Research Reports No.18（2012）

18－01

Studies on the effects of food additives on molecular chaperones

Akira Murakami
Graduate School of Agriculture, Kyoto University

Molecular chaperones play some important roles in repair and refolding of biological proteins, whilst the effects of food factors on their expressions have yet to be fully elucidated. Thus, in the present study I investigated the effects of nutrients, phytochemicals, and food additives. A total of 43 food ingredients were selected and added to Hepa1c1c7 mouse hepatocytes. After incubation, the levels of mRNA expressions of HSP70, a representative inducible chaperone, were quantified by using qRT-PCR. As the results, most of the nutrients, except for all-trans retinal and zinc chloride, were inactive while phytochemicals such as curcumin, ursolic acid、a-humulene, zerumbone, phenethyl isothiocyanate markedly induced HSP70 mRNA. In addition, only benzoic acid among food additives showed significant activity. Based on the fact that benzoic acid is a phytochemical, this study proposed the hypothesis that phytochemicals are unique class of plant metabolite which has notable capability of up-regulating molecular chaperones.

18－02

Study on the Advanced Distribution Management Technology of Foods by Effective Use of Food Additives

Toshio Kawano
Faculty of Agriculture, National University Cooperation Kochi University

 For the purpose of preventing from illegally substituting counterfeit foods for distributed genuine ones, new invisible marking technology for foods was proposed, and its effectiveness was investigated. The technology makes use of food additives as the marking ingredients and of a light scanning method as the non-destructive scanner for discriminating between counterfeit and genuine foods. In this study, simple circle-shaped edible markers, which consisted of few kinds of food additives, were made by way of trial.
As the results of visualizing the invisible markers by newly developed image-reconstructing programs, the reconstructed marker images resembled real shape of edible markers on foods with sufficient accuracy.

18－03

Analytical studies on the International Trend of the safety assessment for Flavoring Substance

Yoichi Konishi
Professor Emeritus, Nara Medical University
Visiting Professor, Oklahoma Medical Research Foundation

Current International information concerned the trend of safety assessment of flavoring substances (FSs) was collected and analyzed. The information was mainly collected from the articles and meetings of current situation of the safety assessment of FSs in Flavor and Extract Manufacture Association (FEMA), Joint FAO/WHO Expert Committee on Food Additives (JECFA), European Food Safety Authority (EFSA) and Japanese Regulatory Agency. The subjects were focused on the necessity of genotoxicity and 2-year carcinogenicity tests, evaluation and improvement of Threshold of Toxicological Concern (TTC) application, usefulness of in silico for the prediction of toxicity, reliability of Acceptable Daily Intake (ADI) calculated from Non-Observed Adverse Effect Level (NOAEL) and international trends of regulatory agencies. The results suggest that the safety assessment of FSs in Japan is needed to be changed along the movement of that in JECFA, U.S. and Europe.

18－04

Development of inclusion solubilizer for lipophilic antioxidants and its kinetic determination for radical scavenging ability of solubilized antioxidants

Yoshimi Sueishi
Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University

The inclusion complexations of lipophilic antioxidants with cyclodextrins, methylated p-sulfonatocalixarenes, water-soluble paracyclophane, and cucurbit[7]uril were characterized by NMR measurements. We could not observe the inclusion complex formation of representative antioxidant trolox with methylated p-sulfonatocalix[6]arenes, water-soluble paracyclophane and cucurbit[7]uril. Further, using native b-cyclodextrin (b-CD) and heptakis(2,6-di-O-methyl)-b-cyclodextrin (DM-b -CD), we have examined the effect of the cyclodextrin inclusion on the radical scavenging abilities of CD-solubilized antioxidants. Scavenging rates of solubilized lipophilic antioxidants were measured with the use of ESR spin-trapping method. The results indicate that the scavenging rates of catechin and resveratrol were notably dependent on the solubilizer used. But, epicatechin and epigallocatechin gallate showed almost no dependence on the cyclodextrin used. Based on the results, the effects of inclusion on the radical scavenging abilities of CD-solubilized antioxidants were discussed. The depth of inclusion of the functional group determines the extent of the scavenging-rate difference, suggesting that the cavity wall of the cyclodextrin acts like a barrier that hinders the approach of attacking free radicals.

