Research Reports No.24 （2018）

24－01

Development of Antioxidant-Efficiency Assay-Method for Foods

Shin-ichi Nagaoka
Laboratory of Structural Chemistry, Department of Chemistry and Biology,
Faculty of Science and Graduate School of Science and Engineering, Ehime University

Tocopherols (vitamin E analogues), ascorbic acids (vitamin C analogues), tea catechins, carotenes, and so on, which are used as food additives for antioxidants, coloring agents, etc., prevent food degradation caused by oxidation due to free radicals, singlet oxygen, and so forth. Furthermore, they exist in various living tissues and scavenge free radicals, singlet oxygen, etc. to prevent aging. In the present study, we have attempted to develop practical antioxidant-efficiency assay-methods (Aroxyl Radical Absorption Capacity (ARAC), Singlet Oxygen Absorption Capacity (SOAC), Alfa-Tocopherol REcycling Capacity (ATREC) assay methods) for food additives and foods containing the additives.

24－02

Food science studies for prevention of magnesium deficiency by food additives

Akira Ikari
Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University

Japanese people in the modern age have a chronic magnesium deficiency, which is induced by changing in eating habits and excessive stress. Magnesium deficiency may increase the risk of developing lifestylerelated diseases including heart disease and diabetes. However, there is no effective treatment method for magnesium deficiency. The administration of magnesium-containing preparation or supplement in large quantities may cause diarrhea, and thereby further aggravate magnesium deficiency. Therefore, we need to identify a novel regulatory factor which increases magnesium absorption. Transient receptor potential melastatin 6 (TRPM6), which is expressed in the distal tubule of kidney, plays an important role in the regulation of magnesium homeostasis. Because citrates, food additives, increased TRPM6 expression in the renal tubular epithelial cells, we investigated what mechanism was involved in the citrate-induced elevation of TRPM6 expression in the present study. Our data indicated that TRPM6 expression and magnesium influx were up-regulated by the activation of NADPH oxidase and an EGFR/MEK/ERK/c-Fos pathway. Citrates were suggested to have dual function of a pH adjuster and magnesium absorption accelerator.

24－03

Effect of food additives on the periodontal pathogen in vitro

Shigeki Kamitani
Graduate School of Comprehensive Rehabilitation, Osaka Prefecture University

Periodontitis, which commonly appears in the elderly, is a chronic inflammatory disease caused by bacterial infection in the gums, where periodontal pathogen forms oral biofilm. Recently, it has been found that periodontitis is associated with aspiration pneumonia. People with swallowing disorder, which is a risk factor of aspiration pneumonia, usually use the dysphagia diet, which mostly contains food additives, especially artificial sweeteners and mid-chain fatty acids. In general, the development of periodontitis is considered to be affected by foods or constituents in foods, but the effects of food additives on periodontal pathogens remain largely unknown. In this study, we investigated the effect of artificial sweeteners and mid-chain fatty acids on the periodontal pathogen by using Porphyromonas gingivalis.

24－04

Chemical synthesis of carotenoids for reference standards

Yosuke Demizu
Division of Organic Chemistry, National Institute of Health Sciences

For quality assurance of food additives, it is important to establish component standard tests by developing highly accurate analytical and evaluation methods. In this study, chemical synthetic methods for stable and high-quality supply of carotenoids were developed. Many of the carotenoids are supplied from natural raw materials and it is difficult to obtain high-purity standards. Thus, development of analytical methods by establishing total synthetic routes of those carotenoids is required. In this paper, the synthetic methods for crocetin, capsaicin, and capillin were described.

24－05

Studies on effect of food additives on detection of genetically modified food

Kosuke Nakamura
National Institute of Health Sciences, Division of Biochemistry

Effect of food additives on detection of genetically modified (GM) food was analyzed. In this study, the effect of sodium carboxymethyl cellulose (CMC), a food additive used as a thickener and emulsion stabilizer, on detection of GM foods was evaluated. The addition of CMC to processed rice foods at 2% (w/w) concentration inhibited the yield of DNA in the DNA purification step by up to 40% and 70% using ionexchange resin-type DNA purification kit and silica membrane-type DNA purification kit, respectively. The DNA yield from the rice vermicelli commodities containing CMC was significantly lower than that from the CMC-free rice vermicelli commodities. When 2% (w/w) CMC was contained in the rice flour with ＜ 5,000 copies of transgenic genes for GM rice, the false negative rate in the real-time polymerase chain reaction detection targeting the genes was more than 10%. The CMC attenuates the DNA purification efficiency from the rice food samples, and may interfere with the GM rice testing using DNA samples prepared from processed rice foods containing CMC.

