The Research Reports No.31 （issued in 2025）

Abstracts of Research Reports funded by the Foundation's Research Grants in FY2024.

31－01

Synthesis of Biodegradable Polymers Using Food Additives as Safe Catalysts

Feng Li
Division of Applied Chemistry, Faculty of Engineering, Hokkaido University

　In response to the growing need for safer alternatives to conventional metal and organocatalysts in the synthesis of biodegradable polymers, this study explores the use of food additives as catalysts for the ringopening polymerization of aliphatic polyesters. Various organic acids and their sodium salts, including saccharin, malonic acid, citric acid, and EDTA, were evaluated as catalysts for the polymerization of L-lactide (L-LA) and ε-caprolactone (ε-CL). The results showed that organic acids exhibit high catalytic activity for ε-CL polymerization, while sodium salts are more effective for L-LA. These systems enabled the precise synthesis of polymers with narrow molecular weight distributions and were applicable to other cyclic esters and carbonates with different ring sizes. Furthermore, combining organic acids and sodium salts as dual catalysts allowed for the synthesis of sequence-controlled copolymers, such as statistical and block copolymers of L-LA and ε-CL. These findings demonstrate that food-grade additives possess significant potential as safe and effective catalysts for the synthesis of biodegradable polymers.

31－02

New perspective on food additive "lactate"
Exploring the preventive effects of oral intake of lactate combined with exercise on cognitive improvement

Takanori Tsuda
College of Bioscience and Biotechnology, Chubu University

　Exercise has various benefits for the entire body. For example, Exercise is an effective strategy for body weight control because it elevates energy expenditure and improves overall glucose and lipid homeostasis as well as insulin sensitivity. Among them, it is well-known that exercise can prevent cognitive decline. Lactate is not a cause of fatigue, but also is thought to be a signaling molecule. Furthermore, it has been reported that exercise induced elevation of blood lactate concentration results in improvement of cognitive function. Although lactate is daily ingested from various foods, there have been no studies on the physiological function of lactate intake as a food additive. Accordingly, the purpose of this study was to verify the amplification of the beneficial effect by combination of lactate intake and exercise. In our previous study, we confirmed that the most suitable mouse strain, exercise condition and dose of lactate were determined. In the present study, we verified the improvement of cognitive function by combination of oral administration of lactate and exercise. As a result, it was revealed that significant improvement of cognitive function was observed in the lactate + exercise group. Furthermore, the mechanism could be due to upregulation of brainderived neurotrophic factor in the hippocampus.　　

31－03

Development of a Highly Sensitive Analytical Method for Pyrrolizidine Alkaloids in Food Using an Automated Pretreatment System

Shizuka Shida
Division of Foods, National Institute of Health Sciences

　Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins that can cause liver damage when ingested through contaminated foods such as herbal teas. In this study, a highly sensitive and quantitative LCMS/MS method was developed for the determination of 21 PAs in herbal teas (including tea leaves) and honey, employing an automated solid-phase extraction (SPE) system. Extraction conditions, cleanup procedures, and LC-MS/MS parameters were thoroughly optimized to minimize matrix interferences and enable accurate quantification of individual compounds. The developed method involves extraction with 0.1 mol/L sulfuric acid/methanol (1:1) (0.05 mol/L sulfuric acid for honey), followed by automated cleanup using cation- and anion-exchange cartridges prior to LC-MS/MS analysis. At a spiking level of 1 ppb, recovery tests demonstrated satisfactory performance, with recoveries ranging from 80% to 100% and relative standard deviations below 8%, except for one compound in tea samples, which showed a low recovery due to ion suppression. This automated method allows for high-throughput analysis and is expected to serve as an effective tool for monitoring and understanding the contamination status of PAs in food products.

31－04

Elucidation of epigenetic alterations in offspring induced by maternal D-tagatose intake

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

　Recent epidemiological studies suggest that susceptibility to obesity is influenced by both genetic predisposition and environmental factors. Although approximately 60 percent of an individual's body mass index (BMI) is estimated to be determined by genetic factors, the recent global surge in obesity cannot be fully explained by genetics alone. Therefore, epigenetic mechanisms, which refer to heritable changes in gene expression without alterations in the DNA sequence, have garnered attention as key mediators of environmentally induced obesity. Fibroblast growth factor 21 (FGF21), a hormone primarily secreted by the liver and a key regulator of glucose and lipid metabolism, has been identified as a potential "epigenomic memory gene." The author has previously demonstrated that FGF21 suppresses sugar intake behavior. Furthermore, the author found that administering d-tagatose, a known inducer of Fgf21, to maternal mice reduced sugar preference in their offspring during adulthood. Based on these findings, the present study hypothesizes that maternal intake of d-tagatose induces epigenetic modifications in the Fgf21 gene and its promoter region in the offspring, leading to a phenotype with reduced sugar preference, which is essentially a lower propensity for obesity. This study aims to elucidate the underlying epigenetic mechanisms and to evaluate the potential of maternal nutritional interventions in promoting resistance to obesity and preventing etabolic disorders such as diabetes.

