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Postbiotics: A global perspective on regulatory progress

September 13, 2024/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.

2023 in Review: Highlights in the Field of Biotic Science

December 15, 2023/in ISAPP Science Blog /by KC

By Kristina Campbell, Prof. Colin Hill PhD, Prof. Sarah Lebeer PhD, Prof. Maria Marco PhD, Prof. Dan Merenstein MD, Prof. Hania Szajewska MD PhD, Prof. Dan Tancredi PhD, Prof. Kristin Verbeke PhD, Dr. Gabriel Vinderola PhD, Dr. Anisha Wijeyesekera PhD, and Marla Cunningham

Biotic science is an active field, with over 6,600 scientific papers published in the past year. The scientific work that emerged in 2023 covered many diverse areas – from probiotic mechanisms of action to the use of biotics in clinical populations. In parallel with the scientific advancements, consumer interest in gut health and biotics is at an all-time high. A recent survey showed that 67 percent of consumers are familiar with the concept of probiotics and 51 percent of those who consume probiotics do so with the aim of supporting gut health.

Several ISAPP-affiliated experts took the time to reflect on 2023 and identify the most important directions in the fields of probiotics, prebiotics, synbiotics, postbiotics, and fermented foods. Below are these experts’ picks for the top developments in biotic science and application during the past year.

Increased recognition of biotics as a category

After ISAPP’s publication of the recent synbiotics and postbiotics definitions in 2020-2021, board members and others began referring to probiotics, prebiotics, synbiotics, and postbiotics collectively as “biotics”. 2023 has seen the term being used more widely (for example, in article headlines and communications from major organizations) to refer to these substances as a broad group.

Steps forward and steps back in the regulation of live microbial interventions

The actions of regulators have a profound impact on how biotic science is applied and how products can reach consumers. On the positive side, 2023 heralded the regulatory approval of two live microbial drug products for recurrent C. difficile infection by the US Food and Drug Administration (FDA). Both products are derived from fecal samples, but one is delivered to the patient gastrointestinal (GI) tract by enema, and the other is delivered orally.

Meanwhile, a case of fatal bacteremia in a preterm infant who had been given a probiotic product prompted the FDA to issue a warning letter to healthcare practitioners about probiotics in preterm infants, as well as warning letters to two probiotic manufacturers. These actions had the concerning effect of reducing access to probiotics for this population, despite the accumulated evidence that probiotics effectively prevent necrotizing enterocolitis in preterm infants. As outlined in ISAPP’s scientific statement on the FDA’s actions, the regulatory decision weighting the risks of commission over omission did not reflect the wealth of evidence for probiotic efficacy in this population and the low risk of harm.

Wider awareness of the postbiotic concept and definition

Scientific discussions on postbiotics continued throughout 2023, with several debates and conference sessions devoted to discussion of the postbiotic concept – including the status of metabolites in the definition. According to ISAPP board member Dr. Gabriel Vinderola PhD, who was a co-author on the definition paper and an active participant in many of these debates, the ISAPP definition is gaining traction and the debates have been useful in pinpointing further areas of clarification for the sake of regulators and other stakeholders. As shared with the audience at Probiota Americas 2023 in Chicago, Health Canada became the first regulatory agency to address the definition, and has started considering the term postbiotics under the ISAPP definition.

Advances in technologies for analyzing different sites in the digestive tract

When studying how biotics interface with the host via the gut microbiota, the science has relied mainly on analysis of fecal samples, with the majority of the GI tract remaining a ‘black box’. But a 2023 paper by Shalon et al., which was discussed at the ISAPP meeting in Denver, describes a device able to collect intestinal samples from different regions in the GI tract. Analysis of the metabolites and microbes indicated clear regional differences, as well as marked differences between samples in the GI tract versus fecal samples (for example, with respect to bile acids); an accompanying paper revealed novel insights into diet and microbially-derived metabolites. Efforts are underway across the world to develop smart pills or robotic pills that take samples all along the GI tract. Some devices have sensors that immediately signal to a receiver and others have been engineered to release therapeutic contents. Although these technologies may need more validation before they are useful in research or clinical contexts, they may greatly expand knowledge of the intestinal microbial community and how it interacts with biotic substances.

First convincing evidence linking intake of live microbes with health benefits

When an ISAPP discussion group in 2019 delved into the question of whether a higher intake of safe, uncharacterized live microbes had the potential to confer health benefits, it spurred a program of scientific work to follow. Efforts of this group in subsequent years led to the publication of an important study in 2023: Positive Health Outcomes Associated with Live Microbe Intake from Foods, Including Fermented Foods, Assessed using the NHANES Database. Researchers analyzed data from a large US dietary database and found clear but modest health benefits associated with consuming higher levels of microbes in the daily diet.

The benefits of live dietary microbes are being explored further in the scientific literature (for example, here, here, and here) and are likely to remain an exciting topic of study in the years ahead, building evidence globally for the health benefits of consuming a higher quantity of live microbes.