24－06

Stimulation of GLP-1 (Glucagon-like peptide-1) secretion by food additives and elucidation of the mechanism

Takanori Tsuda
College of Bioscience and Biotechnology, Chubu University

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Here, we hypothesized that food additives (curcumin) induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. In our previous study, we showed that curcumin significantly increased GLP-1 secretion in vitro. In the present study, we investigated stimulatory pathway of GLP-1 secretion by curcumin, and demonstrated that curcumin induces GLP-1 secretion in vivo. Our results indicated that curcumin-mediated GLP-1 secretion is due to elevation of intracellular calcium ion and activation of CaMKII protein. Moreover, we demonstrated that administration of curcumin significantly stimulated GLP-1 secretion in vivo. These findings provide possible abilities of curcumin to prevent and treat diabetes via the stimulation of GLP-1 secretion.

24－07

Development of asparaginase from lactic acid bacteria for suppression of acrylamide formation in processed food and verification of its usability as a food additive

Mamoru Wakayama
Department of Biotechnology, College of Life Sciences, Ritsumeikan University

Semi-comprehensive screening of potential lactic acid bacteria producing asparaginase usable for mitigation of acrylamide generation in processed food has been performed. In this study, lactic acid bacteria of sixteen species in eight genera were investigated for their asparaginase production. Consequently, all lactic acid bacteria examined except for Pediococcus pentosaceus showed asparaginase activity but low specific activity. Among them, Lactobacillus sakei, Streptococcus thermophiles, Lactobacillus brevis and Lactobacillus delbruekii showed a relatively higher activity than the others. Judging from SDSPAGE, low asparaginase activity of crude extract was considered to be due to the low expression of asparaginase gene but not its low molecular activity. Genes of asparaginases from S. thermophiles and L.brevis have been cloned and expressed in Escherichia coli. Characterization of purified recombinant enzymes have been performed.

24－08

Action research for domestic production of safe and high quality saffron: application for agritherapy and contribution to local resident's health

Kayoko Takaura
The Museum of Osaka University

Saffron (Crocus sativus L.), has been valued as medicine and spice from ancient times. It is highly economical farm product because of complicated processing, but most of the domestic consumption in Japan is compensated by imports from Spain and Iran. Taketa city (Oita, Japan) is known as its major production area, where "Taketa-Saffron Cultivation Method" has been developed, but the number of saffron producers is decreasing because of economy and aging. In this study, we aim to investigate the benefit of that special method, record its experiences and skills, and encourage the technique succession. First, we thoroughly surveyed literature to investigate the development and spread process of the method. Then, we interviewed and recorded the farming work throughout the year, and tried to edit the movies as the training materials. We collected saffron samples from Taketa city and abroad, then revealed the high quality of saffron produced in Taketa city through the morphological investigation.

24－09

The development of high-value added functional food with enzymatically modified isoquercitrin

Hiromasa Uchiyama
Laboratory of Formulation design and Pharmaceutical Technology Osaka University of Pharmaceutical Sciences

Flavonoid compounds contained in functional food tend to show the poorly soluble property. Therefore, it is important challenge to improve the dissolution property to enhance the absorption of them. The objective in this study is to investigate whether enzymatically modified isoquercitrin (EMIQ) can act as the functional additive to improve the dissolution property of poorly soluble compounds. Quercetin (QUE) was used as poorly soluble flavonoid. When amorphous particles of EMIQ and QUE were prepared by spraydrying method, they showed the dramatically improved solubility of QUE compared to untreated QUE powder. Interestingly, the concentration of dissolved QUE increased with an increase in the loading ratios of EMIQ. A linear relationship was obtained between the amounts of dissolved QUE and the concentration of EMIQ. There was no cytotoxicity to Caco-2 cells at levels of 10 % EMIQ solution. Furthermore, the amounts of QUE permeated through the Caco-2 cell monolayers was enhanced with EMIQ. The permeated amount of QUE from spray-dried particle with EMIIQ was 5-fold compared to that of untreated QUE powder. In conclusion, EMIQ is a promising functional food additive to improve the dissolution property of poorly soluble compounds.

