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Yakult’s future research into the beneficial health effects of and

AUTHORS ore than 80 years ago, Dr Minoru from individuals across age groups using Mitsuhisa Kawai, Kan Shida, Akira Kushiro Shirota, the founder of Yakult, YIF-SCAN® has shown that although the genus and Kouji Miyazaki M advocated two health-related Enterobacteriaceae was dominant immediately Correspondence: Mitsuhisa Kawai principles: ‘preventive medicine’, which after birth, in individuals over three years of age Yakult Central Institute aims to protect humans against diseases gut microbiota were dominated by obligate Yakult Honsha Co., Ltd. rather than treating symptoms; and ‘healthy anaerobes including . 5-11 Izumi, Kunitachi intestines for longevity’, which works to keep A human foetus has an axenic intestine, but Tokyo 186-8650 Japan the human intestine healthy throughout a variety of microbes begin to colonise the life. Using these principles, Yakult scientists gut shortly after birth. We asked the question: Email: [email protected] have been conducting research on intestinal What is the source of Bifidobacterium, the most and probiotics that are involved in dominant genus in infants? Our colleagues at homeostasis. Here we describe our efforts to Yakult Honsha European Research Centre for address the ultimate challenge: understanding Microbiology in Ghent, Belgium, found that the significance of gut microbiota in strains of bifidobacteria are transferred from maintaining human health and the results of the intestinal tracts of mothers to their babies. studies. To carry out the research, the team compared bifidobacteria isolated from the faeces of 12 antenatal mothers and from each newborn The symbiotic relationship using multilocus sequence typing (MLST). between humans and The researchers recorded identical strains intestinal bacteria of bifidobacteria from each mother–infant pair1. Some of the babies had the same strain Human lifespan and gut microbiota detected in their faeces from shortly after birth At the Yakult Central Institute in Tokyo, we until they were three months old, suggesting have been isolating and collecting that those strains were vertically transmitted bacteria and bifidobacteria and studying from the intestinal tracts of their mothers. them as part of our research and development Vertical transmission was not observed in into probiotics that are beneficial for human the five mother–infant pairs who had given health. Additionally, as part of our studies on birth by caesarean section, suggesting that intestinal bacteria, we have isolated, identified transfer of intestinal microbiota occurs when and numerated microbes by using the culture an infant passes through the birth canal2 method or YIF-SCAN® (Yakult Intestinal (Figure 1). Babies delivered by caesarean Flora Scan) analyses based on quantitative section exhibited delayed colonisation of polymerase chain reaction (PCR) and reverse bifidobacteria compared with babies who transcriptase polymerase chain reaction were spontaneously delivered. In babies (RT-PCR), characterising many types of intestinal born by caesarean, bifidobacteria began to bacteria. Because a considerable number of colonise the gastrointestinal tract one month microbes are difficult to cultivate, we have been after birth. Infants delivered by caesarean using 16S ribosomal RNA (rRNA) gene segments at the same hospital did not share identical analysis to comprehensively sequence specific strains of bifidobacteria; the origins of regions of 16S rRNA genes in all faecal microbes bifidobacteria in the caesarean section infants to accumulate data on intestinal bacteria. were not identified. The influence of bacterial For example, examining gut microbiota colonization of the gastrointestinal tract

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the intestinal tract, deploy immune cells in response to a variety of risks. Living systems also take advantage of stimuli from the external environment along the intestinal tract to ensure sustained function. We are researching how the immune system within the intestinal

tract is crucial for health maintenance, and SPONSOR FEATURE are working on the challenges of using the activities of intestinal bacteria and probiotics to modulate immune functions and ensure good health.