18－05

Study on evolution and color properties of pyranoanthocyanin from red wine.

Masashi Hisamoto
Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi

Anthocyanins are the major components responsible for the color of young wines. However, they are relatively unstable and tend to form anthocyanin derivatives during maturation and aging, some of them are more stable than anthocyanins. In this study, the evolution of anthocyanin derivatives content, as measured by UPLC-DAD-TOFMS, was studied in Cabernet Sauvignon (CS) and Muscat Bailey A (MBA) wines during bottle storage, as well as to establish the contribution of the major pigment families to wine color. CS and MBA wines produced in Japan from grapes of the same vineyard in vintages (2000-2008). It is found that anthocyanins and flavanol-anthocyanin acetaldehyde-mediated condensation products were decreased, and pyranoanthocyanins and polymeric pigments were increased in wines. These results indicate that the chemical reactivity of red pigments in wine is influenced by the grape variety and the chemical structure of each anthocyanin derivatives during aging under bottle storage.

18－06

Studies on regulatory effect of food additives containing flavonoids on the blood glucose level

Hitoshi Ashida
Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University

Dietary intake of flavonoids is suggested to be potentially beneficial for the prevention of various diseases. To prevent hyperglycemia and diabetes mellitus, regulation of the postprandial blood glucose level is one of the important issues. However, the underlying molecular mechanisms are not fully understood yet. In this study, we investigated that the effects of four flavonoids, quercetin, quercetin 3-glucoside (isoquercitrin), quercetin 3-O-beta-rutinoside (rutin), and enzymatically modified isoquercitrin (EMIQ), on hyperglycemia in mice. Our results demonstrated that oral administration of EMIQ and quercetin suppressed the postprandial hyperglycemia after glucose ingestion, accompanying with promotion of glucose transporter 4 (GLUT4) translocation in the plasma membrane of skeletal muscle of ICR mice. EMIQ and quercetin also promoted GLUT4 translocation in skeletal muscle of C57BL/6 and ICR mice fed a flavonoids-supplemented diet for 7 or 14 days, respectively. In these beneficial effects, EMIQ was more effective than quercetin. Moreover, EMIQ and quercetin suppressed high-fat diet-induced hyperglycemia and glucose intolerance through promotion of GLUT4 translocation in skeletal muscle of C57BL/6 mice. Similarly, isoquercitrin and rutin also suppressed high-fat diet-induced hyperglycemia through promoting GLUT4 translocation. On the other hand, these effects of flavonoids were independent of a-glucosidase inhibition in the small intestine. These results indicate that quercetin and its related compounds are beneficial for improvement of glucose intolerance by promoting GLUT4 translocation to the plasma membrane of skeletal muscle.

18－07

International comparison of Halal systems on foods and food additives

Ryoichi Namikawa
Faculty of Policy Study, Chukyo University

The food markets of Islamic countries are becoming attractive because of their large populations and rapid economic growth. Food companies from non-Islamic countries, to enter food market in Islamic countries, are required to clear the Halal system, which controls trade, distribution and production of nonstandard foods according to the religious principles. They experience difficulties entering the markets because an internationally harmonized Halal standard is not established. From careful observation of the Halal systems of four countries in Asia-Pacific region, the author argues discrepancy among these systems. The author finds that there is not clear discrepancy in the scope of regulations but some differences in strictness of the regulations. The author also shows that such differences come from rather the social and industrial structure in those countries than religious principles.

18－08

Elucidation of the anthocyanin biosynthetic pathway of purple carrot (Daucus carota L.).