24－10

Risk assessment of arsenic in food: analysis of mechanisms of arsenic carcinogenicity

Min Gi
Osaka city university graduate school of medicine, Department of molecular pathology

We previously demonstrated that prenatal exposure to dimethylarsinic acid (DMA), which is the main metabolites of inorganic arsenic, induced lung and liver tumor in male offspring. The purpose of the present study is to clarify the carcinogenic mechanism by which prenatal exposure of DMA induce lung carcinogenesis in male offspring. DMA was administered to female CD-1 mice at the dose of 0 or 200 ppm for 10 days from 8 to 18 days of gestation, and the lungs of male newborn were analyzed. A significant increase in the cell proliferative activity was observed in the lungs of the male newborns of DMA-treated mice as compared with the controls. Analysis of arsenic metabolites in the lungs using HPLC/ICP-MS revealed that prenatal exposure to DMA significantly increased two thioarsenicals DMMTA and DMDTA but not TMAO which is an ultimate metabolite of DMA. Furthermore, methyl donor S-adenosylmethionine (SAM), expression of histone methyltransferase G9a and histone H3K9me3 was significantly increased in the lungs of newborns of DMA-treated mice. These findings suggested that that abnormal histone methylation may responsible for the prenatal DMAV carcinogenicity in lung.

24－11

Survey on food additives used as the functional substances of Foods with Functional Claims

Sayaka Masada
Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences

Food additives are substances generally added to food to preserve flavor or enhance its taste, appearance, or other qualities. Some food additives are recently used as food substances in "foods with functional claims." In this study, we tried to illuminate the actual states of food additives used in foods with functional claims to clarify and discuss the points of their regulation. Twenty-five kinds of food additives were found to be registered as the main substance to provide special function to the foods with functional claims. The multivariate analysis of LC/MS data from food with functional claims and food substances derived from Licorice did not indicate their chemical equivalence in opposition to our previous reports. Additionally, we evaluated the quantity of functional substance in foods with functional claims and carried out the elution test with foods with functional claims to obtain the primary data about elution pattern of the food additives. More investigation should be needed to discuss for establishing the novel regulation of food additives as functional substances in foods.

24－12

Study on preparation of reference materials for analysis of ciguatoxins, principal toxins of ciguatera fish poisoning

Naomasa Oshiro
Division of Biomedical Food Research, National Institute of Health Sciences

Availability of ciguatoxins, causative agents of ciguatera fish poisoning, are extremely limited. To prepare the reference materials of CTXs, we collected fish specimens of Gymnothorax javanicus from the Waters of Okinawa and Amami. While the polar congeners of CTX1B, CTX1B, 52-epi-54-deoxyCTX1B, were detected from flesh specimens, amount of the CTXs were lower than those of the fishes including Lutjanus bohar, Variola louti and Variola albimarginata.
And also, fish powder was prepared from toxic flesh of V. louti and stability and properties are studied.

24－13

Refinement of hepatocarcinogenicity evaluation for furans based on the biological characterization of rat preneoplastic lesions

Shinji Takasu
Division of Pathology, Biological Safety Research Center, National Institute of Health Sciences

Furan induces hepatocellular tumors in rodent. It has been reported that the mutant frequencies of reporter gene are not changed in the liver of furan-treated gpt delta rats. In contrast, the number and area of glutathione S-transferase placental form (GST-P)-positive foci are significantly increased. We previously reported that GST-P positive foci induced by diethylnitrosamine were increased after cessation of the treatment, those by furan being decreased. In present study, to establish the method to distinguish two different types of GST-P positive foci in terms of their kinetics after cessation of carcinogen treatment, we investigated the kinetics of GST-P positive foci induced by 2-methylfuran (2-MF) or 2-pentylfuran (2-PF). 6-week old male F344 rats were given 30 mg/kg/day 2-MF or 100 mg/kg/day 2-PF by gavage for 13 weeks. Number and area of GST-P positive foci in the liver of rat treated with 2-MF or 2-PF were significantly increased. These results indicated that 2-MF and 2-PF might be similar to furan in terms of formation of GST-P positive foci. We are investigating the fate of GST-P positive foci induced by 2-MF or 2-PF after cessation of treatment.

