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Archive Highlight: The role of microbes in gut-brain communication, with Prof. Emeran Mayer MD

/in Podcast, Season Three /by Laura

 

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Continuing our series on the microbiota-gut-brain axis, we are highlighting Episode 26 from our archives. In this episode, ISAPP podcast host Prof. Dan Tancredi PhD welcomes guest Prof. Emeran Mayer MD, a gastroenterologist and researcher at University of California Los Angeles. They talk about the microbiota-gut-brain axis, covering its evolutionary origins and how this complex system works in the human body to support overall health.

Key topics from this episode:

  • Microbiota-gut-brain communication has a long evolutionary history: microbes have been around for billions of years and they stored a lot of information in their genes. At some point in evolution microbes got inside the digestive tube of a primitive marine animal called hydra and it proved advantageous for this animal.
  • The hydra shows the origin of the human enteric nervous system (ENS): microbes live inside this tube and transfer genes to the nerve cells of this digestive tube, showing the origin of neurotransmitters.
  • Today in humans the neurotransmitters influence gene expression of microbes and change the microbial behaviors; the metabolites produced feed back to the brain.
  • Prof. Mayer’s initial interest as a gastroenterologist was the ENS and how it regulates motility. Subsequently the ENS was found to regulate many gut functions. The gut also houses a large part of the immune system and a complex hormonal system, and all these systems are connected with each other and communicate with the brain.
  • There is an increasing understanding that many chronic diseases relate to Inappropriate engagement of the immune system, starting in the gut.
  • When Prof. Mayer started in the field, the term “gut health” did not exist. Now it’s a ubiquitous term which has associations with wellbeing, acknowledging the gut has influence on many other body systems.
  • The associations between gut (microbiota) and brain health started with provocative animal experiments from Cork, Ireland, in which researchers manipulated the gut microbiome and found changes in emotion-like behaviors of animals. However, it has been difficult to translate to human interventions.
  • How do microbiome-targeted dietary interventions affect the brain? We do know the “Standard American Diet” (deficient in fiber) has changed the gut microbes in a way that compromises the production and maintenance of the gut barrier. 
  • There are many misconceptions about “leaky gut”, but basically contact between beneficial microbes and immune system sensors stimulate the immune system of the gut to low-grade inflammation. This can alter the tight junctions, making the gut more permeable, and ultimately this can affect the brain. Diet can affect the role of microbes in maintaining an effective gut barrier.
  • Prof. Mayer describes how he ended up studying the microbiota-gut-brain axis – he would not have predicted how important and popular this field would become.
  • In the future, there will be more sophisticated and personalized interventions. He sees a paradigm shift happening from reductionist approaches in medicine to systems biological approaches. This field is making us acknowledge that diet will play a major role.

Episode links:

About Prof. Emeran Mayer MD:

Emeran A Mayer is a Gastroenterologist, Neuroscientist and Distinguished Research Professor in the Department of Medicine at the David Geffen School of Medicine at UCLA, the Executive Director of the G. Oppenheimer Center for Neurobiology of Stress & Resilience and Founding Director of the Goodman Luskin Microbiome Center at UCLA. He is one of the pioneers and leading researchers in the bidirectional communication within the brain gut microbiome system with wide-ranging applications in intestinal and brain disorders. He has published 415 scientific papers, co edited 3 books and has an h-index of 125. He published the best selling books The Mind Gut Connection in 2016, the Gut Immune Connection in June 2021, and the recipe book Interconnected Plates in 2023. He is currently working on a MasterClass and a PBS documentary about the mind gut immune connection. He is the recipient of numerous awards, including the 2016 David McLean award from the American Psychosomatic Society and the 2017 Ismar Boas Medal from the German Society of Gastroenterology and Metabolic Disease.

Postbiotics: A global perspective on regulatory progress

/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola PhD, CONICET, National University of Litoral, Argentina

While the conceptualisation of postbiotics varies among scientists, some recent actions may suggest that regulatory agencies around the world are starting to align with the ISAPP definition (Salminen et al. 2021), understanding postbiotics as preparations of inanimate microorganisms able to confer a health benefit.

Before the May 2021 publication of the postbiotic consensus definition by an expert panel convened by ISAPP, a search in www.pubmed.com using the term postbiotics rendered around 320 entries in the period 1975-2021. Three years after the ISAPP publication, by August 2024, almost 1200 entries could be found. However, it is still to be examined how many of these entries use the term postbiotics to refer to (1) administered metabolites, (2) metabolites produced by the gut microbiota or (3) inanimate microbial preparations, the three most prevalent conceptualizations of the term. A future bibliometric analysis of the literature could be performed to shed light on this. Meanwhile outside academia, the discrepancy in postbiotic definitions continues: some companies market specific metabolites as postbiotics whereas other companies use the term postbiotics to refer to heat-inactivated lactobacilli.

The first movement I noticed towards potential regulatory adoption of the term was made by Health Canada, as suggested in a presentation by an officer at Probiota 2023 in Chicago last year. The presentation shared the ISAPP definition, stating that postbiotics would fall under the Natural and Non-Prescription Health Products Directorate (NNHPD), that some probiotic specifications may apply (strain specification, antibiotic resistance, etc,), and that quantification, in principle would be based on milligrams, expecting that more sophisticated and accurate methodologies would arise over time. This issue was addressed further in a Discussion Group in the recent ISAPP meeting at Cork (Ireland) – see the annual meeting report here. Presently, there is only one entry for the word postbiotics in the Health Canada webpage, where it is stated that “gut modifiers as livestock feed are products that, once fed, have a mode of action in the gastrointestinal tract of an animal. The gut modifier category can encompass a variety of feed ingredients, these ingredient types may include, but are not limited to viable microbial strains, prebiotics, postbiotics, enzymes, organic acids and essential oils”. However, no further indications of the meaning of the term postbiotic are stated on the website.

In January 2024, the trade journal Nutraingredients announced that the China Nutrition and Health Food Association (CNHFA) had decided to draft industry standards for quantifying postbiotics or inactivated cells and were rallying industry players and the public to take part in the draft process through a public consultation. The National Institutes for Food and Drug Control (NIFDC) was leading the process and it had drafted flow cytometry standards to measure postbiotics composed of inactivated cells of lactic acid bacteria. In addition, a fluorescent quantitative PCR detection method had been drafted for inactivated Bifidobacterium lactis cultures. In correspondence with the NIFDC, it was discussed that a direct counting method using a standard microscope for single culture postbiotics was being explored.

The TGA (Therapeutic Goods Administration) is the Australian body that regulates medicines, medical devices and biologicals. The TGA recently published a guidance to provide information for applications relating to microorganisms as active ingredients for use as new substances in listed medicines (the category which includes the majority of dietary supplements marketed in Australia), or as active ingredients in registered complementary medicines (RCM). Listed medicines and RCM containing microorganisms as active ingredients are generally referred to as probiotics or postbiotics. For the purpose of this guidance, microorganisms are whole and intact cells of bacteria and fungi (including yeasts) that are live or non-viable. This guidance is intended for the premarket assessment of new live and whole/intact non-viable microorganisms potentially used as probiotics and postbiotics. Interestingly, the guidance does not include cell fragments, which have different pharmacokinetics within the gut. It is worth noting that Australia is part of the ACCESS Consortium, consisting of Australia’s TGA, Health Canada, the UK’s MHRA, Swissmedic from Switzerland and Singapore’s Health Sciences Authority. However, it’s not yet known whether the ACCESS Consortium will take inspiration from the Australian guidance.

Which scientific publications may be influencing these regulatory directions? At the beginning of this blog I discussed the possibility of conducting a bibliometric analysis of the literature in order to find out how the term postbiotic has been used so far in relation to the different conceptualizations it may have. Surprisingly to me, a bibliometric analysis was published as a preprint last February at www.preprint.org and entitled “Who is qualified to write a review on postbiotics? A bibliometric analysis”. Authors indicated that between November 2021 and December 2023, 76 review articles were published on postbiotics, with a mean of almost 3 reviews per month. Authors concluded that a portion of this collection of work was written by first authors with no previous engagement with related research and lacking colleagues or mentors involved with microbiome/probiotics research to support them as senior authors. Our article “The Concept of Postbiotics”, in collaboration with Dr. Mary Ellen Sanders PhD and Prof. Seppo Salminen PhD ranks in third place among the top 10 publications according to the number of citations received.

While the academic and scientific sphere still debate the proper meaning of the term postbiotics, it seems the regulatory landscape for postbiotics is progressing to consider them to be preparations of inanimate microorganisms able to confer a health benefit, as proposed by ISAPP.

