Research Reports No.19 Abstract (2013)

19-01

DNA identification of a sweet-potato cultivar for anthocyanin production

Makoto Tahara
Graduate School of Environment and Life Science, Okayama University

In order to develop cultivar specific DNA markers of sweet-potato for anthocyanin production, the genome-wide insertion sites of two active retrotransposon families in sweet-potato were studied over 39 cultivars using the next generation sequencer, MiSeq. After processing the MiSeq data of the insertion site sequences by the two families, the sequences of 2,204 sites were determined, which cover most of the insertion sites among 39 cultivars. Most of the insertion sites are polymorphic among the cultivars and the site present only at a single cultivar counts 18.5% of the identified sites. Once an active retrotransposon family is discovered, the cultivar specific insertion sites that can be easily converted to DNA markers are identified with the high throughput next generation sequencing. The cultivar specific insertion markers have proved to be quite practical in identifying the source cultivars in processed foods. The techniques presented in this study will play an important role in developing cultivar specific DNA markers.

19-02

Analytical Studies on the International Trend of the Safety Assessment for Flavoring Substance

Yoichi Konishi
Professor Emeritus, Nara Medical University

Currently, various international foods are widely distributed to the entire world. On the other hand, it is well known that each food contain chemicals including flavoring substances; however, the method for safety assessments of flavoring substances is not internationally harmonized. In this special issue, the author believes now is good timing and has requested five internationally distinguished doctors from different countries to describe the present situation of safety assessment of flavoring substances with the aim of identifying possible directions for international harmonization. After the reading of each article, this author's impression is that the assessment method of JECFA is the favored procedure and its high level of quality is proposed to be internationally recognized. Unfortunately, the method presently used in Japan is behind the times and that affects the export business of flavor substances. A disturbing point of the assessment methods in Japan is that flavoring substances are categorized the same as other food additives, not independent, and TTC is not effectively used but is still dependent upon the test results of genotoxicity and repeated-dosed toxicity in animals. Further, EFSA recently started to use in silico methods of QSAR and read-across for the improvement of applicability of the TTC. To smoothly improve international business and obtain exact information on safety for general consumers, this special issue proposes further active involvement of regulatory agencies in the international harmonization of flavoring substance assessments.

19-03

Development of a novel web-database that enables to retrieve microbial growth inhibition of food additives

Shigenobu Koseki
National Agricultural Research Organization, National Food Research Institute

ComBase is a large database of microbial responses to food environments and has attracted the attention of many researchers and food processors. Although ComBase contains a vast amount of data, it is not easy to obtain desired information from the retrieved data. In the present study, we developed a new ComBase- derived database (Microbial Responses Viewer, MRV) consisting of microbial growth/no growth data. The MRV provides information concerning growth/no growth boundary conditions and the specific growth rates of queried microorganisms. The revised MRV allows to retrieve effects of bacterial growth inhibition of food additives such as lactic acid, nitrite, acetic acid, and sorbic acid, etc. The MRV enables users to recognize the effect of food additives on the bacterial growth intuitively. Using the MRV, food processors can easily identify appropriate food design and processing conditions.

19-04

Fundamental Safety Assessment of Natural Flavoring Substances-3

Toru Akita	Nippon Shinyaku Co. Ltd., Functional Food Company
Masanori Kuroyanagi	School of Pharmaceutical Sciences, University of Shizuoka
Kimiko Kobayashi	Kobayashi Clinic
Motoyoshi Satake, Reiko Nakamura	Institute of Environmental Science for Human Life, Ochanomizu University
Yukihiro Shoyama*	Faculty of Pharmaceutical Sciences, Nagasaki International University
Setuko Sekita	Faculty of Pharmaceutical Science at Kagawa Campus, Tokushima Bunri University
Takashi Morimoto	Saneigen FFI., Inc.
Kunitoshi Yoshihira	University of East Asia
Kohmei Wani	West Japan Dietary Culture Research Group
	* Chairman

