The Research Reports No.29 （issued in 2023）

Abstracts of Research Reports funded by the Foundation's Research Grants in FY2022.

29－01

Development of semi-automagical analytical methods for residual high polar pesticides in foods

Hiroshi Akiyama
Department of Analytical Chemistry, Hoshi University

　 We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of residual glyphosate, glufosinate, and their metabolites N-acetylglyphosate (Gly-A),
3-methylphosphinicopropionic acid (MPPA) and N-acetylglufosinate (Glu-A) in honey using a mixed mode column of reversed-phase and anion exchange without derivatization. The target analytes were extracted from honey samples using water, cleaned up on a reverse phase C18 cartridge column and an anion exchange NH2 cartridge column, and quantified using LC-MS/MS. Glyphosate, Glu-A, Gly-A, and MPPA were detected in negative ion mode based on deprotonation, whereas glufosinate was detected in positive ion mode. The coefficients of determination (R²) of the calibration curve, calculated in the range of 1-20 μg/kg for glufosinate, Glu-A, and MPPA, and 5-100 μg/kg for glyphosate and Gly-A, were higher than 0.993. The developed method was evaluated using honey samples spiked with glyphosate and Gly-A at 25 μg/kg and glufosinate, and MPPA and Glu-A at 5 μg/kg, based on the maximum residue levels. The validation results show good recoveries (86-106%) and precision (< 10%) for all target compounds. The limit of quantification of the developed method is 5 μg/kg for glyphosate, 2 μg/kg for Gly-A, and 1 μg/kg for glufosinate, MPPA and Glu-A. These results suggest that the developed method is applicable for quantifying residual glyphosate, glufosinate, and their metabolites in honey in compliance with Japanese maximum residue levels. Moreover, the proposed method was applied to the analysis of honey samples and glyphosate, glufosinate, and Glu-A were detected in some samples. The proposed method will be a useful tool for the regulatory monitoring of residual glyphosate, glufosinate, and their metabolites in honey.

29－02

Development of an Accurate Quantitative Gas Chromatography-Flame Ionization Detector Method Using Relative Molar Sensitivity for Monoterpenes

Naoko Masumoto
Division of Food Additives, National Institute of Health Sciences

　The relative molar sensitivity (RMS) method is a quantitative method in which the response factor of the analyte is different from that of the reference material, allowing the analyte to be quantified without an analytical standard. Recently, there has been an increase in the number of applications where valuable components contained in natural products are quantified despite analytical standards being difficult to obtain. However, few studies have investigated the factors that affect RMS in gas chromatography-flame ionization detector (GC-FID). In this study, monoterpenes were used as model compounds to investigate the GC-FID factors that affect RMS versus a reference standard. The results showed that the liquid stationary phase of the column, the carrier gas, and the makeup gas each affected RMS by about 10% for some monoterpenes, suggesting that the RMS should be determined whenever these factors are changed. By contrast, when the flow rate of the carrier gas was varied, changes in RMS were within 2%. 　　　

29－03

Elucidating the effects of vitamin C on the immune response of CD8+ T cells against pathogens

Kenta Kondo
Department of Biochemistry and Molecular Biology, Shiga University of Medical Science

　Recent studies have reported that vitamin C promotes DNA demethylation by activating TET protein. Gene expression induced by DNA demethylation is important for the optimal immune response of CD8+ T cells. However, it is unclear whether the DNA demethylation by vitamin C is involved in the optimal immune response of CD8+ T cells.
　In this study, we examined the effects of vitamin C on CD8+ T cells by culturing CD8+ T cells in the presence of vitamin C. We found that vitamin C promotes DNA demethylation, leading to increasing IFNγ expression in CD8+ T cells. In addition, vitamin C increased the expression of Batf3 and enhanced the via bility and immune response against Listeria monocytogenes of CD8+ T cells. Thus, vitamin C may optimize the immune responses of CD8+ T cells through DNA demethylation.
　These findings may lead to the development of functional foods using vitamin C.