Episode 35: Investigating gut microbiome links to chronic diseases, with Dr. Purna Kashyap MBBS

/in Podcast, Season Three /by Laura

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In this episode, the ISAPP hosts discuss the gut microbiome’s role in chronic diseases with Dr. Purna Kashyap MBBS, from Mayo Clinic in Rochester, Minnesota, USA. Dr. Kashyap talks about how to discover the complex factors that trigger and perpetuate chronic diseases such as inflammatory bowel disease, zeroing in on the gut microbiome as a contributor to different aspects of gastrointestinal (GI) tract physiology.

Key topics from this episode:

  • Dr. Kashyap became interested in some of the initial studies linking the gut microbiome to chronic diseases around 2007-2008, and subsequently began to study the molecular mechanisms that underlie changes in GI tract physiology.
  • How can scientists figure out causality in chronic diseases and the role of gut microbes? Dr. Kashyap sees causality as an ongoing cascade of events in the GI tract, with no single causal factor. Both the initial triggers and the perpetuating factors can be considered part of what causes these diseases.
  • Microbes can help perpetuate a certain state in the host because once they establish themselves they serve to make the environment more conducive to their survival. In chronic diseases, the factor that triggers the microbial community configuration may not be as important as the factor(s) that perpetuate it on an ongoing basis.
  • The gut microbiome is changeable but not easy to change. Scientists need to know how the microbial community sustains itself and intervene there to change the community.
  • Even small microbiome studies can be informative if you look at who responds to the intervention and why. This information can be valuable for informing which treatments might work for which subgroups of people.
  • Dr. Kashyap encourages combining three types of research: large-scale studies on microbial metabolites and potential drug targets; clinical studies on the metabolites present in various subgroups; preclinical models studying the effects of individual metabolites.
  • Diet, microbes, and host uptake all contribute to the physiological effects of different metabolites. And for example, if a metabolite is low, knowing which microbes are present is not enough information to explain why it’s low.
  • In gastroenterology, clinicians primarily care about the gut microbiome in relation to the new treatments it makes possible. Now that FDA-approved treatments exist (standardized fecal microbiota transplants for recurrent C. difficile), clinicians may start paying more attention.
  • Does Dr. Kashyap recommend interventions to patients based on their gut microbiomes? A high-fiber diet is good for the gut microbiome and also for overall health, so he advises patients to adhere to dietary recommendations for their daily fiber intake.

Episode abbreviations and links:

Additional resources:

Why researchers need to understand more about the small intestinal microbiome. ISAPP blog.

About Dr. Purna Kashyap:

Dr. Purna Kashyap is practicing gastroenterologist and Professor of Medicine and Physiology, the Bernard and Edith Waterman Director of the Microbiome program, and Director of the germ-free mouse facility in the Center for Individualized Medicine at Mayo Clinic, Rochester, MN. The NIH funded Gut Microbiome laboratory led by Dr. Kashyap is focused on delineating the complex interactions between diet, gut microbiome, and host gastrointestinal physiology.  The laboratory uses germ-free mouse models in conjunction with measures of gastrointestinal physiology in vitro and in vivo to investigate effects of gut microbial products on host gastrointestinal function. In parallel, they use a systems approach incorporating multi-omics, patient metadata, and physiologic tissue responses in human studies, to aid in discovery of novel microbial drivers of disease. The overall goal of the program is to develop novel microbiota-targeted therapies. Dr. Kashyap has published nearly 100 peer reviewed articles including journals like Cell, Cell Host Microbe, Science Translational Medicine, Nature Communications, and Gastroenterology. He was inducted to American Society of Clinical Investigation in 2021. He has previously served on the scientific advisory board of American Gastroenterology Association Gut Microbiome Center, and on the council of American Neurogastroenterology and Motility Society. He now serves on the council and the research committee of AGA, in an editorial role for Gut Microbes and as an ad hoc reviewer on NIH study sections.

Clarifying the role of metabolites in the postbiotic definition

/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola PhD, Instituto de Lactología Industrial (CONICET-UNL), Faculty of Chemical Engineering, National University of Litoral, Santa Fe, Argentina and and Prof. Colin Hill PhD, School of Microbiology and APC Microbiome Ireland, University College Cork, Cork, Ireland

ISAPP published a definition for the term postbiotics in 2021 that states that “a postbiotic is a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host” (Salminen et al., 2021). This 19-word definition had to distill the content of the accompanying article that ran to over 9,000 words (not including references) and so obviously a lot of nuance was lost. A reading of the full paper should dispel any misconceptions, but we thought it might be timely to discuss what is perhaps the most common misunderstanding.

Some of the previous definitions included metabolites (purified or semi-purified) under the postbiotic concept. We did not agree with this interpretation. For us, the term postbiotics refers to preparations that consist largely of intact microbial cells, or preparations that retain some or all of the microbial biomass contained in microbial cells. This latter concept was captured in the phrase “and/or their components” The first column of page 3 of Salminen et al., 2021 elaborates on this; “The word ‘components’ was included because intact microorganisms might not be required for health effects, and any effects might be mediated by microbial cell components, including pili, cell wall components or other structures. The presence of microbial metabolites or end products of growth on the specified matrix produced during growth and/or fermentation is also anticipated in some postbiotic preparations, although the definition would not include substantially purified metabolites in the absence of cellular biomass. Such purified molecules should instead be named using existing, clear chemical nomenclature, for example, butyric acid or lactic acid”. So, taken in context, the scope of the ISAPP definition covers inanimate, dead, non-viable microbes; either as intact whole dead cells or in the form of “their components”. We do not consider microbial metabolites to be postbiotics. Such an interpretation would, for example, make butyrate or other end-products of fermentation postbiotics (once shown to have a health benefit). The ISAPP definition does not exclude the likelihood that microbial metabolites will be present in a postbiotic preparation, but it does require that dead microbes or microbial cell fragments or structures should be present to qualify as a postbiotic.

Why did the ISAPP definition exclude purified or semi-purified metabolites in the absence of cellular components? We fully accept that metabolites or other microbe-generated functional ingredients such as lactate, butyrate, bacteriocins, defensins, neurotransmitters, and similar compounds can be present in a postbiotic preparation. But as you can see from this list, these compounds already have names that are clearly understood. The ISAPP definition of postbiotics focuses on the beneficial role of inanimate microbes and/or their components, a category that did not have a clear definition. Postbiotics are simply one category of substances that provide microbe-associated health benefits. In terms of semantics, dictionaries define the prefix ‘post’ as meaning ‘after’ and the word ‘biotic’ as meaning ‘living things’, and so a postbiotic in that context is something that was living and is now after-life, or inanimate. Metabolites are derived from living things, but never had an independent ‘life’ of their own. As a thought experiment, let us imagine a microbe that has been shown to have a health benefit and therefore qualifies as a probiotic. If the same microbe is inactivated and continues to show a health benefit, this new formulation is no longer a probiotic and qualifies as a postbiotic. If this postbiotic preparation can be further purified and it is shown that a metabolite or metabolites in the absence of cells or their components can provide the same health benefit it ceases to be a postbiotic and becomes a health-promoting metabolite. We could imagine microbially-produced vitamins as an example.

Ideally, definitions should be clear without supplemental explanation. But short, simply worded definitions that describe complex concepts must be read in a context. There is a background, they have a scope, there are things that are covered by that definition and things that are not, and of course definitions have their limitations. It would be hard, if not impossible, to include the scope, the background, the coverage and the limitations in a 19-word definition. For instance, the 15-word probiotic definition is “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al, 2014). This does not include the idea that probiotics are strain-dependent, a fact that is widely accepted by the field. Other criteria for probiotics not stated in the definition include the fact that that they may be of any regulatory category, that their health benefits must be demonstrated in well-controlled trials in the target host, and that they must be safe (Binda et al. 2020).

In closing, we believe that the postbiotic concept can be an incredibly important scientific, regulatory and commercial concept. That is why we spent the time and effort to arrive at what we hope is a workable definition. We accept that the definition is not perfect but we do think it is useful, and we urge those interested in the future of this important field to read the accompanying paper carefully and to place the definition in its proper context.

See ISAPP’s Postbiotics infographic here.

 

Episode 26: The role of microbes in gut-brain communication

/in Podcast, Season Two /by Laura

 

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The Science, Microbes & Health Podcast 

This podcast covers emerging topics and challenges in the science of probiotics, prebiotics, synbiotics, postbiotics and fermented foods. This is the podcast of The International Scientific Association for Probiotics and Prebiotics (ISAPP), a nonprofit scientific organization dedicated to advancing the science of these fields.

