The Research Reports No.27 （issued in 2021）

Abstracts of Research Reports funded by the Foundation's Research Grants in FY2020.

27－01

Elucidation of the pathogenesis of type 2 diabetes through the intestinal-liver linkage of trans-fatty acids

Takuro Okamura
Department of Endocrinology and Metabolism,
Kyoto Prefectural University of Medicine, Graduate School of Medical Science

Many nutritional and epidemiological studies have shown that high intake of trans-fatty acids has several adverse effects on human health, including cardiovascular diseases, diabetes, and cancer. In this study, we investigated the effects of trans-fatty acids on innate immunity in the gut by observing mice fed a diet high in trans-fatty acids. The methods were as follows: C57BL6/J mice were fed a normal diet (ND) or a high fat and high sucrose diet (HFHSD) or a high trans fatty acid and high sucrose diet (HTHSD) for 12 weeks. 16S rRNA gene sequencing was performed on mouse stool samples, and flow cytometry, real-time PCR, and lipidomics analysis were performed on mouse serum and liver samples. for in vitro studies, RAW264.7 cells were used. Mice fed with HTHSD had significantly higher blood glucose levels and more advanced fatty liver and intestinal inflammation compared to mice fed with HFHSD. Furthermore, compared to the mice fed HFHSD, the mice fed HTHSD had significantly increased expression of CD36 and decreased expression of IL-22 in the small intestine. Furthermore, the population of ILC1 and T-bet positive ILC3 was significant- ly increased in the intrinsic layer of the small intestine in mice fed HTHSD. Finally, the relative abundance of the family Desulfovibrionaceae, which belongs to the phylum Proteobacteria, was significantly higher in mice fed HFHSD or HTHSD than in those fed ND, and was slightly higher in the HTHSD group than in the HFHSD and HTHSD groups. This study revealed that, compared with saturated fatty acid intake, trans fatty acid intake significantly aggravates metabolic diseases such as diabetes and fatty liver.

27－02

Application 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 tis- sues and scavenge free radicals, singlet oxygen, etc. to prevent aging. In the present study, we have attempt- ed to apply practical antioxidant-efficiency assay-methods (Aroxyl Radical Absorption Capacity (ARAC), Singlet Oxygen Absorption Capacity (SOAC), Alfa-Tocopherol REcycling Capacity (ATREC) assay meth- ods) to estimation of the antioxidant-efficiency for food additives and foods containing the additives.

27－03

Identification of the mechanism underlying curcumin-mediated regulation of circadian clock

Jun Hirayama
Department of Clinical Engineering, Faculty of Health Sciences, Komatsu University

The circadian clock generates daily physiological rhythms that have been proposed to control the timing of sleep and metabolism. The circadian clock is established by cell-autonomous oscillators called cellular clocks, which are present in every cell of a living organism. Curcumin is used as a spice and food additives, and, especially in Japan, it is designated as a coloring agent as a turmeric pigment. In addition, curcumin has been reported to regulate gene expression. Previous study found that curcumin is able to regulate cellular clocks' activity. This study aimed to reveal the mechanism underlying curcumin-mediated regulation of cel- lular clocks.

27－04

Improving Operability and Visibility of Embryo Transfer Using Food Additive Dyes

Hiroyuki Imai
Laboratory of Veterinary Anatomy, Joint Faculty of Veterinary Medicine, Yamaguchi University

Most commercial culture media have a light red color with the addition of phenol red. On the other hand, when embryo transfer is performing using these culture media, the color tone of the media is similar to that of the uterus in mice. As a result, it is difficult to check with naked eye and is not easy to operate. The aim of the present study was to improve the visibility and usability of embryo culture and transfer media using known food addictive dyes. We identified two types of dyes that can alter color tone while maintaining the function of the embryo culture media. Using these candidate dyes, we are currently investigating the pos- sibility of applying them to embryo transfer media. Furthermore, we seek to expand the range of application of these dyes.

