Kirin Holdings Company, Limited Daisuke Fujiwara Ph.D

1. From a brewing tradition to functional food innovation: Kirin’s novel approach for ageing population Kirin Holdings Company, Limited Daisuke Fujiwara Ph.D

2. History of Kirin Brand • Born as a Japanese beer brand in 1888, KIRINhas been loved by consumer for over 100 years in alcohol and soft drink business. • High quality brand image has been nurtured throughout the history of the company and it has become an important, intangible asset of the Kirin Group. 1949 1889 （Origin of the label today) 1888 Recent 1957

3. Business Overview Successfullytransformed to a global company with portfolio of alcohol, soft drink, and pharma. 2016 1980 Rev. $19.6bn Rev. $7.3bn

4. Future vision of Kirin’s business domains Pharmaceutical Business domain Food & beverage Businesses bridging Businesses Alcoholic beverage Non-alcoholic beverage Establish and foster Pharmaceutical Health and Well-being Social Responsibility Community Engagement The Environment

5. Capturing the trend in ageing market Medical treatment Consumer with no health concerns Prevention / Control B to C marketing food products B to B business functional ingredients

6. History & Portfolio of Kyowa Hakko Bio • World’s first invention of the process of amino acid (L-Glutamic acid) production by fermentation. 1956 Today Dr. S. Kinoshita

7. What is Glutathione? Kyowa’s Glutathione • Strong technology background since 1960s • The only supplier of both reduced and oxidized form • Global standard quality • GRAS and TGA (Australia) certified • Clinically studied and patented • Strong marketing support

8. Immunity as a fundamental health system Most of the troubles in the human body are related to the immunity. ⇒The best way to maintain health is to control the immunity. Infection Eye problem Muscle loss Bone density reduction Metabolic decline Skin deterioration Immunity

9. The Brand “iMUSE” iMUSE＝Kirin’s healthcare brand based on immunology supported by its first discovery in the world. 2017- 2019.10- LactococcusPlasma Lactobacillus KW

10. LactococcusPlasma

11. Emerging risk of viral infection in Asia Dengue Fever Bird flu Dengue Fever, Chikungunya Fever

12. Anti-infection immunity in human Plasmacytoid Dendritic Cell (pDC) Direction/ Order NK Cell Killer T Cell Helper T Cell B Cell Disease causing bacteria/virus rejection/clearance pDC is the “control tower” of anti-infection immunity

13. Attempt to find the activator for pDC ? pDC Conventional Lactic Acid Bacteria (LAB) Direction/ Order NK cell Killer T cell Helper T cell B cell Disease causing bacteria/virus rejection/clearance

14. C G F E D B A Discovery of LAB driving pDC activation For the first time in the world, Kirin discovered the special LAB that activates the “control tower” of the immunity. pDC activation by LABs 400 300 IFN-a ( pg / ml ) Conventional LAB cannot activate. 200 100 control 0 L. Plasma Conventional LABs PLoS One, 2012

15. Kirin’s discovery of Lactococcus Plasma By activation of “control tower” of immunity, L. Plasma can enhance resistance to infection and ageing significantly. LactococcusPlasma After Before

16. Clinical trial against cold & flu Methods Lactococcus Plasma Subjects：Adults From Jan to Mar in 2013 Placebo 100 100 Symptoms of flu Results Feverishness Cough # of patients The number of flu and cold patients decreased and the symptom has also eased. Br J Nutr, 2015

17. Trial of L.Plasma’s effect in flu Where：Shizukuishi town in Iwate When：From Jan to Mar in 2015 What：Rate of absence due to flu Yogurt made with Lactococcus Plasma

18. Flu risk decreased by Lactococcus Plasma Rate of absence caused by flu decreased. Cumulative rate of absence due to flu ■Neighboring town □Shizukuishi total Elementary school Junior high school Health, 2017

19. Arising Risk of Dengue fever • Globally, the number of patientsare increasing • Vaccines are yet developed

20. Potential activity against dengue virus infection Titer (power) of dengue virus was drastically reduced. control L. Plasma 2 weeks Dengue virus 2 days Int J Mol Med, 2019

21. Effect on ageing through lifetime intake control L. Plasma 82 weeks 5 weeks * 100 L. Plasma 80 Control 60 Survival rate (%) 40 20 P = 0.043 log-rank test 0 Weeks 0 20 40 60 80 IntImmunopharmacol, 2018