24－14

Analysis of polychlorinated biphenyls in food using a polychlorinated biphenyl clean-up system

Tomoaki Tsutsumi
Division of Foods, National Institute of Health Sciences

We describe the evaluation of an analytical method for determining polychlorinated biphenyl (PCB) concentrations in fish and shellfish using the PCB clean-up system and gas chromatography tandem mass spectrometry (GC-MS/MS). The trueness and repeatability of the method were assessed by performing replicate analyses (n = 5) of fish and shellfish samples (tuna and clam) spiked with PCB mixtures (Kanechlor 300, 400, 500, and 600) each at a concentration of 100 ng/g. The total PCB concentrations found were 92%-94% of the spiked concentrations and the relative standard deviations were ＜1%. The concentrations of seven indicator PCBs (PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, and PCB180) in the spiked samples were 95%-105% of the spiked concentrations, and the relative standard deviations were ＜5%. A certified reference fish sample was analyzed, and the concentrations of the certified congeners were within the uncertainty limits of the certified concentrations. Finally, the clean-up system was used to determine the total PCB concentrations in 16 fish samples, and the concentrations were compared with concentrations determined using a conventional clean-up method using open columns. The total PCB concentrations determined by both methods correlated very well (r = 0.99), and the slope and y-intercept of the linear regression were close to 1 and 0, respectively. The concentrations of the seven indicator PCBs determined by both methods also correlated very well (r = 0.99). The recoveries of the internal standards were 51%-115% for the new method and similar (51%-113%) for the conventional method. The results confirmed that the total PCB concentrations determined using the new and conventional methods were comparable, and indicate that the new method using the PCB clean-up system will be very useful for rapidly determining total PCB concentrations in fish and shellfish.

24－15

The cultivation of evaluating the safety and effectiveness of food additives by expression of inflammatory cytokines in the peripheral leukocytes

Kazuki Mochizuki
Laboratory of Food and Nutritional Sciences, Department of Local Produce and Food Sciences, Faculty of Life and Environmental Sciences, University of Yamanashi

In this study, we aimed to cultivate the method evaluating the safety and effectiveness of an antioxidant β-carotene, and mulberry leaf powder, which is a food including 1- deoxynojirimycin that reduces postprandial hyperglycemia, by measuring gene expression of inflammatory cytokines and redox proteins in the peripheral leukocytes.
Mongolian gerbils, which has the similar metabolism of β-carotene to human being, were fed the highfat diet with/without β-carotene at concentration of 0. 001% (a lower dose) or 0.004% (a higher dose) in the diet. The intake of a diet with a higher dose of β-carotene induced the fibrosis of the liver and protein level of matrix metalloproteinase-9, which is induced during development of the fibrosis of the liver.
Excessive treatment with β-carotene in monocyte-like THP-1 cells, which were cultured with the highglucose condition, induced expression of an adhesion factor CD11a, which is expressed in activated monocytes, and the redox genes (GSR, PRDX2, PRDX6, TXNRD1 and TXNRD3).
Feeding the diets including the mulberry leaf powder at lower dose (0.2%) or a higher dose (1.0%) in the diet reduced the non-fasted blood glucose and expression of redox genes such as Cat, Gpx1 and Sod1 in peripheral leukocytes of type 2 diabetic NSY mice. The feeding the mice the diet with the higher mulberry leaf powder tended to induce expression of an inflammatory cytokines interleukin 1b in peripheral leukocytes.
Combined with these results, intake of optimal doses of antioxidants or food factors reducing the postprandial hyperglycemia can reduce the risks of developing life-style related diseases, however excessive intakes could induce the development of these diseases. Furthermore, our current study suggests that determination of mRNA levels of inflammatory cytokines and redox genes in peripheral leukocytes is useful for the evaluation of the safety and effectiveness of food additives, and assessment of the liver disorder of the Mongolian gerbils is useful for the evaluation of the safety and effectiveness of the β-carotene.

