■Program & Abstracts

November 17, 2021 (Wednesday)

13:00 - 16:50

Hosted by: Euglena Co., Ltd.

Co-hosted by: Health Longevity Food Research and Development Platform (Room O-09)

Supported by: Ministry of Agriculture, Forestry, and Fisheries - The Gathering and Utilization of Knowledge

■Greetings

We are pleased to announce the "Second Ergothioneine and Selenoneine Research Conference," organized by Euglena Co., Ltd., which will be held online on November 17, 2021.

Ergothioneine is a valuable antioxidant molecule often referred to as the "longevity vitamin" from a nutritional perspective. It is abundant in our diets as a component of foods such as mushrooms and fermented products. Selenoneine, structurally similar to ergothioneine, is another antioxidant molecule with emerging distinct properties and is found in abundance in the blood of fish like tuna. In recent years, research on the physiological functions of ergothioneine and selenoneine has been actively pursued both in Japan and worldwide, revealing their potential contributions to human health, including cognitive function and inflammation control, among others. As a result, these compounds are being increasingly utilized as functional ingredients in products such as cosmetics, pharmaceuticals, and health foods that are part of our daily lives. It is anticipated that the industrial and market structure focusing on ergothioneine and selenoneine will see significant growth in the near future, accompanied by increased competition in product development and market share acquisition.

Today, we find ourselves in a pivotal period where the "Ergothioneine and Selenoneine Industry" is poised for growth. Recognizing this opportunity, we established the "Ergothioneine and Selenoneine Research Conference" last year and held the inaugural session as a kickoff event. Fortunately, leading researchers and corporate developers from Japan and beyond came together, providing a valuable platform for the presentation and discussion of cutting-edge research, development, and practical utilization ideas spanning various fields. We hope that this initiative has contributed to fostering collaboration within the industry-academia community and raising awareness among the general consumer population.

To take the next step, we have successfully organized the second edition of this research conference. This year's lineup of speakers includes key figures from the industry who, we understand, will present a wide range of new and timely insights, including hot topics and achievements that have emerged in the past year.

In conclusion, we believe that the Ergothioneine and Selenoneine community in Japan, consisting of stakeholders from academia, industry, and government, can come together at this socially, scientifically, and industrially significant moment. We hope that, as part of an effort to spread awareness and understanding among the general public, each of you will actively engage in your respective endeavors. We have faith that this research conference will serve as a meaningful cornerstone for the future of human society.

■Second Ergothioneine and Selenoneine Research Conference Organizing Committee　Chairperson:

Dr. Iwao Otsu (Associate Professor, Division of Life and Environmental Sciences, University of Tsukuba)

■Operational Committee

Chairperson:

Kengo Suzuki (Executive Officer, Research and Development, Euglena Co., Ltd.)

Committee Members:

Dr. Iwao Otsu (University of Tsukuba, Division of Life and Environmental Sciences)

Yusuke Kono (University of Tsukuba, Division of Life and Environmental Sciences)

Kazuo Nishimura (Euglena Co., Ltd., Product Development Department)

Yuka Hashimoto (Marukawa Euglena Research and Development Department)

Masanobu Horiuchi (Euglena Co., Ltd., Product Development Department)

Chika Toyokawa (Euglena Co., Ltd., Research and Development Department)

Kyoko Mori (President, Aikyo Industry Co., Ltd.)

Conference Moderator:

Chika Toyokawa (Euglena Co., Ltd., Research and Development Department)

■Producer of the Health Longevity Food Research and Development Platform (Room O-09)

Producers:

Kyoko Mori (President, Aikyo Industry Co., Ltd.)

Dr. Iwao Otsu (Associate Professor, College of Life and Environmental Sciences, University of Tsukuba)

Koichiro Mori (Development Research Section Chief, Hokuto Corporation)

Hideaki Oike (Senior Researcher, Animal Production Research Division, National Agriculture and Food Research Organization)

■Program

13:00 – 13:10 Opening Address (Committee Chairperson)

Kengo Suzuki (Executive Officer, Research and Development, Euglena Co., Ltd.)

13:10 – 13:40 Special Lecture: "Development of Ergothioneine High-Production Smart Cells - Toward the Commercialization of Fermentation-Derived High-Purity EGT"

Goh Nakatani (Nagase & Co., Ltd., Nagase R&D Center, Core Technology Section Manager)

13:40 – 13:45 　　　　　　　　 　——– Break (5 minutes)——–

13:45 – 14:05 Invited Lecture 1: "Fermentation Production of Ergothioneine Using Corynebacteria and Budding Yeast"

Takashi Hirasawa (Associate Professor, School of Life Science and Technology, Tokyo Institute of Technology)

14:05 – 14:25 Invited Lecture 2: "Mail-In Testing and the Potential of Ergothioneine"

Yosuke Takimoto (President, Healthcare Systems Co., Ltd.)