The role of microbes in gut-brain communication, with Prof. Emeran Mayer MD

Episode summary:

In this episode, ISAPP podcast host Prof. Dan Tancredi PhD welcomes guest Prof. Emeran Mayer MD, a gastroenterologist and researcher at University of California Los Angeles. They talk about the microbiota-gut-brain axis, covering its evolutionary origins and how this complex system works in the human body to support overall health.

Key topics from this episode:

  • Microbiota-gut-brain communication has a long evolutionary history: microbes have been around for billions of years and they stored a lot of information in their genes. At some point in evolution microbes got inside the digestive tube of a primitive marine animal called hydra and it proved advantageous for this animal.
  • The hydra shows the origin of the human enteric nervous system (ENS): microbes live inside this tube and transfer genes to the nerve cells of this digestive tube, showing the origin of neurotransmitters.
  • Today in humans the neurotransmitters influence gene expression of microbes and change the microbial behaviors; the metabolites produced feed back to the brain.
  • Prof. Mayer’s initial interest as a gastroenterologist was the ENS and how it regulates motility. Subsequently the ENS was found to regulate many gut functions. The gut also houses a large part of the immune system and a complex hormonal system, and all these systems are connected with each other and communicate with the brain.
  • There is an increasing understanding that many chronic diseases relate to Inappropriate engagement of the immune system, starting in the gut.
  • When Prof. Mayer started in the field, the term “gut health” did not exist. Now it’s a ubiquitous term which has associations with wellbeing, acknowledging the gut has influence on many other body systems.
  • The associations between gut (microbiota) and brain health started with provocative animal experiments from Cork, Ireland, in which researchers manipulated the gut microbiome and found changes in emotion-like behaviors of animals. However, it has been difficult to translate to human interventions.
  • How do microbiome-targeted dietary interventions affect the brain? We do know the “Standard American Diet” (deficient in fiber) has changed the gut microbes in a way that compromises the production and maintenance of the gut barrier. 
  • There are many misconceptions about “leaky gut”, but basically contact between beneficial microbes and immune system sensors stimulate the immune system of the gut to low-grade inflammation. This can alter the tight junctions, making the gut more permeable, and ultimately this can affect the brain. Diet can affect the role of microbes in maintaining an effective gut barrier.
  • Prof. Mayer describes how he ended up studying the microbiota-gut-brain axis – he would not have predicted how important and popular this field would become.
  • In the future, there will be more sophisticated and personalized interventions. He sees a paradigm shift happening from reductionist approaches in medicine to systems biological approaches. This field is making us acknowledge that diet will play a major role.

Episode links:

About Prof. Emeran Mayer MD:

Emeran A Mayer is a Gastroenterologist, Neuroscientist and Distinguished Research Professor in the Department of Medicine at the David Geffen School of Medicine at UCLA, the Executive Director of the G. Oppenheimer Center for Neurobiology of Stress & Resilience and Founding Director of the Goodman Luskin Microbiome Center at UCLA. He is one of the pioneers and leading researchers in the bidirectional communication within the brain gut microbiome system with wide-ranging applications in intestinal and brain disorders. He has published 415 scientific papers, co edited 3 books and has an h-index of 125. He published the best selling books The Mind Gut Connection in 2016, the Gut Immune Connection in June 2021, and the recipe book Interconnected Plates in 2023. He is currently working on a MasterClass and a PBS documentary about the mind gut immune connection. He is the recipient of numerous awards, including the 2016 David McLean award from the American Psychosomatic Society and the 2017 Ismar Boas Medal from the German Society of Gastroenterology and Metabolic Disease.

Postbiotics: debate continues and the ISAPP definition gains support

/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola PhD, Instituto de Lactología Industrial (CONICET-UNL), Santa Fe, Argentina

The publication of a new definition for the term “postbiotics” by ISAPP in 2021 (Salminen et al., 2021a) spurred discussion on a variety of platforms, including scientific journals, social media and in-person debates organized at industry and scientific meetings. A couple of months after the publication of the definition, a group of scientists expressed their disagreement about the new definition (Aguilar-Toalá et al., 2021), and this was followed by a reply in support of the ISAPP definition (Salminen et al., 2021b). An example of the debate on social media is reflected in this post on LinkedIn. The comments that followed the post highlighted points of disagreement and misunderstandings about the ISAPP definition. These reactions were helpful to me in preparing for panels and debates scheduled at 2023 meetings in Amsterdam, Chicago and Bratislava, discussed more fully below.

Prior to the ISAPP panel, many terms were used to refer to non-viable microorganisms that confer a health benefit when administered in adequate amounts: heat-killed probiotics, heat-treated probiotics, heat-inactivated probiotics, tyndallized probiotics, ghost-probiotics, non-viable probiotics, paraprobiotics, cell fragments, cell lysates or postbiotics. ISAPP proposed that going forward, the single term “postbiotic” be used in scientific communications, marketing, regulatory frameworks and to counter the difficulty in tracking of papers for comprehensive systematic reviews. ISAPP’s goal was to bring focus and clarity to the term postbiotic, provide criteria for proper use of the term and set the stage for future innovation in the field.

Two competing terms

When considering preparations of non-viable microorganisms that confer a health benefit, two terms seem to have emerged most dominantly:

The term paraprobiotic was coined by Taverniti and Guglielmetti (2011) and defined as non-viable microbial cells (intact or broken) or crude cell extracts (i.e. with complex chemical composition), which, when administered (orally or topically) in adequate amounts, confer a benefit on the human or animal consumer.

The term postbiotic as proposed by Salminen et al. (2021a) refers to a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host.

The definition of paraprobiotics is limiting in that it does not clarify the scope for metabolites to be present alongside non-viable cells, and this may be problematic as most products of this type developed and marketed so far contain microbial metabolites along with non-viable cells. Further, the definition of paraprobiotics refers to conferring a benefit, but not a health benefit, a divergent way of conceptualizing a ‘biotic’ substance. Probiotics, prebiotics, synbiotics, and as defined above, postbiotics, all stipulate the requirement of conferring a health benefit. In addition, embedding the term ‘probiotic’ into the term paraprobiotic may mislead some to conclude that a paraprobiotic is a dead probiotic, which places a significant burden on any live microbial precursor to first meet the probiotic definition.

Finally, the authors (Taverniti and Guglielmetti 2011) state in their paper: “In addition, once a health benefit is demonstrated, the assignation of a product into the paraprobiotic category should not be influenced by the methods used for microbial cell inactivation, which may be achieved using physical or chemical strategies, including heat treatment, or UV ray deactivation, chemical or mechanical disruption, pressure, lyophilisation or acid deactivation”. Since inactivation technology may have a significant impact on the functionality of a dead microbe, disassociating a paraprobiotic with the method used to inactivate the microbes makes it impossible to know if any given paraprobiotic preparation will be effective.

The definition of postbiotics by Salminen et al. (2021a) anticipates that metabolites may be optionally present in the finished product, requires a health benefit and does not suggest, at any point in the wording, that the progenitor strain of a postbiotic must be a probiotic. Further, although not explicitly stated in the definition, the supporting documentation for the proposal of this definition states that the process to make the postbiotic must be delineated specifically, the progenitor microorganism must be clearly identified and characterized and the final product must be safe for its intended use. This definition encompasses a meaningful and useful scope.

To add to the complexity of the existing landscape, prior to the ISAPP definition of postbiotics, six other definitions of the term postbiotic were proposed in the literature. While these are reviewed in detail in Salminen et al (2021b Supplementary information), many shared the commonality that their focus was bacterial byproducts or metabolites.

Questions about the ISAPP definition of postbiotic

A common question is, “Why did the ISAPP panel choose the term postbiotic to refer to inactivated microbes?” In short, the word seemed most appropriate since post means ‘after’ and biotic means ‘life’.  Further, the panel recognized that although microbial metabolites might contribute to the health benefit conferred by a postbiotic, a preparation containing metabolites alone could be encompassed by a different term. Further, such metabolites (to the extent they are purified from the microbes that produce them) are readily referred to by their chemical names. Microbial metabolites may be present in a postbiotic preparation, but they are not required. The core of the definition of postbiotics is non-viable microbes, either as whole intact cells, disrupted cells or cell fragments. The life termination technology used to manufacture a postbiotic preparation should be stipulated. It cannot be assumed that heat inactivation, radiation, high pressure or any other technology will necessarily render an equally functional inanimate microbe.

Why use the descriptor “inanimate”? This is another common question. This word – meaning lifeless – reflects that the microorganisms should be dead, non-viable, no longer able to grow, to replicate, or, from an applied point of view, to form visible colonies in an enumeration medium or to be detected as live cells in flow cytometry techniques. It was preferred over the term “inactivated” only to call attention to the fact that postbiotics must confer a health benefit and in that sense, are active. For all practical purposes, non-viable can be used as an appropriate synonym.