27－05

Elucidation of regulating mechanisms on tumor microenvironment in a colorectal carcinogenic mouse model by fucoxanthin-added biscuit

Masaru Terasaki
School of Pharmaceutical Sciences, Health Sciences University of Hokkaido

Fucoxanthin (Fx) is a carotenoid abundantly contained in brown algae experienced in food culture such as Undaria pinnatifida (wakame) and has potent anticancer, anti-obesity and anti-diabetes. Recently, Fx has been processed and developed as food additives such as pie, shortbread and scraper, expected to have its functionality. However, there is little information on the functional effect of Fx-added foods. In the present study, we have investigated the cancer chemopreventive effect by a Fx-added biscuit using azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model. As a result, administration of Fx-added biscuit sig- nificantly prevented colorectal carcinogenesis in AOM/DSS mice. In addition, the many genes involving in growth and inflammation significantly were altered in the colorectal mucosal tissue. Next future, the mo- lecular mechanisms underlying the cancer chemopreventive effect by Fx-added biscuit in the mice will be investigated.

27－06

Analyzing the effect of food additives using a new monkey intestinal organoid culture

Ken Iwatsuki
Tokyo University of Agriculture, Department of Nutritional Science and Food Safety

The gastrointestinal tract not only digests and absorbs nutrients but also has multiple function such as barrier, immune and endocrine. These functions are exerted by highly specialized epithelial cells. The endo- crine cells are one of cells that secretes hormones upon detecting nutrients and maintain homeostasis of the body. From previous reports, endocrine cells secrete incretins that regulate blood glucose level. These days there exist a trend that consumers chose high-intensity sweeteners to replace the position of the sugar be- cause high-intensity sweeteners are generally zero- or low-calorie. However, there are few reports to show whether high-intensity sweeteners affect endocrine cell growth or function. We therefore aimed to analyze the effect of high-intensity sweeteners on endocrine cells in intestinal organoids derived from mouse or monkey.
By now, our results indicate that sucralose could affect endocrine differentiation in mice but not in mon- keys.

27－07

Study about cooking conditions to reduce acrylamide formation in foods

Hiromi Nabeshi
Division of Foods, National Institute of Health Sciences

We examined effects of soaking of potato in various solutions to reduce acrylamide (AA) formation in French fries. Thin strips of potato soaked for 20-180 min in various solutions such as water and vinegar water were used to make French fries in a conventional deep fryer and an air fryer. AA concentrations in the French fries were determined by LC-MS/MS analysis. Compared to unsoaked controls, water soaking suppressed AA formation in both fryers. This suppression effect tended to increase with prolonged soaking time. Under the same soaking time, the use of warm water, vinegar water and salt water all more effectively suppressed AA formation in French fries compared to room temperature water. Changing the water also ef- fectively suppressed AA formation. On the other hand, AA concentrations in the French fries cooked by the air fryer were higher than those cooked by the conventional deep fryer. This may be attributable to higher heating temperatures, longer heating times and larger heating irregularities in the air fryer compared to the conventional deep fryer. Furthermore, AA concentrations in French fries cooked by the air fryer without oil were markedly higher than those cooked with oil. It may be necessary to pay attention to the possibility of increased AA formation in French fries cooked by air fryers (particularly non-oil cooking).

27－08

Molecular Structure of Gardenia Blue Pigments using solid-state NMR

Kaname Tsutsumiuchi
Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University

Genipin was reacted with several amino acids to prepare gardenia blue (GB). GBs with amino acids (GB- AAs) were characterized by both liquid and solid-state NMR measurements. Interestingly, many significant peaks appeared in the solid-state NMR spectra, although the ¹³C NMR spectra from solution samples did not show any distinct peaks. Therefore, we determined that GB-AAs had an alternating copolymer struc- ture composed of methyne and 5H-2-pyrindine, which was substituted by amino acids at N atom and linked with methyne at 5 and 7 positions. To confirm this molecular structure, the pyrolysis gas chromatography- mass spectrometry (GC-MS) measurement of GB-AAs was carried out, and 5H-2-pyrindine and its methyl derivatives were observed as main pyrolysis products from the polymer chains. Furthermore, we performed ultraviolet−visible (UV−vis) spectral calculations with time-dependent density functional theory (TDDFT) to confirm that the molecular structure of GB is reasonable to provide the blue color.