14:25 – 14:45 Invited Lecture 3: "Edible Mushrooms and Ergothioneine"

Koichiro Mori (Development Research Section Chief, Hokuto Corporation)

14:45 – 14:55 　　　　　　　　 　——– Break (10 minutes)——–

14:55 – 15:15 Invited Lecture 4: "Ergothioneine Production Using Rice Koji"

Ken-Ichi Yoshitomi (President, Sakayoshi Co., Ltd.)

15:15 – 15:35 Invited Lecture 5: "Development of Functional Food 'Memory Guardian' Containing Ergothioneine as an Active Ingredient"

Satoshi Matsumoto (Executive Officer, Manufacturing and Development Department, L Corporation)

15:35 – 15:55 Invited Lecture 6: "Construction of Ergothioneine Production Systems Through Microbial Breeding"

Takamasa Ishiguchi (Master's Program, Graduate Program in Biological Resource Sciences, University of Tsukuba)

15:55 – 16:05 　　　　　　　　 　——– Break (10 minutes)——–

16:05 – 16:25 Invited Lecture 7: "Research on the Improvement of Pre-Illness Conditions in Humans by Selenoneine"

Takuya Seko (Fisheries Research and Development Division, Environmental and Applied Division, Fisheries Research Agency)

16:25 – 16:45 General Discussion

Moderator: Kengo Suzuki (Executive Officer, Research and Development, Euglena Co., Ltd.)

16:45 – 16:50 Closing Remarks (Representative of Co-host)

Kyoko Mori (Producer, Health Longevity Food Research and Development Platform)

■Special Lecture

Development of High-Yield Ergothioneine Smart Cells

Presented by: Nagase & Co., Ltd.

Ergothioneine (EGT) is a natural amino acid with excellent antioxidant properties found in trace amounts in mushrooms and other sources. It is expected to have a wide range of applications in various fields such as food, cosmetics, and pharmaceuticals. EGT cannot be synthesized in the body, but specific EGT transporters like OCTN1, which are expressed in various tissues, have been discovered to transport dietary EGT into cells. Recent research has indicated that EGT can eliminate reactive oxygen species and may slow down the development of conditions such as wrinkles and cognitive decline associated with aging. Moreover, there is a correlation between reduced levels of EGT in the body and conditions like dementia, mild cognitive impairment, frailty, Parkinson's disease, and cardiovascular diseases. The discovery of OCTN1 and its relationship with these diseases has led to the proposal of EGT as one of the "longevity vitamins" that should be obtained through diet, increasing expectations for EGT utilization in the market.

When we began our development efforts, EGT extraction from natural sources and chemical synthesis were known methods for EGT production, but the cost was often in the millions of yen per kilogram in terms of pure product equivalents. This made it challenging to produce EGT at a price point that could be used in supplements and cosmetics. Additionally, there was a need for high-purity products for ease of use. In response, Nagase R&D Center has been working on the development of an environmentally friendly, bio-based production process using fermentation to produce cost-effective and high-purity EGT since 2014. Furthermore, since 2016, we have been involved in NEDO's Smart Cell Project, accelerating our efforts to increase EGT production using cutting-edge biotechnology. Over the years, we have increased production efficiency by over 1000 times compared to the production strain at the start of our research, successfully surpassing our commercialization goals. Alongside strain development, we have completed the development of cultivation and advanced purification processes. Currently, we are actively engaged in planning for commercialization starting next year.

In this presentation, we aim to share our research on EGT production and raise awareness among the general public about the remarkable substance EGT represents. We will also discuss what our company can do to contribute to the growth of EGT as an industry.

■Invited Lecture 1

Fermentation Production of Ergothioneine Using Corynebacteria and Budding Yeast

Presented by: Kei Hirazawa, Tokyo Institute of Technology, School of Life Science and Technology

Ergothioneine (EGT) has garnered attention due to its significant physiological effects based on its high antioxidant capabilities. To meet the demand for increased EGT supply for industrial applications, we have focused on the fermentation production of EGT using microorganisms. This presentation introduces our efforts in producing EGT through fermentation using recombinant strains of Corynebacterium glutamicum, commonly used for the fermentation production of valuable compounds like amino acids, and Saccharomyces cerevisiae, a budding yeast used in alcohol fermentation.