Questions arise also about the breadth of definition, with concerns that “anything can be a postbiotic”. But broadness of a definition should not be seen as a disadvantage, as long as the limits to the definition are clear. Any microorganisms may be used as a postbiotic, as long as the identity is provided to the strain level, a life termination process is deliberately applied and safety and efficacy are demonstrated in a trial in the target host. Further, a postbiotic is not simply a dead probiotic. A probiotic is shown to confer a health benefit alive and it cannot be assumed that this property is retained when it is dead. Clearly, not anything can be a postbiotic.

Reflections on three recent conferences where the concept of postbiotics was debated

The first debate took place at the Beneficial Microbes conference in Amsterdam in November 2022. The outcomes were reported in a previous blog.

The second panel discussion took place in Chicago, at the Probiota 2023 conference in mid-June. After my talk, an audience poll was taken. Seventy-six out of around 250 attendees voted by an app in their cell phones to the question, How do you define a postbiotic? 68% selected the ISAPP definition, 9% said postbiotics were metabolites produced by probiotics, 4% chose the option “metabolites produced by the gut microbiota”, 14% said “none of the above” (I was curious to know what it would be for them), whereas 4% were not sure. Thus, the ISAPP definition was preferred by the majority. It is interesting to note the composition of the panel debate: three industry representatives and myself. Two of the companies represented presently market products referred to as postbiotics and containing non-viable microbes, whereas for the third company, postbiotics are “molecules created by bacteria”, according to their webpage. A discrepancy in the industry towards what postbiotics are was embodied on the stage. The preference for these meeting participants for the term postbiotic over the term paraprobiotic could be deduced from the meeting program, as the first term was mentioned 56 times, while the second had not one entry.

At Probiota 2023, an officer from Health Canada announced that the regulatory body will start considering the term postbiotics, which was defined in his presentation using the ISAPP definition. As for the quantification units for postbiotics, he indicated that milligrams would be considered currently, although he anticipated the development of more refined methodologies. The topic of what and how to quantify postbiotics is a commonly heard question. I intend to lead a Discussion Group on this topic comprising academic and ISAPP member company representatives at the 2024 ISAPP meeting July 9-11 in Cork, Ireland. If you are an academic expert or an industry member interested in joining the discussion, please reach out to me at gvinde@nullfiq.unl.edu.ar.

Panel discusson on postbiotics at the Bratislava International Probiotic Conference, 2023

A third panel discussion took place late in June in Bratislava at the 16th edition of the International Probiotic Conference. Before the debate, presentations were made by Arthur Ouwehand (IFF Health, Finland), Wilbert Sybesma (Yoba For Life Foundation, The Netherlands) and Eva Armengol (AB-BIOTICS, Spain). These speakers presented examples of postbiotics as they perceived them, which in all cases referred to administered non-viable microbes, in most cases containing microbial metabolites, thereby fitting the ISAPP definition. The fourth speaker, Simone Guglielmetti, proposed separate terms for non-viable microbes, which he proposed to call paraprobiotics, and for metabolites, which he proposed to call postbiotics, according to previous definitions (Taverniti and Guglielmetti, 2011; Tsiliringi and Rescigno, 2013).

There was also a sense of agreement that definitions should encompass current science but not unduly restrict future innovation. Some examples of products presently available in the market that contain non-viable microbes, and have efficacy studies with a clinical endpoint or biomarker enhancement, are:

 

Species or strain/s Composition Reference
B. bifidum MIMBb75 Heat inactivated bacteria https://pubmed.ncbi.nlm.nih.gov/32277872/
Akkermansia muciniphila Heat inactivated bacteria https://pubmed.ncbi.nlm.nih.gov/31263284/
L. fermentum CNCM MA65/4E-1b and L. delbrueckii CNCM MA65/4E-2z Heat inactivated bacteria plus metabolites https://pubmed.ncbi.nlm.nih.gov/33281937/
B. breve C50 and S. thermophilus 065 Heat inactivated bacteria plus metabolites https://pubmed.ncbi.nlm.nih.gov/32629970/
Aspergillus oryzae Heat inactivated fungi plus metabolites https://pubmed.ncbi.nlm.nih.gov/33742039/
L. paracasei MCC1849 Heat inactivated bacteria plus metabolites https://pubmed.ncbi.nlm.nih.gov/33787390/
L. sakei proBio65 Bacterial lysate plus metabolites https://pubmed.ncbi.nlm.nih.gov/32949011/
S. cerevisiae Heat inactivated yeasts plus metabolites https://pubmed.ncbi.nlm.nih.gov/21501093/
Vitreoscilla filiformis Bacterial lysate plus metabolites https://pubmed.ncbi.nlm.nih.gov/34976852/
Mixture of pathogens Bacterial lysate plus metabolites https://pubmed.ncbi.nlm.nih.gov/34976852/

 

These ten examples of commercial products based on non-viable microbes all fit the definition of postbiotics conceptualized by Salminen et al. (2021). Only the first two fit the Taverniti and Guglielmetti (2011) definition, as these contain just non-viable microorganisms, without metabolites. This may suggest that products in the current marketplace are best described by the Salminen et al. (2021) concept, which encompasses products based on non-viable microbes, which may or may not also contain microbial metabolites.

Conclusions

In conclusion, I suggest that the term postbiotic and the definition of Salminen et al. (2021a) be used for non-viable microbes (with or without metabolites) able to confer a health benefit, as reflected by the present state of the art and products developed and marketed. If deemed useful by the field, there is room yet for a new term to encompass products developed with microbial metabolites only (devoid of cells). If we consider definitions that mutually exclude non-viable microbes or metabolites, then the vast majority of products present today in the market would not be covered, as most of them deliver non-viable microorganisms and metabolites simultaneously. My overall sense after attending the Chicago and Bratislava meetings is that the meaning of the term postbiotic as mentioned by speakers, included in the meeting programs, seen in posters (future products) and in commercial products presented in booths, refers to the ISAPP definition of non-viable microbes. Time will tell how this term and definition evolves and if a broader consensus can be reached.

 

References

Aguilar-Toalá, J. E., Arioli, S., Behare, P., Belzer, C., Berni Canani, R., Chatel, J. M., D’Auria, E., de Freitas, M. Q., Elinav, E., Esmerino, E. A., García, H. S., da Cruz, A. G., González-Córdova, A. F., Guglielmetti, S., de Toledo Guimarães, J., Hernández-Mendoza, A., Langella, P., Liceaga, A. M., Magnani, M., Martin, R., … Zhou, Z. (2021). Postbiotics – when simplification fails to clarify. Nature reviews. Gastroenterology & hepatology, 18(11), 825–826. https://doi.org/10.1038/s41575-021-00521-6

Salminen, S., Collado, M. C., Endo, A., Hill, C., Lebeer, S., Quigley, E. M. M., Sanders, M. E., Shamir, R., Swann, J. R., Szajewska, H., & Vinderola, G. (2021a). The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nature reviews. Gastroenterology & hepatology, 18(9), 649–667. https://doi.org/10.1038/s41575-021-00440-6

Salminen, S., Collado, M. C., Endo, A., Hill, C., Lebeer, S., Quigley, E. M. M., Sanders, M. E., Shamir, R., Swann, J. R., Szajewska, H., & Vinderola, G. (2021b). Reply to: Postbiotics – when simplification fails to clarify. Nature reviews. Gastroenterology & hepatology, 18(11), 827–828. https://doi.org/10.1038/s41575-021-00522-5

Taverniti V, Guglielmetti S. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept). Genes Nutr. 2011 Aug;6(3):261-74. doi: 10.1007/s12263-011-0218-x. Epub 2011 Apr 16. PMID: 21499799; PMCID: PMC3145061.

Tsilingiri K, Rescigno M. Postbiotics: what else? Benef Microbes. 2013 Mar 1;4(1):101-7. doi: 10.3920/BM2012.0046. PMID: 23271068.

Episode 25: The effects of metabolites in the colon

/in Podcast, Season Two /by Laura

 

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The Science, Microbes & Health Podcast 

This podcast covers emerging topics and challenges in the science of probiotics, prebiotics, synbiotics, postbiotics and fermented foods. This is the podcast of The International Scientific Association for Probiotics and Prebiotics (ISAPP), a nonprofit scientific organization dedicated to advancing the science of these fields.

The effects of metabolites in the colon, with Prof. Kristin Verbeke PhD

Episode summary:

In this episode, the ISAPP podcast hosts talk about colonic metabolites with Prof. Kristin Verbeke PhD, from KU Leuven, Belgium. She talks about characterizing microbial metabolism in the colon and the consequences of producing various metabolites, both beneficial ones (such as short-chain fatty acids) and potentially detrimental ones.

