The Research Reports No.28 （issued in 2022）

Abstracts of Research Reports funded by the Foundation's Research Grants in FY2021.

28－01

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. In FY2020, we identified two candidate dyes that can alter the color tone while maintaining the function of the embryo culture media. In this study, we verified the feasibility of the use in actual embryo transfer, and attempted to expand the application to cryopreservation of embryos using known food addictive dyes.

28－02

Exploration and application of rare sugars with inhibitory effects on simple sugar preference

Sho Matsui
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology,
Graduate School of Agriculture, Kyoto University

　Diabetes and obesity are increasing worldwide. We previously reported fibroblast growth factor 21 (FGF21)-oxytocin (OXT) system controlling sugar intake. Substances-inducing FGF21 is expected to lead to the development of new dietary therapies that allow for a reasonable restriction of sugar intake. Therefore, we identified the FGF21-inducing substances and tested their effect on sugar intake. We screened for FGF21-inducing rare sugars by adding 46 rare sugars to primary hepatocytes of wild-type (WT) mice, and measuring the concentration of FGF21 in the supernatants. FGF21 secretion from primary hepatocytes was significantly induced by D-sorbitol, D-psicose, and D-tagatose. Moreover, oral administration of these rare sugars to WT mice significantly increased blood FGF21 levels, and oral administration of D-sorbitol, D-psicose, and D-tagatose also decreased preference for sugar. Taken together, these data uggest that D-sorbitol, D-psicose, and D-tagatose, which may stimulate the FGF21-OXT system, could suppress sugar intake.

28－03

A Systematic Review of the Safety Evaluation of Food Additives without Animal Testing

Hajime Kojima, Ph.D.
Div. of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Japan

　With the worldwide acknowledgement of the '3Rs' in animal testing (reduction, refinement, replacement), institutional acceptance of safety evaluations using non-animal testing is proceeding. Accordingly, this study investigated safety evaluations using the New Approach Methods (NAM) to determine a system to evaluate and regulate the safety of food additives without using animal experiments. Assuming a systematic toxicity using NAM, information on toxicokinetic and non-animal toxicity studies was collected through interviews with domestic and foreign experts. The current status of safety evaluations using NAM in domestic and foreign administrative organizations was summarized. Collaboration occurred within the International Workshop on Alternatives to Animal Testing Project (in the domain of foods) held in November, 2021 by the International Life Sciences Institute (ILSI), Japan. Based on the above information, the relevant literature on the safety evaluation of food additives and functional ingredients was retrieved and systematically reviewed.　This study will assist Japan in setting targets for the safety evaluation of food additives that will reflect the international situation, facilitate the development of new food additives, and encourage other countries to use original Japanese food additives that have not yet been made available in the international market.

28－04

Flavonoid profiling and alkaloid detection of Citrus fruits by metabolomic techniques

Takashi Tsujimoto
Faculty of Technology, Tokyo University of Agriculture and Technology

　Citrus plants are used to traditional oriental natural medicines. Recently, Citrus plants are processed and sold in markets as health foods. Against the health merits, Citrus fruits accumulate one of alkaloids, synephrine, the content of which differs among Citrus species. When Citrus fruits are processed, the origin of fruits of the species is hard to be identified. It is known that Citrus plants can hybrid among interspecies, causing to the complexity of the origin of species using molecular biological techniques. This research examined metabolomic approach for species identification could apply to detect synephrine coupled with flavonoids as species identification markers.

28－05

Elucidation of the Mechanism of Improved Glucose Metabolism by Sugar Alcohols Using Metabolomics Approach

Naoko Nakanishi
Department of Endocrinology and Metabolism,Kyoto Prefectural University of Medicine,
Graduate School of Medical Science

　Erythritol, a type of sugar alcohol, is widely used in the diets of diabetic and obese patients as a sugar substitute. The purpose of this study was to investigate the effects of erythritol on metabolic abnormalities induced by a high-fat diet in 57BL/6J mice, focusing on changes in innate immunity. Mice fed a high-fat diet and treated with water containing 5% erythritol (Ery group) showed significantly lower body weight, improved glucose tolerance, and significantly higher energy expenditure compared to control mice (Ctrl group) (n=6). Furthermore, the Ery group showed less fat accumulation in the liver, smaller adipocytes, and significantly better inflammatory findings in the small intestine compared to the Ctrl group; the concentrations of short-chain fatty acids (SCFA) such as acetic acid, propanoic acid, and butanoic acid in serum, feces, and white adipose tissue were significantly higher in the Ery group compared to the Ctrl group. In flow cytometry experiments, the number of group 3 innate immune system lymphocytes (ILC3) in the small intestine-specific layer and the number of ILC2 in white adipose tissue were markedly increased in the Ery group compared to the Ctrl group. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that Il-22 expression in the small intestine of the Ery group was markedly higher than in the Ctrl group. Erythritol markedly ameliorates metabolic disorders such as diet-induced obesity, glucose intolerance, dyslipidemia, and fatty liver by increasing SCFA and modulating innate immunity.