24－16

Study on the metabolite of curcumin produced by human feces

Toshio Niwa
Faculty of Health and Nutrition, Shubun University

We have isolated a metabolite of curcumin produced by human feces. From the analytical data, we considered the structure as 3-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane. We could not use the NMR analysis due to the overlapped signals. We herein synthesized the metabolite to determine the structure. A product, which had same profiles to that obtained from the methylation of metabolite, was prepared from hexahydrocurcumin.

24－17

Metabolites produced by intestinal bacteria exhibit the anti-inflammatory activity in the presence of chondroitin sulfate

Kyohei Higashi
Graduate School of Pharmaceutical Sciences, Chiba University

We have previously found that fecal supernatants decreased the levels of Th1 cytokines including IL-2 and IFN-γ produced by solenocytes. In addition, it was also found that supernatant from feces treated with ampicillin decreased the immunomodulatory activity. We have believed that immunomodulator in feces was involved in anti-inflammatory effect of chondroitin sulfate (CS). In this study, we explored the immunomodulatory in feces. At first, effects of supernatant of feces on the cytokine production were carefully studied by the Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay kit. As results, productions of IL-1α, IL-2, IL-3, IL-4, IL-9, IL-17, GM-CSF, IFN-γ, KC (CXCL1), MIP-1α, MIP-1β and RANTES were remarkably decreased by fecal supernatant and this effect was inhibited by ampicillin. Thus, metabolome analysis of feces treated with or without ampicillin was performed using CE-TOFMS, and it was found that levels of 26 components in feces were substantially changed by ampicillin. Thus, 26 components were added to the culture medium, however, IL-2 production by splenocytes were not influenced. Based on the observations, it is thought that small oligosaccharides or fatty acids produced by gut microbiota may be responsible for the inhibition of Th1 activity.

24－18

Effects of sodium alginate and lactic acid bacteria on invasion and infection of food pathogenic bacteria

Takashi Kuda
Department of Food Science and Technology, Tokyo University of Marine Science and Technology

Low molecular weight sodium alginate (LM-Na) has low viscosity and used for functional beverages and foods. Lactobacillus plantarum Tennozu-SU2 was isolated from acorns. In this study, inhibitory effect of LM-Na and Tennozu-SU2 against Salmonella Typhimurium (ST) in human enterocyte-like HT-29-Luc cells and BALB/c mice was determined. In addition, the effect of Tennozu-SU2 on gut microbiota in normal BALB/c mice was determined. Both LM-Na and Tennozu-SU2 suppressed adhesion and invasion of ST to HT-29-Luc cells, but no synergistic effect was observed. Tennozu-SU2 in drinking water also suppressed ST colonization and ST invasion to liver in BALB/c mice. However, the effect of LM-Na and synergistic effect were not observed. In normal BALB/c mice administered Tennozu-SU2, a part of gut indigenous L. plantarum and L. reuteri were increased or decreased. It is considered that the correlation of these changes of indigenous lactic acid bacteria and ST suppression should be studied in the future

24－19

Inhibitory effects and mechanism of food additives derived from polyphenols on the bacterial superantigen toxin

Yuko Shimamura
School of Food and Nutritional Sciences, University of Shizuoka

Staphylococcal enterotoxin A (SEA) is a toxin protein, and is the most common cause of staphylococcal food poisoning. Polyphenols, such as catechins, are known to interact with proteins. In this study, we investigated the binding of the food additives derived from polyphenols Teavigo (a green tea polyphenol preparation containing 94% (−)-epigallocatechin gallate (EGCG)) to SEA using SPR (Biacore), Nuclear magnetic resonance, (NMR), fourier transform infrared spectroscopy (FT-IR), isothermal titration calorimetry (ITC), and protein-ligand docking. Furthermore, genes whose expression varied by SEA were analyzed using DNA microarray. We found that EGCG could strongly bind to SEA. According to thermodynamic parameters, a negative ΔG indicated that the interaction between EGCG and SEA was spontaneous, and the electrostatic force accompanied by hydrophobic binding forces may play a major role in the binding. Data from Western blot analysis and docking simulation suggest that the hydroxyl group at position 3 of the galloyl group in the catechin structure was responsible for binding affinity with the Y91 of the A-6 region of SEA active sites. From the result of microarray analysis, exposure of SEA to mouse spleen cells markedly induced the response of Th1 cell. Currently, we analyze the effect of Teavigo on SEA-induced toxin activity and the response induced by Th1 cell.

