated with calcium ionophore A-23187 (10^-6M) or thrombin (1U), or without either agonist in the presence or absence of preservatives. Platelets were also pretreated with an individual preservative and then subjected to incubation with agonists. Commercially used amount of each preservative was added to drinking water and administered to a rabbit for 5 days, then washed platelets were prepared and subjected to incubation. Incubation lasted for 5 min at 37℃ with vigorous shaking and was terminated by placing the reaction mixture on ice followed by centrifugation. The magnitude of platelet activation was determined by the amount of thromboxane B₂ (TXB₂) synthesis and platelet aggregation. Among lipid soluble preservatives, Butyl p-hydroxybenzoate had a strong inhibitory effect on both agonists' induced TXB₂ synthesis (Fig. 1A, 2A), and this effect revealed to be irreversible in vitro (Fig. 3, 4). On the other hand, water soluble preservatives (sodium dehydroacetate, sodium benzoate, potassium sorbate, and sodium propionate) inhibited A-23187 induced TXB₂ synthesis irreversibly at the maximum concentrations used (Fig. 1B, 5), and exerted concentration dependent reversible inhibitory effect against thrombin induced TXB₂ synthesis in vitro (Fig. 2, 6). In an ex vivo experiment, in contrast to in vitro experiment, hinokithiol and sodium dehydroacetate inhibited both agonist induced TXB₂ synthesis. But butyl p-hydroxy benxoate had no effect on TXB₂ synthesis. Platelet aggregation was inhibited or suppressed in those samples prepered from administration of lipid soluble preservatives (Fig. 9, 10), but any effect was observed in samples from administration of water soluble ones. There were no significant differences of blood biochemical assay data between pre and post administration of preservatives (Table 1).

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A 90-day repeated dose toxicity study of tamarind pigments cocoa brown TRSP(B) was conducted in Sprague-Dawley SPF rats [Crj:CD(SD)] by dietary administration at dose levels of 0 (powdered basal diet), 1.25, 2.5 and 5.0%. The mean intake of test article for the 1.25, 2.5 and 5.0% groups during the administration period was 803.2, 1669.2 and 3278.1 mg/kg/day in females, respectively. During the entire period of administration, there were no test article related changes in the clinical signs, body weights, food consumption, food effeciency, ophthalmology, urinalysis, hematology, blood chemistry or pathology. Based on these results, it was concluded that the nontoxic dose of tamarind pigments cocoa brown TRSP(B) is 5.0%, the maximum concentration by dietary administration, for each sex (3278.1 mg/kg/day for males and 3885.1 mg/kg/day for females) in the present study.

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To investigate antibacterial action of fermented milk against food-poisoning bacteria, overnight culture of Salmonella serotype Enteritidis NIHJ G14 (SE), Salmonella serotype Typhimurium LT2 (ST) and clinically isolated Vibrio parahaemolyticus V17 (VP) cells were incubated at 37℃ with a liquefied yoghurt product and survival of the cells were monitored by CFU assay. Viable SE cells in the product dramatically dereased by 2 logs after 2 h incubation and could not be detected at 4 h incubation. The product also showed bactericidal effect against ST cells. Decline rate of the cells was faster than that of SE. Bactericidal effect of fermented milk against VP was further remarkable because the viable VP cells could not be detected within 5 min. These bactericidal activities were not affected when lactic acid bacteria in the product were completely inactivated by heating or removed by membrane filtration. On the other hand, SE, ST or VP cells in neutralized samples slightly grew in the early stage of incubation, then reversed themselves to decline rapidly by production of organic acids including lactic acid by lactic acid bacteria in the product. The skim milk solution whose pH was adjusted at 4.0 by lactic acid, also exhibited bactericidal activities against these bacteria. However, this phenomenon was not obvious when pH of the skim milk solution was adjusted by hydrochloric acid instead of lactic acid. These results suggest that free form of lactic acid molecule is a major factor of the potent bactericidal activity and affects viability of the cells. This molecule must permeate into bacterial cells through cell membrane and perturb cellular pH to become acidic. Furthermore, the fermented milk samples represented a marked zone of inhibition against Staphylococcus aureus by an agar-diffusion method. Taken together, the fermented milk must exhibit bactericidal activity against broad spectrum of bacteria.