Key topics from this episode:

  • Prof. Verbeke is a pharmacist by training, and now leads hospital breath testing and carries out research on microbial metabolites in the gastrointestinal tract, including how prebiotics and probiotics can change bacterial metabolism.
  • The majority of protein in the diet is digested in the small intestine, but about 5% of animal protein and 10-15% of plant protein reaches the large intestine to be fermented by the microbiota. This produces metabolites, which are shown in vitro to be toxic. However, in vivo there is less evidence of toxicity; the negative effects of these metabolites may be reduced by the interactions of different compounds in the colon.
  • Short-chain fatty acids (SCFAs) are produced when the body digests dietary fiber, and Prof. Verbeke’s group and others are investigating whether they are responsible for the benefits of eating fiber.
  • Most SCFAs are quickly absorbed in the large intestine, and they serve as an energy source for the cells. They then travel to the liver via portal circulation, where they have additional functions. What’s left over reaches systemic circulation.
  • The difficulty is knowing how many SCFAs are produced in the colon, and how many reach systemic circulation. In one experiment, they labeled the SCFAs that were administered to the colon via capsule; 36% ended up in systemic circulation. Further, when SCFAs were administered at physiological doses the subjects receiving them (compared to placebo) showed a lower cortisol response to stress.
  • SCFAs also affect fat oxidation and fat synthesis in the liver. Their relevance to non-alcoholic fatty liver disease are being investigated.
  • It’s important to eat fiber, and lots of different types. After fiber consumption, SCFAs increase in a sustained manner and take about 8h to get back to baseline. But with SCFA delivery via capsule they spike quickly and then disappear.
  • As for coatings to deliver to the colon, some coatings are time-dependent, pH dependent, etc. and this is an area for further exploration.

Episode links:

About Prof. Kristin Verbeke PhD:

Kristin Verbeke graduated from the KU Leuven, Belgium as a pharmacist in 1991. She obtained a PhD in Pharmaceutical Sciences at the Laboratory of Radiopharmaceutical Chemistry in 1995 and subsequently spend a postdoctoral period in developing radioactively labelled compounds. In 2002, she was appointed at the department of gastroenterology of the Medical Faculty of the Leuven University where she got involved in the use of stable isotope labelled compounds to evaluate gastrointestinal functions. Within the University Hospitals Leuven, she is responsible for the clinical application of diagnostic 13C- and H2-breath tests. Her current research interest specifically addresses the microbial bacterial metabolism in the human colon. Her team has developed several analytical techniques based on mass spectrometry and stable isotope or radioisotope technologies to evaluate several aspects of intestinal metabolism and function in humans (transit time, intestinal permeability, carbohydrate fermentation, protein fermentation, metabolome analysis). Collaborative research has allowed showing an aberrant bacterial metabolism in patient groups with end stage renal failure, inflammatory bowel diseases, irritable bowel disorders and alcohol abuse. These collaborations all have resulted in high quality peer-reviewed papers. In addition, she showed the impact of dietary interventions (modulation of macronutrient composition, pre- or probiotic interventions) on the microbial metabolism and its impact on health. As a PI, she acquired grant support from the university and different funding bodies and successfully completed these projects. Similarly, she supervised several PhD projects that all resulted in the achievement of a PhD degree. Her research resulted in over 200 full research papers. Together with colleague Prof. J. Delcour, she was the beneficiary of the W.K. Kellogg Chair in Cereal Sciences and Nutrition (2010-2020). She is the president of the Belgian Nutrition Society, the vice-chair of the Leuven Food Science and Nutrition Center, and the co-chair of the Prebiotic task force at ILSI Europe. Furthermore, Kristin Verbeke is the editor of the journal Gut Microbiome and member of the editorial board of Gastrointestinal Disorders. Kristin joined the ISAPP Board of Directors in 2023.

Can we use fermented foods to modulate the human immune system?

/in ISAPP Science Blog, Consumer Blog /by KC

By Dr. Paul Gill PhD, Monash University

Fermented foods have grown in popularity in recent years, marketed for their purported health effects, including on the gut microbiome and immune system. Many of us have had a family member or friend recommend to us kombucha or sauerkraut based on a claim of curing their ailments. However, a reliable recommendation goes beyond anecdotal evidence and the science of how fermented foods confer any health benefits is often poorly understood. We often associate health effects of fermented foods with bacteria such as lactobacilli or Bifidobacterium, but what is lesser known is the role of microbial metabolites. These have sparked recent interest, particularly amongst researchers.

Many fermented foods naturally contain a mixture of live microorganisms and metabolites, such as phenolic compounds and short-chain fatty acids (SCFA). All of these components have the potential to impact host immunity, through two main mechanisms. Firstly, by directly interacting with local gut immune cells that have receptors for bacterial components such as lipopolysaccharide or peptidoglycan. Secondly, by modulating gut microbiota composition or function that will lead to indirect changes to host immunity. Together, these mechanisms are important for regulation of gut barrier integrity and immune homeostasis. Furthermore, bacterial metabolites such as SCFA are also absorbed by the portal vein and reach peripheral circulation, suggesting that they may also play a role in regulating systemic immune responses.

Although many of these findings are based upon observations from in vitro studies or pre-clinical models, several pilot studies in humans have also reported similar effects. A recent trial in a small cohort of healthy people found that consumption of an average of six servings of fermented foods per day for 10 weeks was associated with reduced serum inflammatory markers. Furthermore, consumption of a diet that included three servings of apple cider vinegar each day for three weeks, increased levels of plasma short-chain fatty acids and reduced subsets of circulating lymphocytes in a group of 20 healthy people. Taken together, these studies highlight the potential anti-inflammatory effects of fermented foods and postbiotics.

It remains a challenge to attribute consumption of fermented foods to alterations in host immunity, particularly due to the complex nature of these foods. This is particularly the case for traditional fermented food products that are not well characterised. After isolation and identification of individual metabolites within fermented foods, characterisation of how these compounds are absorbed and interact within the body is also necessary to determine how frequently they should be consumed to have meaningful effects on the immune system. Future studies need to be designed of sufficient duration, with a realistic dietary intervention and optimal timing of biological sampling is crucial to validate observations from exploratory trials. Finally, studies in patients with immune deficiencies will be needed to assess safety and potential therapeutic benefit. Alternatively, studies in healthy people during an immune challenge, such as during vaccination, are another desirable approach to investigate immune and therapeutic effects of fermented food consumption.

The scientific and medical communities, alongside the food industry, are continuing to improve our understanding of how fermented foods may benefit our health and immune system, including which components are responsible for any health benefits. Future studies are still needed to confirm if these may be of therapeutic benefit, and who may benefit the most from consuming these products. As our knowledge evolves, it is important that we continue to follow expert groups such as ISAPP to keep well informed and correctly communicate this information to patients and the public.

How metabolites help us to understand the effect of gut microbes on health

/in ISAPP Science Blog /by KC

By Dr. Anisha Wijeyesekera, University of Reading, UK

Much literature relating to the gut microbiota has focused on microbial composition (for example, using culture-dependent and -independent molecular biology approaches). Composition is important; knowing which microbes are present in a community enables us to gain insight into population dynamics and how these may be affected by disease, lifestyle and environmental factors (including diet). However, composition does not provide information on microbial function, and considering the gut contains the most metabolically active microbial community in the whole body, it is thus equally as important to be able to answer the question “what are the microorganisms doing”? This is of particular importance with respect to better understanding the impact of dietary interventions such as prebiotics, probiotics and other ‘biotics on health, where health benefits conferred on the host are mediated via the gut microbiota.

Investigating microbial function

Advances in phenotypic analytical technologies (for example, high-throughput sequencing, biochemical analysis, as well as bioinformatics and other multivariate data analysis approaches), have resulted in a stepwise change in our understanding of microbial function. Metabolic phenotyping (also referred to as metabolomics, metabonomics or metabolic profiling) is an exciting field in systems biology that provides information on the multiple metabolic mechanisms taking place in a system, at a given moment in time (see here). This top-down approach enables high-throughput detection and quantification of low molecular weight molecules present in a biological sample at the time of sampling, without a priori knowledge of metabolites present. Hence, it is ideally suited to augment and complement information obtained from microbial profiling approaches such as metataxonomics, to gain deeper insight into microbial function.