The natural resource of flavoring additives 513 items were listed in 2006. Since there are various kinds of complicated materials like animals, complexes, products, components, mushrooms, seaweeds, resins and materials related to the regulations, we removed such species from this project and relisted up 450 items. We suggested that 450 items can be covered by three years during 2010 to 2012 resulted that we pided 150 items in a year. In order to make sure the safety of flavoring additives, the following investigations have been done.
1) Since all original plant species of flavoring additives have not been determined, we confirmed and determined their scientific names inpidually.
2) The survey of eating experience as food of the natural sources is important for the confirmation of safety. Therefore, the accumulated scientific data regarding eating experiences published in various fields were incorporated in this project resulting in evaluation of safety.
3) Recently so many natural compounds have been isolated and structurally elucidated depending on the development of purification and structural elucidation technique. We therefore prepared data base of components contained in inpidual flavoring additive.
4) It becomes evident that the above data base promoted the scientific safety data of flavoring additives obtained from RTECS data.

19-05

Ochratoxin A does not induce oxidative stress responses for formation of karyomegaly and cell cycle aberrations in renal tubular cells in rats

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

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells located at the outer stripe of the outer medulla (OSOM). However, it is unclear whether the carcinogenic process of OTA involves oxidative cellular responses. This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA. We examined changes on oxidative stress and cell cycle-related parameters in the OSOM of rats treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or 疝och-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1 and Hmox1. OTA also did not change the levels of thiobarbituric acid-reactive substances and oxidized glutathione/reduced glutathione. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption at the M phase and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

19-06

Functional evaluation of essential oils-derived chemicals for health promotion

Hiroyasu Inoue, Rieko Nakata
Department of Food Science and Nutrition, Nara Women' University

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin biosynthesis, plays a key role in inflammation and circulatory homeostasis. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily and are involved in the control of COX-2 expression, and vice versa. Several natural chemicals such as resveratrol have been identified as suppressors of COX-2 expression and activators of PPARs. These two properties targeted to COX-2 and PPARs will be useful in evaluating functional food components against lifestyle-related diseases. In this study, we evaluated spice essential oils using our established assays for COX-2 and PPARs. We found that spice essential oils derived from cinnamon bark, caraway seed, cinnamon leaf, cumin and cardamom suppress COX-2 promoter activity, and activate PPARa, b/d and g, in cell-based transfection assays using bovine arterial endothelial cells. Moreover, from cinnamon bark oil, we identified trans-cinnamaldehyde as a major component of the suppressor of COX-2 expression and an activator of PPARa, b/d and g. PPARg-dependent suppression of COX-2 promoter activity was observed in response to trans-cinnamaldehyde treatment. These results may be important for understanding the anti-inflammatory and anti-lifestyle-related disease properties of trans-cinnamaldehyde.

19-07

Prevention against zinc deficiency by the use of food additives increasing zinc absorption in the intestinal epithelial cells

Taiho Kambe
Graduate School of Biostudies, Kyoto University

Shortage of dietary zinc put human health at risk, because zinc is required for multiple physiological functions. Hence, a food-based strategy enabling efficient zinc absorption should be extensively explored. In the small intestine, the zinc transporter ZIP4 (Slc39a4) functions as an essential component for zinc absorption, which is mutated in humans with the rare, pseudo-dominant, lethal genetic disease acrodermatitis enteropathica. As exogenous expression or overexpression of ZIP4 leads to increase the cellular zinc levels, food components with the activity of increasing ZIP4 expression may be a potential enhancer of zinc absorption in the intestinal epithelial cells. In this study, we examined the efficacy of food additives to increase ZIP4 expression by the use of the screening system using mouse Hepa cells that we have established. Several food additives have the activity to enhance ZIP4 expression. We are now trying to partially purify component(s) from such food additives, and examining the mechanism how they enhance ZIP4 expression.