The relationship between gut microbiota and health The part played by gut microbiota in the Figure 1 | Mother-to-child transmission of intestinal bacteria (bifidobacteria).Among infants development and function of the immune born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant’s system is a field of study to which we have intestine shortly after birth. made a significant contribution. We realized that to analyse the functions of certain types on a child’s health is not yet known, and no fatty acid receptor GPR41. GPR41 activated the of intestinal bacteria, molecular biological consensus has been reached on the risks of a sympathetic nervous system and promoted approaches do not work and instead the child developing asthma or allergies following energy consumption in living systems, bacteria must be isolated and cultivated. a particular mode of delivery — these would while contrarily controlling a ketone body Using the intestinal tracts of mice and rats be areas for future research. β-hydroxybutyrate, which is synthesized in we succeeded in separately propagating I. B. Jeffery and colleagues at University the liver. In addition, they found that those and maintaining segmented filamentous College Cork, Ireland, reported that elderly fatty acids suppressed insulin signals through bacteria (SFB), which cannot be cultivated individuals had a decreased diversity of gut GPR43 expressed in adipose cells and played in vitro, and found that they are important in microbiota compared with younger adults, a role in inhibiting lipid accumulation. Thus, IgA production and in the development of and that those residing in care facilities SCFAs generated as by-products when intestinal intraepithelial lymphocytes6. SFB are or consuming a high-fat diet had specific intestinal bacteria convert a source involved in diverse types of important immune modules of gut microbiota3. In addition, into adenosine triphosphate (ATP) are not responses through inducing Th17 cells, a subset YIF-SCAN® confirmed that individuals aged only used as a nutrient source for intestinal of CD4-positive T cells secreting IL-17. We over 60 had fewer Bifidobacterium and more epithelial cells but also have systemic effects; have provided SFB to more than 40 research Enterobacteriaceae as they grew older. Further this suggests that intestinal bacteria are institutes in Europe, the United States and Asia, studies are needed to monitor intestinal important partners for living systems. as well as within Japan. bacteria in different people and to assess The part played by gut microbiota in Research on the relationship between gut microbiota in a cross section of age groups; maintaining human health has not been microbiota, immune function and health constructing a database of results should help defined. Intestinal bacteria were categorised as maintenance is now one of the most important to advance research in this field. either beneficial or harmful. But the view now subjects in the field of life sciences. In this is that the diversity and balance of microbiota context, much attention has also been paid The significance of intestinal bacteria are tightly linked to human health. Loss of to probiotics — specific bacteria that are In living systems such as human, mouse diversity and an imbalance of gut microbiota proactively delivered into the intestines. and rat, the beneficial nutritional effects of are deeply associated with obesity, allergies Researchers across the world use various intestinal bacteria include the production of and autoimmune diseases. A well-balanced strains of probiotics to conduct studies aimed essential amino acids, vitamins and short- existence of diverse intestinal microbes is more at the prevention and control of immunity- chain fatty acids (SCFAs). But research in important than a dominance of specific ones, associated diseases. casei the past three years has suggested that gut just as a good diet includes eating a broad, strain Shirota (LcS) is a probiotic strain that microbiota might have a greater impact on the well-balanced range of foods. has immunomodulatory functions and has health of living systems than had previously been consumed as a fermented drink been thought. since 1935. In particular, SCFAs, which are metabolites Intestinal functions and produced by intestinal bacteria, were found microbes Probiotics could assist the immune system to be directly involved in energy metabolism. Studies investigating the effects of LcS on I. Kimura and colleagues at Kyoto University, Probiotics and the immune system the immune system began with those on Japan4,5, showed that the SCFAs were used as Living systems have evolved sophisticated cancer prevention and infection protection an energy source for intestinal epithelial cells. immune systems to protect against disease and through activation of biophylactic functions, Some of the SCFAs were absorbed into the maintain homeostasis. Mucosal surfaces that whose impact was described in a research body and were recognized by the short-chain border the external environment, particularly article7 in 1981. Research on activation

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mechanisms of the innate immune system mediated by LcS went further, and the strain has been identified not only as an activator of Probio?cs biophylactic functions, but also as a potential