22. Difference in appearance in ageing model Control L. Plasma

23. Difference in skin tissue in ageing model Skin Dermis Control L. Plasma 表皮 真皮 Intimmunopharmacol, 2018

24. Publication list of Lactococcus Plasma 1. Jounai K, Ikado K, Sugimura T, Ano Y, Braun J, Fujiwara D. (2012) Spherical lactic acid bacteria activate plasmacytoid dendritic cells immunomodulatory function via TLR9-dependent crosstalk with myeloid dendritic cells. PLoS One 7: e32588. 2. Sugimura T, Jounai K, Ohshio K, Tanaka T, Suwa M, Fujiwara D. (2013) Immunomodulatory effect of LactococcuslactisJCM5805 on human plasmacytoid dendritic cells. ClinImmunol 149: 509–518. 3. Jounai K, Sugimura T, Ohshio K, Fujiwara D. (2015) Oral administration of Lactococcuslactis subsp.lactisJCM5805 enhances lung immune response resulting in protection from murine parainfluenza virus infection. PLoS One. 6: e0119055. 4. Fujii T, Tomita Y, Ikushima S, Horie A, Fujiwara D. (2015) Draft genome sequence of Lactococcuslactissubsp. lactisJCM5805T, a strain that induces plasmacytoiddendritic cell activation. Genome Announc 3 : e00113-15 5. Sugimura T, Takahashi H, Jounai K, Ohshio K, Kanayama M, Tazumi K, Tanihata Y, Miura Y, Fujiwara D, Yamamoto N. (2015) Effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus. Br J Nutr. 3:1-7. 6. Suzuki H, Kanayama M, Fujii T, Fujiwara D, Sugimura K（2015）Effects of the beverage containing Lactococcuslactissubsp. lactisJCM5805 on anti‒viral immune responses and maintenance of physical conditions -a randomized, double‒blind, placebo‒controlled, parallel‒group Trial―JpnPharmacolTher. 43:1465‒72. 7. Tanaka K, Suzuki H, Kanayama M, Fujii T, Fujiwara D, Nozawa H, Sugimura K.(2015)The safety evaluation of long‒term or excessive intake of the beverage containing Lactococcuslactissubsp. lactisJCM 5805 and resistant maltodextrin-a randomized, double‒blind, placebo‒controlled, parallel‒group trial―JpnPharmacolTher. 43:1711‒27. 8. Suzuki H, Ohshio K, Fujiwara D. (2016) Lactococcuslactis subsp. lactisJCM5805 activates natural killer cells via dendritic cells. BiosciBiotecBiochem. 80: 798-800 9. Shibata T, Kanayama M, Haida M, Fujimoto S, Oroguchi T, Sata K, Mita N, Kutsuzawa T, Ikeuchi M, Kondo M, Naito K, Tsuda M, Nishizaki Y, Ishii N. (2016) LactococcuslactisJCM5805 activates anti-viral immunity and reduces symptoms of common cold and influenza in healthy adults in a randomized controlled trial. J Func Food. 24: 492-500. 10. Sakata K, Sasaki Y, Jounai K, Fujii T, Fujiwara D. (2017) Preventive effect of Lactococcuslactissubsp.lactisJCM 5805 yogurt intake on influenza infection among schoolchildren. Health 9: 756-762. 11. FujiiT,Jounai K, Horie A, Takahashi H, Suzuki H, Oshio K, Fujiwara D, Yamamoto N. (2017) Effects of heat-killed Lactococcuslactissubsp. lactisJCM 5805 on mucosal and systemic immune parameters, and antiviral reactions to influenza virus in healthy adults; A randomized controlled double-blind study. J Func Food. 35: 513-521. 12. Jounai K, Sugimura T, Morita Y, Ohsio K, Fujiwara D. (2018) Administration of Lactococcuslactisstrain Plasma induces maturation of plasmacytoiddendritic cells and protection from rotavirus infection in suckling mice. IntImmunopharmacol., 56: 205-211. 13. Kanayama M, Kato Y, Tsuji T, Konoeda Y, Hashimoto A, Kanauchi O, Fujii T, Fujiwara D. (2018) Enhancement of immunomodulative effect of lactic acid bacteria on plasmacytoid dendritic cells with sucrose palmitate. Sci Rep., 8: 3147. 14. SugimuraT, Jounai K, Ohshio K, Suzuki H, Kirisako T, Sugihara Y, Fujiwara D. (2018) Long-term administration of pDC-stimulativeLactococcuslactisstrain decelerates senescence and prolongs the lifespan of mice. IntImmunopharmacol., 58:166-172. 15. Tsuji R, Komano Y, Ohshio K, Ishii N, Kanauchi O. (2018) Long-term administration of pDCstimulative lactic acid bacteria, Lactococcuslactis strain Plasma, prevents immune-senescence and decelerates individual senescence. ExpGerontol., 111: 10-16. 16. Suzuki H, Jounai K, Ohshio K, Fujii T, Fujiwara D. (2018) Administration of plasmacytoid dendritic cell-stimulative lactic acid bacteria enhances antigen-specific immune responses.BiochemBiophys Res Commun., 503: 1315-1321. 17. Kato Y, Kanayama M, Yanai S, Nozawa H, Kanauchi O, Suzuki S. (2018) Safety evaluation of excessive intake of Lactococcuslactissubsp. lactisJCM 5805: A randomized, double-blind, placebo-controlled, parallel-group trial. Food NutrSci., 9:403-419. 18. Komano Y, Shimada K, Naito H, Fukao K, Ishihara Y, Fujii T, Kokubo T, Daida H. (2018) Efficacy of heat-killed LactococcuslactisJCM 5805 on immunity and fatigue during consecutive high intensity exercise in male athletes: a randomized, placebo-controlled, double-blinded trial. J IntSoc Sports Nutr., 15:39. 19. Suzuki H, Tsuji R, Sugamata M, Yamamoto N, Yamamoto N, Kanauchi O. (2019) Administration of plasmacytoid dendritic cell stimulative lactic acid bacteria is effective against dengue virus infection in mice. Int J MolMed., 43:426-434 20. Tsuji R, Yamamoto N, Yamada S, Fujii T, Yamamoto N, Kanauchi O. (2018) Induction of anti-viral genes mediated by humoral factors upon stimulation with Lactococcuslactis strain Plasma results in repression of dengue virus replication in vitro. Antiviral Res., 160:101-108. 21. Kokubo T, Komano Y, Tsuji R, Fujiwara D, Fujii T and Kanauchi O. (2019) Plasmacytoid dendritic cell-stimulative lactic acid bacteria, Lactococcuslactisstrain Plasma, relieves exercise-induced fatigue and aids recovery via immuno-modulatory action. Int J Sport NutrExerMetabol., in press. 22. Horie A, Tomita Y, Oshio K, Fujiwara D, Fujii T. (2019) Characterization of genomic DNA of lactic acid bacteria for activation of plasmacytoid dendritic cells. BMC Microbiol., in press. 23. Tsuji R, Fujii T, Nakamura Y, Yazawa K, Kanauchi O. (2019) Staphylococcus aureus epicutaneous infection is suppressed by Lactococcuslactisstrain Plasma via IL-17A elicitation. J Infect Dis., in press. 24. Sugimura T, Jounai K, OhshioK, Fujiwara D. (2019) Plasmacytoiddendritic cell dysfunction caused by heat stress is improved by administration of Lactococcuslactisstrain Plasma in mice. BiosciBiotecBiochem. , in press. 24 theses including 9 human clinical trials

