The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila

Resin glycosides are secondary metabolites exclusive to the convolvulaceous plants. In this study, crypthophilic acids A–C (1–3), the first resin glycosides occurring in another family (Scrophulariaceae), and the other constituents of Scrophularia cryptophila were examined for in vitro antiprotozoal and antimycobacterial potentials. Except for crypthophilic acid B (2), all tested compounds exhibited growth-inhibitory effect against Trypanosoma brucei rhodesiense, with l-tryptophan (6) and buddlejasaponin III (7) being the most potent ones (IC₅₀'s 4.1 and 9.7 μg/ml). In contrast, the activity towards Trypanosoma cruzi was poor, and only crypthophilic acid C (3), 6 and 7 were trypanocidal at concentrations above 40 μg/ml. With the exception of 2 and 6, all compounds were active against Leishmania donovani. Harpagide (4) and 3 emerged as the best leishmanicidal agents (IC₅₀'s 2.0 and 5.8 μg/ml). Only compounds 3, 6 and 7 showed antimalarial activity against Plasmodium falciparum with IC₅₀ values of 4.2, 16.6 and 22.4 μg/ml. Overall the best and broadest spectrum activity was presented by compounds 3 and 7, as they inhibited all four parasitic protozoa. None of the isolates had significant activity against Mycobacterium tuberculosis (MICs >100 μg/ml) or were toxic towards mammalian (L6) cells. This is the first report of antiprotozoal activity for natural resin glycosides, as well as for harpagide (4), acetylharpagide (5), tryptophan (6) and buddlejasaponin III (7).     

Effect of Calcium and Zinc on the Activity and Thermostability of Superoxide Dismutase

The effect of calcium and zinc ions on superoxide dismutase (SOD) from four plant species (Taxus baccata, Pinus sylvestris, Medicago rigidula, and Zea mays) was followed at three temperatures: optimal (20 °C), increased (50 °C), and high, inhibiting temperature (70 – 80 °C). At 20 and 50 °C in vitro added calcium increases SOD activity, but the degree was different for the plants investigated. The effect of zinc ions at the same temperatures varied in the investigated plants from activation to inhibition. An inhibiting effect of high temperature on SOD activity was diminished in the presence of calcium or zinc ions. It was shown that calcium and zinc ions can increase activity and thermostabilize different SOD isoforms.     

Effects of temperature on acaricidal and insecticidal activities of the benzoylureas flucycloxuron and diflubenzuron

Effects of temperature on the activity of flucycloxuron on larval stages of Panonychus ulmi (Koch), based on LC₅₀ values, were highly significant (P < 0.001) with temperature coefficients of-1.7 in both the ranges of 15° to 25°C and 20° to 30°C. The slopes of probit regression lines at 15° and 20°C were significantly steeper than those at 25° and 30°C. As a consequence the temperature coefficients based on LC₉₀ values were-4.4 and-2.2, for the 2 temperature ranges. The ovicidal activity of flucycloxuron on P. ulmi was low and was only statistically detectable at 20°C (LC₉₀ of 84 mg a.i./l). In studies with larvae of Aedes aegypti (Linnaeus), Leptinotarsa decemlineata (Say), Plutella xylostella (Linnaeus), Spodeptera exigua (Hübner) and Spodoptera littoralis (Boisduval) probit regression lines were parallel over temperature. The activity of flucycloxuron on these five insect species was not affected by temperature. Based on LC₅₀ values, diflubenzuron showed positive temperature coefficients on P. xylostella of + 2.1 at 15° to 25°C and + 2.5 at 20° to 30°C. For S. littoralis the temperature coefficient was positive (+ 2.4) at 15° to 25°C but negative (-1.9) at the 20° to 30°C range. Temperature coefficients of diflubenzuron were neutral for A. aegypti, L. decemlineata and S. exigua. In the design and analysis of these studies special allowance was made for date effects and variation in natural mortality over temperature.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

Organic acids and water-soluble phenolics produced by Paxillus sp. 60/92 together show antifungal activity against Pythium vexans under acidic culture conditions

Ectomycorrhizal fungi can produce antifungal compounds in vitro as well as in symbiosis with the host plant that can reduce root diseases. The objective of this study was to isolate antifungal compounds from culture filtrate of Paxillus sp. 60/92, which can form mycorrhizas with Picea glehnii seedlings. Culture filtrate of Paxillus sp. 60/92 showed antifungal activity against Pythium vexans at pH 3–4 but not at pH 5–10, although sterile MMN-b liquid medium (pH 3–10) did not show antifungal activity. Upon separation of antifungal compounds in the culture filtrate, antifungal activity was detected in the organic acid and water-soluble phenolics fractions adjusted to pH 3. Although antifungal activity of individual fractions was lower than that of the culture filtrate, a mixture of these fractions showed antifungal activity similar to that of the culture filtrate. Furthermore, antifungal activity of oxalic acid, which is known to be produced by Paxillus involutus, was increased by mixing with the water-soluble phenolic fraction. Our findings indicate that Paxillus sp. 60/92 produces organic acids and water-soluble phenolics that together show antifungal activity at pH 3–4 against P. vexans.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.     