19-08

Evaluation of immunotoxicity of nanomaterials in foods

Yasuo Yoshioka
Laboratory of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University

A nanomaterial is defined as a substance that has at least one dimension of less than 100 nm in size. Nanomaterial has been applied in various kinds of fields. For example, amorphous silica nanoparticles (nSP) have been already used in various foods as anticaking agents. Therefore, we cannot avoid uptake of nSP by oral route. Under this circumstance, there is increasing the importance of ensuring the safety of nanomaterial in foods. We previously revealed that nSP could penetrate the intestinal barrier, however, did not induce significant general toxicity. On the other hand, the intestinal tract is always exposed foreign bodies, such as food antigens and viruses. Therefore, intestinal tract constructs the unique immune system and is known as the greatest immune organ. When we are exposed to nSP, many food antigens are also exposed simultaneously. Thus, to ensure the safety of nSP, it is important to evaluate the effects of the oral exposure to nSP on immune response to food antigens. Here, we evaluated the effects of oral administration of nSP with a diameter of 30 nm on immunologic response to the food antigen. C3H/HeJ mice were orally treated with ovalbumin (OVA) or OVA plus nSP on days 0, 7, 14 and 21. Seven days after last treatment, we measured OVA-specific antibody levels. Significant induction of OVA-specific IgG levels was not observed in OVA group compared with control. On the other hand, the mice treated with OVA plus nSP had significantly increased OVA-specific IgG levels compared with control groups. These results suggested that uptake of food antigens with nSP might induce excessive immune response to food antigens and then induce the abrogation of immune tolerance to food antigens.

19-09

Functional interaction of artificial food colorants with amino acids and proteins

Yoshimasa Nakamura
Graduate School of Environmental and Life Science Okayama University

In this study, we investigated the effect of amino acids and bovine serum albumin on phloxine B (PhB)-induced hydrogen peroxide formation. PhB with light irradiation significantly increased the hydrogen peroxide concentration in RPMI 1640 medium but not in phosphate buffered saline (PBS), suggesting that certain medium constituents are involved in this photochemistry. Thus we performed the screening of amino acids for enhancers of PhB-induced photosensitization. Four amino acids, including cysteine, methionine, tyrosine, and tryptophan, were identified as potential enhancers of PhB-induced hydrogen peroxide formation in PBS. Combined treatment with these amino acids synergistically increased the hydrogen peroxide concentration compared with each treatment. These results suggest that amino acids having a redox-active moiety might take part in the reduction of photo-excited PhB, prerequisite for the Type I reaction-dependent hydrogen peroxide formation. In addition, bovine serum albumin significantly inhibited PhB-induced hydrogen peroxide formation and photocytotoxicity in human acute promyelocytic leukemia HL-60 cells. This report attempts to provide biological evidence that artificial food colorants can functionally interact with amino acids and proteins.

19-10

The effects of the glycosylated hesperidin for improving postprandial hyperlipidemia and the underlying mechanism of action

Daisaku Masuda
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine

Recent studies have shown that fasting and postprandial hypertriglyceridemia (PHTG) are risk factors for atherosclerotic cardiovascular diseases. PHTG is caused by the postprandial accumulation of chylomicrons (CMs) which are produced from the intestine and CM remnants which are the hydrolyzed products of CMs. Since the accumulation of CM remnants were highly atherogenic, therefore, many drugs were investigated whether they could improve fasting and PHTG. Recently, Hesperidin is a flavonoid compound in citrus fruit peel, oral administration of glycosylated hesperidin (G-hesperidin) lowers serum TG levels in patients with hypertriglyceridemia. In this study, we investigated the effect of G-hesperidin on PHTG using an animal model of PHTG, CD36 knockout (CD36KO) mouse. We used wild-type (WT) mice fed a high-fat diet and CD36KO mice fed a normal chow diet for four weeks, these diets were mixed with (Hes- group, n=8) and without (Hes+ group, n=8) G-hesperidin (5g/kg). G-hesperidin did neither reduced plasma TG levels during fasting and after the oral fat load (OFL) using olive oil (17μl/g body weight) in CD36KO mice nor in WT mice, instead of what we have expected. And HPLC analysis of lipoprotein profiles showed no significant difference between two groups both in plasma and intestinal lymph after OFL. Inhibition of lipoprotein lipase (LPL) using triton WR-1339 indicated that hesperidin decreased in TG production from the liver. Quantitative RT-PCR analyses of enzymes and transporters showed that G-hesperidin decreased expressions of apoB in intestine and those of SREBP1c in liver. And there was no difference in glucose metabolism between two groups. In conclusion, G-hesperidin did not improve PHTG in this animal study since it might decrease TG production from the liver.