Nobuhiro Sasaki
Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology

Cyanidin 3-O-[6''-O-(sinapoyl-6'''-O-glucosyl)-2''-O-xylosyl)]-galactoside (CySGXGal), which is the main anthocyanin of purple carrot (Daucus carota), are used as a food additive because of its stable coloring. The several enzymatic reaction involved in CySGXGal biosynthesis had not been clarified. In this study, we found that the glucosyltransferase activity toward to cyanidin 3-xylosylgalcoside using acyl-glucoses as the glucosyl-donor in the crude protein prepared from cell suspension culture of carrot. This acyl-glucose dependent glucosyltransferase more preferred vanillyl- and feluroyl-glucose than the other acyl-glucoses. The pH optimum of this enzyme was 5.5-6.5 implying it could be work in vacuole and the enzyme remained more than 90% residual activity after storage at 4oC for a week. This is the first report of the enzyme that catalyzes transferring glucosyl moiety from acyl-glucose to the glycosyl moiety conjugating with the anthocyanin basic skeleton.

18－09

Development of High Performance and Powerful Preconcentration System for the Sensitive Analysis of Residue Drug in the Meat and Dairy Products

Yoshitaka Takagai
Cluster of Science and Technology, Faculty of Symbiotic Systems Science, Fukushima University

Ivermectin B1a (IVM) remarkably effects for veterinary animals to control parasitic disease.  Many international public health agencies have been requiring a close monitoring of veterinary drug residues in edible animal tissues increasing in global demand for safety and health toward food.  However, the ordinary and conventional pretreatment steps for chromatographic techniques are very complicated, so that simple and rapid protocol is required in worldwide.
In this report, a simple and effective approach of alternative pretreatment protocol of IVM for gas chromatographic and/or liquid chromatographic mass-spectrometric analysis is presented.  In particular, we focus on a preconcentration of extracts (organic solvent containing IVM) after biological tissue sample was treated using organic solvent. The extracts was preconcentrated utilizing a phase separation phenomenon based on ternary solvents (triphasic) component system (water / water-miscible organic solvent / water-immiscible organic solvent); it is so-called the homogeneous liquid-liquid extraction ternary solvent system (HoLLE).  The pertinent experimental factors (the combination of solvent type, moisture (water) percentage in 2nd phase, concentration of salt and solution pH) that impact the HoLLE parameters will be elucidated.  For instance, 99.2 % extractive efficiency of IVM was achieved in combination of acetone and carbon tetrachloride. In the attempt to enhance the analytical sensitivity of IVM, the HoLLE was applied to GC-MS and/or LC-MS.  In GC-MS protocol with HoLLE, whereas the trimethylsilization was required, it exhibits almost close detection limit value of LC-MS. The detail of TMS reaction also will be summarized in the presentation. In addition, IVM analysis in actual edible meat was successfully demonstrated. Present pretreatment and preconcentration protocol would be expected to utilize in many fields such as food analysis and biological tissue sample.

18－10

Fundamental Safety Assessment of Natural Flavoring Substances-2

The natural source 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 divided 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 individually.
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 individual flavoring additive.
It becomes evident that the above data base promoted the scientific safety data of flavoring additives obtained from RTECS data.

18－11

Concomitant apoptosis and regeneration of liver cells as a mechanism of liver-tumor promotion by a-naphthoflavone involving TNFb-signaling due to oxidative cellular stress in rats
-Mechanistic study of early-stage carcinogenesis involving oxidative stress responses
　through analysis of modifying effect of antioxidants-