Metabolic phenotyping is conducted by applying analytical chemistry technologies (typically, 1H-nuclear magnetic resonance spectroscopy, and/or mass spectrometry often with chromatographic separation techniques such as gas chromatography and liquid chromatography (for prior separation of molecules followed by detection)) to capture a biochemical snapshot of a sample. In human samples (e.g. urine, blood plasma/serum and stool), metabolites detected using metabolic phenotyping are low molecular weight molecules and include intermediate and end by-products of endogenous host metabolic pathways (e.g. TCA cycle, amino acid metabolism), but also exogenous signals arising from diet, drugs and other lifestyle and environmental stimuli, including products of microbe-host co-metabolism, which provide insight into host-gut microbiota interactions. These include short-chain fatty acids (predominantly acetate, butyrate and propionate, which have a key role in host energy metabolism), bile acids (involved in the gut-liver axis), biogenic amines (involved in the gut-brain axis) and vitamins. Metabolic phenotyping, which provides functional assessment of the gut microbiota and captures information on microbial metabolic activity following ‘biotics intervention, can aid in forming hypotheses about microbial activity that may lead to health benefits.

Challenges in determining the functions of microbes

Nevertheless, functional assessment of the microbiota remains analytically challenging. For example, human metabolic phenotypes contain information relating to different forms of optically active isomers, such as lactate and amino acids (where D- forms originate from bacteria). These enantiomers cannot be differentiated using standard metabolic phenotyping experiments, and it would be important particularly in studies identifying potential biomarkers of disease, to understand the origin of these compounds. Hence, we and others have also conducted mechanistic studies using in vitro human gut model systems (e.g. the model developed by Macfarlane et al., 1998, which  has been validated against gut contents from sudden death victims and give a very close analogy to bacterial activities and composition in different areas of the hindgut), Metabolic screening of fermentation samples using metabolic phenotyping approaches provides a unique opportunity to capture dynamic microbial metabolism that is reflective of the gut microbiome in vivo, and removes contributions derived from host physiological processes (see here).

Unravelling the close interplay between microbes and host, using approaches such as metabolic phenotyping, not only provides insight into host-gut interactions, but aids our understanding of the alterations in gut microbiota mediated mechanisms that result in disease, and which demonstrate potential as therapeutic targets. More research in this area will aid in deepening understanding of the role of the gut microbiota in health and disease, and aid in the design of interventions targeting the gut microbiota (for example, the development of functional foods) for therapeutic benefit.

Bifidobacteria in the infant gut use human milk oligosaccharides: how does this lead to health benefits?

/in ISAPP Science Blog /by KC

By Martin Frederik Laursen, Technical University of Denmark, 2022 co-recipient of Glenn Gibson Early Career Research Prize

Breast milk is the ‘gold standard’ of infant nutrition, and recently scientists have zeroed in on human milk oligosaccharides (HMOs) as key components of human milk, which through specific interaction with bifidobacteria, may improve infant health. Clarifying mechanisms by which HMOs act in concert with bifidobacteria in the infant gut may lead to better nutritional products for infants.

Back in early 2016, I was in the middle of my PhD studies working on determinants of the infant gut microbiota composition in the Licht lab at the National Food Institute, Technical University of Denmark. I had been working with fecal samples from a Danish infant cohort study, called SKOT (Danish abbreviation for “Diet and well-being of young children”), investigating how the diet introduced in the complementary feeding period (as recorded by the researchers) influences the gut microbiota development 1,2. Around the same time, Henrik Munch Roager, PostDoc in the lab, was developing a liquid chromatography mass spectrometry (LC-MS)-based method for quantifying the aromatic amino acids (AAA) and their bacterially produced metabolites in fecal samples (the 3 AAAs and 16 derivatives thereof). These bacterially produced AAA metabolites were starting to receive attention because of their role in microbiota-host cross-talk and interaction with various receptors such as the Aryl Hydrocarbon Receptor (AhR) expressed in immune cells and important for controlling immune responses at mucosal surfaces 3,4. However, virtually nothing was known about bacterial metabolism of the AAAs in the gut in an early life context. Further, the fecal samples collected from the SKOT cohort were obtained in a period of life when infants are experiencing rapid dietary changes (e.g. cessation of breastfeeding and introduction of various new foods). Thus, we wondered whether the AAA metabolites would be affected by diet and whether these metabolites might contribute to the development of the infant’s immune system. Our initial results quickly guided us on the track of breastfeeding and bifidobacteria! Here is a summary of the story, published last year in Nature Microbiology5. (See the accompanying News & Views article here.)

We initially looked at the data from a subset of 59 infants, aged 9 months, from the SKOT cohort. Here we found that both the gut microbiome and the AAA metabolome were affected by breastfeeding status (breastfed versus weaned). It is well established that certain bifidobacteria dominate the bacterial gut community in breastfed infants due to their efficient utilization of HMOs – which are abundant components of human breastmilk 6. Our data showed the same, namely enrichment of Bifidobacterium in the breastfed infants, but also indicated that the abundance of specific AAA metabolites were dependent on breastfeeding.

Trying to connect the gut microbiome and AAA metabolome, we found striking correlations between the relative abundance of Bifidobacterium and specifically abundances of three aromatic amino acid catabolites – namely indolelactic acid (ILA), phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (4-OH-PLA), collectively aromatic lactic acids. These metabolites are formed in two enzymatic reactions (a transamination followed by a hydrogenation) of the aromatic amino acids tryptophan, phenylalanine and tyrosine. However, the genes involved in this pathway were not known for bifidobacteria. Digging deeper we discovered that not all Bifidobacterium species found in the infant’s gut correlated with these metabolites. This was only true for the Bifidobacterium species enriched in the breastfed infants (e.g. B. longum, B. bifidum and B. breve), but not post-weaning/adult type bifidobacteria such as B. adolescentis and B. catenulatum group.

We decided to go back to the lab and investigate these associations by culturing representative strains of the Bifidobacterium species found in the gut of these infants. Indeed, our results confirmed that Bifidobacterium species are able to produce aromatic lactic acids, and importantly that the ability to produce them was much stronger for the HMO-utilizing (e.g. B. longum, B. bifidum and B. breve) compared to the non-HMO utilizing bifidobacteria (e.g. B. adolescentis, B. animalis and B. catenulatum). Next, in a series of experiments we identified the genetic pathway in Bifidobacterium species responsible for production of the aromatic lactic acids and performed enzyme kinetic studies of the key enzyme, an aromatic lactate dehydrogenase (Aldh), catalyzing the last step of the conversion of aromatic amino acids into aromatic lactic acids. Thus, we were able to demonstrate the genetic and enzymatic basis for production of these metabolites in Bifidobacterium species.

To explore the temporal dynamics of Bifidobacteria and aromatic lactic acids and validate our findings in an early infancy context (a critical phase of immune system development), we recruited 25 infants (Copenhagen Infant Gut [CIG] cohort) from which we obtained feces from birth until six months of age. These data were instrumental for demonstrating the tight connection between specific Bifidobacterium species, HMO-utilization and production of aromatic lactic acids in the early infancy gut and further indicated that formula supplementation, pre-term delivery and antibiotics negatively influence the concentrations of these metabolites in early life.

Having established that HMO-utilizing Bifidobacterium species are key producers of aromatic lactic acids in the infant gut, we focused on the potential health implications of this. We were able to show that the capacity of early infancy feces to in vitro activate the AhR, depended on the abundance of aromatic lactic acid producing Bifidobacterium species and the concentrations of ILA (a known AhR agonist) in the fecal samples obtained from the CIG cohort. Further, using isolated human immune cells (ex vivo) we showed that ILA modulates cytokine responses in Th17 polarized cells – namely it increased IL-22 production in a dose and AhR-dependent manner. IL-22 is a cytokine important for protection of mucosal surfaces, e.g. it affects secretion of antimicrobial proteins, permeability and mucus production 7. Further, we tested ILA in LPS/INFγ induced monocytes (ex vivo), and found that ILA was able to decrease the production of the proinflammatory cytokine IL-12p70, in a manner dependent upon both AhR and the Hydroxycarboxylic Acid (HCA3) receptor, a receptor expressed in neutrophils, macrophages and monocytes and involved in mediation of anti-inflammatory processes 8,9. Overall, our data reveal potentially important ways in which bifidobacteria influence the infant’s developing immune system.

Figure 1 – HMO-utilizing Bifidobacterium species produce immuno-regulatory aromatic lactic acids in the infant gut.

Our study provided a novel link between HMO-utilizing Bifidobacterium species, production of aromatic lactic acids and immune-regulation in early life (Figure 1). This may explain previous observations that the relative abundance of bifidobacteria in the infant gut is inversely associated with development of asthma and allergic diseases 10–12 and our results, together with other recent findings13–15 are pointing towards aromatic lactic acids (especially ILA) as potentially important mediators of beneficial immune effects induced by HMO-utilizing Bifidobacterium species.