19-11

Bioavailability of the pectin masked trace contaminants in food

Yoshiko Konishi
National Institute of Health Sciences

We aimed to develop a new food-processing approach using low-methoxyl pectin (LMP) to reduce gastrointestinal absorption of mycotoxins. With Ca²⁺, LMP forms an egg box-like structure gel.
In previous report, we found that the gel-formed pectin was able to trap a low molecule compounds, such as mycotoxins and established the gastrointestinal digest model in vitro using Caco-2 transwell and then revealed the bioavailability of mycotoxin in the pectin gel. LMP maintained gel form and masking ability under the acidic condition, but could not under alkaline condition. We also demonstrated that thegel formed pectin possessed the masking effect on patulin, aflatoxin M1 and fumonisins. For application approach, we examined permeability in intestinal cells of mycotoxin and other integrant in gel-formed foods using LMP, such as an apple juice jelly containing patulin and a milk jelly containing aflatoxin M1. The results obtained from in vitro model suggested that gelation would not prevent the absorption of integrant as well as mycotoxin in intestine after satisfactory digestion.　　This study demonstrated that gel-formed pectin is effective for mycotoxin which is absorbed in stomach and upper intestine to prevent absorption.

19-12

Chemopreventive effect of antioxidants on non-alcoholic steatohepatitis and hepatocarcinogenesis in rats

Aya Naiki-Ito
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences

Non-alcoholic steatohepatitis (NASH) have the potential leading to develop cirrhosis and hepatocellular carcinoma. It has been suggested that oxidative stress is implicated in the progression of NASH and flavonoids have been reported to possess antioxidant activities. Luteolin, a flavonoid extracted from seed of labiate such as perilla, is one of antioxidant. We previously demonstrated that transgenic rats (Tg) carrying a dominatnt negative mutant of connexin 32 (Cx32) have decreased capacity for gap junctional intercellular communication, and have high susceptibility to hepatocarcinogenesis. In this study, we aimed to clarify a role of luteolin and Cx32 on the development of NASH and on the progression of hepatocarcinogenesis. Male Tg and wild-type (nTg) rats at 10-wk were feeded a methionine-choline deficient diet (MCDD) or MCDD added to luteolin for 12 weeks 2 days after giving an intraperitoneal injection of diethylnitrosamine. MCDD induced fatty change, inflammation and fibrosis in livers, and the degree of these lesions were severe in Tg compared to nTg rats. In addition, luteolin suppressed these pathological findings and the number of GST-P-positive foci in Tg rats. Moreover, luteolin and Cx32 suppressed oxidative stress in the liver of Tg and nTg rats. These results suggest that luteolin and Cx32 potentiate to prevent NASH and to suppress the progression of hepatocarcinogenesis.

19-13

Assessment of nano-food additives to repair and healing of gastric ulcers

Akira ONODERA¹, Atsumi HIROUCHI¹, Ayaka IWASAKI ¹, Ai HOUSHO¹, Shigenobu YONEMURA², Yuichi KAWAI¹
1. Department of Pharmaceutical Sciences, Kobegakuin University　2. RIKEN CDB

