Impact Objectives • Gain a greater understanding of gamma amino butyric acid (GABA), the main inhibitory neurotransmitter in the mammalian central nervous system and the key to understanding the science behind the human taste bud

• Develop a broad array of low-salt recipes for a range of dishes, which would enable the elderly and hospital patients to enjoy nutritional meals that also taste good

• Ultimately work to commercialise a range of low-salt diet recipes

The science of salt

Dr Hiroshi Ueno is investigating GABA-synthesising enzyme and working on commercialising a low-salt diet recipe in research that is pleasing to the taste buds

Could you begin was believed that GAD mainly locates in Our research is trying to achieve progress by describing how the brain and nervous system. A major in developing tasty low-salt dishes. We you came to work shift occurred in 1991 with the discovery still need to screen salt enhancers from in the field of of isoform for GAD, meaning humans thousands of spices and natural products. biochemistry? have two sets of GAD, namely GAD65 and We use an enzymatic tool that involves a GAD67, located in different chromosomes. spice extract with glutamate decarboxylase I studied quantum In addition, GAD65 is the target protein for 67 enzyme to measure enzymatic activity. If chemistry in Professor Kenichi Fukui’s self-immune disease of type I diabetes. the extract exhibits activation of the enzyme, laboratory at Kyoto University, Japan. We By using an immuno-histochemical method, it is a candidate salt enhancer. learned that chemistry explains detailed we found the localisation of GAD65 in the chemical reactions that can be immediately stomach, intestine and skin. Because of the Could you explain a little about the human applied to the mystery of life where our main limitations of the method, it was difficult taste tests you have conducted? energy sources are created by the chemical to find other locations. However, another reactions. At this point, my interests breakthrough came from transgenic mouse, Targeting young ladies at college level, shifted from chemistry to biochemistry. GAD67/GFP knock-in mouse, developed we test the tastes for miraculin and low Brandeis University in the US accepted my by Professors Kunihiko Obata and Yuchio salt bread, soup and some cooked foods. application to study at its graduate school. Yanagawa at the National Institute for Changing acidity (pH change) and acidic Brandeis offered a very strong biochemistry Physiological Sciences, Japan. In collaboration materials such as hydrochloric acid, citric programme, during which two eminent with Professors Obata and Yanagawa, with acid, acetic acid and other organic acids biochemists, W P Jencks and R H Abeles, Professor Masahito Watanabe at Osaka were compared for miraculin. For salt taught a one-year biochemistry course. At Medical College, Japan, and my PhD student effect, low salt food was compared with and that time I was interested in how coenzymes Yumi Nakamura, we found GAD67 localisation without small amounts of spice, as well as function. at type III taste buds. It is known that type III with the original recipe. taste buds express salt and acid receptors. Could you discuss your project ‘Search for It was also shown that GABA is produced in How do you see your research progressing salt-enhancement materials from natural the type III taste buds and GABA receptors in the next five to 10 years? resources by using GABA-synthesising are also expressed. GABA receptor is known enzyme, development of low-salt content as chloride ion channel, which participates in Looking ahead, we wish to commercialise foods, and study of salt signal transduction salt sensation. The discovery was the initial our low-salt diet recipe. We also hope to mechanism’? step as to why we started exploring the salt unearth new mechanisms of how salt mechanism. sensation travels from receptor to taste We first explored where GABA-synthesising nerve within the taste bud. enzyme, L-glutamate decarboxylase (GAD) What gaps in understanding is your locates in the body. Up until this point, it research trying to fill?

www.impact.pub 71 Palatable research

At Ryukoku University, Japan, researchers are exploring the science behind taste and working on low-salt dishes that can be enjoyed by the elderly and hospitalised patients