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

Enzymatically modified isoquercitrin (EMIQ) is effective as an antioxidant in vivo and has chemopreventive potential against the development of hepatocellular preneoplastic lesions. β-Naphthoflavone (BNF) is a strong inducer of cytochrome P450 1A enzymes, and exerts liver tumor promoting activity through enhancement of oxidative stress responses in rats. This study investigated the role of the tissue environment surrounding hepatocellular preneoplastic lesions in the early tumor-promotion stage by BNF, using enzymatically modified isoquercitrin (EMIQ) as an anti-oxidative chemopreventive agent. Male F344 rats were fed a diet containing BNF (0.5%) for 6 weeks, with or without EMIQ (0.2%) in the drinking water, 2 weeks after initiation with N-diethylnitrosamine, and were subjected to two-thirds partial hepatectomy 1 week after starting BNF-promotion. BNF-treatment increased concentrations of liver thiobarbituric acid-reactive substances, single liver cells expressing glutathione S-transferase placental form or heme oxygenase (HO)-1, and concomitant apoptosis and proliferation of liver cells. Transcript upregulation of anti-oxidative enzymes (Aldh1a1 and Nqo1), cell cycle-related molecules (Cdc20 and Cdkn2b) and inflammation-related molecules including proinflammatory cytokines (Ccl2, Col1a1, Il6, Nos2 and Serpine1) was also evident. Furthermore, BNF increased HO-1-expressing Kupffer cells and liver cells expressing tumor necrosis factor receptor 1 (TNFR1) and the TNFR1-associated death domain. Most of these BNF-induced fluctuations disappeared or were suppressed by EMIQ in conjunction with suppression of tumor-promotion. Tnf transcript levels with BNF were also suppressed by EMIQ. These results suggest that BNF-induced oxidative stress causes single liver cell toxicity, allowing subsequent concomitant apoptosis and regeneration involving inflammatory responses including TNFα-signaling, contributing to tumor promotion. Kupffer cells may act to protect against inflammatory stimuli induced as a result of oxidative cellular stress by BNF, causing proinflammatory cytokine level fluctuations.

18－12

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

Kazuo Ohtake
Division 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. KKAy 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. KKAy mice were divided 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 KKAy 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 KKAy 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 KKAy mice followed by restoration to the same levels as C57BL/6 control groups with nitrite supplementation. Compared with KKAy mice without nitrite treatment, nitrite treatment to KKAy significantly decreased mean adipose tissue size. Additionally our study showed that in skeletal muscle nitrite treatment to KKAy mice increase GLUT4 translocation to plasma membrane following PI3-kinase regulation subunit p85 expression and remarkably enhanced Akt phosphorylation . Our study demonstrate that nitrite treatment to KKAy 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 KKAy mice.

18－13

Evaluation of the inhibitory effect of hydrocolloid-type polysaccharide thickener on the food allergen absorption

Tatsuya Moriyama
Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University

In recent years, food allergy has become a serious problem. Food allergy suffering patients has increased dramatically. In general, major type of food allergy is caused by the absorption of undigested food into blood at the intestinal tract, followed by the antigen sensitization (IgE production) and the onset of allergy symptom. Therefore, by inhibiting this process properly, it might be possible to suppress the risk of food allergy developing. In this study, we verified by using an in vivo evaluation system using mice the effects of hydrocolloid-type polysaccharide thickener on the food allergen absorption. The allergen absorption into blood was verified by Western blotting and dot blotting methods. Soy milk was orally administered to mice in the presence of a polysaccharide thickener. Soybean allergen levels migrated in the blood were analysed by Western blotting and dot blotting methods. As polysaccharide thickener, gum arabic, xanthan gum, guar gum, and pectin was used.  As a result, in the presence of any of the polysaccharide thickener, the migration of allergen into the blood has been suppressed to about 30 to 10 % compared with the case of distilled water control. These results indicated that hydrocolloid-type polysaccharide thickener may have a beneficial effect by inhibiting the absorption of food allergens into blood flow at the intestinal tract.

18－14

Effect and safety assessment of nano materials used as food additives in development of gastric ulcers

Akira Onodera, Takuya Furuta, Ayaka Iwasaki, Ai Housho, Yuichi Kawai
Department of Pharmaceutical Sciences, Kobegakuin University
Shigenobu Yonemura
RIKEN CDB