27－09

Evaluation of the effects of simultaneous ingestion of food-derived PPAR ligand components and therapeutic agents using the nonalcoholic steatohepatitis (NASH) model medaka

〇Masaya Uchida ¹, Hiroshi Ishibashi ², Masashi Hirano ³, Ryoko Yamamoto ⁴
1 Department of Creative Engineering, National Institute of Technology, Ariake College
2 Graduate School of Agriculture, Ehime University
3 School of Agriculture, Tokai University
4 Faculty of Agriculture and Life Science, Hirosaki University

The number of patients with lifestyle-related diseases such as dyslipidemia, diabetes, and nonalcoholic steatohepatitis (NASH) has recently been increasing worldwide. The evaluation of functional foods contain- ing biologically active ingredients is required for preventing and managing these diseases, and furthermore alternatives to rodents are urgent issues to reduce the number of experimental animals. In this study, we examined the potential of medaka as an alternative model organism in food function research using the me- daka NASH model. 8-week-old fish were fed a high-fat diet (HFD) for 12 weeks to induce NASH. Fish that were fed the HFD exhibited significantly greater body, liver weight, and hepatosomatic index (HSI). HFD- administered fish also showed abdomen distention, and the presence of swelling and whitening of the liver. Thus, we confirmed the formation of a fatty liver. To evaluate the interaction of functional components with medaka PPARγ, the transcriptional activation of medaka PPARγ by citral and resveratrol was investigated using in vitro reporter gene assay. Treatment with resveratrol induced medaka PPARγ-mediated transcrip- tional activity in a concentration-dependent manner. The concentration range of this response was similar to that of human PPARγ. Taken together, the present study provides the basic knowledge on alternative models to experimental animals in food function research. To efficiently generate NASH models, the conditions of HFD administration will be needed in the future. Furthermore, we plan to investigate the effects of food- derived PPAR ligands/therapeutic agents taken alone or in combination using the medaka NASH model.

27－10

Improvement of the reliability for anthocyanin determination by external calibration qNMR

Yuzo Nishizaki
Department of Food Additives, National Institute of Health Sciences

External calibration quantitative NMR (EC-qNMR) for anthocyanin purity determination was examined. Using cyanidin chloride (Cy chloride) and cyanidin 3-glucoside chloride (Cy3G chloride) supplied from re- agent companies as model samples, deuterated solvents for EC-qNMR were optimized. EC-qNMR showed about 25% purity differences between both six Cy chlorides and seven Cy3G chlorides. Of these, the sample with low purity was found to have a higher Cl content than the other samples by using ion chromatography. From the results, it was guessed that excess Cl was contaminated to the final product during chlorination in the low purity Cy chloride and Cy3G chloride. Calibration curves, corrected by using EC-qNMR purities, of each sample on LC/PDA were close to each other. Therefore, it was supported that the EC-qNMR purity values reflect true values. Among the samples examined, the highest EC-qNMR purity was 96.0% for Cy chloride and 91.9% for Cy3G chloride. The water sorption-desorption analysis showed that the remaining component in Cy chloride (100%-96.0%) and Cy3G chloride (100%-91.9%) seem to be just water.

27－11

Elucidation of the mechanism of alternative splicing regulation by flavonoids used as food additives

Seiji Masuda
Laboratory of Molecular Biology of Bioresponse, Graduate School of Biostudies, Kyoto University

Recently, the splicing process of mRNA has been attracting attention as a target for drug discovery of anti-cancer drugs. Although a number of compounds that can regulate mRNA splicing have been found in food components, detailed analysis of the molecular mechanism has not been performed so far. The purpose of this study was to analyze the mechanism of splicing regulation by food compounds and to present a ba- sic analysis method to elucidate the splicing regulation mechanism by newly discovered food compounds through the analysis. The analysis of splicing changes by next generation sequencing and rMATs, followed by characteristics such as intron length, GC content, 5' and 3' splice site scores, and branch point sequence scores, was shown to be a useful tool for analyzing the molecular mechanism of alternative splicing by food compounds.