Nano-food is developed and produced by processing food materials into nano-size particles with diameter <100 nm. Some coffee-cream packages and powdered soups already include nano-sized silica particles (nSP) as a food additive, and further expansion is expected in the nano-food market. However, various kinds of toxicity have been reported in nano-safety studies involving overexposure to nSP, raising concerns regarding the safety of nano-food. We conducted a study to assess the mechanisms underlying the toxicity of nSP. Our study on the toxicity demonstrated that inflammation triggered by a unique toxicity to nano-materials spreads through the mucous membrane surface. Amorphous nSP particles with a diameter of 70 nm (nSP70) were repeatedly injected into the stomach of gastric ulcer model mice and control mice using a gastric tube. In control mice, remarkable changes were not observed in the gastric mucous membrane. Hence, the unique toxicity to nano-materials was observed only under specific physiological conditions, and it was suggested that the normal gastric mucosa is resistant towards nano-materials.　In the present study, using a gastric ulcer mouse model, we identified the cell type that causes infiltration in the inflammatory reaction to elucidate the toxic mechanism of inflammation by nSP70. Moreover, because substantial intake of nano-food takes place over long periods of time, we evaluated whether nSP was safe by examining blood biochemistry following nSP70 injections three times a week for up to four weeks. Azan-positive cells of fibroblastic lineage and atrophy of glandular organization were observed on the ulcerated gastric mucous membrane surface. On the other hand, in control mice, after repeated injections of nSP70 for four weeks, mucous membrane inflammation was not observed, and the blood biochemistry parameters related to liver, kidney and heart function were normal. Our results suggest that nano-particle cause similar to pathological conditions of atrophic gastritis if the gastric ulcer develops, on the other hand, nano-particle toxicity will not occur even in high doses if the gastric mucosa is normal.

19-14

Effect of nitrite on the development of insulin resistance in obese mice

Kazuo Ohtake
pision of Pathophysiology, Department of Clinical Dietetics and Human Nutrition, Faculty of Pharmaceutical Science, Josai University

Insulin resistance plays a primary role in the development of type-2 diabetes. The onset of which is known to have been involved in inflammatory adipocytokine secreted by hypertropic adipocyte. In such condition, in target organs such as skeletal muscle insulin sensitivity is declined. In skeletal muscle insulin enhanced glucose transporter type 4 (GLUT4) translocation via PI3/Akt pathway. Thus, insulin promote glucose uptake. However, it is not well known how nitric oxide is implicated in the impaired signaling process of insulin resistance. We therefore evaluated the effect of nitrite supplementation on insulin resistance and examined a possibility of nitrite-derived nitric oxide (NO) involvement in insulin signaling pathway of diabetic models. KKA^y mice were used as a model of obese insulin-resistant diabetes and C57BL/6 as control. All mice had free access to water and standard chow. Body weight, amounts of food intake, and blood glucose levels were checked. KKA^y mice were pided randomly into 2 groups (control without nitrite, nitrite-treated with 150 mg/L in drinking water), and 2 groups of C57BL/6 (without nitrite and nitrite-treated with 150 mg/L in drinking water). At 8 week,　intraperitoneal Glucose Tolerance Testing (IPGTT) were performed. Blood glucose level were measured After 10 weeks, blood and gastrocnemius muscles were collected following 16h fasting, then applied to calculation of the homeostasis model assessment of insulin resistance (HOMA-IR), and to the measurement of tissue concentrations of nitrite and nitrate (NOx) in gastrocnemius muscles. Histological analysis was performed. Although diabetic KKA^y mice groups showed remarkably increasing body weights along the 10-weeks experimental period compared to C57BL/6 control groups, nitrite treatment had no impact on body weight changes among each group. There were tendencies toward decreased fasting plasma levels of insulin and glucose with nitrite treatment on 10th week, but not statistically significant. Whereas, in diabetic KKA^y mice IPGTT score was significantly improved and HOMA-IR was significantly reduced with nitrite treatment, which was consistent with reduced nitrite levels in the muscle of diabetic KKA^y mice followed by restoration to the same levels as C57BL/6 control groups with nitrite supplementation. Additionally our study showed that in skeletal muscle nitrite treatment to KKA^y mice increase GLUT4 translocation to plasma membrane following PI3-kinase regulation subunit p85 expression and remarkably enhanced Akt phosphorylation. Compared with KKA^y mice without nitrite treatment, nitrite treatment to KKA^y significantly decreased both mean adipose tissue size and expression of TNF- in adipose tissue. Our study demonstrate that nitrite treatment to KKA^y mice improve hypertrophy of adipose tissue and insulin sensitivity in skeletal muscle. Additionally insulin signaling analysis in skeletal muscle show that improvement of glucose tolerance is contributed to ameliorate PI3/Akt pathway and increase GLUT4 translocation to plasma membrane. These results suggested a possible involvement of NO and its bioavailability in insulin resistance in diabetic KKA^y mice.