31－05

Research on the preparation of high molecular mass condensed tannin standards for the quality evaluation of food additives

Yoshiaki Amakura
College of Pharmaceutical Sciences, Matsuyama University

　We investigated whether a high molecular weight condensed tannin fraction can be isolated from grape skin extract, an existing additive that has been suggested to contain high molecular mass condensed tannins and examined the practicality of creating a standard product characterized by its molecular weight and unit content. To obtain this fraction, separation and purification was attempted using Diaion HP-20 and Sephadex LH-20 column chromatography analyses, and three broad fractions were obtained as intended by HPLC analysis. ¹³C-NMR spectra of the obtained fractions showed signals characteristic of procyanidin B-type, which has a flavan-3-ol as a base unit. To chemically confirm each constituent unit, phloroglucinol degradation was attempted, resulting in epicatechin and epicatechin (4→2) phloroglucinol, suggesting that the structure is mainly a B-type polymerized epicatechin unit. In addition, to clarify the molecular weight, GPC analysis (using polystyrene standards) yielded three fractions with different weight-average molecular weights of 10,945, 25,551, and 34,285.

31－06

Analysis of change in physical forms of silver nanoparticle after oral exposure

Kazuya Nagano
School of Pharmaceutical Sciences, Wakayama Medical University

　It is reported that the physical forms of nanoparticles are changed, such as aggregation, ionization and reconstitution. However, these changes in physical forms of nanoparticles in vivo has not been sufficiently clarified. In this study, we tried to analyze the changes in hysical forms of silver nanoparticles by using our single-particle ICP-MS method for biological samples.
　Since nAg70 may change the physical forms during gastrointestinal absorption, the physical forms in vivo was analysed after nAg70 was orally administered to mice. The results showed that about 20% of Ag in the small intestinal contents were ionised, correlating with the in vitro results, although ionisation was not enhanced in the gastric contents compared to the in vitro results. In addition, nAg70 was distributed as almost ions not only in the peripheral blood, but also in the liver, in which foreign substances such as nanoparticles are taken up before they reach the circulating blood. Therefore, the data suggested that nAg70 changes the physical forms during gastrointestinal absorption, even in vivo.

31－07

Immunomodulatory functions of taste food additives

Osamu Kaminuma
Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University

　To clarify the immunomodulatory function of taste food additives, analysis of taste receptors expressed on immune cells and their functions, and generation of taste receptor-deficient mice were performed. Singlecell analysis of human immune cells showed that bitter taste receptors, TAS2Rs, were expressed on B cells, plasma cells, T cells, and NK cells. Mouse T cells did not express Tas2rs, but each T cell subset expressed different levels of Tas1rs. IL-4 expression in human T cells was enhanced by bitter receptor agonists. Antigen-induced sneezing response and nasal eosinophil infiltration in a mouse model of allergic rhinitis were suppressed by bitter receptor inhibitor treatment. Knockout (KO) mice lacking the Tas1r1, Tas1r2, and Tas1r3 genes were generated using the CRISPR/Cas9-based genome editing. They were further genomeedited to generate Tas1r1/Tas1r3 and Tas1r2/Tas1r3 double KO mice. In addition, flox mice targeting the Tas1r gene were generated and crossed with CD4-specific Cre recombinase-expressing mice. Quantitative RT-PCR analysis of the Tas1r genes in the organs of each mouse strain was performed to confirm their genetic defects. Future plans are to investigate whether taste food additives can help prevent or treat allergic and other diseases by regulating T-cell and other immune functions via specific taste receptors.

31－08

Antioxidant activity and concomitant nutritional enhancement through the additives specified in working inside the human intestine

Yasuhiro Funahashi
Department of Chemistry, Graduate School of Science, the University of Osaka

　Eliminating reactive oxygen species, which cause aging and various diseases, through the action of antioxidants and enzymatic reactions is most important for maintaining the health of dioxygen-breathing humans. High-purity silicon microparticles have demonstrated excellent antioxidant effects in animal experiments and have been shown to have dramatically high recovery and health-promoting effects on a wide range of diseases. This study revealed that the silicon microparticles would not only generate dihydrogen (H₂) but also process biologically relevant reactions to supply electron carrier molecules with reducing ability in the intestinal tract during food digestion. Additionally, we conducted chemical studies of the effects of pH condition on the silicon microparticles, accompanied with biomimetic studies of Si⁴⁺ complex formation of a chelating agent such as siderophores secreted by E. coli. Based on these results, it is suggested that the reaction of silicon microparticles would be followed by the metabolic reactions, producing the bioactive substances with higher antioxidant activity for elimination of the reactive oxygen species within the human body. Therefore, these processes are expected to improve the oxidative stress, thereby promoting health. Furthermore, the silicon microparticles are also anticipated to have future applications as food additives that enhance human health.

