N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM) was synthesized enzymatically from highly concentrated N-(benzyloxycarbonyl)-L-aspartic acid (ZA) and L-phenylalanine methyl ester (PM). An approximate linear relationship was obtained between the initial reaction rate and the substrate concentration up to 1000 mmol·dm-3. The highest reaction rate obtained at the concentration ratio of ZA and PM was 0.5. When the ratio of 0.5 ([ZA]/[PM]=400 mmol·dm-3/800 mmol·dm-3) was employed, it was found that the high reaction rate and the conversion of ZAPM synthesis could be performed at pH 6.0, 40°C, with 10 g·dm-3 NaCl addition.
The objective of this research was to determine the microflora of full fat and reduced fat Edam cheese during maturation. Reduced fat (16% fat) and full fat (28% fat) Edam cheese was stored up to 20 weeks at 6°C after manufacture. The cheeses were analyzed for pH, moisture, total solids, fat and protein content. Reduced fat cheese had higher protein and water content than full fat. We determined total bacteria, lactococci, and lactobacilli counts of reduced and full fat cheese during 5 months of ripening. Results revealed that total bacteria counts were about 1.9×105/g at one day for reduced fat cheese and 8×106/g for full fat cheese. Total counts decreased during maturation in both cheeses. Initial lactococcal counts were about the same in both products.
A precooling step is necessary to convert heated soymilk to freeze-gel. In order to estimate the significance of precooling, soymilk was precooled at various temperatures and then frozen. Based on the appearance and stress-strain curve of thawed soymilk which was heated, precooled and freeze-stored, precooling at —5°C was most suitable for freeze-gelation. Although the effects of precooling temperatures on precipitation levels, syneresis and SDS-PAGE patterns were relatively small, there were great differences in freezing curves and ice crystal morphology. It was very important that the precooling temperature be lower than the freezing point of soymilk in order to form small and spherical ice crystals. The uniform distribution of these small ice crystals was considered to be important for the gel-like texture.
Linoleic acid was encapsulated with gum arabic, pullulan or maltodextrin at various weight ratios of the fatty acid to the wall material, and the fraction of the linoleic acid extracted from the microcapsule with chloroform was observed as a function of time. A certain amount of the linoleic acid could not be extracted even if the microcapsule was immersed in chloroform for a long period. The fraction of extractable linoleic acid was lower for the microcapsule having the lower weight ratio, and significantly changed between the weight ratios of 0.5 and 1.0 for gum arabic and pullulan. It was also shown that the fraction of extractable linoleic acid correlated fairly well with the fraction of easily oxidizable linoleic acid. An increase in the fraction was observed with 1-day storage of linoleic acid encapsulated with gum arabic at the ratio of 0.5, and the initial extraction rate linearly increased with the period of storage.
An apparent specific heat model was introduced to predict three dimensional heat transfer in food thawing by far-infrared radiation. A finite element method was applied to solve the fundamental equation and was used to examine the influence of the temperature of heater, and the influence of auxiliary heater attached beneath the sample. To keep the food from overheating, control of the surface temperature was discussed. Experiments of far-infrared heating were carried out to verify the method. Temperature distributions in samples were measured and corresponded with theoretical predictions.
The first step of the extraction of alcohol acetyltransferase (AAT) from fruits is usually achieved by preparation of free-cells with enzymatic maceration of tissues. The free-cells from strawberry tissue were suspended in a suitable buffer and disrupted by centrifugation. The supernatant held a strong activity of AAT. The addition of 0.1% Triton X-100 to the extraction buffer indicated that a minor part of AAT was bound-form. Strawberry pulp was grated in isotonic solution (0.5 M sorbitol and 2.5% ficoll), then fractionated through a sucrose-gradient layer (20-65%) by centrifugation (82,200×g, 3 h). The distribution of AAT in the subcellular fraction of the grated pulp was similar to those of glucose-6-phosphate dehydrogenase (cytosolic enzyme) and anthocyanin (vacuolar pigment). Protoplasts were prepared by enzymatic digestion, and fractionated by stepwise gradient sucrose centrifugation, into the collapsed vacuole part and miniprotoplasts. Miniprotoplasts consisting only of cytosol, were formed from protoplasts during the preparation. Since most activities of AAT were detected in the miniprotoplast fraction, we concluded that most AAT was located in the cytosol.