Nanotechnology is progressively used not only in the industry and the medical field but also in the food industry. In the food industry, many products are marketed for enhancing food quality, improving usage efficiency of food constituents, taste masking, and packing. On the other hand, acquisition of a new function by nanosizing a material has resulted in the onset of a strange toxicity called nanotoxicity.
We analyzed the safety of a nanoscale material relevant to food for promoting the effective and safe use of nanotechnology in the food industry. The model compound Nano uses the amorphous nanosilica with a particle diameter of 30 nm and 70 nm compared with the amorphous silica of microscale particle diameter of 300 and 1000 nm. Candidate of toxic evaluation is the stomach which stores food in the first place; especially we analyzed risk of development of gastric ulcer. We employed the following three procedures: administration of each silica particle resulting in an excessive dose due to repeated injection by a gastric tube in normal mouse (Verification-1), an excessive dose of a single injection to the gastric mucous membrane surface in normal mouse (Verification-2), or an excessive dose of repeated injection by a gastric tube in alkylating agent induced gastric ulcer mouse model (Verification-3). A remarkable toxicological change in the gastric mucous membrane surface was not observed in Verification-1 and 2. However, in Verification-3 the nanoscale-dependent delay of ulcer healing and fibrosis by inflammation that spread in the entire gastric area was observed. Further, to clarify the delay of ulcer healing by nanosilica, we demonstrated the effect of wound healing by nanosilica using an in vitro model. As a result, nanoscale-dependent prevention of cell migration was observed. This suggested that the prevention of cell migration to the ulcer part of epithelial cells or fibroblasts is one of the causes for delayed ulcer healing by nanosilica.
Although the nanoscale material in the food field was not observed to cause independent toxicity, the nanopeculiar toxicity revealed prevention of ulcer healing.

18－15

Bioavailability of the pectin masked trace contaminants in food

Yoshiko Konishi
National Institute of Health Sciences

We have found out that low methoxyl pectin gel has ability to reduce the absorption of trace contaminant in food, mycotoxin due to mask the contaminants from in vitro and in vivo experiments.  Since mycotoxins are known to cause adverse effects on human and animal health, the reduction of intake mycotoxins is final goal in order to prevent the adverse effect. 
In this study, we established the gastrointestinal digest model in vitro using artificial saliva juice, stomach juice, intestinal juice and Caco-2 transwell to clarify the bioavailability of mycotoxin in the pectin gel.  We also examined the masking effect of pectin gel on patulin, aflatoxin M and fumonisins. The experiments revealed that low methoxyl pectin maintained gel form and masking ability after saliva juice and stomach juice treatment, but after the treatment of intestinal juice the pectin gel changed form to liquid due to breakdown by pH change.  The low methoxyl pectin gel showed masking effect for patulin , aflatoxin M and fumonisins as well as deoxynivalenol.  These results suggest that the pectin gel would be useful to mask and reduction of absorption of mycotoxins which absorb in stomack mainly, such as deoxynivalenol, patulin , aflatoxin M and fumonisins.

18－16

Analysis of metabolic activation of chemicals by human cytochrome P450 proteins for establishment safety evaluation system of food additives

Tatsushi Goto
Research Center for Environmental Genomics, Kobe University

The umu test using rat liver S9 fraction is presently accepted as a general method for convenient detection of mutagenic compounds but includes inconsistency with the test using for human liver S9 fraction. We attempted to improve the given umu test and to apply it to safety evaluation of food additives that are added to foods for the purpose of the quality preservation or the quality enhancement. We prepared Escherichia coli-generated human CYP isoforms which account for 80% of total hepatic CYPs; CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. The umu test assay of known mutagens (2-aminoanthrathene, aflatoxin B1 and benzo[a]pyrene) in the presence of these CYP isoforms showed that the mutagenic response of 2-aminoanthrathene and aflatoxin B1 were enhanced by CYP1A2 and CYP3A4 isoform, respectively. This result indicated that the modified umu test was useful for the mutagenicity detection and for the specification of the relating CYP isoform. The food additives examined in the modified umu test did not show the evidence of DNA damage.