29－04

Total synthesis of natural blue pigments for use as food dyes and evaluation of properties of their nanoparticles

〇Ryuju Suzuki, Kiyotaka Maruoka
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University

　Growing concern for safety in the food industry has led to the increasing use of natural pigments instead of synthetic chemicals as coloring agents. Currently, the major carotenoids used for magenta and yellow are fat-soluble. However, natural blue food colorants are currently limited to three types: anthocyanins, phycocyanins, and gardenia pigments, and they are all water-soluble. Hence, the food industry continues to search for candidate compounds to enable coloring of a wider range of foods. An azulene derivative isolated from Lactarius indigo, an edible blue mushroom, is a potential candidate for a new blue colorant. As this pigment is fat-soluble, it can be used to uniformly color fatty foods. In addition, its fine particle dispersion has the potential to color aqueous systems. However, the colorant properties of this compound remain unexplored. Therefore, in this study, we succeeded in the total synthesis of azulene derivatives for the first time in the world by combining the Ziegler-Hafner method to construct the azulene skeleton and carbometalation using zirconium complexes to convert an ethynyl group to isopropenyl group. Moreover, to examine the colorant applications, we prepared an aqueous dispersion of the nanoparticles of the azulene derivative and analyzed its visible spectra. The compound exhibited a deep-blue color with an absorption peak at ~600 nm in both organic solvents and nanoparticle dispersions.

29－05

Development of an analytical method for determination of furan and its derivatives in processed foods using solid-phase microextraction

Tomoaki Tsutsumi
Division of Foods, National Institute of Health Sciences

　We investigated an analytical method for four furans (furan, 2-methylfuran, 3-methylfuran and 2,5-dimethylfuran) in processed foods using headspace solid-phase microextraction coupled to gas chromatog raphy-mass spectrometry (HS-SPME/GC-MS). The four furans were quantified by GC-MS measurement operation in SIM mode and using isotope dilution technique with deuterated homologue internal standards. A medium polarity column, Rxi-624 Sil MS was used to achieve good separation for 2,5-dimethylfuran and its structural isomer, 2-ethylfuran. The coffee samples mixed with saturated NaCl solution were incubated at 40°C for 20 min and extracted at 40°C by the SPME Arrow fiber with a divinylbenzene/ polydimethylsi loxane coating for 5 min. This gave the most sensitive and stable determination of the analytes. Calibration curves for the four furans had good linearity (r > 0.999). The limits of quantification for the method were 1 ng/g for each of the four furans. A quality control coffee sample was analyzed by the method, and the ten tative concentrations of the four furans were within acceptable limits. Overall, these results suggested the present method is sensitive and useful for analysis of the four furans in processed foods.

29－06

A Systematic Review of the Safety Evaluation of Food Additives without Animal Testing, Part 2

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

　With the worldwide acknowledgement of the '3Rs principles' 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 Methodologies (NAM) to determine a system to evaluate and regulate the safety of food additives without using animal experiments. Last year, the relevant literature on the safety evaluation of food additives and functional ingredients was retrieved and systematically reviewed.
　This year, the NAM case studies of Caffeine and Coumarin were reviewed in the literature and clarified their usefulness and applicability domain of the case study. As a result, the information of the database used human metabolism and in silico were useful without POD (the point on a toxicological dose-response curve established from experimental data or observational data generally corresponding to an estimated low effect level or no effect level) based on in vitro data that not validated. Regarding the utilization of NAM, I pro pose the necessity of independent peer review for each NAM case study strongly.

29－07

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

　The age-associated 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 healthy aging. Skeletal muscle mass is negatively regulated by several transforming growth factor (TGF)-β superfamily members, including myostatin. Since blocking myostatin activity can increase muscle mass, myostatin is a therapeutic target to prevent sarcopenia. Recent studies show that in addition to myostatin, GDF11, which shares a high amino acid sequence similarity with myostatin, induces muscle atrophy at supraphysiological levels. Importantly, higher circulating GDF11 levels are associated with frailty in humans. However, little is known about whether pathophysiological levels of GDF11 activate Smad2/Smad3 signaling and induce muscle atrophy. The aims of this study were to assess the effects of pathophysiological levels of GDF11 on muscle atrophy using iPSC-derived myocytes and to identify small molecules and food factors that can prevent GDF11- and myostatin-dependent muscle atrophy. Our results showed that pathophysiological levels of GDF11 induce muscle atrophy through the GDF11-FOXO1 signaling and that blocking FOXO1 prevents GDF11/ myostatin-dependent muscle atrophy. We also find that a vitamin-related compound X attenuates GDF11-dependent muscle atrophy in human iPSC-derived myocytes.

29－08

Effects of aspartame on the regulation of immune cells' function by mammalian target of rapamycin complex 1

Akira Onodera
Department of Pharmaceutical Sciences, Kobe Gakuin University

　Aspartame (APM), an artificial sweetener, is hydrolyzed by esterases and peptidases to form two natural amino acids. These amino acids are thought to be used for protein synthesis through normal metabolic pathways; however, there is a concern regarding the health effects of excessive intake of APM. The human dendritic cells PMDC05 treated with APM for 72 hr showed reduced Interferon (INF)-alpha2 and INF-beta mRNA expression by the toll-like receptor (TLR) 7 ligand Gardiquimod. Moreover, the addition of the mechanistic target of rapamycin (mTOR) inhibitor Everolimus synergistically reduced these mRNA expressions. These cells also showed a reduced uptake of 750 nm fluorescent polystyrene particles demonstrating a function of phagocytosis. The TLR7 is localized at the lysosome, which is also the complex including mTOR activation site. An excessive APM intake is thought to impair the dendritic cells' function by reducing the lysosomal homeostasis.