suppressor of excessive immune reactions that Phagocytes may also be effective against inflammatory SPONSOR FEATURE bowel disease, allergy and autoimmune Down-regulate disease. Research on the immunomodulatory Up-regulate Healthy effects of LcS is unusual in that its efficacy is not limited to specific diseases, but that it Immune response exerts diverse effects by restoring decreased immune functions and suppressing excessive Compromised Excessive immune reactions8. Ongoing research into the immunomodulatory functions of many other probiotics is conducted in discrete fields, such Figure 2 | Immunomodulatory functions expected from probiotics. Probiotics can help to modify as inflammatory bowel disease, allergy and the immune system’s homeostatic functions by either upregulating or downregulating reactions to infection protection. Primarily, we should not keep the immune system healthy. expect efficacy on particular diseases from probiotics consumed as food. Probiotics will have crucial roles in helping living systems Neuroendocrine system to cope with varied health risks by ensuring maintenance and recovery of normal immune Vulnerability Physical functions (Figure 2). The flexible effects of LcS ResilienceResilience Healthy symptoms on the immune system should be consistent with those views above. Risk factors Positive factors The extent to which LcS affects the immune system is not understood in detail, but the effects are thought to be attributed to their specific cell wall structures. Phagocytes Gut-brain interaction deployed throughout every tissue in the body, including the intestinal tract (which Microbiota have pattern-recognition receptors such as Toll-like receptors (TLRs)), rapidly capture Dysbiosis Synbiosis microbes invading the living system, recognize constructs presented on the microbes or newly Probiotics exposed peptidoglycan digests and nucleic acids that are digested within the phagocytes, and start adequate immune reactions. Figure 3 | Neuroendocrine-modulatory functions expected from probiotics. Probiotics directly or However, LcS has two types of neutral indirectly act on the brain through gut–brain interaction and maintain function of the neuroendocrine polysaccharides densely covering the cell system, thereby enhancing resilience and enabling responses to multiple risks (physical or wall surface that are not recognized by TLRs9. psychological stressors). These polysaccharides may not only conceal the potential TLR ligands of LcS, but may also Introducing the microbiota–gut–brain The effect of gut microbiota on the prevent their exposure by conferring tolerance interaction neuroendocrine system to enzymatic digestion that lyse cell wall The nervous, endocrine and immune systems Over the past ten years, research on the effects peptidoglycans in the phagosome in which it maintain homeostasis in complex living of gut microbiota on human physiological was captured10. At the same time, LcS keeps its systems. The concept of ‘gut–brain interaction’, functions has moved on from establishing three-dimensional cellular structures within which refers to the two-way communication links with physiological homeostasis, a phagocyte for some time; LcS stimulates between the brain and the intestinal tract, was including immunomodulation, to looking at the phagocyte by using its structure and the proposed in a primitive way in 1880s and has psychological connections involved in anxiety stimuli may, rather than induce a specific evolved to become the ‘microbiota–gut–brain or depression. unidirectional response to the phagocyte, interaction’. We assume that normalisation of Notions that gut microbiota have effects on enhance immune reactions to maintain gut microbiota might positively influence the the neuroendocrine system have been derived homeostasis. development and maturation of the nervous from basic research using germ-free mice. For We expect that efforts to explore the and endocrine systems, as well as those of the example, R. D. Heijtz and colleagues at the immunomodulatory functions of LcS will immune system, and are conducting research Karolinska Institute in Stockholm, Sweden11, enable us to determine the ideal probiotics to on the relationship between the intestine and showed that commensal microbiota were help the immune system. human health. important for the development of the cranial

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Gut Microbiota Database Clinical Applications group showed development and exacerbation of physical symptoms (abdominal and cold

Probiotics symptoms) accompanied by increase of stress markers, but the LcS-consuming group showed suppression of the onset of these

