Research Reports No23 Abstract (2017)
23-01
Reproductive endocrinology studies aimed to establish the safety of soybean isoflabons for infants
Mariko Shirota Laboratory of Comparative Toxicology, School of Veterinary Medicine, Azabu University
A synthetic estrogen, 17α-ethynylestradiol (EE), is widely used as a positive control for assays to evaluate the estrogenic activity of xenobiotics. We have found that neonatal exposure to EE affects subsequent female reproductive functions, and its severity and elapsed time to be apparent are dependent on the neonatal doses of EE. To determine whether the relationship between the neonatal dose of EE and subsequent effects could be applied to the safety evaluation of soy isoflavone intake by infants, we orally administered daidzein (DZ) or its vehicle and EE (positive control) to female Sprague-Dawley rats for 5 days from postnatal day 1. As a result, ovulation rate at the vaginal opening was slightly reduced in the DZ-treated group, whereas that of the EE-treated group was remarkably reduced because of its higher estrogenic potency than DZ. These results suggest the possibility that the relationship between neonatal doses of EE and subsequent effects on female reproductive functions could be applicable to the estimation of effects of neonatal exposure to soy isoflavones, based on the relative estrogenic activity of these compounds to EE, although detailed analyses are required.
23-02
Influences of non-sugar sweeteners in milk production ability in mammary epithelial cells during pregnancy
Ken Kobayashi
Laboratory of Cell and Tissue Biology, Research Faculty of Agriculture, Hokkaido University
Breast milk contains appropriate and abundant nutrition for suckling infants and is secreted by mammary epithelial cells (MECs) in lactation. The milk production ability of MECs is regulated by a large variety of endogenous and exogenous factors. Inappropriate secretion of breast milk causes stunted growth in sucking infants. In this study, we investigated whether non-sugar sweeteners influences in milk production ability of MECs. MECs were isolated from mammary glands of virgin ICR mice. Milk production ability of MECs was induced by using the differentiation medium containing prolactin and dexametasone, and the lactating MECs were then treated with 4 types of non-sugar sweeteners (saccharin, sucralose, acesulfame K, stevioside). The influences of them in β-casein production, expression of tight junction proteins were evaluated by western blotting and immunostaining. Sucralose treatment caused the decrease in intracellular and secreted β-casein in MECs in dose-dependent manners. MECs treated with stevioside also showed the increase in CLDN4, which is one of the tight junction components. On the other hands, saccharin or acesulfame K didn't show the detectable influences in MECs in this study. These results suggest that lactating MEC changes the milk production ability in response to sucralose and stevioside.
23-03
The relation of anthocyanin structure and colors in carrot
Taira Miyahara
Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology
Black carrot extraction, that using food dye, includes anthocyanin components shows more than 5 anthocyanin structures in the major breed varieties. Although some previous reports already have shown anthocyanin colors and its stabilities of the carrot extractions, no reports showed an individual color and stability of the anthocyanin structures. In this report, we isolated the individual anthocyanin molecules and showed its absorbance and the heat stability. The results demonstrate the anthocyanin structure modified by an aromatic residue causes high stability and takes on deep color. Moreover, to investigate how to produce the high stability anthocyanin in vivo, we established the methods to salvage the candidate anthocyanin biosynthesis enzyme genes from carrot genome sequences.
23-04
Development of safety evaluation system of food additives based on the change of expression level of adipokines
Makoto Nishizuka
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University
It is very important to prevent the onset of obesity and obesity-related disease such as diabetes mellitus. Day to day diets have considerable effect on the onset of these diseases. Therefore, it is very important to evaluate the effect of food additives on bodily function. In this study, we development the safety evaluation systems of food additives based on the promoter activity of adiponectin by measurement of luciferase activity. Furthermore, we found that several food additives have an influence on promoter activity of adiponectin. In addition, we revealed that several food additives regulate the expression of adipocytokines including adiponectin, MCP-1 and resistin in mature adipocytes.
23-05
Molecular phylogenetical analysis of fungi causing the fumonisin contamination in the Japanese domestic grape berries for wine brewing, and a study on the mechanism of fumonisin production
Hiroyuki Nakagawa
National Agriculture and Food Research Organization (NARO), Food Research Institute
Among the Fusarium sp. fungi isolated from a Japanese domestic winery, 2 strains of F. fujikuroi showing higher fummonisin production on rice medium were examined for their gene similarity to those strains known as the causal strains of bakanae disease, based on the molecular genetical analysis. These strains were further inoculated on grape berries used for wine brewing to investigate the infection activity and fumonisin production on the fruits.
