ISSN 2436-7362

Vol. 51 No.1, 20211      The Fifty-fourth Annual Meeting ofThe Japanese Association of Germfree Life and Gnotobiology
January 21-22, 2021
TokyoPresident

Kazuhiro Hirayama

Zoom

2

無菌生物 J. germfree life gnotobiol.

Vol. 51 No.1, 2021

3

4

無菌生物 J. germfree life gnotobiol.

7梅﨑昌裕

 9 12141618 20

Vol. 51 No.1, 2021

5 00 00

Listeria monocytogenes

Lactobacilllus casei

Clostridium perfringens

 00・00・ 00

6

無菌生物 J. germfree life gnotobiol.

・・・・・・・・・00

Campylobacter jejuni
市川拓磨，平山和宏

・・・・・・・・・・・・・・・・・・ 00（東京大学農学部獣医公衆衛生学教室）

・・・・・・・・・・・・・・・・・・・・・・・・・・00 ・・・・・・・・・・・・・・・・・・・・・・・・・・・00

・・・・・・・・・・・・・・・・・・・00

Vol. 51 No.1, 2021

7

8

無菌生物 J. germfree life gnotobiol.

Low protein adaptation among Papua New Guinea Highlanders
MASAHIRO UMEZAKI

Department of Human Ecology, Graduate School of Medicine, The University of Tokyo, Tokyo
The inconsistency between Papua New Guinea Highlanders' protein-deficient diet and theirmuscular physique is well known. Moreover, although their protein intake is less than biologicallyadequate, protein-deficiency related disorders have rarely been reported in this setting. We havespeculated that the populace have adapted to a low-protein diet by utilizing air nitrogen fixed byintestinal bacteria and by intensive "recycling" of urea that passes into the gastrointestinal tract.
The recent emergence of genome science has enabled us to examine the unsolved question of
Papua New Guinea Highlanders' adaptation to a low protein diet in more direct way. In our project,
we evaluated gut microbiota for healthy individuals in four Papua New Guinea communities
subject to different degrees of protein deficiency. Association of gut microbiota and variation ofprotein intake and nutritional biomarkers were investigated. In addition, fecal samples ofindividuals from one of least modernized communities in Papua New Guinea Highlands weretransplanted into germ-free mice, which were then fed with low protein diet. I will present andoverview of the project as well as preliminary findings from the analysis.

Vol. 51 No.1, 2021

9,,,,

IAAO,,,,

de novo,
IAAO,,SPF

IAAO
1,SPF 16-18,
BALB/c,,3, 6, 12,18%,

L-[1-13C] phenylalanine
13CO,SPF

2, 3%, SPF

2,13CO

2,3, 4

SPF

,,,,,
IAAO

,
IAAO
IAAO
,,

10

無菌生物 J. germfree life gnotobiol.

Effect of intestinal bacteria on host amino acid metabolism
YOSHIE SAITO
Co-Creation Center, Meiji Holdings Co., Ltd.,Tokyo
Gut microbiota have diverse metabolic activities and play an important role in host nutrition.
Regarding amino acid metabolism, gut microbiota provide amino acids to the intestinal tract
through degradation of undigested proteins, as well as de novo synthesis of some amino acid. On
the other hand, intestinal bacteria utilize substantial amounts of amino acids for the synthesis of
their cellular components and as an energy source. Thus, amino acid metabolism by gut bacteria
may have an influence on amino acid homeostasis in the host. Indeed, it has been reported thatgerm-free mice have a different amino acid profile in the gut and blood compared to SPF miceand that perturbations in microbial amino acid metabolism are associated with malnutrition in thehost. The effects of microbial amino acid metabolism on the host are complex, and it has been
unclear how they affect the amino acid requirements of the host. To elucidate this, we applied theindicator amino acid oxidation (IAAO) method in germ-free mice. The IAAO method can estimate
the amount of amino acid pool in the body and protein metabolism by measuring the surplus ofingested indicator amino acids, which are not used for protein synthesis and are oxidized andexcreted as carbon dioxide in the breath. In this presentation, we will focus on the results of theIAAO method in germ-free mice.