31－09

Isolation of Fusarium spp. from corn borer larval feces and estimation of the relationship with mycotoxin contamination diffusion

〇Hiroyuki Nakagawa¹, Shohei Mitsuhashi^{2, 3}, Maiko Watanabe^4
1 National Agriculture and Food Research Organization (NARO), Core Technology Research Headquarters, Research Center for Advanced Analysis
² National Agriculture and Food Research Organization (NARO), Institute of Livestock and Grassland Science
³ National Agriculture and Food Research Organization (NARO), Core Technology Research Headquarters
⁴ National Institute of Health Sciences (NIHS), Division of Microbiology

　Larvae of the corn borer were collected from the cornfield, and isolation of Fusarium species fungi from their excrement was performed. Identification of the obtained strains was conducted through molecular phylogenetic analysis, and the strains identified as Fusarium were further examined for mycotoxin (fumonisin) production.

31－10

Design study of single reference compounds for quantitative evaluation of food additives

Genichiro Tsuji
Division of Organic Chemistry, National Institute of Health Sciences

　Conventional quantitative analysis requires a reference material that is identical to the analyte being analyzed, but in some cases the reagents are expensive, unstable, or difficult to synthesize, making it difficult to obtain, and alternative methods are needed. In this study, we investigated a design method of "single-reference (SR) compounds" for use in the quantification of food additives by HPLC. In this study, based on the bisaryl-maleimide skeleton, which is easy to develop a structure for functionalization, we introduced various aromatic rings (Ar) to search for compounds that show absorption bands similar to those of the compounds to be measured. Furthermore, the retention time on HPLC could be controlled by adjusting the hydrophobicity by changing the functional group of the tail in the molecule. In addition, we were able to find some compounds as a promising SR candidate by designing compatible compounds based on both UV absorption properties and retention time on the HPLC chromatogram, oriented toward the annatto dye, bixin as a target for quantification. This compound design method is expected to enable efficient synthesis of SR candidate compounds with tunable UV absorption wavelength range and elution position on HPLC chromatograms.

31－11

Functional metabolites of tannins listed as existing food additives

Hideyuki Ito
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University
(Current Affiliation: Center for Private-Public-Academic Collaboration Research, National Institute of Health and Nutrition, National Institutes of Biomedical Innovation, Health and Nutrition)

　As part of a search for functional metabolites of extracts containing tannins listed in existing food additives, we have evaluated the detailed in vivo behavior of metabolites in biological fluids after administration of Trapa bispinosa extract (TBE). After oral administration of TBE, which is rich in ellagitannins and gallotannins, to rats, we quantified 12 types of urolithin-related metabolites and 6 types of gallotannin-related metabolites in plasma and urine samples by HPLC-ESI-MS/MS. As a result, the blood concentration of ellagitannin-related metabolites was found to be maximum at about 24 h after administration, whereas the blood concentration of gallotannin-related metabolites was found to be maximum at about 1 h after administration. In urine, ellagitannin-related metabolites were excreted in a sigmoid curve up to 72 h after administration of TBE, and most of the gallotannin-related metabolites were excreted within 24 h after administration. Ellagitannin-related metabolites exhibit delayed absorption and excretion, whereas gallotannin-related metabolites are absorbed and eliminated rapidly, indicating that these metabolites display distinct in vivo behaviors.
　We herein focused on the existing additive Eucalyptus extract and investigated the in vivo behavior of oenothein B, which is known as its main polyphenol component. Recent research has revealed that oenothein B has various biological activities such as antioxidant, anti-inflammatory, antifungal, anti-hepatitis C virus, and anti-tumor effects, and it has also attracted increasing attention as a functional tannin compound. Furthermore, oenothein B is a unique ellagitannin dimer with a macrocyclic structure, and has two galloyl and two valoneoyl acyl groups. In particular, the behavior of the valoneoyl group in the body, such as absorption and metabolism, is completely unknown. In this study, we investigated the urinary metabolites following oral administration of oenothein B to rats, as well as fecal metabolites after in vitro incubation, to explore novel functional metabolites derived from tannins listed as existing food additives.