28－06

Study on effects of vitamin-related food additives on the prevention of skeletal muscle atrophy using human iPS cell-derived myocytes

Yoshio Yamauchi
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo

　Skeletal muscle is the largest organ in the body, accounting for 40% of body weight. Skeletal muscle plays critical roles not only in locomotion but also in whole-body metabolism and energy homeostasis. The age-associated skeletal muscle atrophy known as sarcopenia is a risk factor that leads the elderly to disability. Therefore, its prevention is one of the most important issues for extending healthy life expectancy. The transforming growth factor (TGF)-β superfamily member myostatin is a negative regulator of skeletal muscle mass. Since evidence shows that blocking myostatin signaling increases muscle mass, its suppression is considered a plausible strategy to prevent sarcopenia. We have previously selected food-derived compounds that inhibit myostatin signaling in HEK293 cells. This study aims to examine the effects of vitamin-related compounds on myostatin-induced atrophy using human iPS cell-derived myocytes. Our results suggest that certain vitamin-related compounds have the ability to protect human iPS cell-derived myocytes from myostatin-induced myotoxicity.

28－07

Malabaricone C derived from nutmeg inhibits lipid mediator synthesis and ameliorates chronic inflammatory diseases

Toshiko Yamamoto
Department of Nutritional Science, Faculty of Health and Welfare Science,
Okayama Prefectural University

　Prostaglandin (PG) E₂ and leukotrienes (LTs) are pro-inflammatory lipid mediators metabolized from arachidonic acid and have pathophysiological activities in various inflammatory diseases. In inflammation with the hyperproducion of PGE₂ and LTs, microsomal PGES (mPGES)-1 is an inducible terminal enzyme in the synthesis of PGE₂ and 5-lipoxygenase is a key enzyme in the synthesis of LTs. The present study demonstrated that malabaricone C derived from nutmeg (Myristica fragrans) inhibited the both enzymes, exhibiting a non-competitive inhibition of mPGES-1 (IC₅₀ of 40.5 μM) and a competitive inhibition of 5-lipoxygenase (IC₅₀ of 0.2 μM). In the psoriasis-like mouse skin induced by imiquimod application, malabaricone C significantly ameliorated hyperplasia and inflammatory cell infiltration and suppressed the expression of the psoriasis-associated genes S100a9, Krt1, Il17a and Il22. Lipidmic analysis showed that malabaricone C markedly decreased the level of LTB₄ but did not trigger excessive synthesis of other lipid mediators in the psoriasis-like mouse skin. Thus malabaricone C derived from nutmeg might be a potentially useful for preventionof the LTs-related inflammatory diseases.

28－08

Development of fluorescent-labeled artificial sweeteners and application for safety research

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

　There is a broad negative perception of artificial sweeteners (AS), including exacerbation of allergic reactions and weakening immunity. Also, safety research from various perspectives is desired. This study attempts to develop a Fluorescein isothiocyanate (FITC)-labeled form of aspartame (APM), one of the representatives AS used in Japan, for establishing and applying safety study based on intracellular and in vivo kinetics of AS. APM has an amino group (NH₂) and a methoxy group (CH₃O), which are considered capable of labeling FITC, and we succeeded in synthesizing FITC-APM and APM-CH₂NH-FITC. It was revealed in the analysis using cultured cells that these FITC-labeled APMs were taken up in a short time, there are strong/high FITC uptake (FITC^High) and weak/low FITC uptake (FITC^low), and uptake of APM-CH₂NHFITC is involved in macropinocytosis. The in vivo kinetics of these FITC-labeled APMs was mostly present in ileal feces about 1 h after oral administration by gastric sonde. This paper outlines the details of these analyses and their usefulness for safety research.