Vol.51 No.1, 2021

11References
1. NEIS, PJ., G., EVELIEN, CORNELIS, HC., DEJONG, & SANDER, S., RENSEN. :The role of microbial amino acid metabolism in host metabolism”. Nutrients, 7(4), 29302946, 2015.
2. MISHIMA, E., FUKUDA, S., MUKAWA, C., YURI, A., KANEMITSU, Y., MATSUMOTO,Y., AKIYAMA, Y., FUKUDA, N., N., TSUKAMOTO, H., ASAJI K., SHIMA, H., KIKUCHI,K., SUZUKI, C., SUZUKI, T., TOMIOKA, Y., SOGA, T., ITO, S. & ABE, T. : "Evaluationof the impact of gut microbiota on uremic solute accumulation by a CE-TOFMS–based
metabolomics approach." Kidney International, 92 (3), 634-645, 2017.
3. SMITH, MI., YATSUNENKO, T., MANARY, MJ., TREHAN, I., MKAKOSYA, R.,
CHENG, J., KAU, AL., RICH, SS., Concannon, P., Mychaleckyj, JC., Liu, J., Houpt, E., Li,JV., Holmes, E., Nicholson, J., Knights, D., Ursell, LK., Knight, R., Gordon, JI. : "Gutmicrobiomes of Malawian twin pairs discordant for kwashiorkor." Science, 339, 6119, 548554, 2013.
4.

KUMAR, M., JI, B., BABAEI, P., DAS, P., LAPPA, D., RAMAKRISHNAN, G., FOX, TE.,HAQUE, R., PETRI, WA., BÄCKHED F., & NIELSEN, J. : "Gut microbiota dysbiosis isassociated with malnutrition andreduced plasma amino acid levels: lessons from genomescale metabolic modeling." Metabolic Engineering, 49, 128-142, 2018.

12

無菌生物 J. germfree life gnotobiol.

BK-4
PXR
K-4

K-4K-4K-4-cAMP

KcAMP-44-4-4-4

Vol.51 No.1, 2021

Nutritional research using germfree animals – vitamins
HITOSHI SHIRAKAWA YUSUKE OHSAKI, YOSHIHIKO MINEGISHI andMICHIO KOMAI
Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai
Numerous gut microorganisms synthesize several vitamins including B-group vitamins andvitamin K. Produced vitamins are absorbed by the host and contribute to a part of host's nutritional
requirement. Vitamin deficient model using germfree animals has great advantage compared tothe model induced by the administration of antibiotics and metabolic antagonists because druginduced models cannot exclude off target effects completely. For clarifying novel functions ofvitamin K, we developed vitamin K deficient model using germfree animal. In this paper, weintroduce novel function of vitamin K revealed in this model. Vitamin K is one of the fat-soluble
vitamins and acts as a cofactor for -glutamyl carboxylase, which catalyzes posttranslationalmodification (called Gla formation) of blood coagulation factors and bone proteins. There are twotypes of naturally occurring vitamin K, phylloquinone derived from plants and menaquinones
mainly produced by microorganisms. Menaquinones have isoprene units of various lengths in theirside-chain structures. Large amount of menaquinone-4, which has four isoprene units, is observed
not only in the liver and bone, but also in the pancreas, brain, and testis. Menaquinone-4 in thesetissues is endogenously synthesized from dietary phylloquinone and microbial-derived
menaquinones, and has unique functions other than Gla formation. We explored further functionsof menaquinone-4 using germfree rats fed vitamin K deficient or supplemented diets. In testis, wefound mRNA level of steroidogenic gene and testosterone level were reduced under vitamin K
deficient state. Further analysis indicated that menaquinone-4 can stimulate adenylate cyclase andactivate downstream pathway toward testosterone production. This novel property of
menaquinone-4 could contribute to prevent age-related diseases and further prolong healthy life
expectancy.

1314

無菌生物 J. germfree life gnotobiol.

(multiple sclerosis(Takewaki et al., PNAS. 2020).
T,,,,.

.

,,198040,20,,,MS.

.