Biochemical characterisation of the esterase activities of wine lactic acid bacteria

Esters are an important group of volatile compounds that can contribute to wine flavour. Wine lactic acid bacteria (LAB) have been shown to produce esterases capable of hydrolysing ester substrates. This study aims to characterise the esterase activities of nine LAB strains under important wine conditions, namely, acidic conditions, low temperature (to 10°C) and in the presence of ethanol (2–18% v/v). Esterase substrate specificity was also examined using seven different ester substrates. The bacteria were generally found to have a broad pH activity range, with the majority of strains showing maximum activity close to pH 6.0. Exceptions included an Oenococcus oeni strain that retained most activity even down to a pH of 4.0. Most strains exhibited highest activity across the range 30–40°C. Increasing ethanol concentration stimulated activity in some of the strains. In particular, O. oeni showed an increase in activity up to a maximum ethanol concentration of around 16%. Generally, strains were found to have greater activity towards short-chained esters (C2–C8) compared to long-chained esters (C10–C18). Even though the optimal physicochemical conditions for enzyme activity differed from those found in wine, these findings are of potential importance to oenology because significant activities remained under wine-like conditions.     

Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73

An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Trehalase activity in mycorrhizal and nonmycorrhizal roots of leek and soybean

Root extracts of leek (Allium porrum L.) and soybean (Glycine max L. Merr.) showed trehalase activity which was inhibited by phloridzin and was several times higher than the activity of general -glucosidase. The activity had an acidic optimum. Trehalase activity in extracts of sporocarps and extraradical mycelium of the arbuscular mycorrhizal fungus Glomus mosseae Nicol. & Gerd. (Trappe & Gerd.) was higher than in root extracts and had an optimum at pH 7. Following inoculation with G. mosseae, trehalase activity increased in mycorrhizal roots above the levels observed in nonmycorrhizal roots. Irrespective of fungal colonization, root trehalase activity increased in the presence of Mg²⁺, decreased in the presence of Mn²⁺ and Zn²⁺, and was unaffected by Na₂EDTA.     

A Comparative Analysis of the Ice Nucleation Activity of Pseudomonad Cells and Lipopolysaccharides

The paper deals with the study of the ice nucleation activity of the cells, extracellular lipopolysaccharides (ELPSs), lipopolysaccharides (LPSs), and LPS structural components (lipid A, core oligosaccharide, and O-specific polysaccharide) of Pseudomonas fluorescens, P. syringae, P. fragi, and P. pseudoalcaligenes. Aqueous suspensions of intact cells of P. syringae IMV 1951 and IMV 185 began to freeze at –1 and –4°C, respectively. This suggests that these cells possess ice nucleation activity. Aqueous cell suspensions of two other strains, P. fluorescens IMV 1433 and IMV 2125, began to freeze at lower temperatures than did distilled water (–9°C), which suggests that the cells of these strains possess antifreeze activity. The ice nucleation activity of the bacterial strains studied did not show any correlation with their taxonomic status. The ice nucleation activity of ELPSs depended little on their concentration (within a concentration range of 0.2–0.4%). In most cases, the ice nucleation activity of ELPSs, LPSs, and LPS structural components differed from that of the intact cells from which these biopolymers were obtained. This may indicate that the biopolymers under study play a role in ice nucleation but this role is not crucial. The relationship between the structure of LPSs and their effect on ice nucleation is discussed.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Comparative evaluation of antioxidant, nitrite scavenging, and antitumor effects of Antrodia camphorata extract

The effects of decoctions prepared from Antrodia camphorata on antioxidant, nitrite scavenging, and antitumor activities were investigated. Glutathione (GSH) production was 18.2 μM/g of rat liver under the influence of a methanol extract, a result similar to with the effect of silymarin administration. GSH peroxidase activity was about 2.0-fold higher than with silymarin administration. Superoxide dismutase activity was 18.9 U/mg protein, about 2.5-fold higher than the control group. The hot water extract (1000 μg/mL) showed the highest nitrite scavenging activity at 98.1%, similar to those observed with BHA and vitamin E. Among a variety of human cancer cell lines, when the concentration of hot water extract was increased from 31 to 500 μg/mL, the viability of Hep3B cell was decreased from 100 to 60.7%. On the other hand, in the case of methanol extract, it was decreased from 98.4 to 10.0%. These results supported the conclusion that the antioxidant, nitrite scavenging, and antitumor activities of this A. camphorata methanol extract indicate a potential source for the development of various health supplements and pharmaceutical and nutraceutical applications.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.