29－09

Effects of non-caloric sweeteners on the blood-milk barrier during lactation

Ken Kobayashi
Laboratory of Cell and Tissue Biology, Research Faculty of Agriculture, Hokkaido University

　Breast milk contains appropriate and abundant nutrition for suckling infants. Appropriate breastfeeding contributes to the health of the lactating mother and suckling infants. However, many mother suffer from breast milk problems such as hypolactation and abnormal milk secretion. In lactating mammary glands, milk is produced by mammary epithelial cells (MECs). MECs also form less-permeable tight junctions (TJs) to function as blood-milk barrier. Our previous studies have revealed that MECs express the sweet taste receptor T1R3. In this study, we investigated whether a non-carbohydrate sweetener sucralose affects the abilities of milk production and TJ formation in MECs. The results showed that in the absence of glucose, sucralose activates STAT5, which is a main transcriptional factor for milk production in MECs. In contrast, sucralose in the presence of glucose inactivated STAT5 concurrently with reducing casein production. There was little effect of apical sucralose on TJ proteins, and the effects of basolateral sucralose on MECs were weaker than those of apical sucralose. These findings suggest that oral sucralose has little effect on blood-milk barrier of MECs.

29－10

Bioregulatory Function of Myricitrin-containing Red Bayberry (Myrica rubra) Cyclodextrin Clathrate Considering Pharmacokinetics

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

　Myricitrin (Myr) is a flavonol glycoside which prevents hyperglycemia at high concentration. However, its functionality is low due to its low water solubility and poor stability. In this study, it was used readily water-soluble myricitrin (W-Myr), γ-cyclodextrin complex of Myr, to enhance stability and water solubility. We investigated that comparison of pharmacokinetics between Myr and W-Myr in mice, then the biological regulatory ability in cultured cells. The amounts of Myr and myricetin (Mce), an aglycone of Myr, were quantified by HPLC in serum and issues of mice. Our results demonstrated that the amounts of Myr and Mce in the cecal epithelium of W-Myr-dosed mice were significantly higher than those in Myr-given ones. On the other hand, this difference does not observe in the ileal epithelium and liver. Oral administration of these compounds suppressed postprandial hyperglycemia, and W-Myr showed higher effect than Myr. In cultured cell studies, these compounds showed no effect on the expression of antioxidant enzymes and suppression of fat accumulation in hepatocytes, adipocyte differentiation, and inhibition of muscle atrophy. However, Myr promoted the secretion of glucagon-like peptide-1 from the intestinal secretory cells. Therefore, it strongly suggests that W-Myr and Myr suppress hyperglycemia through the function in gastrointestinal tract.

29－11

Safety evaluation of food additive grade titanium dioxide E171 in glycolipid metabolism

Daisuke Matsumaru
Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University

　Food additive grade TiO₂ (E171) is known to contain nanoparticles of TiO₂ (TiO₂-NPs). Since TiO₂-NPs can induce liver steatosis and increase blood glucose level, there is concern that E171 may also show same effects on the glycolipid metabolism. In this study, we performed daily oral administration of E171 for 60 days and anlyzed the metabolic phenotypes and liver morphology. E171-treated mice did not show abnormal plasma markers. Although the accumulation of Ti was observed by E171 treatment, the liver morphology was comparable to that in control mice. Moreover, the comprehensive gene expression analysis showed no critical alterations in the glycolipid metabolism related genes. Therefore, the current study suggests that E171 has little or no toxic effects. We further analyzed the possible pathogenic mechanisms of liver steatosis induced by TiO₂-NPs. We performed TiO₂-NPs treatment for 28 and 60 days. The omprehensive gene expression analysis in the treated liver showed no upregulation in the expression of glycolipid metabolism related genes. We then analyzed the TiO₂-NPs treated intestine and revealed augmentation of proinflammatory cytokine and antioxidant genes. Supplementation of N-acetyl-l-cysteine reversed the intestinal inflammation and liver lipid accumulation. The current results suggest that, at least, some phenotypes observed in TiO₂-NPs treated liver are derived from oxidative stress in the gastrointestinal tract.