Accumulation of Human Gut Microbiota Data Intestine symptoms and had significantly suppressed - Healthy subject and patients salivary cortisol levels that otherwise elevated SPONSOR FEATURE Gut Microbiota Research 16 Basic and Clinical Research immediately before examinations . A basic Immune system study with stress model animals confirmed that intake of LcS reduced hypersecretion of a Homeostasis stress hormone under stressful conditions, and reduced stress reactivity in the brain16. Nervous system Endocrine system Thus, probiotics beneficial for humans pidemiologic study E should, by preventing disturbances of gut microbiota, maintain homeostasis of the Health Well-being & neuroendocrine and immune systems, enhance Longevity resilience inherent in living systems, and sustain healthy minds and bodies (Figure 3). However, Preventive medicine Whole the relationship between gut microbiota or Body A healthy intestinal tract probiotics and the neuroendocrine system is leads to a long life still not fully understood and further research is recommended. Figure 4 | Probiotic research to which the Yakult Central Institute is oriented. Daily intake of probiotics enhances the body’s natural healing ability and resilience, recovery from disturbed homeostasis, and helps to maintain health. Healthy intestines lead to longevity nervous system associated with motor control Imbalanced gut microbiota and anxiety behaviour. In another study, We are currently undertaking research into Research on intestinal bacteria for health N. Sudo and colleagues at Kyushu University whether imbalanced gut microbiota affects the Research into gut microbiota and probiotics in Fukuoka, Japan12, worked with germ-free health of the neuroendocrine system. We have has helped us to understand the close mice to show that commensal microbiota were conducted a large number of analytical studies relationship between healthy infants and involved in the development and maturation on gut microbiota and confirmed that LcS is bifidobacteria, and has led to the idea of of the hypothalamic–pituitary–adrenal axis. a beneficial probiotic that recovers disturbed harnessing bifidobacteria to maintain intestinal These studies suggest that gut microbiota gut microbiota. D. Benton and colleagues at health in infants and children. In addition, provide stimuli directly or indirectly from Swansea University in Wales14 asked healthy the development of techniques such as the the intestinal tract, and are deeply involved individuals to consume LcS-containing milk culture method and gnotobiotic animal in the development and maturation of the drinks for three weeks. The study reported that technology (which can produce animals cranial nervous and endocrine systems. Using the individuals who had impaired moods or containing a known strain of bacteria from model mice that display features of autism emotions at the start of the study reported an germ-free animals) to analyse gut microbiota spectrum disorder, E. Y. Hsiao of California improvement in mood by the end of the study. has helped to advance research into the effects Institute of Technology in the United States In another study, A. V. Rao of the University of probiotics on the intestinal environment. and colleagues13 found that disturbances in of Toronto, Canada, and colleagues15 asked The generation of strain-specific primers and gut microbiota were related to psychological people with chronic fatigue syndrome to monoclonal antibodies has enabled analysis of and physical symptoms. They reported that the consume LcS-containing milk drinks for eight intestinal and faecal probiotics at a strain level, model mice showed characteristic dysbioses, weeks and found that the number of faecal which has helped understand the mechanisms and that treatment with Bacteroides fragilis lactic acid bacteria and bifidobacteria increased of probiotic survival and in vivo dynamics. altered their gut microbiota and ameliorated and the participants’ anxiety symptoms were An important topic in basic gut microbiota autistic spectrum-like symptoms. alleviated. Several studies using stress animal research is the advance of 16S rRNA gene Gut microbiota seem to be important models have suggested that gut microbiota segments analysis techniques using next- in the development and maturation of the in the intestines would become imbalanced generation sequencers. Technical advances in cranial nerves and the endocrine system, and if placed under stress. In collaboration with 16S rRNA gene segments analysis targeting disturbances may have negative psychological researchers at Tokushima University in specific regions of 16S rRNA genes in all faecal effects. Research on the ways in which Japan we conducted a placebo-controlled, microbes have markedly developed gut- microbiota can affect neurodevelopmental double-blind study in which we asked healthy microbiota research and helped to evolve what disorders in humans is in its early stages. undergraduate medical students who were was once a niche field of research into a major At Yakult Central Institute we analyse gut revising for examinations to consume LcS- one. However, the analysis is suitable for major microbiota data from people with and without containing milk drinks for eight weeks prior microbes but not for minor microbes in faeces. neurodevelopmental disorders. to their exams. We found that the placebo Therefore, the combination of 16S rRNA gene