27－12

Development on a new quantitative analysis method for alginic acid using a novel exo-type alginate lyase

Toshiyuki Shibata
Department of Life Sciences, Graduate school of Bioresources, Mie University
Seaweed Biorefinery Research Center, Mie University

4-Deoxy-L-erythro-5-hexoseulose uronic acid (DEH) is a monosaccharide produced by the action of exo- type alginate lyase (Exo-Aly) from alginic acid. DEH does not originate from glycuronan other than alginic acid, so it can be considered as the only compound that can be used as a standard for alginic acid. The pur- pose of this study is to develop a quantitative analysis method for alginic acid that combines the decompo- sition of alginic acid into DEH using a novel Exo-Aly (AlyFRB) and the quantitative analysis of DEH by LC/MS. The marine bacterium Falsirhodobacter sp. alg1 produces a novel Exo-Aly (AlyFRB) with strong alginic acid degrading property. A recombinant protein (rAlyFRB) of the Exo-Aly was prepared and the condition for the enzymatic reaction required for the complete degradation of alginic acid (polymannuronate) was established. It was found that a mixture of alginate lyases was able to degrade alginic acid into DEH in the presence of thickening polysaccharides (pectin, λ-carrageenan, and sodium carboxymethylcellulose). As a result of a test using a commercially available beverage including sodium alginate (food for specified health use) as a sample, it was showed that a method that combines enzyme treatment with rAlyFRB and quantitative analysis method of DEH using LC/MS can be applied to quantitative analysis of alginic acid.

27－13

Absorption, distribution and metabolism of dietary glycerophosphocholine

Noriyuki Yanaka
Graduate School of Integrated Sciences for Life, Hiroshima University

In recent years, choline deficiency is shown to be associated with the development of liver diseases and dementia, however choline intake in developed countries has not been sufficient. On the other hand, it has been recently reported that high choline intake increases the risk of cardiovascular disease (CVD). Dietary choline is converted to trimethylamine (TMA) by gastrointestinal microflora, and then metabolized to tri- methylamine N-oxide (TMAO) in the liver. Blood TMAO level has been discussed to be associated with the development of CVD.
In the present study, we focused on glycerophosphocholine (GPC), a deacylated form of phosphatidylcho- line, as a nutritional material for choline supply, and found that GPC intake increased choline and betaine levels not only in the blood and liver, also in the hippocampus. However, the blood TMAO concentration was shown to be higher in the GPC intake group. In this study, GDE5 flox mice, which have loxP sequences at both ends of exon11, which encodes the active center of the GDE5 gene involved in GPC metabolism, were generated using the PITCh method, and mated with villin-cre (Vil cre) transgenic mouse, which ex- presses Cre recombinase specifically in gastrointestinal epithelial cells. We generated Vil cre+/-, GDE5 flox/- mice and have a plan to analyze the GPC degradation activity and the absorption mechanism of GPC.

27－14

Study of the antifungal activity of ε-poly-L-lysine, a food additive, by its inhibitory effect on cell cycle

Yoshiharu Inoue
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

ε-Poly-L-lysine (ε-PL) exhibits antifungal activity against fungi including the budding yeast Saccharo- myces cerevisiae. In this study, we investigated the possibility that the antifungal activity of ε-PL is due to cell cycle inhibition. Comprehensive analysis of gene expression profiles following treatment with ε-PL was performed in wild-type and mpk1∆ strains by DNA microarray analysis. As a result, we categorized the genes in three groups: category #1 (genes whose expression levels increased in the wild strain but not in the mpk1∆ strain), category #2 (genes whose expression levels decreased in the wild strain but not in the mpk1∆ strain), and category #3 (genes whose expression levels did not change in the wild strain but increased in the mpk1∆ strain). The gene group in category #1 included SLR3, which is a target of the cyclin-dependent kinase Cdc28 and is involved in transcriptional repression at the start of the G1/S transition of the cell cycle. Gene-disrupted mutants in this category showed ε-PL susceptibility, suggesting that these genes are required for resistance to the antifungal action of ε-PL in an mpk1-dependent manner. Category #2 included CLB6, a B-type cyclin in the S phase of the cell cycle, and RNR1, a major isoform of a subunit of ribonucleotide re- ductase essential for DNA synthesis. An rnr1∆ mutant showed hypersensitivity to ε-PL. The gene-disrupted strains in category #3 did not show any particular phenotype with respect to sensitivity to ε-PL. ε-PL in- duced phosphorylation of Cdc28 at Tyr19, suggesting that it may inhibit the G2/M transition.