For EGT fermentation production with C. glutamicum, we introduced the EGT biosynthetic genes egtABCDE from Mycobacterium smegmatis into the wild-type strain of C. glutamicum. After two weeks of cultivation, we successfully produced approximately 20 mg/L of EGT. We also achieved about a twofold increase in EGT production by using a cysteine-producing strain of C. glutamicum. This approach showed that introducing the egtA gene, responsible for catalyzing the formation of γ-glutamylcysteine, led to higher EGT production levels.

In the case of EGT fermentation production using budding yeast S. cerevisiae, we introduced the EGT biosynthetic genes egt1 and egt2 from Schizosaccharomyces pombe, another yeast species. After two weeks of cultivation, we confirmed the production of approximately 40 mg/L of EGT. By adding essential amino acids (histidine, cysteine, and methionine) for EGT biosynthesis to the culture medium, we achieved a twofold increase in EGT production. However, introducing the egtABCDE genes from M. smegmatis did not result in EGT production in the recombinant strain of S. cerevisiae.

■Invited Lecture 2

Mail-In Testing and the Potential of Ergothioneine

Presented by: Yosuke Takimoto (Healthcare Systems Co., Ltd.)

Ergothioneine (EGT) has gained significant attention as a next-generation functional food ingredient due to recent research findings on its various physiological functions within the body and its absorption dynamics. We have been conducting research on the usefulness of EGT, particularly in improving sperm motility and addressing male infertility, based on its antioxidant properties. Through experiments involving the addition of EGT to boar semen and human clinical trials, we have obtained promising results.

Animals and plants cannot synthesize EGT themselves, so humans obtain EGT produced by mushrooms and certain bacteria through their diet. This suggests that individuals may have varying levels of EGT in their bodies depending on their dietary habits.

We are developing biomarkers for sub-health conditions closely related to lifestyle and diet and aiming to promote science-based behavioral changes using mail-in testing kits that can be easily used by the general public. To date, we have launched testing kits for oxidative stress markers, gut bacterial metabolites of dietary components, vitamins, minerals, and more, providing test results to over 500,000 users.

Currently, we are developing a testing kit to measure EGT sufficiency, with the hope of enabling many people to easily assess their own EGT levels in the near future. We also aspire to contribute to large-scale research efforts, such as studies on the relationship between diet and EGT and cohort studies, by leveraging the convenience of mail-in testing.

■Invited Lecture 3

Edible Mushrooms and Ergothioneine

Presented by: Koichiro Mori, Hiroshi Hanayama, Kaori Tsuchii, Yoshinori Asaki, Aya Kawai

(HOKUTO Corporation, Development Research Division)

Currently, ergothioneine (EGT) sourced from mushrooms such as Hypsizygus marmoreus (Bunashimeji) and Lyophyllum decastes (Maitake) is primarily used as a raw material for food products. However, common edible mushrooms like Pleurotus eryngii (King Trumpet Mushroom), Pleurotus ostreatus (Oyster Mushroom), and Hypsizygus marmoreus (Bunashimeji) are also rich sources of EGT. As a mushroom producer, we aim to promote the widespread use of EGT and expand the market. To achieve this, we have been conducting research on the EGT content in edible mushrooms and processed products, engaging in educational activities about EGT through various channels, and working on the functional characterization of EGT-containing mushrooms.

Our research indicates that EGT is often abundant in mushrooms belonging to the Pleurotus genus. Starting this fiscal year, we have begun emphasizing the EGT content of Bunashimeji mushrooms, which have the highest EGT content among our fresh mushroom products. We hope to raise awareness about EGT among consumers.

EGT is believed to have various physiological effects, including antioxidant properties that may contribute to the prevention of conditions such as dementia, frailty, and cardiovascular diseases. We are currently conducting joint research with Professor Masao Kato of the Medical and Health Sciences Research Area at Kanazawa University to investigate the inhibitory effects of EGT on skin disorders. Experiments using a UVB-induced skin disorder mouse model have shown that administration of Bunashimeji mushrooms leads to an increase in EGT concentrations in both plasma and skin tissues. This is accompanied by the suppression of skin barrier deterioration, reduced skin moisture levels, and epidermal thickening caused by UVB irradiation. We are continuing experiments to elucidate the molecular mechanisms behind these effects.