24－20

Enzymatic characterization of LPH (Lactase-phlorizin hydrolase) involved in intestinal absorption of natural product glycosides

Kazuyoshi Terasaka
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University

LPH (lactase-phlorizin hydrolase) is an important hydrolase in the brush border of the small intestine.LPH would play important roles in intestinal absorption of quercetin glycosides. The aim of the present study is to characterize the glycosidase activity of LPH towards natural product glycosides. The recombinant enzymes corresponding to human and rat LPH were used for glucosidase assays with various glycosides as a substrate. The recombinant LPH exhibited low glucosidase activity toward several glucosides. As for the regioselectivity of glucosylation positions, recombinant LPH preferentially hydrolyzed quercetin-4'-Oglucoside. These data suggest that LPH has the substrate preference toward natural product glycosides.

24－21

Effects of sweeteners on the pathosis and underlying mechanisms of lifestyle-related diseases for the molecular target control of such diseases

Noriko Kemuriyama
Department of Nutritional Science and Food Safety, Faculty of Applied Biosciences, Tokyo University of Agriculture

The aim of the present study is to examine effects of sweeteners on the pathosis and underlying mechanisms of lifestyle-related diseases using animal models. In the present study, we chose nonalcoholic steatohepatitis (NASH) and inflammatory bowel disease (IBD) as the diseases, Luo Han Guo fruit extract as a sweetener, and rat and mouse models featuring a chronic feeding of a choline-deficient, methionine-lowered, L-amino acid-defined (CDAA) diet and murine dextran sulfate sodium colitis model as animal models. Luo Han Guo (Siraitia grosvenorii) fruit is well-known for its sweet taste, and its extract is widely used as a sweetener and a component of health-care products, such as antitussive and anti-sore throat agents. In southern China, Luo Han Guo has been believed to aid longevity. We conducted 3 experiments in the fiscal year of 2017, and in experiment 1, we evaluated phenotypical and genotypical strain differences between male Hsd: Sprague Dawley (Hsd) and Fischer 344 (F344) rats, both were fed the CDAA diet for the NASH manifestation. The increase of plasma activities of aspartate and alanine aminotransferases, and the development of glutathione S-transferase (GST-P)-positive, preneoplastic hepatocellular foci were observed only in F344 rats with severe liver fibrosis. In experiment 2, we developed a mouse NASH model by feeding a modified CDAA (mCDAA) diet (Mice are resistant to the original CDAA diet), and examined effects of Luo-Han-Guo fruit extract dissolved in the drinking water at concentrations of 0, 0.2, 0.6 and 2%. In the mice fed the mCDAA diet for 7 months, plasma activities of hepatic aminotransferases were increased, and nodular lesions were diffusely observed in the liver. Coadministered Luo-Han-Guo fruit extract did not cause remarkable changes, but detailed assessments are still going on. In experiment 3, we used a dextran sulfate sodium (DSS)-induced colitis models of in mice, in which acute damage is induced in the colonic epithelia. This model is useful to explore stress response and pathways that maintain gut integrity in the presence of a biological insult, and therapeutics that protect intestinal epithelial cells from the damage. DSS was administered in the drinking water for 1 week (an acute study) or intermittently 4 weeks (a subacute study). During the DSS dosing period, Luo-Han-Guo fruit extract was administered by admixing into a standard chaw at concentrations of 0.15, 0.5 and 1.5%. In the 1-week acute study, Luo-Han-Guo fruit extract exerted amelioration on mucosal damage, inflammation, regeneration in the colon, survival rate and body weight loss. On the other hand, in the 4-week subacute study, Luo-Han-Guo fruit extract did not exert remarkable changes. It is thus indicated that it will be meaningful in the next fiscal year to assess effects of Luo-Han-Guo fruit extract on the CDAA dietinduced rat NASH model using Fischer 344 rats, the strain more sensitive than Hsd rats used in the last year, that effects of Luo-Han-Guo fruit extract on the mCDAA diet-induced mouse NASH model should further be assessed, and that effects of Luo-Han-Guo fruit extract on the DSS-induced mouse colitis model also should further be assessed, while the extract may be protective toward DSS-induced colitis at least its acute phase.