,, MS

unmet medical needs”,RRMS
MS (secondary progressive MS,, SPMS.

,,,.

,MS,.

,.

, SPMS

MS,(,,)DNA,.

MS (relapsing remittingMS,Eubacterium,,pathwa.

Vol. 51 No.1, 2021

Gut microbiome research in patients with multiple sclerosis
DAIKI TAKEWAKI
Department of Immunology, National Center of Neurology and Psychiatry, Tokyo
The number of patients with multiple sclerosis (MS) in Japan has been rapidly increasing fordecades. This may be linked with westernization in lifestyle and subsequent alterations of the gut
microbiome. Notably, recent achievements established the bidirectional causality between gut
microbiomes and development of MS. As the next step, many researchers try to overcome "unmet
medical needs" of MS through the gut microbiome. MS generally starts as relapsing remitting MS(RRMS), but often shifts into secondary progressive MS (SPMS). SPMS represents a moreadvanced stage of MS, characterized by accumulating disabilities and refractoriness tomedications. Recently, we revealed the characteristics of gut microbiomes associated with each ofRRMS and SPMS based on the microbial composition, metagenomic functional, and metaboliteanalyses (Takewaki et al., PNAS. 2020). In microbial composition analysis, we identified specificbacteria whose abundance was significantly increased in SPMS compared with that in RRMS andpositively correlated with clinical severity score of the patients. Next, we have isolated thisspecific species from the fecal sample of a SPMS patient and are doing in vivo analysis to verify
the functional significance of this species. In metagenomic functional analysis, we revealed anenhancement in microbial DNA mismatch repair in SPMS, which was consistent with excessive
fecal oxidation shown in sulfur metabolomic analysis. In this seminar, I will talk about thebackground of MS microbiome research and significance of the obtained results and discuss thefuture therapy for intractable neurological diseases focusing on the gut microbiome.

15

16

無菌生物 J. germfree life gnotobiol.

Vol. 51 No.1, 2021

17

18

無菌生物 J. germfree life gnotobiol.

1990

100
100 200600MPa

400500 MPa

MPa

Vol. 51 No.1, 2021

KAZUTAKA YAMAMOTO and YOSHIKO NAKAURA

Conventionally, food has been processed by thermal treatments such as heating and cooling.
Since 1990, treatment with high hydrostatic pressure (HHP) ranging from 100 to 600 MPa has
been applied to food processing as a nonthermal process. Since HHP processing suppresses
chemical reactions in food, flavor, color, and nutrients can be maximally retained. In addition,
HHP induces physical changes such as protein denaturation and thus it lethally or sublethally
inactivates pathogenic and/or spoilage microbes, particularly bacteria. Therefore, HHP foodprocessing has attracted great attention of food technologists for producing high quality safe foods.
Safety of HHP-processed foods is the highest priority in the food industry, and thus HHP-induced
microbial inactivation has been studied intensively. This paper will overview HHP inactivation ofmicrobes with focuses on HHP-induced injury of bacteria and subsequent recovery as well as
suicidal germination induced to bacterial spores by HHP-heating combination.

19

20

無菌生物 J. germfree life gnotobiol.

Vol. 51 No.1, 2021

21

22

無菌生物 J. germfree life gnotobiol.

Vol. 51 No.1, 2021

23

24

無菌生物 J. germfree life gnotobiol.

Vol. 51 No.1, 2021

25

26

無菌生物 J. germfree life gnotobiol.

Kazuhiro Hirayama

2

Low protein adaptation among Papua New Guinea Highlanders
Masahiro Umezaki

7

Effect of intestinal bacteria on host amino acid metabolism
Yoshie Saito

9

Nutritional research using germfree animals – Vitamin
Hitoshi Shirakawa et al.

12

Gut microbiome research in patients with multiple sclerosis
Daiki Takewaki

14

Study of food and physiological functions using germ-free animals and management of the experimental facility
Akira Hosono

16Microbial inactivation by high hydrostatic pressure treatment
Kazutaka Yamamoto

18

Next-generation sericulture and germ-free silkworm
Takashi Kiuchi

20

Guideline for authors

22