23-06
Stimulation of GLP-1 (Glucagon-like peptide-1) secretion by food additives and elucidation of the mechanism
Takanori Tsuda
College of Bioscience and Biotechnology, Chubu University
Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that food additives (anthocyanins and curcuminoids) induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) and curcumin significantly increase GLP-1 secretion in vitro. The results of anthocyanins suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. In the case of curcuminoids, the results suggested that at least one methoxyl moiety at the aromatic ring and the β-diketone structure are essential for significant stimulation of GLP-1 secretion. In addition, we established evaluation system of GLP-1 secretion by candidate food additives using animal. These findings provide possible abilities of food additives to prevent and treat diabetes via the stimulation of glucagon-like peptide-1 secretion.
23-07
Studies on color development and stability of anthocyanins from edible plants
Yoshida Kumi
Grauate School of Informatics, Nagoya University
Anthocyanins attract attention as a natural food colorant having several biological activities against liferelated diseases. Nowadays, the anthocyanins from various edible plants are used as food colorant, but still not few problems remain; the price, stability, the odorous and variety of color development. Particularly, about the blue color development, there is no colorant with anthocyanins to date. We have been studied chemical structure and the color development of anthocyanins and found that simple anthocyanins could develop stable blue color by coexistence with co-pigment and metal ions. In this study, we tried to make a procedure of a large-scale purification of simple anthocyanins from edible beans. Furthermore, we experimented the stability and blue color development using these pigments. We established a large-scale purification protocol of black turtle bean (Phaseolus vulgaris L.) and black soybean (Glycine max L) as follows: extraction with hydrochloric acid-methanol, crude purification by Amberite XAD-7 gel and Amberite XAD-7column chromatography, and final purification with ODS-LC fractionation to give pure anthoyanins (ca. 1 g). Using thee anthocyanins we tested the stability in various pH conditions. As a co-pigment we used flavocommelin from blue petals of Commelina communis, beause, the compound freely soluble in water. Addition of 10 equivalent flavocommelin gave the bathochromic shift and color stability of the solution in pH 1, 5 and 7. Furthermore, we found that glucosylation at 3-OH of anthocyanins gave a strong stability at pH 1. We obtained a stable blue solution by mixing delphinidin 3-glucoside (Dp3G) with 1 eq. of Al3+ and 2 eq. of 5-O-caffeylquinic acid (5CQ) as a co-pigment. The concentration of delphinidin 3-glucoside was 100 μm and the color was stable more than 1 week. Using the blue solution, we recorded ESI-TOF Mass. We detected a molecular ion peak attributable to Dp3G-Al3+-5CQ = 1:1:1. We are expecting a practical use of the blue colorant forming with simple anthocyanins with copigment and metal ion in near future.
23-08
Effects of sweeteners or preservative consumption in early stage of life on microbiome and systemic metabolism
Takashi Uebanso
Department of Preventive Environment and Nutrition, Institute of Biomedical Sciences, Tokushima University Graduate School
Xylitol inhibit the growth of some bacterial species including Streptococcus mutans. It used as a food additive to prevent a caries. Recent progress in the research field of gut microbiota revealed that gut microbiota produces a lot of bioactive compounds from dietary components and regulates host metabolisms. We previously showed that 1.5 to 4.0 g/kg BW/day xylitol improve lipid metabolism in high fat dirt (HFD) fed rats. However, effect of more low dose dietary xylitol, as we daily consume, on gut microbiota and lipid metabolism is unclear. In the present study, we examined effect of 40 to 200 mg/kg BW/day xylitol intake on gut microbiota and lipid metabolism in mice. We found that administration of low or medium dose xylitol (40 or 194 mg/kg BW/day) significantly altered composition of the feces microbiota. Especially, relative abundance of Bacteroidetes phylum and Barnesiella genus were reduced in feces of mice fed 194 mg/kg BW/day xylitol as compared with those of control mice with both control diet and high fat diet. In contrast, relative abundance of Firmicutes phylum and Prevotella genus were increased in only HFD with 194 mg/kg BW/ day xylitol feeding group as compared with those of control mice. Contrary to significant alteration of gut microbiota, body composition, hepatic and serum lipid parameters and luminal metabolites did not different between with or without xylitol consumption. In conclusion, we found that 40 or 194 mg/kg BW/day xylitol, induced grateful changes in the gut microbiota but not in lipid metabolism in mice.