27－15

Qualitative and quantitative analyses of the influence of artificial sweetener on immune tissue and cell functions

〇Akira Onodera, Daichi Arao, Haruna Mizuno, Yuichi Kawai
Department of Pharmaceutical Sciences, Kobe Gakuin University

Artificial sweeteners (AS) are included in soft drinks and confectioneries as low-calorie alternatives, though there are concerns about the health risks associated with long-term and excessive consumption of AS. The goal of this study was to elucidate the molecular mechanisms AS use to influence immune function and response. We used a human monocyte-like cell line (THP-1) and an allergic contact dermatitis mouse model to examine how AS influence cell function and T cell response. Acesulfame K (Ace K), Aspartame (APM), and Sucralose (SCL) all decreased proliferation, and APM decreased phagocytosis when exposed to THP-1. In vivo exposure of APM to mice promoted the expression of CD8-positive cytotoxic T cells, which are not known to play a role in the development of allergic dermatitis. These results show that AS either directly or indirectly influences the immune system, though further studies clarifying how immune cells rec- ognizes and respond to AS are necessary

27－16

Study of the three-dimensional culture organization and toxicity evaluation based on the analysis of the toxicity mechanisms on the intestinal injury induced by chemical substances in mice

Katsuhiro Miyajima
Department of Nutritional Science and Food Safety, Faculty of Applied Biosciences,
Tokyo University of Agriculture

The aim of the present study is to establish a safety evaluation system for food ingredients by an in vitro test system of intestinal organs based on the underlying mechanisms from the evidence of the in vivo stud- ies. In the fiscal year of 2020, we evaluated toxicity of Deoxynivalenol (DON) and inflammatory changes induced by dextran sulfate sodium (DSS) in male C57BL/6J or Lgr5-EGFP Tg mice.
Organoid cultures are three-dimensional culture systems that maintain the features of tissues or organs in vivo by constructing specific tissues consisting of multiple cells in vitro. In organoid cultures, function of the stem cell and differentiated tissue is reproduced, and it is also possible to observe the interaction between the constituent cells, and the cells and extracellular matrix. Because incorporate many of the physiologically relevant characteristics as living tissues physically, molecularly and physiologically mimic organs or tissues, intestinal organoid culture is also good model for evaluation in vitro.
Using intestinal organoid cultures that isolated from intestinal stem cells, grew and differentiated in gels, and cultured them three-dimensionally, we investigated the effects of various chemical substances including food ingredients on intestinal function, and the usefulness of organoid culture systems in intestinal research was also demonstrated.
Specifically, the effects of chemical substances such as DON and DSS on organoid cultures derived from the intestinal tract of mice were evaluated the mucosal changes including their formation rate, intestinal stem cell properties, related gene expression. In addition, mice were orally administered such chemicals and their effects on the intestinal mucosa were also evaluated in vivo and we compared the results of both experi- mental systems.