 

References

  1. Laursen, M. F. et al. Infant Gut Microbiota Development Is Driven by Transition to Family Foods Independent of Maternal Obesity. mSphere 1, e00069-15 (2016).
  2. Laursen, M. F., Bahl, M. I., Michaelsen, K. F. & Licht, T. R. First foods and gut microbes. Front. Microbiol. 8, (2017).
  3. Zelante, T. et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 39, 372–385 (2013).
  4. Sridharan, G. V. et al. Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat. Commun. 5, 1–13 (2014).
  5. Laursen, M. F. et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat. Microbiol. 6, 1367–1382 (2021).
  6. Sakanaka, M. et al. Varied pathways of infant gut-associated Bifidobacterium to assimilate human milk oligosaccharides: Prevalence of the gene set and its correlation with bifidobacteria-rich microbiota formation. Nutrients 12, 71 (2020).
  7. Keir, M. E., Yi, T., Lu, T. T. & Ghilardi, N. The role of IL-22 in intestinal health and disease. J. Exp. Med. 217, (2020).
  8. Peters, A. et al. Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3. PLoS Genet. 15, e1008145 (2019).
  9. Peters, A. et al. Hydroxycarboxylic acid receptor 3 and GPR84 – Two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells. Pharmacol. Res. 176, (2022).
  10. Fujimura, K. E. et al. Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation. Nat. Med. 22, 1187–1191 (2016).
  11. Stokholm, J. et al. Maturation of the gut microbiome and risk of asthma in childhood. Nat. Commun. 9, 141 (2018).
  12. Seppo, A. E. et al. Infant gut microbiome is enriched with Bifidobacterium longum ssp. infantis in Old Order Mennonites with traditional farming lifestyle. Allergy Eur. J. Allergy Clin. Immunol. 76, 3489–3503 (2021).
  13. Meng, D. et al. Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine. Pediatr. Res. 88, 209–217 (2020).
  14. Ehrlich, A. M. et al. Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells. BMC Microbiol. 20, 357 (2020).
  15. Henrick, B. M. et al. Bifidobacteria-mediated immune system imprinting early in life. Cell 184, 3884-3898.e11 (2021).

Do polyphenols qualify as prebiotics? The latest scientific perspectives

/in ISAPP Science Blog, Consumer Blog /by KC

Kristina Campbell, Consulting Communications Director, ISAPP

When the ISAPP scientific consensus definition of ‘prebiotic’ was published in 2017, the co-authors on the paper included polyphenols as potential prebiotic substances. At the time, the available data on the effect of polyphenols on the gut microbiota were insufficient to show a true prebiotic effect.

An ISAPP webinar held in April 2022, aimed to give an update on the health effects of polyphenols and their mechanisms of action, along with how well polyphenols fit the prebiotic definition. Prof. Daniele Del Rio from University of Parma, Italy, and Prof. Yves Desjardins from Université Laval, Canada, presented the latest perspectives in the field.

What are polyphenols?

Polyphenols are a group of compounds found in plants, with over 6000 types identified to date. They can be divided into two main categories, flavonoids and non-flavonoids.

Polyphenols are absorbed in two different ways in the body. A very small fraction is absorbed in the small intestine, but 95% of them reach the lower gut and interact with gut microbiota. Although polyphenols have a special capacity to influence the activities of microorganisms, some resident microorganisms, in turn, can change the chemical structure of polyphenols through enzymatic action. These interactions produce a unique array of metabolites, which may be responsible for some of polyphenols’ prebiotic effects.

What are the health effects of polyphenols?

Epidemiological studies show that polyphenols in the diet are associated with many health benefits, including prevention of cardiovascular disease, certain cancers, and metabolic disease. These effects occur through various mechanisms. However, association is not proof of causation. So how good is the evidence that polyphenols can lead to health benefits?

Numerous human studies exist, but the most robust study to date for the health benefits of polyphenols is a randomized, controlled trial of over 20,000 adults, published in 2022, which showed supplementation with cocoa extract reduced death from cardiovascular events (although it did not reduce the number of cardiovascular events).

What are the mechanisms of action for polyphenols?

Polyphenols have multiple mechanisms of action. Del Rio focuses on the metabolites produced from dietary polyphenols called flavan-3-ols, which are found in red wine, grapes, tea, berries, chocolate and other foods. Along with colleagues, he showed that the metabolites produced in response to a polyphenol-rich food occur two ‘waves’: a small wave in the first 2 hours after ingestion, and a larger wave 5-35 hours after ingestion. The second wave is produced when flavan-3-ols reach the colon and interact with gut microbiota.

Work is ongoing to link these metabolites to specific health effects. Along these lines, Del Rio described a study showing how cranberry flavan-3-ol metabolites help defend against infectious Escherichia coli in a model system of bladder epithelial cells. These polyphenols are transformed by the gut microbiota into smaller compounds that are absorbed—so the health benefit comes not from the activity of polyphenols directly, but from the molecule(s) that the gut microbiota has produced from the polyphenols.

How else do polyphenols work? Ample evidence suggests polyphenols interact in different ways with gut microbes: they have direct antimicrobial effects, they affect quorum sensing, they compete with bacteria for some minerals, and/or they modify ecology, thereby affecting biofilm formation. Desjardins explained that these interactions may occur in parallel: for example, polyphenols may exert antimicrobial effects when they reach the colon, and at the same time, microorganisms in the gut begin to degrade them.

The mode of action of polyphenols Desjardins studies is the prebiotic mode of action—or as he describes it, “prebiotic with a twist”. A landmark paper from 2015 showed how cranberry polyphenols had protective effects on metabolism and obesity through the creation of mucin in the intestine, which formed a good niche for Akkermansia muciniphila, a keystone bacterial species for good metabolic health. Other polyphenols have since been shown to work the same way: by stimulating production of mucin, thereby providing ideal conditions for beneficial bacteria to grow. In this way, polyphenols appear to show small-scale effects comparable to the effects of probiotics, by inducing a host response that alters the bacterial niche.

Are the effects of polyphenols individual?

Del Rio offered some evidence that the health effects of polyphenols, via metabolites, is personalized: a study showed the existence of three distinct patterns of metabolite production in response to dietary polyphenols (ellagitannins). These may depend on the particular microbes of the gut and their ability to produce the relevant metabolites—so in essence, in each case the gut microbiota is equipped to produce a certain set of metabolites in response to polyphenols. More work is needed, however, to be able to personalize polyphenol intake.

Do polyphenols qualify as prebiotic substances?

Polyphenols clearly interact with gut microbiota to influence human health. The definition of a prebiotic is “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Given the available evidence that polyphenols are not metabolized or utilized by bacteria in all cases in the same direct way as carbohydrate prebiotics, Desjardins sees them as having a “prebiotic-like effect”. Rather, polyphenols are transformed into other biologically active molecules that ultimately provide health benefits to the host. These prebiotic-like properties of polyphenols are nicely summarized in a 2021 review paper and include decreasing inflammation, increasing bacteriocins and defensins, increasing gut barrier function (thereby reducing low-grade inflammation), modulating bile acids, and increasing gut immuno-globulins.

Overall, the speakers showed that polyphenols exert their health effects in several ways—and while the gut microbiota are important for their health effects, polyphenols, as a heterogenous group, may not strictly meet the criteria for prebiotics. Clearly, more research on polyphenols may reveal other mechanisms by which these important nutrients influence the gut microbiome and contribute to host health, and they may someday be regarded as prebiotics.

Watch the replay of the ISAPP webinar here.

Behind the publication: Understanding ISAPP’s new scientific consensus definition of postbiotics

/in ISAPP Science Blog /by KC

A key characteristic of a probiotic is that it remains alive at the time of consumption. Yet scientists have known for decades that some non-living microorganisms can also have benefits for health: various studies (reviewed in Ouwehand & Salminen, 1998) have compared the health effects of viable and non-viable bacteria, and some recent investigations have tested the health benefits of pasteurized bacteria (Depommier et al., 2019).

Since non-viable microorganisms are often more stable and convenient to include in consumer products, interest in these ‘postbiotic’ ingredients has increased over the past several years. But before now, the scientific community had not yet united around a definition, nor had it precisely delineated what falls into this category.

An international group of scientists from the disciplines of probiotics and postbiotics, food technology, adult and pediatric gastroenterology, pediatrics, metabolomics, regulatory affairs, microbiology, functional genomics, cellular physiology and immunology met in 2019 to discuss the concept of postbiotics. This meeting led to a recently published consensus paper, including this definition: “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”.