23-09
Induction mechanism of cell proliferation contribute to the mutagenicity of estragole, a hepatocarcinogen
Yuji Ishii
Division of Pathology, Biological Safety Research Center, National Institute of Health Sciences
Estragole (ES) is a hepatocarcinogen in rodents. In spite of ES-specific DNA adducts being formed in a dose-dependent manner, the mutagenicity along with induction of cell proliferation was observed only at the high dose. These results indicate that induction of cell proliferation is prerequisite in the pathways leading from ES-specific DNA adducts to gene mutations and that ES at the lower dose has no potential for mutagenicity. Given that humans are actually exposed to ES at very low doses, to clarify the detailed mechanisms including molecular signaling pathways in induction of cell proliferation is very important in terms of extrapolation to humans. In this study, we examined time-dependent changes of mutagenicity-related factors in the livers of gpt delta mice treated with low-dose ES. Eleven-week-old female B6C3F1 gpt delta mice were given ES at a single dose of 100 mg/kg by gavage. The livers collected after 1, 2, 3, 5, 7 and 14 days were examined. At day 1, the formation of ES-specific DNA adducts was observed. However, there were no histopathological changes including hepatocyte apoptosis and mitosis in the livers of mice. In addition, cleaved caspase3 and PCNA positive cells and increases in the expression of TNFα and cell cycle-related genes were not observed. Therefore, hepatocyte apoptosis observed in the mice treated with high-dose ES might be responsible for ES-induced proliferation. These data indicate that there is a threshold for ES-induced proliferation.
23-10
Development of physical-chemical information retrieval system for food additives III
Naoki Sugimoto
Division of food additives, National Institute of Health Sciences
We launched a retrieval service for spectrum data of food additives containing about 730 data of products on our Web site. This data set for this service includes the data of about 230 products extracted from the database of food additives constructed by Dr. Yoshihira's research group in University of East Asia and the newly added our qNMR data. In addition, we also launched another retrieval service for standards and criteria of food additives. The data set is consisted of the monographs of food additives and reagents for assays that will be officially specified in Japan's Specifications and Standards for Food Additives. Since the content of the 9th Edition Food Additives Official Document has not been confirmed, this service is open to some limited users at the present time. As soon as the content of the document is officially confirmed, the data will be updated and planned to be released to the all users on our web site. And then we plan to link them to the information on EU, JECFA and others.
23-11
Analysis of polychlorinated biphenyls in food using a polychlorinated biphenyl clean-up system
Tomoaki Tsutsumi
Division of Foods, National Institute of Health Sciences
We evaluated the application of the PCB clean-up system (MIURA Co. Ltd.) in determining PCB concentrations in fish. The fish samples were digested with ethanolic KOH; then, the resulting alkaline solutions were extracted with n-hexane. Extracts were purified by the clean-up system based on a sulfuric column followed by an alumina/metal-supported alumina column, then subjected to gas chromatography tandem mass spectrometry (GC-MS/MS). Recovery tests in which 209 PCB congeners were added to fish extracts resulted in acceptable recoveries (70%-100%), indicating no significant loss of any of the PCB congeners during the clean-up process. Additionally, recovery tests using fish extracts spiked with PCB mixtures (Kanechlor 300-600) also showed good recoveries for total PCBs in two kinds of extracts (92% and 94%). The performance of the analytical method was evaluated using fish spiked with the PCB mixtures at 100 ng/g (n = 5). The trueness and repeatability of the method for total PCBs were estimated to be 93% and 0.9%, respectively. Finally, we used the clean-up system to determine the total PCBs of 10 fish samples compared with those obtained by the conventional clean-up method using open columns. The total PCB concentrations produced by both clean-up methods showed excellent correlations for total PCB concentrations (r = 0.99), with the slope and y-intercept of the linear regression equation being close to 1 and 0, respectively. Overall, our results indicated that the PCB clean-up system is a useful method for rapidly determining total PCB concentrations in fish.