18－17

Exploring the transporters involved in the intestinal absorption of anthocyanins in gastrointestinal tract

Katsuhisa Inoue
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University

It has recently been suggested that carrier-mediated transport is involved in the intestinal absorption of anthocyanins, which belong to a large and widespread group of water-soluble phytochemicals and exhibit potent antioxidative and anti-inflammatory properties. The carrier involved in anthocyanins uptake may be one of those which play important roles in absorption of flavonoids, but it has not been identified yet. In this study, we conducted studies to develop a sensitive assay method for the assessment of the functionality of facilitative transport systems of flavonoid glycosides including anthocyanins, using MU-glucosides as a fluorescent probe substrate, in the HEK293 cells stably expressing GBA3, a cytosolic beta-glucosidase. Furthermore, we attempted to identify transporters involved in the absorption of flavonoid glycosides. It was found that the uptakes of MU-Glc and MU-Gal in HEK293 cells stably expressing GBA3 were detectable with a highly pH-dependent and concentration-dependent (saturable) manner, suggesting the involvement of an endogenous carrier in the HEK293 cells. According to kinetic analysis, the Michaelis constant for MU-Glc was 8.5 lochM, which is much lower than that of GBA3, suggesting the process of carrier-mediated transport is a rate-limiting step for the entire events for the uptake and metabolism. However, the exogenous expression of several candidate transporters such as GLUT1, GLUT2, GLUT5 and GLUT10 in the HEK293 cells stably expressing GBA3 did not alter the uptake activities. These results suggest that the carrier of flavonoid glycosides is a facilitative transporter driven by a proton gradient, but distinct from glucose transporters such as GLUTs.

18－18

Effectiveness of preservatives concerning food-borne fungi

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.
Assessment of microbial was mainly described for bacteria such as Escherichia coli.
On the contrary, food fungi have not treated as important microbes because of lacking in regulatory field. Therefore, we investigated on the effectiveness of representative 6 preservatives (calcium propionate, sodium propionate, 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, Eurotium herbariorum FCR 322, Chaetomium globosum FCR 322, Fusarium oxysporum FCR 452, Rhizopus stolonifer FCR 411 and Wallemia sebi FCR176. 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 200 ppm or so. However, we investigated much more detail study on 6 preservatives that the effectiveness of 6 preservatives examined static anti-fungal activities at 3, 5, 7, 10, 14, 21 and 28 days. Finally we showed the exact evaluation. That is, the static anti-fungal activities were shown the effective and sensitive value for short time as 3 or 5 day evaluation.  The fungal morphological changes treated with 2 representative preservatives (calcium propionate and p-hydroxy benzoic acid ethyl) were tried with 3 fungi. These fungi were shown the resistant forms as the arthrospore like hyphae and the cell wall with dark concentrated colored.
As the results, the effectiveness of preservatives concerning the food-borne fungi must be investigated and evaluated at diversified studies not only MIC value evaluation.

18－19

Studies on effective use of an antibacterial peptide “nisin A” in neutral pH range

Yasushi Kawai
Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University

 Nisin is an antimicrobial peptide (bacteriocin) produced by Lactococcus lactis, a lactic acid bacterium for dairy farming, and has been used as food additives in higher than 50 countries containing Japan. However, the antibacterial effect of nisin drastically decreases at neutral pH because of low solubility. In this study, we searched effective availability of nisin at neutral pH range and tested the antibacterial effect and mode of action against an indicator strain, Lactobacillus delbrueckii subsp. bulgaricus JCM1002T. Nisin had the high heat stability (95°C, 1h) and showed the synergistic effect in 20% ethanol. Metabolome analysis clarified that nisin increases the antibacterial effect by accumulation of organic acids such as lactic acid in intracellular of bacterial indicators in addition to action mechanism of general bacteriocins such as pore formation in target cell membranes and efflux of low molecular weight substances from the membrane resulting in death. And high accumulation of some organic acids in the indicator cells was observed by nisin in 20% ethanol.

18－20

Flavor Release from Spray-dried Powder by Ramping　with Dynamic Vapor Sorption-Gas Chromatography

Hidefumi Yoshii
Department of Applied Biological Science, Kagawa University

The ramping method in humidity at constant temperature was used to evaluate the release of d-limonene from spray-dried powder by using the handmade dynamic vapor sorption (DVS) instrument. The release fluxes of d-limonene from spray-dried powder at two different feed liquid temperatures, 40°C and 80°C could rapidly evaluate the flavor release properties of spray-dried powder.  The flavor fluxes for 40°C of feed liquid temperature were faster than those for 80 °C at all humidity range. The powder properties such as flavor release rate and collapse could be measured by the humidity ramping method.　The storage relative humidity at which collapse of matrix occurs in a spray-dried powder could easily be identified by the humidity ramping method.