Thus, a postbiotic must include some non-living microbial biomass, whether it be whole microbial cells or cell components.

Below is a Q&A with four of the paper’s seven ISAPP-linked authors, who highlight important points about the definition and explain how it will lay the groundwork for better scientific understanding of non-viable microbes and health in the years ahead.

Why was the concept of postbiotics needed?

Prof. Seppo Salminen, University of Turku, Finland:

We have known for a long time that inactivated microorganisms, not just live ones, may have health effects but the field had not coalesced around a term to use to describe such products or the key criteria applicable to them. So we felt we needed to assemble key experts in the field and provide clear definitions and criteria.

Further, novel microbes (that is, new species hitherto not used in foods) in foods and feeds are being introduced as live or dead preparations. The paper highlights regulatory challenges and for safety and health effect assessment for dead preparations of microbes.

Can bacterial metabolites be postbiotics?

Prof. Gabriel Vinderola, National University of Litoral, Argentina:

Postbiotics can include metabolites – for example, fermented products with metabolites and microbial cells or their components, but pure metabolites are not postbiotics.

Can you expand on what is not included in the category of postbiotics?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

The term ‘postbiotic’ today is sometimes applied to components derived from microbial growth that are purified, so no cell or cell products remain. The panel made the decision that such purified, microbe-derived substances (e.g. butyrate) should be called by their chemical names and that there was no need for a single encompassing term for them. Some people may be surprised by this. But microbe-derived substances include a whole host of purified pharmaceuticals and industrial chemicals, and these are not appropriately within the scope of ‘postbiotics’.

For something to be a postbiotic, what kinds of microorganisms can it originate from?

Prof. Gabriel Vinderola, National University of Litoral, Argentina:

A postbiotic must derive from a living microorganism on which a technological process is applied for life termination (heat, high pressure, oxygen exposure for strict anaerobes, etc). Viruses, including bacteriophages, are not considered living microorganisms, so postbiotics cannot be derived from them.

Safety and benefits must be demonstrated for its non-viable form. A postbiotic does not have to be derived from a probiotic (see here for a list of criteria required for a probiotic). So the microbe used to derive a postbiotic does not need to demonstrate a health benefit while alive. Further, a probiotic product that loses cell viability during storage does not automatically qualify as a postbiotic; studies on the health benefit of the inactivated probiotic are still required.

Vaccines or substantially purified components and products (for example, proteins, peptides, exopolysaccharides, SCFAs, filtrates without cell components and chemically synthesized compounds) would not qualify as postbiotics in their own right, although some might be present in postbiotic preparations.

What was the most challenging part of creating this definition?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

The panel didn’t want to use the term ‘inactive’ to describe a postbiotic, because clearly even though they are dead, they retain biological activity. There was a lot of discussion about the word ‘inanimate’, as it’s not so easy to translate. But the panel eventually decided it was the best option.

 Does this definition encompass all postbiotic products, no matter whether they are taken as dietary supplements or drugs?

Prof. Hania Szajewska, Medical University of Warsaw, Poland:

Indeed. However, as of today, postbiotics are found primarily in foods and dietary supplements.

Where can you currently find postbiotics in consumer products, and what are their health effects?

Prof. Hania Szajewska, Medical University of Warsaw, Poland:

One example is specific fermented infant formulas with postbiotics which have been commercially available in some countries such as Japan and in Europe, South America, and the Middle East for years. The postbiotics in fermented formulas are generally derived from fermentation of a milk matrix by Bifidobacterium, Streptococcus, and/or Lactobacillus strains.

Potential clinical effects of postbiotics include prevention of common infectious diseases such as upper respiratory tract infections and acute gastroenteritis. Moreover, fermented formulas have the potential to improve some digestive symptoms or discomfort (e.g. colic in infants). In addition, there is some rationale for immunomodulating, anti-inflammatory effects which may potentially translate into other clinical benefits, such as improving allergy symptoms. Still, while these effects are likely, more well-designed, carefully conducted trials are needed.

What do we know about postbiotic safety?

Dr. Mary Ellen Sanders, ISAPP Executive Science Officer, USA:

Living microbes have the potential, especially in people with compromised health, to cause an infection. But because the microbes in postbiotics are not alive, they cannot cause infections. This risk factor, then, is removed from these preparations. Of course, the safety of postbiotics for their intended use must be demonstrated, but infectivity should not be a concern.

What are the take-home points about the postbiotics definition?

Prof. Seppo Salminen, University of Turku, Finland:

Postbiotics, which encompass inanimate microbes with or without metabolites, can be characterized, are likely to be more stable than live counterparts and are less likely to be a safety concern, since dead bacteria and yeast are not infective.

Read the postbiotic definition paper here.

See the press release about this paper here.

View an infographic on the postbiotic definition here.

See another ISAPP publication on postbiotics here.

New publication co-authored by ISAPP board members gives an overview of probiotics, prebiotics, synbiotics, and postbiotics in infant formula

/in News, ISAPP Science Blog /by KC

For meeting the nutritional needs of infants and supporting early development, human milk is the ideal food—and this is reflected in breastfeeding guidelines around the world, including the World Health Organization’s recommendation that babies receive human milk exclusively for the first six months of life and that breastfeeding be continued, along with complementary foods, up to two years of age or beyond. In certain cases, however, breastfeeding is challenging or may not even be an option. Then, parents rely on alternatives for feeding their infants.

A group of scientists, including three ISAPP board members, recently co-authored an article in the journal Nutrients entitled Infant Formula Supplemented with Biotics: Current Knowledge and Future Perspectives. In the review, they aimed to highlight the new technologies and ingredients that are allowing infant formula to better approximate the composition of human milk. They focused on four types of ingredients: probiotics, prebiotics, synbiotics, and postbiotics.

Co-author Gabriel Vinderola, Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at Dairy Products Institute (CONICET-UNL) in Santa Fe, Argentina says, “Modern technologies have allowed the production of specific microbes, subtrates selectively used by the host microbes, and even non-viable microbes and their metabolites and cell fragments—for which scientific evidence is available on their effects on infant health, when administered in adequate amounts. Thus, this current set of gut modulators can be delivered by infant formula when breastfeeding is limited or when it is not an option.”

The authors say a well-functioning gut microbiota is essential for the overall health and proper development of the infant, and components of human milk support the development of this microbiota. They list important human milk components and the novel ingredients that aim to mimic the functions of these components in infant formulas:

  • Human milk oligosaccharides (HMOs)

HMOs are specialized complex carbohydrates found in human milk, which are digested in the infant colon and serve as substrates for beneficial microbes, mainly bifidobacteria, residing there. In recent years, prebiotic mixtures of oligosaccharides (e.g. short-chain GOS and long-chain FOS) have been added to infant formula to recapitulate the effects of HMOs. But now that it’s possible to produce several types of HMOs synthetically, some infant formulas are enriched with purified HMOs: 2’-fucosyllactose (2’FL) or lacto-N-neotetraose (LNnT). Even 3′-galactosyllactose (3′-GL) can be naturally produced by a fermentation process in certain infant formulas.

  • Human milk microbiota

Human milk has a complex microbiota, which is an important source of beneficial bacteria to the infant. Studies support the notion that the human milk microbiota delivers bioactive components that support the development of the infant’s immune system. Probiotic strains are sometimes added to infant formula in order to substitute for important members of the milk microbiota.

  • Bacterial metabolites

Human milk also contains metabolic byproducts of bacteria called “metabolites” in addition to the bacteria themselves. These components have not been fully studied to date, but bacterial metabolites such as butyrate and other short-chain fatty acids may have important health effects for the overall development of the infant. A future area of nutritional research is likely to be the addition of ‘postbiotics’ — non-viable cells, their metabolites and cell components that, when administered in adequate amounts, promote health and well-being — to infant formulas. (ISAPP convened a scientific consensus panel on the definition of postbiotics, with publication of this definition expected by the end of 2020.)

 

The precise short- and long-term health benefits of adding the above ingredients to infant formula are still under study. One pediatric society (the ESPGHAN Committee on Nutrition) examined the data in 2011 and at that time did not recommend the routine use of infant formulas with added probiotic and/or prebiotic components until further trials were conducted. A systematic review concluded that evidence for the health benefits of fermented infant formula (compared with standard infant formula) are unclear, although improvements in infant gastrointestinal symptoms cannot be ruled out. Although infant formulas are undoubtedly improving, review co-author Hania Szajewska, MD, Professor of Paediatrics at The Medical University of Warsaw, Poland, says, “Matching human milk is challenging. Any alternative should not only match human milk composition, but should also match breastfeeding performance, including how it affects infant growth rate and other functions, such as the immune response.”