19-15

Studies on the effect on the masking of bitterness by
the sweet-tasting protein, thaumatin

Tetsuya Masuda
pision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University

Sweet-tasting protein, thaumatin elicits a sweet taste at 50 nM. Extensive sweet sensations of thaumatin are widely used as sweeteners in food industries. Besides their unique properties as sweeteners it has also been suggested thaumatin potentially seems to mask a bitter taste and enhance flavour of food stuck. However, such additional properties on thaumatin besides sweeteners have not fully understood. In the present study, to clarify the effect of the masking of bitterness by thaumatin, we prepared the HEK cells stably expressing bitter receptors and then we investigated the effect of the masking of bitterness.

19-16

Metabolites of ellagitannins and their anti-inflammatory effects

Hideyuki Ito
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University

Various pharmacological activities of ellagitannins such as antioxidant, antiviral, and antitumor activities suggest importance of their beneficial effects in human health. However, there are few definitive studies on the absorption and metabolism of ellagitannins. We recently reported that seven urinary and intestinal microbial metabolites in rats were isolated after the ingestion of geraniin, and their structures were determined to be dibenzopyran derivatives. Ellagitannins also contain several food additives including Pimento, Melaleuca, Eucalyptus, and Pomegranate. Since pomegranate is abundant in punicalagin having a novel gallagyl group, we investigated metabolism of gallagic acid by gut microflora. The intestinal microbial transformation of gallagic acid prepared from punicalagin was evaluated by using DAD-HPLC. Ellagic acid was slightly observed after 1 h anaerobic incubation with rat fecal suspension, suggesting that gallagic acid was converted to ellagic acid by microflora. After 120 h incubation, the production of ellagic acid was not displayed, and then the peaks with shorter retention times were detected, implying that metabolites of gallagic acid were produced by longer incubation. Identification of gut microbial metabolites of gallagic acid is under investigation.

19-17

Molecular Behavior of Natural Food Additives, Glycyrrhizn on Lipid Rafts Biomembrane Model

Faculty of Pharmaceutical Sciences, Nagasaki International University
Seiichi Sakamoto

An interaction of glycyrrhizin (GC) with lipid rafts biomembrane model consisting of N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM), 1,　2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL) has been systematically studied by the Langmuir monolayer technique. In order to construct the lipid rafts model, the surface pressure (p)-molecular area (A) and surface potential (ﾄV)-A isotherms for three-component (PSM/DOPC/CHOL) systems on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) have primarily been measured as a function of their inpidual composition. A thermodynamic parameter and interaction parameter for binary PSM/DOPC and PSM/CHOL systems reveal that the PSM interacts more strongly with CHOL than DOPC. In addition, the morphological analysis performed with Brewster angle microscopy (BAM) and fluorescence microscopy (FM) disclosed the optimal ratio of PSM/DOPC/CHOL (1/1/1, by mole) as a model of lipid rafts. Second, the interaction of GC with the ternary PSM/DOPC/CHOL monolayers has been investigated on the Tris buffer solutions containing different GC concentrations (1, 5, 10, 25, and 50 lochM). In BAM and FM images, microdomains are found to become smaller by increasing the GC concentration in the subphase, suggesting that GC regulates the size of the raft domains, which provide dynamic scaffolding for numerous cellular processes. More interestingly, the distinctive GC striped regions are formed at the interface at 50 lochM, which shows that GC pides the ternary monolayer into pieces. This phenomenon is observed only in the presence of CHOL in monolayer. These results suggest CHOL plays an essential role in the interaction with GC, which cause one of the saponin's major activities, membrane disruption.