23-12
Research on diuretic action and reduction of lipogenesis in rats received high fat diet and enzymatically modified isoquercitrin
Toshinori Yoshida
Tokyo University of Agriculture and Technology, Graduate School, Division of Animal Life Science, Institute of Agricultural Science
We determined effects of enzymatically modified isoquercitrin (EMIQ) and a diuretic potassium canrenoate (PC) on hepatic steatosis-related early carcinogenesis in rats fed high fat diet (HFD) subjected to a twostage hepatocarcinogenesis model. Rats were fed with basal diet or HFD co-administering with PC and/or an antioxidant EMIQ. In glucose tolerance test during the study, the HFD increased blood glucose level as compared with control basal diet-fed rats, the effects was prevented by combined treatment of PC and EMIQ. After 10 and 11 weeks of the study, blood samples, and liver and adipose tissue weight were collected. The HFD increased body weight and intraabdominal adipose tissue weight, and plasma Fe and transferrin saturation in blood samples as compared with the control rats. Although there was an decrease in liver weight, HFD increased score of non-alcoholic fatty liver disease (NAFLD) activity score (NAS) and slightly increased the number and area of a preneoplastic glutathione S-transferase placental form (GST-P)+ foci, where higher expression of an apoptosis marker cleaved caspase-3+ cells was confirmed. PC treatment decreased body and adipose tissue weights and NAS, with or without EMIQ supplement, although there were no effects on food intake. PC treatment, however, increased GST-P+ foci, which were reduced by EMIQ in association with decreased cleaved caspase-3+ cells. In gene expression analysis, HFD and/or PC altered gene expression related to lipid, iron, and oxidative stress including NADPH oxidase, and EMIQ in combination with PC increased Catalase gene expression. The results suggested that EMIQ in combination with PC had a potential of suppressing HFD-increased obesity, liver steatosis, and preneoplastic liver lesions though an antioxidant effect.
23-13
Genetic and breeding study for increase and stabilization of yield of food additive color from safflower
Tsuneo Sasanuma
Faculty of Agriculture, Yamagata University
In order to increase pigment yield of safflower, basic traits such as flowering day and plant height, petalrelated traits such as total petal length in capitula, petal yield by hand picking, and pigment content in petals measured by absorbance were investigated for 18 accessions of safflower genetic resources. Comparative expression analyses of a candidate gene related to pigment biosynthesis among organs, among developmental stages and among varieties with different flower colors and determination of full length cDNA of the candidate gene by RACE were also performed. In the trait investigations, it was revealed that Mogamibenibana, a variety of Yamagata prefecture, has good traits due to annual stability and petal yield, but even in overseas varieties, there is a variety with more petals and higher proportion of 3mm over petals than Mogamibenibana, suggesting the high potential for future breeding. In the pigment content survey, Mogamibenibana did not show particularly good values, and an overseas accession showed more than 80 times more red pigment content than other accessions. This high value, however, is probably caused by the harvesting conditions, suggesting that the harvesting conditions seriously affect pigment content. In the expression analyses of clone 3, the candidate gene related to pigment biosynthesis, it was shown that the expression in the bud is the most pronounced, but it is expressed not only in petals, but also in roots, stems and leaves. There was no significant difference in expression among the lines with different flower colors. The RACE method successfully determined the 1,116 bp full length cDNA sequences of clone 3. Since this sequence has multiple stop codons in any open reading frame, it is deduced that this gene is not translated into protein even though transcribed, that is, it was one of the long non-coding RNA. Together with the results of the expression analyses, the clone 3 gene is not considered to be a gene directly involved in the safflower petal pigment synthesis pathway.
23-14
Effects of chronic intake of a non-caloric artificial sweetener on blood glucose control in mice
Yasunobu Yasoshima Division of Behavioral Physiology, Graduate School of Human Sciences, Osaka University
In many countries, caloric and non-caloric sweeteners are widely used in our daily feeding behavior. Previous studies suggest that increased intake of the caloric sweeteners induces overeating, obesity and some lifestyle diseases. Various foods and beverages contain non-caloric artificial sweeteners to avoid caloric intake increase. However, some reports indicate that non-caloric artificial sweeteners caused maladaptation of body weight regulation and food intake. To address the issue, we examined whether chronic intake of a non-caloric sweetener, saccharin, induced dysregulation of blood glucose in mice. We also examined effects of sucrose overconsumption on secretion of peptide YY, a gut peptide hormone, in mice. The present results indicated that chronic intake of saccharin affected regulation of blood glucose in some animals. In addition, peptide YY secretion in mice with sucrose consumption decreased in comparison to animals that experienced sucrose without its overconsumption. It remains unclear whether saccharin intake causes a similar dysfunction in gut hormone system. The present study suggests that chronic saccharin intake is a risk factor to deteriorate physiological feeding regulation such as blood glucose. Further study needs to examine saccharin's effects on gut hormone secretion.