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A method for the simultaneous analysis of eight kinds of preservatives and sodium saccharin (SacNa) in food products using ion-pair high performance liquid chromatography (HPLC) was developed. The preservatives of interest were dehydroacetic acid (DHA), sorbic acid (SoA), benzoic acid (BA), ethyl p-hydroxybenzoate (Et-PHBA), propyl p-hydroxybenzoate (n-Pro-PHBA), isopropyl p-hydroxybenzoate (iso-Pr-PHBA), butyl p-hydroxybenzoate (n-Bu-PHBA) and isobutyl p-hydroxybenzoate (iso-Bu-PHBA). These food additives were separated on an inertsil ODS-2 column (150 x 4.6 mm I.D.) using 50 mM monosodium dihydrogenphosphate-acetonitrile solution (66:34) containing 2 mM cetyltrimethylammonium bromide as the mobile phase and detected with a photodiode array detector at 305 nm for DHA, 254 nm for SoA, Et-PHBA, n-Pr PHBA, iso-Pr-PHBA, n-Bu-PHBA and iso-Bu-PHBA, and 230 nm for BA and SacNa. Comparison of the measured spectra with reference spectra allowed qualitative analysis (Fig. 1 and 2). The combination of dialysis extraction and liquid extraction with organic solvent is often used for pretreatment. However, purification with large amounts of organic solvents after dialysis extraction is a serious concern in terms of inductrial hygiene. In addition, the use of many types of analytical instruments causes operational complexity. For these reasons, a new method for concentrating and purifying dialysis extracts was developed. A comparison of four types of mini-columns revealed the Sep-Pak PS-2 column to have the best retainability (Fig. 3). Ten ml of methanol was used as the eluent from this column (Fig. 4). When 3, 0.5 and 0.01 g/kg of standard substances were added to the samples, excellent average recoveries of 99.1% were achieved (Table 1). The results obtained from 21 samples, preservatives or SacNa being detected were in excellent agreement with the values of dialysis-gas chromatography (Table 2 and Fig. 5). Thus, combination of dialysis extraction and solid phase extraction facilitates handling of many samples of various types, reducing the work load and the amounts of organic solvents required.

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A novel determination method of acidic-protease was developed by using casein-gellan gum gel in a microplate. Acidic casein solution (in 0.72% lactic acid, pH2.6) was mixed with gellan gum at 60℃ and solidified in wells of a microplate. Casein micelle was dispersed in the gellan gum gel as colloidal perticle. By adding pepsin as a standard acidic endo-protease on the well, OD₆₅₅ of the casein dispersed gel was decreased by the pepsin action. The intensity of the pepsin activity was in logarithmic proportion to regression coefficient of the reaction degree and the reaction time. The results of acidic protease activity determined by present method gave good agreement with the values obtained by the conventional method. By using present method, it may be possible to determine quick acidic-protease activity and/or multi-sample treatment by an automatic devise.

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Axenic shoot culture of Mentha piperita was established from young shoots of plants cultivated in a field, and essential oil and phenolic production of M. piperita shoots cultured on Murashige-Skoog (MS) solid medium was investigated. The main compound of M. piperita cultivated in a field is normaly menthol, but carvone was detected as a main compound in the shoots cultured on phytohormone-free MS solid medium under 16h/day light for 6 weeks. In contrast menthol was not detected by GC-MS analysis in the shoot culture. M. piperita shoots cultured on phytohormone-free MS solid medium in the dark for 6 weeks produced only carvone. M. piperita plantlets obtained by culturing in vitro for 3 weeks were transplanted to soil, and essential oil was examined. Carvone was detected as a main compound during cultivation period (until 7 weeks after transplantation), while production of menthol started at week 3 after the transplantation. Rosmarnic acid (RA), caffeic acid derivative, was detected in M. piperita shoots cultured on MS solid medium under 16h/day light and in the dark, but other polyphenols such as lithospermic acid and lithospermic acid B were not detected by HPLC analysis. Potted plants, regenerated from shoot cultures, accumulated RA (ca. 4%) at a relatively high level as a main compound after 7 weeks of cultivation.

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Polyphenol contents in eight Cornus plants (C. kousa var. chinensis, C. kousa 'Milky Way', 'Gold Star', 'Satomi', 'Snowboy', C. capitata 'Mountain Moon', C. drummodii 'Eddie's White Wonder' and C. officinalis) were determined. C. capitata leaves in vivo contained large amount (1.46% as dry weight) of hydrolyzable-type tannin 1,2,3,4,6-penta-O-galloyl-β-D-glucose, which level was 2-10 times larger than those of the other Cornus species. Shoot cultures of C. capitata 'Mountain Moon', C. kousa var. chinensis and C. kousa 'Milky Way', were established and the polyphenol production in the tissues was also investigated. The major polyphenol constituent was mono-galloylglucose (β-glucogallin) and the content of the other polyphenols was not so high. Micropropagation of two Cornus species (C. capitata 'Mountain Moon' and C. kousa var. chinensis) was succeeded by the method of shoot culture using two different media i. e. BW solid medium with NAA-BA (C. capitata 'Mountain Moon') or BA (C. kousa var. chinensis) for shoot proliferation and 1/2 BW solid medium with NAA-IBA (C. capitata 'Mountain Moon') or IBA-NAA (C. kousa var. chinensis) combination and activated charcoal for root proliferation. The subculture periods of the two plants were totally 1281 days (C. capitata 'Mountain Moon') and about 4 years (C. kousa var. chinensis), respectively. The maximum number of the subculturing shoots reached to 2463 (C. capitata 'Mountain Moon') and 869 (C. kousa var. chinensis), respectively. Acclimatization of the shoots was succeeded at a high rate and the plantlets grew well in a greenhouse. The high contents of galloylglucoses of Cornus plants will open new demand for these plants as a new resource for the production of useful natural polyphenols which would be applicable as anti-oxidative food ingredients.