19-18

Studies toward the enhancing effects on the absorption of acylated anthocyanins

Shigenori Kumazawa
University of Shizuoka, School of Food and Nutritional Sciences

Eight anthocyanins were isolated from edible purple yam Dioscorea alata and determined their structures. Four compounds in the eight anthocyanins were new acylated anthocyanins. Anthocyanins with organic acid or catechol moiety in the structures possessed high antioxidant activity in vitro. Furthermore, the enhancing effects of polysaccharides in purple yam on absorption of acylated anthocyanins were investigated by evaluation of in vivo antioxidant activity using rats. The plasma of the rats after orally administration of the extracts of purple yam increased the antioxidant activity. This result suggested that polysaccharides in purple yam increase the bioavailability of acylated anthocyanins.

19-19

Analyzing the Effects of Flavonoids on the inhibition of intestinal cholesterol transport

Shoko Kobayashi
Research Center for Food Safety, Graduate School of Agricultural and Life Sciences, The University of Tokyo

Elevated blood cholesterol levels contribute to atherosclerotic coronary heart disease. Cholesterol homeostasis in the body is mainly balanced by intestinal absorption, endogenous biosynthesis, and biliary/intestinal excretion. Niemann-Pick C1-Like 1 (NPC1L1) is a critical mediator of cholesterol absorption, and ezetimibe is a potent inhibitor of NPC1L1 that has been approved for treating hypercholesterolemia.
Epidemiological studies have revealed a correlation between polyphenols and decreased risk of atherosclerosis, which is known as the "French paradox"; however, the mechanisms of this paradox have yet to be clarified. The present study screened several flavonoids for specific inhibitors of intestinal cholesterol absorption and determined the inhibitory effects of selected flavonoids on intestinal cholesterol absorption.
Luteolin and quercetin potently inhibited cholesterol micellization and absorption to the Caco-2 (human colonic adenocarcinoma) cell monolayer, which is a model of intestinal epithelial cells. Luteolin and quercetin also inhibited cholesterol absorption to human embryonic kidney 293 cells expressing NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels via inhibiting cholesterol micellization and intestinal cholesterol absorption mediated by NPC1L1.

19-20

Study on the relationship between the chemical structure and antimicrobial activity in herbal flavor constituents

Takao Koeduka
Institute for Chemical Research, Kyoto University

Many compounds that belong to the phenylpropene class differ from one another in two aspects: (i) the substitution pattern (OH, OCH₃ and so on) of the benzene ring (C₆) and (ii) the position of the double bond in the C₃-side chain. Because of their characteristic aroma and antimicrobial properties, human beings have historically used plant materials containing phenylpropenes as food preservative, spices and cosmetics. However, despite many previous investigations on the essential oils containing phenylpropenes from medicinal herbs and spices, there are few reports regarding a relationship between the chemical structure and their biological activities. In this study, we examined the antifungal activities to investigate the structure-activity relationship of phenylpropenes. In addition, we investigated the properties of flavor and fragrance in a variety of phenylpropenes. The structure-activity relationship of a series of eugenol derivaties showed that specific phenylpropenes including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6) with a phenolic hydroxy group had antifungal activities for fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no activities. The results suggested the importance of a phenolic hydroxy group for the structure-based design of new functional properties of phenylpropenes.

19-21

Preparation of profile on sensitive and resistant fungi for preservatives

Kosuke Takatori
Department of Agriculture, Tokyo University of Agriculture
Atsuko Takahashi
Department of Science for Living, Junior College of Kiryu University