An endopeptidase in powdered yam was purified using a combination of anion-exchange and gel-filtration chromatographies. The preparation gave 9.2-fold purification with yield of 19% against the crude extract. The endopeptidase was classified as a serine endopeptidase with inhibitory spectrum, with an optimum temperature and pH of approximately 60°C and 7.1, respectively. It also had high heat stability and high salt resistance.
Carthamin, the red pigment from Carthamus tictorius L. (safflower) is almost water-insoluble, so the compounds which solubilized it were screened from polysaccharides, flavonoids, and so on. When glycosyl hesperidin, which had the highest effect, was added to carthamin at the ratio of carthamin/glycosyl hesperidin=1/5 at pH 5.0, the content of soluble carthamin was about 14 times greater than that without its addition. The color tone of solubilized carthamin became more reddish, while the original one was yellowish red at pH 5.0 according to the Hunter Lab diagram system. On the basis of these results, carthamin solution could be prepared more effectively from the petals of safflower using glycosyl hesperidin instead of cellulose which is now used industrially.
When sarcoma-180 (SR-180) cells were incubated with α- or β-subunit prepared from insoluble ovomucin in the gel fraction of egg white, the cell proliferation rate was apparently reduced by the addition of β-subunit, while it was largely unaffected by α-subunit. Examinations by scanning and transmission electronmicroscopy indicated a dose- and time-dependent cytotoxic effect of β-subunit on SR-180 cells, whereas β-subunit did not injure mouse peritoneum macrophage used as normal cells. Ultrastructurally, SR-180 cells treated with β-subunit showed changes such as swelling and bleb formation of microvilli on cell membrane, irregular clumping of chromatin, irregular nuclear shape, and marked swelling of organelles in cytoplasm associated with cell degeneration in necrotic change. Furthermore, apoptotic analysis of the cultured SR-180 cells also demonstrated that β-subunit did not induce the apoptotic DNA fragmentation.
Changes in chlorophyll (Chl) degrading enzyme activities were determined to clarify the pathway of Chl degradation in stored broccoli (Brassica oleracea L.) florets. Chl content in the florets decreased greatly after 2 days of storage at 15°C, whereas at 3°C they showed almost no change during storage. Chlorophyllase activities decreased gradually during storage at 15°C and 3°C. Chl degrading peroxidase activities showed a sharp increase and the hydrogen peroxide content a slight increase with the advance of floret yellowing at 15°C. Chl oxidase (linolenic or linoleic acid-dependent) activities decreased gradually during storage at 15°C. We suggest that Chl degrading peroxidase could be the main factor involved in Chl degradation in stored broccoli florets.
The apoptosis-inducing effect of fucoxanthin on human leukemic HL-60 cells was investigated. Fucoxanthin, which was obtained from the brown alga Undaria pinnatifida, inhibited the proliferation of HL-60 cells and induced DNA fragmentation on agarose gel electrophoresis, a typical characteristic feature of apoptotic cells. The results of sandwich ELISA using anti-biotin-antibody and anti-DNA-antibody also demonstrated DNA fragmentation in accordance with the increase in fucoxanthin concentration and incubation time. Since the level of DNA fragmentation did not increase after 24 h incubation, fucoxanthin appeared to have some effect on cell cycle. In contrast, β-carotene did not show an apoptosis-inducing effect on HL-60 cells. These results suggested that a carotenoid structure might be crucial for inducing apoptosis.
A total of 125 strains of lactic acid bacteria were isolated from Miso-paste products made in Yamanashi prefecture, Japan and tested for bacteriocin production by modified agar well diffusion method. Only one isolate designated as GM005 showed antibacterial activity against Bacillus subtilis JCM1465T and Staphylococcus aureus JCM12544T. This suggested that the antibacterial substance produced by strain GM005 could be of a proteinaceous nature and that the antibacterial action was a bacteriocin. The bacteriocin was stable even after autoclaving at 121°C for 15 min in the pH range of 3 to 7, and showed a broad antibacterial spectrum among tested lactic acid bacteria. Strain GM005 was identified as a lactic acid bacterium belonging to the genus Enterococcus. This is the first report dealing with a bacteriocin produced by lactic acid bacteria isolated from Miso-paste.