There are five basic recognised tastes – Composition Data Base for Food’, and sweetness as well as umami. This effect is salty, sweet, bitter, sour and umami, and general biochemical approaches toward called “contrast effect.” If you control GABA taste receptors are distributed among two glutamate decarboxylase, histidine levels in the cell, it should affect umami different cell types, type II for sweet, bitter decarboxylase and serine carboxypeptidase taste. Low-salt food is often criticised due and umami, and type III for salty and sour. Y. He has a special interest in gamma to the loss of umami taste, which may be Combinations of these receptors contribute amino butyric acid (GABA), which is the prevented if we trick the contrast effect. to different perceptions of taste. Taste is main inhibitory neurotransmitter in the To do so, we add a small amount of spice clearly important, preparing the body for mammalian central nervous system. It when salt levels are reduced.’ digesting food by triggering the salivary is produced from L-glutamic acid in the glands and, ultimately, we want to eat food decarboxylation reaction catalysed by ACTIVATING GAD ACTIVITY that tastes good. However, nutrition is also glutamic acid decarboxylase (GAD). GABA In another line of investigation, the important. So how do we get the best of sends chemical messages through the brain researchers are exploring how to control both worlds and ensure the nutritional food and the nervous system and is involved in GAD activity by adding natural products we are consuming also tastes good? regulating communication between brain such as herbs and spices, as Ueno explains: cells. Due to the expected physiological ‘The purpose was to develop drugs to Dr Hiroshi Ueno is leading a project effect of GABA in the suppression of control the nerve system by using safe entitled ‘Activation of GABA synthetic blood pressure increase, diuretic effect materials, like the ones utilised in food.’ enzyme, glutamate decarboxylase, by spice and relaxation effect, it is also being used In this work, the researchers screened and reduction of salt and elucidation of in health food products. However, as the hundreds of herb and spice extracts testing salty taste signalling mechanism’. He is molecular role of GABA and GAD is not fully on GAD activity and observed whether each Professor in the Faculty of Agriculture at understood, Ueno and his team are seeking extract dramatically inhibited or activated Ryukoku University, Japan, with expertise to unravel these molecular mysteries. GAD enzymatic activity. According to Ueno, in applied microbiology and biochemistry. the results of the research represent the Prior to this, he held positions at Japan’s In the team’s project, ‘Search for salt- phenomena of eating. ‘For example, when Nara Women’s University, Kyoto University enhancement materials from natural we eat spicy food, we sweat or sometimes and Osaka Medical College, and Rockefeller resources by using GABA-synthesising have a bad stomach,’ he states. ‘It can be University in the US. His current research enzyme, development of low-salt content explained that food components could bind surrounds the science behind the taste foods, and study of salt signal transduction to specific receptors located in our digestive buds and has included projects such as mechanism’, the researchers began by systems, then those signals received by ‘Food Components Inhibiting Recombinant exploring where GAD locates in the body. the receptors are transmitted further to Human Histidine Decarboxylase Activity’, They used an immuno-histochemical cause various metabolic or physical activity ‘Construction of Free Amino Acids method and identified the localisation of changes. It is reasonable to understand GAD65 in the stomach, intestine and skin. the wide effects we observed for herbs and However, due to the limitations associated spices on GAD activity.’ with this method, the team was unable to identify further locations. Later, when the In this project, Ueno’s PhD student Kumiko team used a GAD67/GFP knock-in mouse, Hisaki and other members of the team the researchers were able to identify GAD67 examined spice extracts for possible salt localisation on type III taste buds, which are enhancer effects on human subjects. The known to express salt and acid receptors, team prepared breads in which the salt while the GABA receptor is known as the content was reduced by half the amount of chloride ion channel and to participate in the original recipe. They found that when salt sensation. Ueno provides an insight spice extract was used as a replacement for into the relationship between GABA and salt, the breads had a normal taste. This taste: ‘In the field of cookery, it is known prompted the team to work on developing GFP expressed on transgenic mouse taste bud that a small amount of salt enhances low-salt recipes for a range of dishes,

72 www.impact.pub Project Insights

FUNDING Funding for this project provided through: JSPS KAKENHI Grant No. JP15K00872; The Salt Science Research Foundation, No. 16D1, 17D1, 18D1; Society for Research on Umami Taste; Japan Society for Bioscience, Biotechnology and Agrochemistry Innovative Research Program Award, 2010; and Urakami Foundation for Food and Food Culture Promotion.

COLLABORATORS Dr Kumiko Hisaki, Osaka International College, Japan • Dr Yoko Nitta, Okayama Prefectural University, Japan • Dr H Komori, Kagawa University, Japan • Dr Yukiko Ueda, Osaka City University, Japan • Prof Emeritus Kunihiko Obata, National Institute for Physiological Sciences • Dr Yuchio Yanagawa, Gunma University, Japan • Dr Kenichi Ito, Ichimaru Pharcos Co., Ltd. • Dr Yumi Nakamura, Suntory • Dr Yoko Inoue (decease) • Dr Satoko Matsukawa, Shionogi & Co., Ltd • Dr M Watanabe, Kansai University of Welfare Sciences, Japan

CONTACT Hiroshi Ueno In this work, the researchers screened hundreds of Project Coordinator

herb and spice extracts testing on GAD activity T: +81 775995686 E: [email protected]; hueno@ and observed whether each extract dramatically cc.nara-wu.ac.jp inhibited or activated GAD enzymatic activity W: http://stein.agr.ryukoku.ac.jp/ueno PROJECT COORDINATOR BIO Dr Hiroshi Ueno studied at Kyoto enabling the elderly and people who are receptors expressed on the cell wall,’ he University, School of Engineering, hospitalised to enjoy eating nutritional says. ‘In addition to this, I was a laboratory majoring in theoretical chemistry meals that also taste good. The researchers member of Professor Stanford Moore at under Prof Kenichi Fukui (1981 Nobel also investigated the salt signal mechanism Rockefeller University, who won the Nobel Chemistry) before moving to Brandeis University, Graduate Department of in the cells. They used the method of Prize in 1972 for his study on ribonuclease Biochemistry for his Master’s and Iowa surface plasmon resonance (SPR), an A. During the study, he and Professor State University, Graduate School to optical technique that measures changes William H Stein developed an automated complete his PhD. He is currently a in index of refraction, to identify specific amino acid analyser. I maintained the first Professor in the School of Agriculture proteins that form a complex with glutamate such machine, we called No. 1, while I was at Ryukoku University, Japan. decarboxylase 67. at Rockefeller University for 10 years and continue to keep the method going.’ COMPREHENSIVE DATABASES In addition to their innovative work Presently, Ueno and the team are still on GABA and creating low-salt dishes, using enzymatic processes to screen salt the researchers are engaged in other enhancing spices and natural materials. The investigations as Ueno highlights: ‘We researchers are also continuing their work are establishing a database that contains to identify the proteins that are involved free-amino acids in various food supplies, in forming complexes with GAD67. In including fruits, vegetables and others. addition, they are exploring using spices Most of the existing amino acid databases already identified for their salt enhancing give amino acid contents after samples have effect to make dishes according to the been acid hydrolysed. Recent advancements original recipe. ‘Testing these dishes for revealed free-amino acids play significant appropriateness is the immediate next step,’ roles in our body functions, including Ueno explains. Ultimately, the researchers tastes, activation and/or de-activation hope to be successful in developing low-salt of various cell activities via amino acid dishes. l

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