18－21

Possibility as enhancer for food additives and characterization of flavoring compounds in citrus peels from Iyo

Yoshiaki Amakura
College of Pharmaceutical Sciences, Matsuyama University

Ehime is a leading prefecture in cultivating citrus fruits in Japan, and various citrus fruits as well as Citrus unshiu are grown as special products.  Among them, there are rare citrus fruits which are in short supply.  Therefore, scientific data on their constituents, including essential oils, are limited.  In this study, the volatile components of citrus peel oil samples from Ehime were analyzed in order to evaluate the characteristic aroma constituents which contribute to the development of the citrus flavor for the first time.  First, the volatile components of 6 citrus (Ogonkan, Setoka, Harehime, Harumi, Kanpei, and Benimadonna) peel oils were characterized by GC-MS. Limonene (63.1%) was the most abundant compound in the Ogonkan peel oil, followed by g-terpinene (12.1%), b-farnesene (2.4%), and a-pinene (2.2%). The Setoka peel oil contained limonene (91.8%), myrcerne (1.4%), and sabinene (1.0%). Limonene (83.1%), myrcene (3.5%), and a-pinene (1.0%) were present in the Harehime peel oil, with limonene (81.2%), g-terpinene (2.9%), linalool, and cymene (1.5% each) in the Harumi peel oil. Also, limonene (78.5%), g-terpinene (6.3%), myrcene (2.9%), a-pinene (1.8%), decanal (1.2%), and linalool (1.1%) were present in the peel oil of Kanpei. Benimadonna peel oil contained limonene (81.7%), myrcene (3.3%), sabinene (1.5%), and a-pinene (1.1%). Next, the contents of 12 constituents (flavanols and their glycosides, polymethoxyflavone, etc.), characteristic citrus components, in the EtOH extracts of 6 citrus peels were analyzed by HPLC. Furthermore, the synergetic effect of each citrus oil and EtOH extract as a food additive (antioxidant) was evaluated, and the influence of the citrus oils and EtOH extracts from Ehime on food additives was discussed.

18－22

Study on Physical Properties of Care Foods for Dysphagic Patients

Hitoshi Kumagai
Faculty of Home Economics, Kyoritsu Women’s University

To determine the appropriate properties as foods for dysphagic patients, relation between the parameters obtained from TPA (Texture Profile Analysis) and the velocity of foods through the pharynx were investigated.  TPA tests were conducted at plunger speeds of 1 and 10 mm/s, and three textural parameters of hardness, adhesiveness, and cohesiveness were obtained from TPA profiles.  The velocity through the pharynx was measured by the ultrasonic pulse Doppler method.  A test speed of 1 mm/s in TPA was suggested to be more suitable than 10 mm/s.  Hardness was correlated best with the maximum velocity through the pharynx , Vmax, which has been reported to be one of the best predictors of aspiration risk, among three textural parameters.  On the other hand, the cohesiveness was not correlated with velocity distribution through pharynx, with changing in sample properties. Hardness is, therefore, considered to be the most suitable index for care foods for dysphagic patients among these three parameters.

18－23

Effect of emulsifying polysaccharides on LPS absorption from the intestine

Kazunari Ushida
Animal Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University

Some of the dietary polysaccharides may adsorb bacterial lipopolysaccharides (LPS) by their emulsifying properties. Adsorbed LPS then excreated from the intestine without significant absorption from the mucosa. Since LPS works as endotoxin after absorption which induces various inflammatory responses, LPS absorption can aggaravate the obesity. Gum arabic has a potential to eliminate LPS from absorption by adsorbing it with hydrophobic protein. In this experiment, we fed rats with various dietary fibers with varying emulsifying capasities to determine the rate of LPS absorption from intestinal mucosa. Gum arabic (lower molecular weight) significantly reduced portal LPS level and as a result it reduced expression levels of inflammatory cytokine expression in the visceral adipose tissue.