A. awamori feruloly esterases, termed FAE-a and FAE-b, were purified to homogeneity by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, DEAE-Toyopearl 650 column chromatography, hydrophobic chromatography on Butyl Toyopearl 650S, and gel filtration on Toyopearl HW55. The molecular weights (Mr) of FAE-a and FAE-b were estimated to be 31 KDa and 29 KDa by SDS-PAGE; these isoelectric points were 3.9 and below 3.4, respectively. Both enzymes were optimally active at 45-50°C and pH 5.0-5.5. FAE-a was stable up to 37°C, and FAE-b was stable up to 50°C. Both enzymes hydrolyzed predominantly the feruloylated carbohydrate ester prepared from pineapple stem residue rather than methyl esters of ferulic acid.
α-Galactosidase II from Mortierella vinacea releases galactose from guar gum. This enzyme was easily prepared from the culture filtrate free of β-mannanase activity. Galactose-depleted guar gum samples with various mannose/galactose (Man/Gal) ratios were prepared by treatment with the enzyme. The samples showed a synergistic interaction with xanthan gum, and formed gels like the locust bean gum. These results indicate that α-galactosidase II from M. vinacea is suitable for modifying the Man/Gal ratio of guar gum and that galactose-depleted guar gum might be a substitute for locust bean gum as a gel promoter.
The intraperioneal administration of β-subunit prepared from egg white ovomucin was found to suppress the growth of subcutaneously xenografted sarcoma-180 cells in mice and cure the tumor. In the present system, β-subunit (5 mg/kg of body weight) was injected to mice daily for 19 days after the 10th day when tumors had grown to 7 mm in diameter. The light and electron microscopic appearances of tumor portions on day 28 showed that β-subunit-treated tumor cells were in the states of degenerated and necrotic cells, and massive accumulations of neutrophils, macrophages and lymphocytes were found at the margin of the degenerated and necrotic tumor tissue area. These findings suggested that β-subunit brought about the regression of tumor, probably by activating the immune system.
The stimulatory effect of wasabi leafstalk (Wasabia japonica MATSUM.) extract on bone calcification in mouse calvaria tissue culture in vitro was characterized. The extract had an appreciable effect on increasing bone calcium content; the effect was great at 10 and 50 μg/ml, but at higher concentrations (100-300 μg/ml) the effect was weaker. Sinigrin (5-20 μg/ml) had no an effect. Wasabi leafstalk extract (10 μg/ml) did not inhibit the effect of parathyroid hormone (PTH; 10-6 M), a bone-resorbing hormone, in decreasing bone calcium content, and the effect of the wasabi extract on bone calcification was stable when heated (100°C) and in acid or alkaline. It was subjected to gel filtration chromatography with a superose 12 column connected with a high pressure liquid chromatography system. The presence of fraction IV (retention time of 160-200 min) in the tissue culture caused a remarkable elevation of the bone calcium content; the effect was great at a concentration of 100 μl/ml of medium. The present study suggests that the small molecular components in wasabi leafstalk extract have a stimulatory effect on bone calcification in vitro.
The composition of cis-trans isomers of β-carotene in fresh vegetables and fruits was studied using high performance liquid chromatography (HPLC) with a photodiode array detector. All-trans-β-carotene was the predominant β-carotene and accounted for 66-96% of total isomers, depending on the vegetables and fruits. The main cis isomer detected in green-colored vegetables and fruits, such as spinach, broccoli, garland chrysanthemum, asparagus and kiwifruit is 9-cis-β-carotene. In contrast, 13-cis-β-carotene is found mainly in yellow and red colored vegetables and fruits, such as loquat, sweet potato, squash, tomato and watermelon.
Seasonal variations of contents of moisture, protein, fat, ash and glycogen, and compositions of fatty acids, and amino acids were investigated in the meat of the surf clam, Spisula sachalinensis. The glycogen content was highest in July (7.3%) and lowest in January (2.8%). The major amino acids were glycine, taurine and alanine. Taurine was detected at the level of 1100 to 2500 mg/100 g throughout the year, and was largest in February. A reverse correlation was observed between free amino acid content and glycogen level. The content of moisture, protein, fat, and ash, the lipid class compositons and fatty acid compositions were almost unchanged throughout the year.