28－09

Effect of 4-Methylimidazole Included in Caramel Coloring III and IV on Gastrointestinal Epithelial Cells

Yusuke Takizawa
Division of Clinical Pharmaceutics, Department of Pharmaceutical Sciences,
Nihon Pharmaceutical University

　Soft drinks are a significant part of the daily diet, and its market continues to grow annually. Since the contents of soft drinks are generally withheld and protected by law, unknown components may affect cell proliferation and the function of gastrointestinal epithelial cells. Class III and IV caramel coloring in cola, which is the best-selling soft drink, contains 4-methylimidazole (4-MeI) and 2-Acetyl-4-tetrahydroxybutylimidazole (THI), which may affect gastrointestinal epithelial cells. Therefore, the present study examined the effects of Caramel coloring III, IV, 4-MeI and THI on gastrointestinal epithelial cells, which are continually exposed to them during the consumption of cola. Whereas, no significant effects on cell proliferation or epithelial barrier function were observed under conditions that could be ingested from soft drinks, changes in barrier function were observed under high concentration conditions of 4-MeI and THI. Since the present study only examined the effects of 4-MeI or THI alone, interactions with the concomitant intake of drugs and other foods need to be considered. Because this information will directly affect the safety of foods consumed on a daily basis, it is important to clarify the detail effects of 4-MeI or THI included in soft drinks, such as Cola, on gastrointestinal epithelial cells.

28－10

Whole-brain mapping of neurons that are activated by the ingestion of artificial sweeteners and drive their preferences

Daisuke Tanaka
Tokyo Medical and Dental University

　The experience of comfort is an essential and important experience that enriches our lives. Artificial sweeteners can provide the experience of delicious comfort, but the neural basis of this experience remains unclear. It is known that mice exposed to the artificial sweetener saccharin show a distinctive taste response ('liking' response), such as tongue protrusion, which is also seen in human neonates, and this 'liking' response is thought to reflect pleasantness. Although cells involved in pleasant emotions are thought to be distributed in the anterior insula, the relationship between the activity of these cells and the intake of artificial sweeteners or the expression of 'liking' responses has not been clarified so far. In the present study, using in vivo calcium imaging, we found that the activity of neurons in the anterior insular cortex is activated by the ingestion of saccharin and sucralose. However, the activity of these cells tended to correlate with the oral stimulus itself, and no correlation with the pleasant response itself was detected. Furthermore, artificially activating these cells did not induce a clear 'liking' response. These results suggest that while neurons in the insular cortex are activated by the ingestion of artificial sweeteners, they are unlikely to be directly involved in the 'liking' response.

28－11

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

　We have previously shown that cell death of intestinal epithelial cells (IECs) and infiltration of eosinophils were significantly increased in the small intestine of mice orally treated with an aluminum-containing food additive.
　In this study, we investigated alterlation of gene expression in small IECs of mice orally treated with an aluminum-containing food additive by performing RNA-sequencing and transcriptome analysis. The results showed that an aluminum-containing food additive increased expression of genes involved in the response to Gram-negative bacteria and inflammatory responses in IECs, including Casp4, a caspase-4 gene involved in inducing inflammatory cell death; Tnf and Il17c, inflammatory cytokine genes; and Nos2, a nitric oxide synthase gene. It also induced Il33 expression, which is involved in promotion of type 2 inflammatory responses. These results suggest that an aluminum-containing food additive may enhance antimicrobial responses against intestinal bacteria, production of inflammatory cytokines TNF-α and IL-17c, inflammatory cell death via caspase-4 activation, and eosinophil infiltration via IL-33 secretion.
　This study revealed that an aluminum-containing food additive increases gene expression involved in inflammatory responses to bacteria and inflammatory cell death in IECs. An aluminum-containing food additive may induce intestinal inflammation by promoting intestinal epithelial cell death and IL-33 release, and Further reserch on its use as a food additive is needed.

28－12

Evaluation of storage stability of Carotenoid Z-isomers in edible media and optimization of antioxidants for improving stability

Kazuya Murakami
School of Food and Nutritional Sciences, University of Shizuoka

　The Z-isomers of carotenoid have attracted attention recently because of their high antioxidant capacity and absorption capability in the human body. However, Z-isomers are unstable and easily isomerized to the all-E-isomer, Hence, a stabilization method for carotenoid Z-isomers must be established. In this work, we investigated the effects of the storing media and the influence of antioxidant addition on the lycopene Z-isomers stability as representative carotenoids. Using storage tests, we clarified the decomposition behavior of Z-isomer-rich lycopene, in 25 different types of edible oils and fats. Additionally, by testing five types of antioxidants in olive, green nut, and sesame oils, we found that the most suitable antioxidant for preserving Z-isomer-rich lycopene depends on the medium. The best combinations of oil and antioxidant for storage were olive oil with butylated hydroxytoluene (BHT), green nut oil with BHT, and sesame oil with α-tocopherol.