23-15
The possibility of combination effects of environmental chemicals and food additives on the expression of immunotoxicity
Sekimoto Masashi
Laboratory of Environmental Hygiene, Department of Environmental Science, School of Life and Environmental Science, Azabu University
We have already demonstrated that the synergistic activation of aromatic hydrocarbon receptor (AhR), an important transcription factor for xenobiotic metabolism, was occurred in human hepatoma HepG2 cells by the complex treatment with food additive (Thiabendazole, TBZ) and environmental chemical (3-methylcholanthrene, MC). Since excessive AhR activation causes immune abnormality, in this study, we examined the possibility of combined effects of TBZ and MC on cytokine gene expression using undifferentiated or macrophage-like differentiated human leukemia THP-1 cells. As a result, synergistic induction of TNFα and IL-8 was observed by combination treatment with TBZ and MC in THP-1 cells differentiated by PMA. Furthermore, we investigated the combined effect of TBZ and MC on AhR-target gene expression using various human cell lines (HepG2, A549, Caco-2, MCF-7, Hela, Ishikawa, and LNCaP). As a result, the similar synergistic effects in HepG2 were observed in these cell lines. These findings suggested that combination treatment of TBZ and MC causes synergistic induction of the expression of cytokine genes in PMAdifferentiated THP-1 cells, and that the combined effect of TBZ and MC is a phenomenon common to many human cell lines.
23-16
Risk assessment of arsenic in food: analysis of mechanisms of arsenic carcinogenicity
Min Gi
Osaka city university graduate school of medicine, Department of molecular pathology
The main source of arsenic exposure is via ingestion of food containing arsenic in the general Japanese population. Accumulating evidence indicates that prenatal exposure to inorganic arsenic, a human carcinogen, can subsequently cause cancer in adulthood in mice. DMA is a major metabolite of inorganic arsenic and organic arsenic compounds contained in food. However, little is known about the carcinogenic risk of prenatal DMA exposure. The purpose of the present study is to determine the carcinogenic effects of prenatal DMA exposure in CD1 mice. Pregnant CD1 mice were given 0 or 200 ppm DMA in drinking water from gestation day 8 to 18. Offspring were observed for up to 84 weeks of age. In male offspring, incidence of hepatocellular adenoma tended to increase and incidence of hepatocellular carcinoma was significantly increased in the prenatal DMA exposure group. In addition, the incidence of hepatocellular tumor (adenoma plus carcinoma) tended to increase in the in prenatal DMA exposure group. Furthermore, the incidence of lung tumor (adenoma plus adenocarcinoma) was significantly increased in the in prenatal DMA exposure group. In contrast, there were not increases in the hepatocellular tumors and lung tumors in the DMA-exposed female offspring mice. These results show that exposure of pregnant CD1 mic to DMA through the drinking water induce hepatocarcinogenesis and lung caricinogenesis in male offspring.
23-17
Study on preparation of reference materials for analysis of ciguatoxins, principal toxins of ciguatera fish poisoning
Naomasa Oshiro
Division of Biomedical Food Research, National Institute of Health Sciences
Availability of ciguatoxins, causative agents of ciguatera fish poisoning, are extremely limited. To prepare the reference materials of CTXs, we collected fish specimens including Variola louti, Variola albimarginata, and Oplegnathus punctatus from Okinawa, Ogasawara and other waters of Japan. Specimens from Okinawa were contained oxidized CTX1B analogs. No CTXs analog was detected from Ogasawara specimens. The specimens of O. punctatus collected off Okinawa and Mainland Japan were also carried out for LC-MS/MS analysis. Of which, only two specimens from Okinawan waters contained CTXs.