In a market where fermentation-based EGT production technology is developing and the use of chemically synthesized EGT is increasing in Europe and the United States, EGT sourced from mushrooms remains dominant in Japan's food industry. We aim to develop efficient and stable EGT production technology using mushrooms, focusing on what will be sought after in the future market. If you have an interest in EGT from mushrooms, we welcome your opinions and requests.

■Invited Lecture 4

Ergothioneine Production Using Rice Koji

Presented by: Kenichi Yoshitomi (SAKICHI. Inc)

Our company is engaged in the production and sale of products using koji mold located in Nagasaki Prefecture. As a member of the Nagasaki Prefecture Health and Longevity Committee, we are committed to creating high-value healthcare services that contribute to extending healthy life expectancy.

In September 2017, we decided to participate in the Japan Food Restaurant Show held in New York and conducted various tests on our product "Frozen Sweet Sake" to promote its functionality. During these tests, we discovered that a very small amount of ergothioneine (EGT) was contained in the product. This discovery led us to initiate research on EGT.

We conducted investigations into the EGT content in our products and confirmed that our sweet sake contained up to 3.7 mg of EGT per 100g. We then continued to focus on the biosynthesis pathway of EGT and examined the cultivation conditions of koji mold. During these experiments, we noticed changes in our skin, hair, and mood as we consumed the product, which led us to initiate experiments involving the topical application of EGT to the skin. In 2019, we introduced "Sakiyoshi Koji Amazake," a cosmetic ingredient that contains 10 mg of EGT per 100g. Additionally, for food applications, we successfully developed "Sakiyoshi Koji," which achieves high EGT content using only rice and koji.

During the first Ergothioneine and Selenoneine Research Conference last year, we initiated a joint research project with Euglena Co., Ltd. Euglena, a microalgae, has a high content of sulfur-containing amino acids, which are precursors of EGT. Euglena itself also contains EGT, and it has been reported that their existing raw material, "Midori Koji," has higher EGT content compared to regular rice koji. Therefore, we believed that adding Euglena to Sakiyoshi Koji as a fermentation substrate could further increase the EGT content. After studying the Euglena concentration and types of koji mold to add, we found that even a small amount of Euglena added to Sakiyoshi Koji could achieve higher EGT content compared to using rice alone as a substrate. In this presentation, we will report the current results of our research.

We will also discuss the potential brought by rice and koji, which are essential components of Japanese cuisine.

■Invited Lecture 5

Development of Functional Food "Guardian of Memory" with Ergothioneine as an Active Ingredient

Presented by: Satoshi Matsumoto

El-S Corporation, Manufacturing and Development Department, Executive Officer

Until now, health functional foods that could display their functions were limited to "Specific Health Use Foods (Tokuhou)" and "Nutrient Function Foods." However, in April 2015, the Functional Labeling Food System was launched. Under this system, businesses can display the functionality of their food products based on rules established by the government. Unlike Tokuhou, businesses are responsible for ensuring that the scientific evidence regarding the safety and functionality of the food is in place. As of November 5, 2021, 4,651 notifications have been accepted, with 406 notifications related to health claims about memory and attention using ingredients such as Ginkgo biloba leaf-derived (flavonoid glycosides, terpene lactones), EPA-DHA, and soybean-derived phosphatidylserine (according to our research). Ergothioneine has been included in two of these notifications.

In this regard, we received approval from the Consumer Affairs Agency for the functional labeling of Japan's first functional food, "Guardian of Memory," which uses ergothioneine derived from Tamogi mushrooms as its active ingredient (Functional Labeling Food Notification No. F682) in January 2021. As it is a new notification for ergothioneine, there are particularly important aspects in the application materials, including safety and the validity of the claimed functionality.

We obtained the validity of the claimed functionality through human clinical trials (randomized, placebo-controlled, double-blind parallel-group comparison trials) and stratified analysis. As a major outcome, we evaluated cognitive function using the Cognitrax cognitive function test.

In this presentation, I will discuss the development process leading up to the application for functional labeling food and aim to raise awareness of ergothioneine as a functional ingredient among the general public, promoting the use of ergothioneine-containing food products.

■Invited Lecture 6

Construction of Ergothioneine Production System Using Microbial Breeding

Presented by: Ryusei Ishiguchi, Yusuke Kawano, Iwao Otsu

(Tsukuba University, Biological Resources and Life Environment)

Ergothioneine (ERG) is a sulfur-containing amino acid with excellent antioxidant properties, and its industrial applications are expected in a wide range of fields, including food, cosmetics, and medicine. While some fungi, bacteria, and certain microorganisms can synthesize ERG, humans cannot produce it. Therefore, humans need to consume foods containing sulfur-containing amino acids to obtain ERG. Currently, ERG available in the market is chemically synthesized, with prices ranging from tens to millions of yen per gram. This manufacturing process is also associated with high environmental impact due to the use of solvents and other chemicals.