27－17

Analysis of release of damage-associated molecular patterns involved in inflammation and allergy after intestinal epithelial damage by potassium aluminum sulphate and immunological safety evaluation

Ayako Wakabayashi
Nippon Medical School, Department of Microbiology and Immunology

Our studies in mice have shown that oral ingestion of aluminum-containing food additives such as potas- sium alum (potassium aluminum sulphate) and ammonium alum (ammonium aluminum sulphate) induces eosinophil infiltration in the intestinal tract. The induction of such eosinophil inflammation may be triggered by the release of IL-33 by inflammatory cell death of intestonal epithelial cells (IECs) accompanied by the formation of apoptosis-related speck-like card proteins (ASC) specks. Gene expression involved in inflam- masome formation and inflammatory cell death such as ASC, Casp1, Casp4, Gasdmd, Il18, Nlrc4, and Nlrp6 was observed in IECs of mice. They were also expressed in the intestinal organoids of mice. Analysis of the molecular mechanisms of the inflammasome formation and inflammatory cell death in IECs stimulated by aluminum-containing food additives using mice and intestinal organoids is expected to contribute to preven- tion and treatment of the induction and progression of food allergy and intestinal inflammation.

27－18

Verification of the importance of arginine intake during anticancer drug treatment and evaluation of pharmacokinetics and tissue distribution in aged mice

Hitoshi Kotani
Shimane University Faculty of Medicine

Myeloid‐derived suppressor cells (MDSCs) play a crucial role in immunosuppression in tumor‐bear- ing hosts. MDSCs express arginase‐I; It suppress T‐cell function by reducing the levels of arginine. We examined the anticancer effects of supplementation of arginine in CT26 and MC38 colon carcinoma‐bear- ing mice. We have previously reported the effect of enhancing the antitumor effect by supplementation of arginine in young mice treated with anticancer drugs. In this year's study, we conducted a study using aged mice that are close to the actual clinical state. As a result, supplementation of arginine tended to enhance the therapeutic effect of multi-anticancer drug (cyclophosphamide, 5-fluorouracil and oxaliplatin) treatment group. Furthermore, the results were obtained in nude mice in which immune cells were involved in the ef- fects of these multi-anticancer drug treatments. Therefore, we conducted an experiment in which mice in the treatment group that also used an immune checkpoint (PD-1) inhibitory antibody sapplementate arginine. As a result, the therapeutic effect was enhanced by supplementation of arginine. Considering that the propor- tion of immune cells changes with aging of mice, the composition of immune cells of mice of various ages was examined. With aging, the proportion of immuno suppressive regulatory T cells and immuno exhausted (PD-1⁺) CD4⁺ T cells increases, the proportion of naïve T cells decreases, and the proportion of effector / memory T cells increases. This study suggests that aging reduces immunity. However, it was suggested that supplementation of arginine at the time of anticancer drug treatment in old age may enhance the cancer therapeutic effect.

27－19

Development of a quantitative method for the determination of high polar compounds derived from a food and its application to the elucidation of their dynamics in blood

Naohiro Oshima
Division of Environmental Chemistry, National Institute of Health Sciences, Japan

Understanding the types and amounts of food-derived constituents that migrate into the body is important for considering the functionality and safety of foods. In this study, plant extracts containing flavone C-gly- cosides in food were orally administered to mice, and the constituents in their serum were quantitively ana- lyzed by high performance liquid chromatography (HPLC). First, we examined a deproteination method that can easily detect flavone C-glycoside. It found that butanol-treatment method showed the highest intensity. Therefore, one of plant extracts, ephedrine alkaloids-free Ephedra Herb extract, was administered orally to mice, and the subsequent serum was butanol-treated and analyzed. Of the peaks in mouse serum, most were serum-derived peaks, but vicenin-2 was found in serum after oral administration of EFE, although at a low intensity. Therefore, a calibration curve for vicenin-2 was prepared using a secondary standard, apigenin, and serum from mice after oral administration of EFE was used for quantification by HPLC. As a result, vicenin-2 was found to be present in serum at 8.12-11.2 ng/mL. We could analyze the constituents in serum using general-purpose HPLC, suggesting that most of the constituents are transferred without being metabo- lized in the body.