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Safe ranges for intake of food additives are expressed as ADIs (acceptable daily intake, in unites of mg of additive/day/kg of body weight). An actual study of personal daily intakes of various types of food additives by Japanese people makes comparisons with the ADI possible, thus confirming whether intakes are within safe levels. Food additives in Japan are conveniently classified into two groups. Group A includes synthetic substances which do not naturally occur in food. On the other hand, Group B additives are naturally-occurring and nature-identical substances which may be contained in food as an ingredient. In the course of our survey of the daily intakes of food additives since 1976, the authors have studied the daily intake of Group B food additives in Fiscal year 1995-1996 in Japan. Table 1 shows the results of the present study. The total daily intake of 160 subject compounds among 56 Group B substances by Japanese people amounted to 16128.6 mg/day, slightly decreasing compared with that of 10 years before. About fifty-seven percent of the total intake came from processed foods and the rest from fresh foods. The highest three additives taken from processed foods were D-sorbitol, glutamic acid and glycerol, on the other hand, those from fresh foods were citric acid, D-sorbitol and malic acid. Intake of substances of Group B additives was high in group 1 (seasonings and beverages) among processed foods, while group 7 (fruits, vegetables, mushrooms and seaweeds) was found to be highest source among fresh foods. In the present study, no significant different intake pattern was found among regions of western, central and eastern Japan. Furthermore, it was demonstrated that daily intakes of 12 substances in the Group B additives filled reasonably the dietary recommendations for Japanese people. In the substances of Group B compounds, especially in those specified the ADI, it was noticeable that the intake of nitric acid extended to 125.4% of the ADI. However, it was clearly suggested that most of the substance was taken from fresh foods as naturally occurring ingredient.

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Industrial Kefiran production was developed in order to use Kefiran as an ingredient of cosmetic or thickning ingredient of beverage. In addition, chemical modification of purified Kefiran was also performed by acylation, since 5% (v/v) ethanol solution containing 2% (w/v) of purified Kefiran was gelificated under standing at 5℃. By a fermentation method of a mixed culture of Lactobacillus kefiranofaciens KF-75 and Torulaspora delbrueckii IFO 1626, Kefiran concentration in culture broth was 3700mg/l, and this concentration was adequate for industrial Kefiran production. Among chemical modified Kefirans, especially, succinyl Kefiran was almost completely prevented gelling. For example, 40% (v/v) ethanol solution containing 2% (w/v) of succinyl Kefiran was able to preserve state of stable solution in spite of standing at 5℃ for 6 months.

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Characteristics of a novel method of α-amylase activity determination were investigated. The Principles of the present method were based on the light absorbance change through a starch-agar gel made in a microplate well by α-amylase action. The sensitivities of β-amylase and glucoamylase with this method were less than 2% to the sensitivity of α-amylase. α-amylase activity in a extract from rice koji and Rhizopus culture liquid were successfully determined and glucoamylase was not affected to determine α-amylase activity as a result. Since the present method can be possible to determine α-amylase activity selectively, this method was considered as an effective for quick and multi-sample treatment by an automatic devise.

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Polyphenol content in leaf blades of five strawberry cultivars (Fragaria x ananassa Duch. cv.: 'Toyonoka', 'Hinomine', 'Nyohou', 'Harunoka', and 'Haruyoi') was determined by HPLC. Among 7 polyphenols, i.e., (+)-catechin (1), (+)-afzelechin-(4α-8)-(+)-catechin (2), procyanidin B3 (3), procyanidin B6 (4), pedunculagin (5), (+)-taxifolin 3-O-α-L-arabinofranoside (6) and (+)-taxifolin (7), the major constituents accumulated were 1 and 3. In all cultivars, the contents (% dry weight) of 1 and 3 were in the ratio of almost 1:1 at every season, which levels changed between the range of 0.1-1% during a year.

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A thermophilic actiomycete, Thermomonospora curvata KP1246 produced extracellular CM-cellulase when cultivated at 55.C on an medium optimized for enzyme pdocution. The enzyme was most active at 68.C for 120 min. The enzyme preparation was found to exhibit activities towards both Avicel (crystalline cellulose and cellobiose. HPLC analysis extablished that glucose was the major end-product of the conversion of cellulose by using this enzyme fraction.