28－13

Development of thickness measurement muddler/viscometer at home

Masayuki Kurose
Department of Physiology, School of Dentistry, Iwate Medical University

　The rapidly-aging population has been shifted the focus of care for the elderly and disabled people to home care. Drinking water is highest risk of aspiration for dysphagia patients, and such risk is reduced by adding thickeners. However, the thickness of solution depends on the qualitative evaluation, and the quantitative evaluation is limited to the facilities where professional equipment can be installed. Therefore, we have started the development of a muddler (viscometer) that easily detects thickness at home, and successfully developed the measurement system was consisted of multi-axis tactile sensor. In this study, the shape of the stirring blade was examined to improve the thickness detection level. The thickness of different concentrations of glycerin and solutions dissolved thickeners were detected using rheometer and our developed-viscometer attaching various types of blades which were made by 3D printer. The detected thickness differs depending on the shape of the blades, and "Oar"-shaped stirring blade was the most detectable shape. As a result, we were able to obtain basic data for the development of a muddler (viscometer) with a view to home use.

28－14

Study of the nutritional assessment and vitamin B12 analogs of edible insects

Tomohiro Bito
Faculty of Agriculture, Tottori University

　Nowadays, "Insect Food" is considered to have the potential to contribute to global food security. Investigations have shown edible insects to be rich in macronutrients such as protein and fats. However, data on their vitamin and mineral content and characteristics is currently limited. While food safety is required, it is important to analyze various nutrients contained in edible insects in order to advocate the introduction of safer insect foods based on scientific evidence.
　Pseudovitamin B₁₂ has negligible vitamin activity in humans because the apparent affinity constant for pseudovitamin B₁₂ binding to the intrinsic factor involved in gastrointestinal absorption of vitamin B₁₂ is very low. Although some studies have described the vitamin B₁₂ content of edible insect products, little is known about the inactive vitamin B₁₂ analogs, such as pseudovitamin B₁₂, potentially contained in these products. Therefore, to more precisely evaluate the nutritional value of edible insects, the total content of vitamin B₁₂ compound of 12 insect products was quantified using a bioassay with subsequent identification of vitamin B₁₂ compounds by ultra-performance liquid chromatography with mass spectrometric detection.

28－15

Effect of edible insect protein on intestinal digestibility, food texture, and anti-microbial activity

Masaru Ochiai
Laboratory of Animal and Human Nutritional Physiology, Department of Animal Science,
School of Veterinary Medicine, Kitasato University

　The in vivo protein digestibility and expected food functionality of edible insects, which are alternative food resources, is not clear. In this study, digestive properties of the protein from migratory locust powder (MLP), an edible insect, was examined by nutritional and biochemical methods. In addition, the effect of MLP on physical properties during bread making and microbial growth during storage was investigated by the food scientific methods. For the digestibility investigation, fasted rats were fed a diet containing 10% MLP or a control diet for 2 hours, and the protein digestibility of the gastrointestinal contents was evaluated. As a result, since the protein is low in digestibility in the gut but highly digestible in the small intestine, MLP can be digested in the digestive tract. Regarding the physical properties of processed foods, migratory locust powder did not affect the physical properties of bread making immediately after production. On the other hand, the growth of general viable bacteria and Staphylococcus aureus was suppressed during the storage of bread. This study suggests that MLP has digestive properties in the digestive tract and is useful as a food resource that contributes to the extension of the shelf life of processed foods.

28－16

Study for the dissemination and expansion of pharmaceutical educational research on quality assurance of food and food additives

Junko Kurokawa
Department of Bio-informational Pharmacology, Faculty of Pharmaceutical Sciences,
University of Shizuoka

　In Japan's ultra-aging society, "extension of healthy life expectancy" and "prevent onset of and becoming serious condition of lifestyle-related disease" are key to maintaining a sustainable and stable society. Japan will be soon facing the reality of ultra-aging society and forced to cope with it. As for contents of education and research activities in Japanese pharmaceutical sciences relevant to primary health care and self-medication, topics on OTC drugs made from natural ingredients or herbal medicines, functional health foods, dietary supplements, and so-called health foods are often chosen and discussed; however, apart from issues on medical supplies mentors and students are seemed not so much interested in these topics. The "Survey of Pharmaceutical Research and Education on Quality Assurance of Health Foods and Health Functional Foods, etc." was conducted through a questionnaire targeting faculties of pharmacy and pharmacy colleges nationwide. Accordingly, either legislative system nor research activities for these products are not well described. In order to revitalize education and research on primary care and self-medication, this study was aimed to provide information for the report on the survey from Science Council of Japan.