23-18
Effects of a high-dose green tea polyphenol on drug-metabolizing enzyme
Nobutomo Ikarashi
Department of Clinical Pharmacokinetics, Hoshi University
In recent years, the intake of functional foods containing high doses of green tea polyphenols (GP) has been increasing. In this study, the long-term safety of high-dose GP was assessed from a pharmacokinetic point of view by focusing on the drug-metabolizing enzyme cytochrome P450 3A (CYP3A). This study revealed that the intake of a high-dose GP results in a liver-specific decrease in the CYP3A expression level and an increase in the blood level of CYP3A substrate drugs. It is also clear that the hepatospecific decrease in the CYP3A expression level observed after a high-dose intake of GP was caused by EGCG. Because EGCG, which is not absorbed from the intestine, causes a decrease in the level of LCA-producing bacteria in the colon, the level of LCA in the liver decreases, resulting in a decrease in the nuclear translocation of PXR, which in turn leads to the observed decrease in the expression level of CYP3A. In the future, caution should be taken in cases where functional foods containing a high-dose GP are concomitantly consumed with a CYP3A substrate drug.
23-19
Research on metabolic mechanism of food additive vitamin K1 hydrogen adduct (2',3'-PKH2)
Yosihisa Hirota
Department of Bioscience and Engineering, College of Systems Engineering and Science Shibaura Institute Technology
In humans and mice, ingested PK is MD which has a side chain cleaved in the small intestine and has no side chains and is converted into MK-4 by UBIAD1 in each tissue. Vitamin K side chain cleavage mechanism has not been elucidated so far and search of side chain cleavage enzymes is also difficult. In Europe and the United States, many foods containing 2',3'-PKH2 reduced PK in food due to hydrogenation to stabilize the shape of processed vegetable oil and butter are distributed. Based on previous studies, it was reported that 2',3'-PKH2 does not increase the blood MK-4 concentration even when ingested in large quantities by humans and rats, and shows little function as vitamin K , So we elucidated the in vivo metabolic mechanism of 2',3'-PKH2. First, 2',3'-PKH2-d7 newly synthesized in this study showed GGCX activity as vitamin K activity similarly to PK-d7. Therefore, 2',3'-PKH2 was given to MG63 cells and C57BL/6J mice, but no conversion activity to MK-4-d7 was observed as in the previous reports. Next, in order to clarify the in vivo metabolic mechanism of 2',3'-PKH2-d7, 2',3'-PKH2-d7 was administered to rats cannulated portal vein or lymphatic vessels. As a result, it was found that 2',3'-PKH2 is a vitamin K derivative not undergoing a side chain cleavage reaction, since MD which is an intermediate of the MK-4 conversion reaction was not detected at all in the lymph fluid . From the results of this study, it is considered that the vitamin K side chain cleavage enzyme selectively recognizes and cleaves double bonds at the 2nd and 3rd positions present in the side chain of PK. If vitamin K side chain cleavage enzyme can be identified in future, it is clarified what kind of mechanism the ingested vitamin K is metabolically absorbed and exercises physiological function. Therefore, this study is considered to be a very important finding to clarify the molecular nutritional importance of vitamin K in living bodies.
23-20
Enzymatic characterization of LPH (Lactase-phlorizin hydrolase) involved in intestinal absorption of natural product glycosides
Kazuyoshi Terasaka
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University
LPH (lactase-phlorizin hydrolase) play a role in the hydrolysis and absorption of disaccharides in the brush border of the small intestine. Glycosyl conjugation of quercetin enhanced the bioavailability of quercetin, and LPH would play important roles in intestinal absorption of quercetin glycosides. The aim of the present study is to characterize the glycosidase activity of LPH towards flavonoid glycosides. The ORFs of the human and rat LPH cDNAs were expressed in mammalian cells. These recombinant proteins were successfully obtained from HEK293 cells. The crude enzyme extracts corresponding to human and rat LPH were used for glucosidase assays with phlorizin as a substrate. The crude enzyme containing recombinant LPH hydrolyzed phlorizin to phloretin. The substrate specificity of recombinant LPH was examined using various flavonoid glycosides. The recombinant LPH exhibited glucosidase activity toward several flavonoid glucosides, but no activity toward either flavonoid galactosides or flavonoid glucuronides. As for the flavonoid aglycone specificity, recombinant LPH exhibited no activity toward flavanones. These data suggest that LPH has the substrate preference toward natural product glycosides.