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The effects of dissolved oxygen on the photo-stability of curcuminoids, such as curcumin (I), demethoxycurcumin (II) and bisdemethoxycurcumin (III) were studied and the following results were obtained. 1) For photo-irradiation, the curcuminoids were less stable after bubbling argon gas through the solution than after bubbling exygen gas. Therefore, it was suggested that dissolved oxygen was involved in the photo-stability of curcuminoids. 2) I, II and III were isolated for independent photo-irradiation. Their absorption maxima were 425 nm, 422 nm and 418 nm, respectively. Namely, the absorption maximum was shifted to longer wave lengths according to the number of methoxyl groups. 3) After the photo-irradiation of II and III, compounds with a new ring formation similar to that formed in I reported in the preceding paper were detected. Furthermore, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and p-coumaric acid were also detected as the decomposed products of III, and vanillic acid, vanillin and ferulic acid were observed in II along with the 3 compounds detected in III.

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Recently, health consciousness has developed with increased incidence of life-style related diseases. In particular, hyperlipidemia due to high caloric intake accounts for about 20 percent of workers and the development of sweeteners which do not elevate blood sugar levels has been awaited. However, sugar alcohol is known to have a laxative effect as a physiological effect on the human body. This should be kept in mind in applying to food. Since xylitol, which is currently mixed in a variety of food products, also has a laxative effect, this study was conducted to estimate its maximum no-laxative oral dose for the purpose of ensuring safety in humans. A total of 40 subjects in their twenties to fifties, 5 males and 5 females in each decade, were studied. Single oral doses of xylitol were administered to investigate the appearance of diarrheal symptoms. 1) The maximum no-effect dose of xylitol in causing diarrhea when given once daily was estimated to be 0.3g/kg body weight in both males and females. The daily maximum no-effect dose calculated on the basis of average body weight of each 20 subjects was 20.8g in males and 15.8g in females. (Figure) 2) The dose to cause diarrhea in 50% of subjects (50% effective dose, ED₅₀) was 0.52g/kg in males and 0.70g/kg in females and, on the basis of average body weight of each 20 subjects, 36.1g/day in males and 36.9g/day in females. 3) The time to the onset of diarrhea after ingestion who 2 to 3 hours and the time to return to normal abdominal condition was 10-12 hours. No significant differences in these time intervals were found between sexes. 4) Main abdominal symptoms associated with ingestion of xylitol were borborygmus, lower abdominal pain and nausea.

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To study whether individual specifications for Hg and Cd should be established for annatto extract and water-soluble annatto in Japan, the contents of Hg and Cd in commercial products were determined. The Hg level in one product (2.25μg/g) exceeded the limit of the EU (1 mg/kg) and those in totally four products were higher than 0.1μg/g. Therefore, it was considered that purity test for Hg may be necessary for annatto extract and water-soluble annatto in Japan. On the other hand, purity test for Cd may not be necessary, because all the Cd levels in the commercial products were lower than one tenth the EU limit (1 mg/kg).

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Kefir is a popular fermented milk produced by a starter, the so-called Kefir grains, in the Caucasus region of Russia. Kefiran contained in Kefir is a polysaccharide with a repeating unit of Glc-Gal-(-Glc)-Gal-Gal-Glc, and its molecular weight is 1,000-4,000 kDa. Lactobacillus kefiranofaciens KF-75 isolated from the Kefir grains was improved to yield and industrial Kefiran production of 2 kg per day of purified Kefiran, at present. An aqueous solution of 1% (w/v) Kefiran was gelatinized automatically by standing at low temperature (5℃), nor aqueous solution of 1% (w/v) Succinyl Kefiran in 40% (v/v) ethanol concentration. Because of the high viscosity (60mPa・s, at 20℃) of 1% (w/v) Succinyl Kefiran aqueous solution (pH6.5), showed useful properties, both as a beverage ingredient for adding viscous accretion property, and as a cosmetic ingredient for human skin for giving moisture and good feeling accretion properties.

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[in Japanese]

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It has been reported that some kinds of perilla extracts had anti-allergic activities. During the investigation of in vitro anti-allergic activity, it was found that red perilla extract had efficieny activity. A water soluble red perilla extract was prepared. And existence of caffeic acid, rosmaric acid and luteorin which have been reported to have anti-allergic activity was confirmed in the extract. It was also found that the red perilla extract had strong SOD-like activity and inhibitory activity for hyalurobidase. Tablets containing the red perilla extract were prepared and given for the person having symptoms of pollinosis. Effects of red perilla extract on the symptoms of pollinosis were examined comparing that of Rubus suavissimua extract. The results suggested that red perilla extract has more efficient effects for relieving the symptoms of sneezing, of rhinoclesis and of rhinorrhea than Rubus suavissimus extract. No side effect was reported.

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