25. Key opinion leaders who supports Lactococcus Plasma For its numerous evidences, top leaders in medical societies approve and praise the value of L. Plasma. Dr. K. Ouchi : Chief Professor of Pediatrics / Kawasaki Medical School Dr. N. Yamamoto : Honorary Professor of Molecular Virology/ Tokyo Medical and Dental University Dr. S. Iwata : President/ Japan Antibiotics Research Association Dr. S. Ohga: Chief Professor of Pediatrics/Kyushu University Dr. S. Fujimura : Professor / Tohoku Medical and Pharmaceutical University Dr. T. Matsumiya : Plastic and Reconstructive Surgeon / TengenjiSoraira Clinic Dr. Y. Ueda : Gynecological Surgeon / TengenjiSoraira Clinic Dr. T. Tanaka : Director of Pediatrics/ Shizuoka Welfare Hospital Dr. A. Hamada : Chief Professor of Traveler’s Medical Center/ Tokyo Medical School Dr. T. Masumoto : Chief Professor of Infectious Disease/ Intl Univ of Health and Welfare Dr. T. Shimizu : Chief Professor of Pediatrics/Juntendo University Dr. N. Yamamoto : Associate Professor of Infection Control/ Juntendo University Dr. K. Sato : Professor of Infection Disease / Miyazaki University Dr. K. Shimada : Associate Professor of Cardiology/Juntendo University Dr. K. Sakata : Professor of Hygiene and Preventive Medicine / Iwate Medical University Dr. J. Braun : Chair and Professor of Pathology and Medicine / David Geffen School of Medicine UCLA Dr. N. Ishi : Professor of Molecular Life Science / Tokai University School Dr. T. Oroshige : Professor of Molecular LifeScience / Tokai University School Dr. S. Abubaker : Professor of Tropical Infectious Disease / Malaya University Dr. T. Matano : Chair of AIDS research center /National Institue of Infectious Diseases Dr. S. Uematsu : Professor of mucosal Immunology / Chiba University Dr. T Shirasaka : Chair of HIV/AIDS / National Hospital Organization Osaka National Hospital Dr. Y. Matsuoka : Lecturer of Dermatology / Chiba University

26. Application of “Lactococcus Plasma” to consumer products • Killed dry powder has same activity as live bacteria form. • Powder is heat-resist, with no taste and no smell. Can be applied to various product formats Yogurt Drink Yogurt Soft Drink Gummies Supplements

27. Concluding remarks • To contribute to the health issues in the era of “ the 100 YEAR LIFE”,Kirin aims to strengthen its business opportunity • in the arena between pharmaceutical and food & beverage. • Immunity is one of the strategic approaches to address global social health issues, such as ageing and infectious diseases. • Kirin welcomes worldwide partners who can spread this benefit of Lactococcus Plasma across the world.