23-21
Effects of sweeteners on the pathosis and underlying mechanisms of lifestyle-related diseases for the molecular target control of such diseases
Noriko Kemuriyama
Department of Nutritional Science and Food Safety, Faculty of Applied Biosciences, Tokyo University of Agriculture
The aim of the present study is to examine effects of sweeteners on the pathosis and underlying mechanisms of lifestyle-related diseases using animal models. In the fiscal year of 2016, we chose non-alcoholic steatohepatitis (NASH), Luo Han Guo fruit extract and a rat model of a chronic feeding of a cholinedeficient, methionine-lowered, l-amino acid-defined (CDAA) diet, for the disease, sweetener and animal model, respectivelty. Luo Han Guo (Siraitia grosvenorii) fruit is well-known for its sweet taste, and its extract is widely used as a sweetener and a component of health-care products, such as antitussive and antisore throat, while in southern China Luo Han Guo is believed to aid longevity. Male Hsd: Sprague Dawley: SD rats, 6 weeks old and 6 animals per group, were fed the CDAA diet for 3 months and administered Luo-Han-Guo fruit extract dissolved in the drinking water at a concentration of 0, 0.06, 0.2, 0.6, 2 or 6 %. Additional rats were fed a basal diet and administered 0, 0.6 or 6 % of Luo-Han-Guo fruit extract. At the end of the experimentation, the rats were necropsied and sampled their livers and blood. In the animals fed the basal diet, Luo-Han-Guo fruit extract did not cause any changes. In the animals fed the CDAA diet, the activities of hepatic aminotransferases were increased in the plasma; and fatty change, fibrosis and hepatocellular apoptoses were diffusely observed in the liver. Co-administered Luo-Han-Guo fruit extract reduced such plasma and hepatic changes, with the exception of fatty liver, shown in the rats fed the CDAA diet in a dose-dependent manner. It is thus indicated that Luo-Han-Guo fruit extract inhibits the NASH-like pathosis in the rats induced by a chronic feeding of the CDAA diet, and suggested that the fruit extract may be potentially beneficial also for people who suffer or are suffering from NASH.
23-22
Construction of conjugate metabolites library for evaluation of metabolic profiling of food additives
Shinichi Ikushiro
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University
Xenobiotic compounds including food additives or dietary chemicals are conjugated by xenobiotic phase II enzymes, UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs) and catechol-o- methyltransferases (COMT) isoforms, and its biological effects depend, in partly, on the formation of the conjugates. We have developed several mammalian UGT, SULT or COMT expression systems to biosynthesize the conjugates as pharmaceutical or dietary metabolites in budding yeast. By selecting suitable conjugating isoforms in yeast, we could obtain the desired conjugates of various dietary compounds with multiple conjugation sites. These whole cell-dependent production systems of mammalian UGT, SULT or COMT in budding yeast cell would be a powerful tool for enzyme-assisted biosynthesis of methylated quercetin conjugates including glucuronides and sulfates.
23-23
Controlling stability of food ingredient suspension by optimizing food excipients and preparation process
Kohei Tahara
Gifu Pharmaceutical University
Many food ingredients with nutrient function claims show poorly water-soluble property. There is difficulty in absorption via oral administration if the ingredients have low solubility in water. The aim of this study was to enhance the dissolution of poorly water-soluble food ingredient, curcumin using nanoparticle suspension prepared with high pressure crystallizer (PureNanoTM, Microfluidics). Curcumin was dissolved in ethanol and the different polymer (stabilizer) solution (in water) were used as an antisolvent. The curcumin solvent and antisolvent were used to prepare nanoparticle suspension by high pressure crystallizer. Nanoparticle formulations were freeze-dried. The characteristics nanoparticles such as particle size and curcumin dissolution based on Japanese pharmacopeia were examined. Nanoparticles suspension of curcumin could be prepared with high pressure crystallizer. The dissolution rate of curcumin in vitro increased after high pressure crystallization.