29－12

Effects of chronic sodium alginate intake on suppressing blood pressure elevation in hypertensive rats

Saki Maruyama
Hygiene and Preventive Medicine, Faculty of Home Economics, Kobe Women's University

　Sodium alginate (SALG) is a substance derived from brown seaweed that has been shown to reduce blood pressure (BP). However, its effects on renovascular hypertension caused by 2-kidney, 1-clip (2K1C) are not yet clear. Previous research suggests that hypertensive rats have increased intestinal permeability, and that SALG improves the gut barrier in inflammatory bowel disease mouse models. Therefore, the goal of this study was to determine whether the antihypertensive effects of SALG involve the intestinal barrier in 2K1C rats. Rats were fed either a 1.0% SALG diet or a control diet for six weeks after being subjected to 2K1C surgery or a sham operation. Systolic BP was measured weekly, and mean arterial BP was measured at the end of the study. Intestinal samples were taken for analysis, and plasma lipopolysaccharide (LPS) levels were measured. The results showed that BP in 2K1C rats was significantly higher than in SHAM rats when fed CTL, but not when fed SALG. The gut barrier in 2K1C rats was improved by SALG intake. Plasma LPS levels also differed depending on the animal model and diet. In conclusion, dietary SALG may alleviate 2K1C renovascular hypertension by altering gut barrier.

29－13

Reduction of microbial contamination through the combined use of sodium bicarbonate and high pressure treatment

Satomi Tsutsuura
Faculty of Agriculture, Niigata University

　High hydrostatic pressure (HHP) processing is a non-thermal food preservation technique that inactivates microorganisms responsible for foodborne diseases and food spoilage by subjecting foods to pressures, with or without heat, that has a minimal effect on taste, texture and nutritional characteristics. Staphylococcus aureus is one of the most barotolerant species to HHP treatment and not inactivated after HHP treatment even at 400-600 MPa. The aim of this study is to clarify the effectiveness of adding NaHCO₃ in HHP treatment on the sterilization of S. aureus and its growth during storage. From these results, it is suggested that NaHCO₃ was effective in the inhibition of the growth of S. aureus under a low temperature condition for a long period of storage after HHP treatment, although it did not affect the bactericidal effect of HHP treatment on S. aureus.

29－14

Physical stability and storage stability of cocrystals consisting of curcumin and food additives during the manufacturing process

Naoto Suzuki
Laboratory of Pharmaceutics, School of Pharmacy, Nihon University

　Cocrystals are crystalline molecular complexes consisting of two or more components and are used in the pharmaceutical field to improve the physicochemical properties of drugs. When cocrystals are used, it is necessary to select cocrystals with high physical stability to avoid dissociation of the cocrystals during the manufacturing process and storage. This study conducted the characterization and storage stability tests on cocrystals consisting of curcumin and five coformers (resorcinol, hydroxyquinol, hydroquinone, catechol and pyrogallol). Five cocrystals consisted of curcumin and each coformer were newly prepared by solvent evaporation method, and it was found that the characterization of these co-crystals was consistent with that of the previously reported cocrystals. Cocrystals consisting of hydroquinone, catechol, and pyrogallol dissociated depending on temperature or humidity, whereas resorcinol and hydroxyquinol were considered relatively stable. Therefore, the cocrystals composed of resorcinol and hydroxyquinol are expected to maintain improved solubility due to cocrystallization without dissociation during manufacture or storage.

29－15

Capabilities of starch degraded rice endosperm protein as an anti-obesity food material

Masatoshi Kubota
Food and Environmental Chemistry, Faculty and Department of Engineering, Niigata Institute of Technology

　Rice is an important food as not only an energy source but also a protein source. Some studies about physiological functions of rice endosperm protein (REP) were conducted using alkali extraction REP. However, there are few reports about physiological functions of starch degraded REP (SD-REP) which is a commercially available protein source. Therefore, we attempted to clarify the physiological functions, especially anti-obesity effects, of SD-REP. Six-week-old male C57BL/6 mice were divided into three groups, mice fed a high fat and sucrose casein diet (HC), a high fat and sucrose SD-REP diet (HR), and a normal casein diet (NC). Mice were fed each experimental diet for 10 weeks. The body weight in the HR group was significantly suppressed after 2 weeks, compared with one in the HC group. The perirenal, epididymal, and mesenteric fat weights were also significantly lower in the HR group. These results strongly suggests that SD-REP has suppressive effects on obesity. In addition, aspartate aminotransferase and alanine aminotransferase in the blood, which are typical liver dysfunction markers, were significantly lower in the HR group, inferring that SD-REP has protective effects on liver functions. Therefore, SD-REP may have the suppressive effects on obesity and liver dysfunctions.