24－22

Research on toxicokinetics and biological effects of enzymatically modified isoquercitrin

Makoto Shibutani
Tokyo University of Agriculture and Technology, Graduate School, Division of Animal Life Science, Institute of Agricultural Science

We determined effects of alpha-glycosyl isoquercitrin (AGIQ), also known as enzymatically modified isoquercitrin against dextran sulphate sodium (DSS)-induced colitis. Colitis was induced by administering 4%DSS in drinking water for 7 days. The effects of 1.5%AGIQ in feed during recovery period for 7 and 14 days were evaluated by measuring relevant clinical symptoms (fecal blood, diarrhea, and body weight loss), colon length, and histopathology. The clinical symptoms were induced by DSS administration and recovered gradually during recovery period. No adverse effects on these symptoms were detected when administered AGIQ in feed. Shortened colon length by DSS was not changed by AGIQ administration. DSSinduced mucosal injuries such as mucosal loss and inflammatory cell infiltration in the distal colon tended to be prevented by AGIQ administration. A regeneration marker, restitution rate demonstrated by rate of length of a monolayer of epithelium and repairing epithelium tended to be reduced by AGIQ administration, while the number of cluster of unrestituted cells per length of repairing epithelium tended to be increased by AGIQ administration. A possible major metabolite, quercetin was detected in plasma samples 1 and 3 hours after 1000 mg/kg AGIQ administration when given an oral single dose, while it was not detected in plasma samples 2 week after 1.5% AGIQ administration in feed. The results suggested that AGIQ had no adverse effects in DSS-induced colitis model and a very weak potential of enhancing mucosal regeneration. Further study is required to determine the relationship between plasma metabolites and effects on colitis-related regeneration in a colitis animal model.

24－23

Inhibitory effects of emulsifiers on the production of acrolein as a toxic chemical during heat-cooking with vegetable oils

Yasushi Endo
School of Bioscience and Biotechnology, Tokyo University of Technology

Inhibitory activity of emulsifiers on the production of acrolein from edible oils during heat-cooking was evaluated. Polyglycerol fatty acid esters such as S-28D and B-100D were supplemented to soybean oil at the concentration of 0.05 to 0.2%, and they were heated at 180°C. Carbonyl value and acrolein level were measured for heated oils. Carbonyl value and acrolein level of heated oils supplemented with polyglycerol fatty acid esters were almost as same as those of control oils. From these results, polyglycerol fatty acid esters such as S-28D and B-100D did not almost show the inhibitory activity on the production of acrolein from edible oils during heating.

24－24

Control of biofilms established with food-borne pathogens by a combined effect of organic acids with other food additives

Tadayuki Tsukatani
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center

A combined method of a colorimetric microbial viability assay based on reduction of a tetrazolium salt and a biofilm formation technique on the 96-pegs of a microtiter plate lid was used to screen the biofilm eradication activities of various food preservatives. Especially, fumaric acid showed potent biofilm eradication activities against both Staphylococcus aureus and Escherichia coli. In addition, fumaric acid in combination with lactic acid and ferulic acid effectively reduced the minimum biofilm eradication concentration of a wide variety of bacteria. Therefore, the combining acidulants and atioxidant will result in an effective and safe inhibitor for use against the biofilms of a wide variety of bacteria.