23-24
Study of complexing method to stably encapsulate taste and flavor components
Kunikazu Moribe
Graduate School of Pharmaceutical Sciences, Chiba University
Previous studies have reported that spicy taste was masked using a γ-cyclodextrin (γ-CD) complex with a lipid, phytosterol ester. However, the detailed molecular states of the components in the complex has been still unclear. In order to elucidate lipid/γ-CD complex structure and the encapsulated state of a guest molecule, a γ-CD complex with cholesteryl oleate (OCE), which is the main component of phytosterol ester, was prepared by solvent diffusion method. Then, the preparation condition and the structure of OCE/γ-CD complex was investigated. Dynamic light scattering method demonstrated that the freshly prepared suspension had the unimodal size distribution with a mean volume diameter of about 200 nm. The particle size distribution was almost maintained after one day storage. Solution-state 1H NMR measurement of the nanosuspension suggested that OCE existed in the internal core of the complex nanoparticle. The nanosuspension showed similar particle size distribution before and after freeze-drying and redispersion into water, indicating its high re-dispersibility. It is concluded that OCE/γ-CD complex nanosuspension with high stability and re-dispersibility was prepared by solvent diffusion method.
23-25
Analysis of the sweet taste signal transduction and recognition using taste reception system and the salivary secretion system
Yuko Kusakabe
Food Research Institute, NARO
We aimed to develop a new sweetness evaluation system that considers sweet taste signal transduction comprehensively, and documents evaluation data in terms of the concentration-response relationship. In this study, we compared the taste receptor response, taste intensity, salivary secretion, and subjects' preferences for six sweeteners. The responses of human sweet taste receptor hT1r2/hT1r3 expressing HEK293 cells were treated as the input signal for sweet taste signal transduction. We treated the sweetness intensity, sweetness preferences, and the salivary secretion responses in human subjects as the output signals. Our results show that the sweet taste receptor, sweetness intensity, and salivary secretion responses could be expressed as concentration-response relationships. As a result, the sweet taste receptor, sweetness intensity, and salivary secretion responses are highly correlated for the non-nutritive sweeteners we evaluated. Moreover, we observed that the palatability of the sweeteners was unrelated to salivary secretion. Therefore, the preferences for the sweeteners were not related to sweet taste intensity or salivary secretion. These results support the view that salivary secretion, as a response to taste stimulation, is an unconditioned reflex. These findings suggest that the sweet taste intensity of non-nutritive sweeteners can be evaluated by salivary secretion. Thus, using this method, it would be feasible for untrained persons to evaluate taste quality without bias.
23-26
Control of flavor release rate by adjusting glass transition temperature of wall material using rare disaccharide
Shuji Adachi
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Lactose and cellobiose were isomerized to their corresponding rare dissacharides, lactulose and cellobiulose in subcritical aqueous ethanol in the temperature range of 170 to 220ºC. The yields of the rare disaccharides were higher at the higher ethanol content. A cation-exchange resin with 5% divinylbenzene content in Ca2+ form was most suitable for chromatographic separation of lactose and lacxtulose. A simple method was proposed for estimating the glass transition temperature of wall material by analyzing the discoloration process under temperature-programmed heating conditions. Kinetic analysis of d-limonene microencapsulated with various saccharides indicated that the glass transition temperature of wall material was an important factor for controlling the release, but it would not be a sole factor affecting the process.
23-27
Development of novel extraction and recovery methods of rare and useful food additives from natural products
Kenji Mishima
Department of Chemical Engineering, Faculty of Engineering, Fukuoka University Academia, Industry and Government Collaborative Research Institute for Composite Materials, Fukuoka University
Citrus has been accepting a special interest in the medical and food industry. Some natural products contain rare physiologically active substances which can be employed as useful food additives, such as nootkatone contained in the pericarp of grapefruit. We have developed novel extraction and recovery methods for these rare and useful food additives from natural products. The bioactivity of the target substances extracted from natural products was evaluated by means of a brain function imaging apparatus that uses near-infrared spectroscopy (fNIRS). This brain imaging technique indirectly measures brain activity by directly measuring cerebral blood flow oxygenation levels. As a first approach, the substances from the pericarp of grapefruit, coffee beans and vanilla were extracted by conventional extraction methods, such as ethanol and water extraction. Components such as limonene, myrcene, nootkatone, cyclotene, maltol, terpenes and vanillin were isolated and analized by HPLC and GC-MS. Secondly, the same substances from natural products, i.e. pericarp of citrus, coffee beans and vanilla, were extracted by using a supercritical carbon dioxide extraction method (SC-CO2 extraction). We concluded that the SC-CO2 extraction method showed a more effective extraction performance than the conventional methods and has the additional advantage of not needing a desolvation process after extraction. Furthermore, we have proposed a more effective SC-CO2 method using supersonic irradiation.