27－20

Study on stability of food additives after opening of food packaging

Yoshichika Hirahara
Department of Food Science and Human Nutrition, Faculty of Agriculture, Setsunan University

As a basic model study to understand the effect of food additives after opening food packaging, the sta- bility of 2 types of antioxidants and 6 types of preservatives during heating and storage was examined us- ing LC-MS/MS technique. Although BHT in water decreased by 50% after one month in storage at room temperature, all others were stable for one month under room temperature, refrigeration and freezing. Heat treatments for 5 minutes in a water bath and for 3 minutes in a microwave reduced BHT in both water by 100%, whereas all other additives were stable for one month under all heating conditions. More than 46% of paraoxybenzoic acids remained after heating on a hot plate at 250°C for 1 minute. From the above results, after opening the food package, the preservative remains during the cooking process and storage, suggesting that it is thought to contribute to ensuring food safety.

27－21

Relationship between the contamination level of food-borne bacteria and the inhibition of bacteria by glycine in cooked rice

Satomi Tsutsuura
Institute for Research Promotion, Niigata University

In this study, the effects of glycine in combination with sodium chloride (NaCl) and heat treatment on the growth of Staphylococcus aureus and production of staphylococcal enterotoxin A (SEA) in cooked rice which was inoculated with different inoculum sizes were investigated. Under the combination of glycine and NaCl, and the condition of low contamination, the number of bacteria decreased in 0.5% glycine-added cooked rice at the early stage of storage depending on the concentration of NaCl, but SEA was not detected. On the other hand, in 1% glycine-added cooked rice, the growth of bacteria was inhibited regardless of NaCl. Even after 24-hr of incubation, the bacteria did not grow, and SEA was not produced. The effect of heat treatment and glycine on the bacteria was also examined. The number of bacteria decreased from about 10⁶ CFU/g to about 10^2-3 CFU/g by heating, and bacteria did not grow even after 24-hr and 72-hr of incuba- tion. The combined use of heat treatment and glycine sufficiently inhibited the growth of Escherichia coli. Under low contamination condition, the growth of Bacillus subtilis spores was sufficiently inhibited even when only 1% glycine was added to cooked rice.

27－22

Development of flavor evaluation technology mimicking the mammalian olfactory response system

Yosuke Fukutani
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology

Flavors consist of combination of various odor molecules. It is thought that animals utilize the responses of multiple olfactory receptors (ORs) to identify complex odors. In this study, I constructed a screening method to identify human ORs that respond to target fragrances using the vapor stimulation assay with heterologous cells expressing each ORs. I tested this system to identify human ORs that responds to cinna- maldehyde as a practical model fragrance, which is the main component of cinnamon aroma. Furthermore, it was examined whether the identified human ORs respond to the vapor phase components volatilized from the cinnamon powder. As a result, some of the human ORs that responded to cinnamaldehyde also respond- ed to the volatile components of cinnamon. These results indicated that the assay system can also be applied to practical solid fragrances.

27－23

Quantitative analysis of synephrine and related alkaloids in citrus plants

Takashi Tsujimoto
Tokyo University of Agriculture and Technology

Citrus plants have been widely consumed as raw foods, processed foods, and food additives, as well as being used as herbal medicines. The functional components of citrus plants are known to include essential oils, flavonoids, and coumarins, and many functional properties have been clarified. Synephrine, which has a chemical structure similar to ephedrine, is known to have stomachic and antitussive effects, while it is also expected to reduce body fat and is marketed as a health food for slimming diets. However, there have been reports of adverse health events in various countries. Thus, the purpose of this study was to develop condi- tions for the detection and qualitative determination of synephrine and related alkaloids in citrus plants, to clarify the differences in accumulation among plant species and their parts, and to use these conditions for the qualitative determination of synephrine and related alkaloids in various processed mandarin foods, food additives, and health foods, thereby contributing to safety assurance. For this purpose, we examined the conditions for alkaloid analysis, analyzed the components of processed citrus plant-derived products, and conducted metabolomic analysis.