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segments analysis for all major microbes and taken in by intestinal antigen-presenting cells receptor GPR43. Nat. Commun. 4, 1829 (2013). YIF-SCAN® analysis for specific minor microbes to stimulate the immune system, but also act 6. Umesaki, Y. et al. Segmented filamentous will help to interpret data from research into on the centre of a living system, the brain, bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and gut microbiota and further our understanding through the neuroendocrine system using induce MHC class II molecules and fucosyl of the mechanisms of probiotics. unknown receptors in intestinal cells. The brain asialo GM1 glycolipids on the intestinal One subject that is dominating gut uses the neuroendocrine and immune systems epithelial cells in the ex-germ-free mouse. SPONSOR FEATURE microbiota clinical research is the development to help maintain homeostasis. In other words, Microbiol. Immunol. 39, 555–562 (1995). of faecal microbiota transplantation (FMT), probiotics act not on single sites, as drugs do, 7. Kato, I. et al. Antitumor activity of a treatment in which faeces taken from healthy but on the whole body through the immune Lactobacillus casei in mice. Gann. 72, 517–523 individuals are transplanted into patients and neuroendocrine systems from multiple (1981). who have gastrointestinal diseases. At the intestinal sites in a direct or indirect way. We 8. Shida, K. et al. Flexible cytokine production by macrophages and T cells in response to University of Amsterdam, E. van Nood and suggest a new hypothesis that daily intake probiotic bacteria. Gut Microbes. 2, 109–114 colleagues17 found that FMT was more effective of probiotics enhances the body’s natural (2011). against recurrent diarrhoea associated with healing ability and resilience, recovery from 9. Nagaoka, M. et al. Structure of Clostridium difficile than traditional treatments disturbed homeostasis and helps to maintain polysaccharide-peptidoglycan complex with antibacterial drugs. P. Moayyedi of health (Figure 4). from the cell wall of Lactobacillus casei YIT McMaster University in Hamilton, Canada, and To prove the hypothesis above, we need 9018. J. Biochem. 108, 568–571 (1990). colleagues18 reported that a placebo-controlled to accumulate additional evidence on 10. Shida, K. et al. Induction of interleukin-12 by study in people with ulcerative colitis showed the function of probiotics and compile a Lactobacillus strains having a rigid cell wall resistant to intracellular digestion. J. that FMT had a high remission rate. None of database of gut microbiota. To do so will Sci. 89, 3306–3317 (2006). the studies reported side effects caused by involve analysing the relationship between 11. Heijtz, R. D. et al. Normal gut microbiota FMT. Although implementing FMT as a therapy gut microbiota and probiotics with various modulates brain development and faces challenges, including those related clinical symptoms, short-term intervention behavior. Proc. Natl. Acad. Sci. U. S. A. 108(7), to safety, it is developing as a treatment for studies and long-term intervention studies. 3047–3052 (2011). intractable gastrointestinal diseases because Retrospective epidemiological studies have 12. Sudo, N. et al. Postnatal microbial of its high efficacy rates when compared to already reported the relationships between risk colonization programs the hypothalamic– conventional treatment. reduction of bladder19 or breast20 cancers and pituitary–adrenal system for stress response in mice. J. Physiol. 558(1), 263–275 (2004). In conclusion, we can say that basic and probiotic intake. 13. Hsiao, EY. et al. Microbiota Modulate clinical research into gut microbiota has Behavioral and Physiological Abnormalities made a huge contribution to human health One final thought Associated with Neurodevelopmental maintenance through probiotics. Disorders. Cell, 155(7), 1451–1463 (2013). We have worked on the relationship between 14. Benton, D. et al. Impact of consuming a The future of probiotics research the maintenance of human health, intestinal milk drink containing a probiotic on mood The history of probiotics research began with bacteria and probiotics. Our dedication to and cognition. Eur. J. Clin. Nutr. 61, 355–361 studies considering phylactic effects and how research will never change as long as Dr Minoru (2007). 15. Rao, A. V. et al. A randomized, double- to improve the intestinal environment and Shirota’s health-related principles are handed blind, placebo-controlled pilot study of includes work on LcS and on ameliorating down, just as genes are passed to offspring. a probiotic in emotional symptoms of symptoms of intestinal diseases, and chronic fatigue syndrome. Gut Pathogens 1:6 extended to research investigating properties REFERENCES doi:10.1186/1757-4749-1-6 (2009). such as cancer-prevention, anti-allergy, 1. Makino, H. et al. Transmission of intestinal 16. Takada, M. et al. Probiotic Lactobacillus casei anti-inflammatory and effects linked to Bifidobacterium longum subsp. longum strain Shirota relieves stress-associated mental health. stains from mother to infant, determined by symptoms by modulating the gut-brain multilocus sequencing typing and amplified Why do probiotics such as LcS exert such interaction in human and animal models. fragment length polymorphism. Appl. a variety of physiological effects? The answer Neurogastroenterol Motil., in press, doi: Environ. Microbiol. 77, 6788–6793 (2011). 10.1111/nmo.12804. (2016). lies in the intestine. The intestine functions to 2. Makino, H. et al. Mother-to-infant 17. van Nood, E. et al. Duodenal infusion of digest and absorb nutrients from food, absorb transmission of intestinal Bifidobacterial donor feces for recurrent Clostridium difficile. strains has an impact on the early water and generate faeces. It is also a protective N. Engl. J. Med. 368, 407–415 (2013). development of vaginally delivered infant’s barrier against bacteria and foreign bodies that 18. Moayyedi, P. et al. Fecal microbiota microbiota. PLoS ONE 8, e78331 (2013). have entered the body through the digestive transplantation induces remission in 3. Jeffery, IB. et al. Composition and temporal tract, and functions to exchange information stability of the gut microbiota in older patients with active ulcerative colitis in a with the brain (gut–brain interaction) — these persons. ISME J. 10, 170–182 (2016). randomized controlled trial. Gastroenterol. perhaps surprising functions of the intestine 4. Kimura, I. et al. Short-chain fatty acids and 149, 102–109 (2015). could exert a variety of physiological functions ketones directly regulate sympathetic 19. Ohashi, Y. et al. Habitual intake of lactic acid bacteria and risk reduction of bladder when combined with beneficial probiotics. nervous system via G protein-coupled receptor 41 (GPR41). Proc. Natl. Acad. Sci. U. S. A. cancer. Urol. Int. 68, 273–280 (2002). Probiotics could help to improve the 108(19), 8030–8035 (2011). 20. Toi M. et al. Probiotic beverage with soy environment within the intestines and enhance 5. Kimura, I. et al. The gut microbiota isoflavone consumption for breast cancer the activities of other organic acid-producing suppresses insulin-mediated fat prevention: a case-control study. Curr. Nutr. bacteria. Furthermore, they are not only directly accumulation via the short-chain fatty acid Food Sci. 9, 194–200 (2013).

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