In this study, we aimed to establish a technology for the large-scale production of ERG with low cost and low environmental impact using microbial fermentation. Previously, we utilized a cysteine (Cys)-producing Escherichia coli strain that we developed (Tanaka et al., Scientific Reports 2019). However, E. coli does not possess ERG biosynthesis genes. Therefore, we created a mutant strain by introducing and overexpressing the egtABCDE genes from Mycobacteria, which are responsible for ERG biosynthesis. We then investigated various culture conditions, including substrate addition, and achieved successful ERG production in E. coli, which was the world's first (Osawa et al., J. Agric. Food Chem., 2018). Typically, bacterial EgtB uses γ-glutamylcysteine as a substrate, but we explored bacterial-type EgtB that utilizes Cys as a substrate (since eukaryotes exist that utilize Cys as a substrate). By replacing EgtB from Methylobacterium with bacterial-type EgtB that uses Cys as a substrate, we simplified the ERG synthesis pathway (details will be presented) and improved ERG production (657 mg/L) (Kamide et al., J. Agric. Food Chem., 2020). While these results were achieved in flask cultures, we also attempted scale-up in fermenters for industrial production. However, the ERG production in fermenters was lower than expected. We then focused on optimizing the culture medium composition by increasing the organic content, and as a result, we were able to achieve ERG production at 1.6 g/L in a relatively short culture time. However, we still observed histidine accumulation. Currently, we are hypothesizing that histidine is a reaction product of ERG production and hydrogen peroxide generated within the cells (Servillo et al., Free Radic. Biol. Med., 2015). We are working on strain improvement and culture system enhancements to address this issue. In this presentation, we will report on these results and discuss them further.

■Invited Lecture 7

Verification of the Pre-disease Improvement Effect of Humans with Selenoneine

Presented by: Takuya Seko, Yumiko Yamashita, Tomoaki Yamashita, Kazushige Usui,　Yusuke Sugishita, Rie Karasawa, Kazuo Yumoto

(Fisheries Research and Education Agency, Division of Environmental and Applied Ichthyology, Aquatic Life Applied Development Department, St. Marianna University School of Medicine - Center for Intractable Disease Treatment)

The antioxidant selenoneine is found in large quantities in the blood, organs, and red muscle of tuna and mackerel. Its strong antioxidant properties have raised expectations for its utilization and impact on health. Accumulation of selenoneine in the red blood cells of people who consume fish frequently or have a habit of eating marine mammals containing a large amount of selenoneine has been reported. In recent years, research on the relationship between fish consumption and health, particularly focusing on memory and attention, has been progressing. However, actual verification of selenoneine accumulation through fish consumption in humans has remained at the level of mouse experiments, and there has been a demand for verification in humans.

Starting in the fiscal year 2021, we initiated joint research to clarify three points: 1) the accumulation of selenoneine from dietary sources in humans, 2) its effect on in vivo antioxidant capacity, and 3) its effect on various health indicators. To examine the potential for extending healthy life expectancy and addressing pre-disease conditions, we began a trial involving the regular consumption of raw red meat or blood muscles from bigeye tuna for three weeks with 100 participants. We are currently focusing on the following aspects: 1) the evaluation of selenoneine, total selenium, various trace elements, and fatty acids by the Fisheries Research and Education Agency, 2) the measurement of oxidative and antioxidative levels in plasma by the Kanagawa Prefecture Fisheries Technology Center, and 3) the evaluation of Sirtuin 2, a protein associated with lifespan extension, and P16, a tumor suppressor, by the Center for Intractable Disease Treatment at St. Marianna University School of Medicine. In this presentation, I will provide an overview of the joint research initiated in fiscal year 2021 and introduce recent research and challenges related to selenoneine.

■Acknowledgment

We sincerely thank the following companies for their cooperation in organizing this research meeting:

Ministry of Agriculture, Forestry and Fisheries, "Knowledge Accumulation and Utilization Platform for Industry-Academia-Government Collaboration"

Az Worldcom Japan Co., Ltd., Mr. Furukawa

We appreciate their support in disseminating information about this research meeting through email newsletters and their website.