Preservatives as food additives is important for food microbial control in food industry. Microbial assessment was mainly described for　food　poisoning bacteria such as Escherichia coli.
On the contrary, even though food microbial contamination was mainly caused by fungi, food borne fungi have not evidence as important microbes because of lacking in regulatory field. Last year in 2011, at initial experiment, minimum inhibitory concentration(MIC) value was evaluated at each preservative for 8 food-borne fungi at 25°C for 10 days. Most preservatives were shown the MIC level of maximum concentration around 200ppm or so. We investigated on the effectiveness of representative 6 preservatives (calcium propionate, sodium propinate, sodium dehydrate acetate, p-hydroxy benzoic acid ethyl, potassium sorbate and ο-hydroxydiphenyl) concerning food-borne fungi. Also, the eight objective fungi isolated from the foods or food environment were selected Cladosporium cladosporioides FCR214, Penicillium citrinum FCR241, Aspergillus ochraceus FCR152 and so on.
In this study, we investigated much more detail study on 9 preservatives for 29 fungi. Finally we decided the exact antifungal spectrum evaluation of these preservatives. Among the 9 preservatives, we found the prominent preservatives. That is, the static anti-fungal activities were shown the effective and sensitive value.
From the results, we expect that the anti-fungal spectrum for preservatives shows the application to food additives industry.

19-22

Verification of the taste masking with food oil by the taste evaluation method using taste receptors

Yuko Kusakabe
National Food Research Institute, National Agriculture and Food Research Organization

The recent identification of specific taste receptors has explained the relationship between each taste substance and its receptor. However, the effects of mixing different taste substances on the response of taste receptors are still not well understood. Here, we measured the change in intensity of the response by taste receptors to pungent or bitter taste substances mixed with cooking oil. No change in the pungent taste response was observed when pungent taste substances were mixed with cooking oils. Conversely, a change in the bitter taste response was observed when bitter taste substances were mixed with cooking oils. Our data showed that the bitter taste response to 3 mM salicin was significantly decreased by mixing with 0.25% and 0.5% olive oil, and that the bitter taste response to 3 mM phenyl β-D-glucopyranoside was also significantly decreased by mixing with 0.25% olive oil. In contrast, the bitter taste response to 10 mM salicin was significantly increased by mixing with 0.5% olive oil. These results suggest the possibility that the proportion of bitter taste substance to cooking oil effects the bitter taste of food products. Moreover, these results indicate that measuring the taste response of culture cells expressing taste receptors is useful for prioritizing the samples used for sensory evaluation tests.

19-23

Development of a Novel Texture Evaluation Method Based on Pressure Distribution in Fracture

Mitsuru Higashimori
Department of Mechanical Engineering, Graduate School of Engineering, Osaka University

This paper proposes a food texture sensing method for nursing-care jelly foods which can be masticated by tongue. The aim of this study is to evaluate a food texture quantitatively by physical measurement. We propose the method for estimating the value of sensory evaluation based on the pressure distribution during compression and fracture of a jelly. For jellies whose values of sensory evaluation have been obtained, we measure the pressure distribution by using an experimental model where two gripper plates are regarded as a tongue and a palate, respectively. Based on the texture analysis for image, we extract feature values of the pressure distribution. We then derive the regression equation modeling the relationship between the feature values and the values of sensory evaluation for each evaluation item. By giving the pressure distribution of an unknown jelly to this model, we can estimate its value of sensory evaluation. Experimental results show that values of sensory evaluation of nine different jellies are accurately estimated.

19-24

Objective evaluation of taste continuity instead of sensory test

Tomiko Asakura
Graduate school of Agricultural and Life Sciences, The University of Tokyo

Taste continuity is mostly evaluated by sensory test which is complicated for training of panelists and reproducibility of results. The condition of each panelist affects the results. Tongue is covered with squamous epithelial cells whose surface is mainly composed of lipid bilayer. Foods are solubilized by saliva to interact with lingual epithelia and taste substances reach the taste buds. We raised the hypothesis that strong interaction of a food component with a lingual epithelium enhances its taste continuity. We tried to construct an analytical system for evaluating interaction with taste substances and construct the device detecting the interaction between lipid bilayer and food substances. A liposome derived from 1,2-Dioleoyl-sn-glycerol-3-phosphocholine was prepared and interaction of liposome with food components was analyzed using isothermal calorimeter. As a result, monosaccharides and oligosaccharides did not show the interaction of liposome. Epigallocatechin gallate (EGCG) cause the bitterness of green tea showed strong interaction. Thermodynamic parameters showed the interaction of EGCG and liposome account for polar bonds such as hydrogen, coordinate and ion bonds.