24－25

Effect of whey protein hydrolysate on the acceleration of color formation by sodium nitrite in meat - Aiming of reduction of using NaNO₂ in food processing

Shiro Takeda
Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University

We investigated the promoting effects of hydrolysates derived from animal food by-products such as whey, white egg, and egg shell membrane proteins on color formation by sodium nitrite (NaNO₂) in meat products. In this study, 80% whey protein concentrates (WPC) and egg white protein (EW) were used. Hydrolytic whey protein peptide (WPP), hydrolytic egg white peptide (EWP), and hydrolytic egg shell membrane peptide (ESP) were also prepared. They were added, separately at 5% weight/weight (w/w), to the sausage mixture, which was composed of pork, 2% sodium chloride (NaCl), and 30 ppm NaNO₂. The sausages were heated at 75ºC for 40 min, and their International Commission on Illumination (CIE) redness (a*) values and color formation ratios were measured. The a* values were significantly elevated in sausages to which WPP, EWP, and ESP were added, as compared to that of the control. Color formation ratios in EWP and ESP sausages were also significantly higher than that of the control. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) of each animal food by-product and hydrolytic product showed different band patterns. The bands of WPP, EWP, and ESP were observed to be of lower molecular weight than the bands of WPC and EW, respectively. Furthermore, WPP, EWP, and ESP were treated with 80% ethanol and lyophilized to extract hydrolytic products that were added to sausages at 5% (w/w). The a* values and color formation ratios were significantly elevated in sausages to which hydrolytic extracts from WPP and ESP were added, as compared to those of control sausages. Hence, the hydrolysates from WPP and ESP, in particular, have the potential to promote color formation by sodium nitrite in meat products.

24－26

Quantitative analysis of secondary metabolites in herbs as affected by different environments in cultivation

Natsuko Kagawa
Center for Environment, Health and Field Sciences, Chiba University

Plant secondary metabolites, originally contained in herbs, are used as flavoring and food additives. Those botanical ingredients are regulated by plant metabolism, therefore the content of each ingredient the plant produces will vary depending on the location and the season in which the plant grows. In this study, a technology for a plant factory with artificial lighting was applied in cultivation of perilla plants, a popular culinary and medicinal herb in Japan, under different growth environments, and we have explored the effects of those environments on the contents of bioactive secondary metabolites appeared in perilla plants as a response to the difference. The knowledge obtained from this study can contribute development of a production technology for plant raw materials used as plant-based foods, achieving high levels of outcome both in quality and quantity.

24－27

Development of a method to evaluate the effect of flavor on sweetness using salivary secretion and salivary component change

Yuko Kusakabe
Food Research Institute, NARO

We aimed to develop a method to easily evaluate the taste and palatability of foods, and analyzed physiological responses by taste and olfactory stimulation. Sweet, umami, salty, and sour taste were used for taste stimuli. Azuki flavor was used for the flavor associated with sweet taste, and Matsutake flavor was used for the flavor associated with umami taste. Last year, we found that the intensity of sweetness correlates with saliva secretion, so we observed change in saliva secretion and salivary component as physiological response.We evaluated the stress response as salivary component change and measured the change in salivary amylase activity. This data was compared with that of sensory evaluation. We analyzed the physiological responses about basic tastes. We observed the amount of saliva secretion was significantly increased by sweet and sour taste stimulations. The amylase activity was significantly increased only by sour taste stimulation, and tended to decrease by salty taste stimulation. Evaluation by addition of flavor showed that addition of azuki flavor to the sweet taste stimulation increased the amount of saliva secretion, and decrease the amylase activity although there was no significant difference. On the other hand, no influence was observed by addition of flavors for umami taste stimulations. Moreover, no effect on physiological response was observed, while the addition of unassociated flavor reduced preference. Analysis of correlation among palatability, salivary secretion, and amylase activity revealed that only 2 points related to sweetness showed correlation. These results showed that the influence of taste and olfactory stimulation on physiological responses was different depending on taste and olfactory quality. We expect that our data will be effective to evaluate food mixed with different taste and flavor as basic knowledge.

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The Study of Regulations towards Azo Colorants in Great Britain

Kyoko Hidaka
Shibaura Institute of Technology

In this study, the background of the regulation in use of azo food coloring in the UK is investigated from the view of color science and food science. In 2007, the research group of Southampton University researchers conducted experiments to give azo food colorants to children for ingestion. In this experiment, the onset of hyperactivity disorder (ADHD) was seen in children aged 8 to 9 years, so these became the banned food colorants in the UK. The research group reported the experiment results to the UK Food Standards Agency; however, its content was covered by a newspaper before it was even peer-reviewed, and then regulated its use as a social problem.