27－24

Investigation of the molecular mechanisms of food additives for the improvement of iron deficiency

Hitomi Sawai
Graduate School of Life Science, University of Hyogo

"Iron" is an essential nutrient to maintain many important physiological functions such as oxygen trans- port, energy production, and synthesis of many bioactive compounds in all living organisms. However, iron accumulation causes formation of reactive oxygen species. Therefore, iron homeostasis is strictly controlled. Since mammals including humans do not have regulatable iron excretion pathways, iron absorption from duodenal enterocytes is the most important step to maintain iron homeostasis. In the lumen side of duodenal enterocyte, dietary iron is firstly reduced by the membrane-integrated ferric reductase, Dcytb. The reduced iron is then transported into the cell by the divalent metal transporter, DMT1. Dysfunction of these mem- brane proteins has been reported to cause iron disorders such as iron deficiency and iron overload but the de- tailed mechanisms are unknown. Therefore, the detailed understanding the functional mechanism of proteins involved in iron metabolism might lead to clarify the mechanism of diseases related to iron metabolism.
In this study, a functional assay system that can reproduce the iron absorption at duodenal enterocytes us- ing a plastic plate setting "human intestinal model cells". Using this system, screening of food additives that affect to iron absorption was performed. The results might help to improve safety and efficiency of orally administered nutritional agents for iron deficiency that afflicts more than 25% (1.6 billion) of the world's population.

27－25

Development of protein drinks with amino acid nutritional additives and polysaccharide thickener added to soy protein drinks for elderly and ill persons

Eri Nakamura
Department of Innovative Food Sciences, School of Food Sciences and Nutrition,
Mukogawa Women's University

High-quality protein supplementation is often required in the elderly and in ill persons. If their digestion and absorption are impaired, however, it is difficult to obtain sufficient dietary protein. Nutritional supple- mentation via a drink containing readily available protein may be an effective solution to this problem.
We have previously developed an essential amino acid-enriched protein (ES) drink in which an ideal bal- ance of seven essential amino acids were blended with soy protein to compensate for the shortcomings of soy protein.
In this study, we investigated whether the addition of pectin, which is a water-soluble dietary fiber, to ES (EPS) can suppress the rapid increase in blood free essential amino acid concentration that occurs immedi- ately after administration of ES using rats with reduced digestive and absorptive function. We also investi- gated whether the addition of glutamine, which is an energy source for the digestive tract, to EPS (EPGS) can improve the digestion and absorption of proteins and amino acids.
We first investigated the change over time in the concentration of free amino acids in portal vein blood when pectin was added to ES (EPS). Maximum blood concentration (Tmax) was delayed in five of the sev- en essential amino acids added, indicating that EPS could suppress the increase in blood free amino acids. However, the total amount of amino acids absorbed showed a decreasing tendency.
We then investigated the change over time in the concentration of free amino acids in portal vein blood when pectin and glutamine were added to ES (EPGS). There was greater absorption promotion of almost all amino acids compared with EPS, with absorption promotion the same or higher that that observed in ES. There was a tendency for the total amount of amino acids absorbed to be higher than that of ES and EPS.
These results show that although the addition of essential amino acids, pectin, and glutamine to soy protein did not suppress the rapid increase in blood free amino acid concentration immediately after ad- ministration, EPGS was an efficient and balanced supplementation in the situation of reduced digestive and absorptive function. This newly developed protein drink for use by the elderly and ill individuals, enables supplementation of amino acids that have been removed.

27－26

Study on New Texture Design Using Fine Bubbles

Takashi Hata
National Institute of Technology, Kochi College

The fine bubble is the name of the International Organization for Standardization's standard of bubbles less than 100 μm in diameter. In addition, bubble with a diameter of 1 to 100 μm is called microbubble, and it with a diameter of less than 1 μm is called ultrafine bubble. In recent years, industrial technologies using fine bubbles have been expanding, but there are still few applications in the food industry. In this study, fine bubbles were investigated for use in the food industry, especially for their effect on texture design. The texture of cereals showed a tendency to soften the food. Furthermore, it was confirmed that the effects of ultrafine bubbles and micro bubbles were different. It was confirmed that the change in the physical proper- ties of water due to the treatment with fine bubbles could be a factor in these results. On the other hand, it was found that the inclusion of flavors in ultrafine bubbles and the prevention of oxidation of substances adsorbed in ultrafine bubbles by nitrogen encapsulation could be expected as new technologies for food pro- cessing and preservation.