31－12

Studies on enzymatic isomerization of carotenoids in mammals and its impact on biological activity

Yuki Manabe
Graduate School of Agriculture, Kyoto University

　Carotenoids are natural lipophilic pigments widely distributed in foods and used as food colorants. However, except for the conversion of provitamin A carotenoids to retinal, the metabolic conversions of carotenoids in the human body, particularly cis-trans isomerization, remain poorly understood. In this study, we investigated the enzymatic cis-trans isomerization of carotenoids in mammals and evaluated antiinflammatory activity of cis isomers. Incubation of various carotenoids, including halocynthiaxanthin and zeaxanthin, with mouse liver S9 fractions resulted in increased levels of cis isomers. This isomerization was not observed with heat-denatured S9 fractions, suggesting an enzymatic reaction. Furthermore, after oral administration of all-trans alloxanthin to mice, increases in its cis isomers were detected in plasma and liver, indicating in vivo conversion. Anti-inflammatory activity was evaluated using MG6 microglial cells, revealing that cis isomers of alloxanthin significantly suppressed nitric oxide production more effectively than the all-trans isomer. These findings suggest that carotenoids undergo enzymatic cis-trans isomerization in vivo and that cis isomers may exert enhanced biological activities. Modulating such isomerization pathways could offer novel strategies to optimize the functional benefits of dietary carotenoids.

31－13

Analysis of properties of pharmacokinetics of hyaluronan oligosaccharide by simultaneous determination using liquid chromatography coupled with tandem mass spectrometry and involvement of hyaluronan oligosaccharide with gut immunity

Yuki Sato
Faculty of Pharmaceutical Sciences, Hokkaido University

　Hyaluronan (HA) is a repeating disaccharide of glucuronic acid and N-acetylglucosamine and has recently been used in foods and food additives, as well as in medicines and cosmetics. We focused on HA oligosaccharides and developed a method for their simultaneous determination of HA 4, 6 and 8-mer. In this study, we aimed to use this method to analyze the pharmacokinetic properties of HA oligosaccharides and to develop an analytical method for HA disaccharides. After oral administration of HA preparations to Wistar rats, the plasma concentrations of HA 4, 6 and 8 mer were determined using LC/MS-MS. In addition, since HA disaccharides exist in unsaturated and saturated forms, various quantitative conditions were examined. The peripheral concentration of HA peaked 1-2 hours after administration and disappeared 12 hours later. It was also confirmed that the plasma concentrations of HA in portal vein continued to increase for up to 2 hours. To detect unsaturated and saturated HA disaccharides, a solid-phase extraction column, OASIS WAX, was used. Some elution and washing conditions were examined and both were detected. However, it was suggested that it was difficult to completely remove peaks derived from impurities, making it difficult to detect the concentrations of HA in plasma using this method.

31－14

Analysis of the effects of propyl gallate, a food additive (antioxidant), on bacteria, yeast, and human cells

Koji Nagata
Graduate School of Agricultural and Life Sciences, The University of Tokyo

　This study evaluated the antimicrobial activity and cytotoxicity of propyl gallate (PG), a commonly used food additive with antioxidant properties. PG exhibited dose-dependent antimicrobial effects against Grampositive bacteria (Staphylococcus aureus subsp. aureus, Streptococcus mutans) and a Gram-negative bacterium (Vibrio parahaemolyticus), with IC₅₀ values of 213 μg/mL, 140 μg/mL, and 7.5 μg/mL, respectively. In contrast, no activity was observed against Saccharomyces cerevisiae, highlighting species-specific differences in susceptibility. Cytotoxicity assessed using a CCK-8 assay in human HEK293 cells revealed an IC₅₀ of 74 μg/mL, indicating an effect on cell viability at relatively high concentrations. These findings suggest that PG has strong antimicrobial potential, particularly against Gram-negative bacteria, but its application as a food preservative should consider concentration-dependent cytotoxic risks.

31－15

Study on a New Method of Extracting Flavor Components Using Fine Bubbles

Takashi Hata
National Institute of Technology, Kochi College

　Fine bubble is the International Organization for Standardization (ISO) standard name for bubbles less than 100 μm in diameter. In addition, bubbles with diameters of 1 to 100 μm are called microbubbles, and bubbles with diameters of less than 1 μm are called ultrafine bubbles. In recent years, industrial technologies using these fine bubbles have been expanding, but there are still few examples of their application in the food industry. In this study, as a study on the use of fine bubbles in the food industry, in particular, a new method of extracting flavor components using ultrafine bubbles was investigated. At first, since water temperature is relevant to the extraction process, the thermal stability of ultrafine bubbles was investigated. As a result, although the ultrafine bubbles decreased (collapsed) when heated, the ultrafine bubbles were maintained at 80% of their creation temperature even at high temperatures such as 100°C. Then, when the ultrafine bubbles were used to extract caffeine from coffee as a flavor component, it was confirmed that the extraction progressed predominantly at low temperatures and low rpm, conditions that are generally not conducive to extraction. Similar results were also obtained in the extraction of catechins from tea leaves under low temperature conditions.