Increased interest in candidate prebiotics

Polyphenols have long been studied for their health benefits, but newer evidence suggests they may have prebiotic effects, achieving their health benefits (in part) through interactions with the gut microbiota. A theme at conferences and in the scientific literature has been the use of polyphenols to modulate the gut microbiota for specific health benefits. More than a dozen reviews on this topic were published in 2023, and several of them focused on how polyphenols may achieve health benefits in very specific conditions, such as diabetes or inflammatory bowel disease.

Another substrate receiving much attention for its prebiotic potential are human milk oligosaccharides (HMOs). HMOs, found in human milk, support a nursing infant’s health by encouraging the growth of beneficial gut microbes. Several articles in 2023 have delved into the mechanisms of HMO metabolism by the gut microbiota, and explored its potential as a dietary intervention strategy to improve gut health in adults.

Sharper focus on evidence for the health and sustainability benefits of fermented foods

Fermented foods are popular among consumers, despite only early scientific knowledge on whether and how they might confer health benefits (see ‘First convincing evidence linking intake of live microbes with health benefits’, above). ISAPP board member Prof. Maria Marco PhD co-authored a review led by Dr. Paul Cotter PhD in Nature Reviews Gastroenterology and Hepatology on the GI-related health benefits of fermented foods. The paper clearly lays out the potential mechanisms under investigation and identifies gaps to be addressed in the ongoing study of fermented foods.

As calls for reducing carbon footprints continue across the globe, plant-based fermented foods are being singled out as an area for innovation and expansion. One example of how these foods are being explored is through the HealthFerm project, a 4-year, 13.1 million Euro project involving 23 partners from 10 countries, which is focused on understanding how to achieve more sustainable, healthy diets by leveraging fermented foods and technologies.

Novel findings related to lactic acid bacteria

Lactic acid bacteria (LAB) are some of the most frequently-studied microbial groups, but scientists have only begun to uncover the workings of this diverse group of bacteria and how they affect a variety of hosts. These bacteria are used as probiotics and are often beneficial members of human and animal microbiomes, and they are also essential to making fermented foods. This year marked the first ever Gordon Research Conference on LAB in California, USA. Attendees showcased the diversity of research on lactic acid bacteria, and the meeting was energized by the early investigators present and by the interest in LAB in other disciplines including medicine, ecology, synthetic biology, and engineering. One example of a scientific development in this area was the further elucidation of the mechanism of Lactiplantibacillus plantarum’s extracellular electron transfer.

Progress on the benefits and mechanisms of action for probiotics to improve the effectiveness of cancer immunotherapies

Researchers have known for several years that the gut microbiota can be a determinant of the efficacy of cancer immunotherapy drugs that involve immune checkpoint blockade, but interventions that target the gut microbiota to improve response to immunotherapies have been slower to develop. This year saw encouraging progress in this important area, with probiotic benefits and mechanisms of action being demonstrated in several papers. Two of the most highly cited probiotics papers of the year centered on this topic: one showing how a tryptophan metabolite released by Limosilactobacillus reuteri (formerly Lactobacillus reuteri — see this ISAPP infographic) improves immune checkpoint inhibitor efficacy, and another paper that reviewed how gut microbiota regulates immunity in general, and immune therapies in particular.

Updated resource available on probiotics and prebiotics in gastroenterology

This year the World Gastroenterology Organisation (WGO) guidelines on probiotics and prebiotics were updated to reflect the latest evidence, with contributions from ISAPP board member Prof. Hania Szajewska MD PhD and former board member Prof. Francisco Guarner MD PhD. The guideline lists indications for probiotic and prebiotic use, and how the use of these substances may differ in pediatric versus adult populations. Find the guideline here.

Clarifying the role of metabolites in the postbiotic definition

August 10, 2023/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.

Postbiotics: debate continues and the ISAPP definition gains support

July 24, 2023/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 16^th 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.

Popular media, misinformation and ‘biotics’

April 7, 2023/in News /by KC

By Mary Ellen Sanders, PhD, Executive Science Officer, ISAPP

Encountering misinformation is all too easy when seeking understanding of probiotics, prebiotics, synbiotics, and postbiotics (collectively, ‘biotics’). It can be perpetuated both by proponents and detractors. Through this lens, I’m prompted to comment on some high profile pieces making news recently. A Washington Post article Probiotic supplements may do the opposite of boosting your gut health was published on March 28, 2023, by Anahad O’Connor. This author was then interviewed for a CBS video story Studies find that probiotics can harm gut health on March 30, 2023. Then, a National Geographic article Probiotics, prebiotics, postbiotics. What’s the difference? was published on the same day.

These pieces appropriately acknowledge the availability of evidence linking probiotics to human benefits. Yet the points raised about potential harms from probiotics and a misunderstanding of what ‘biotic’ substances really are deserve comment.

Harms of probiotics

Amid a backdrop of marketing and media messaging lauding the many benefits of probiotics, reporters are understandably drawn to the counter message that ‘probiotics can harm gut health’. Safety must always be rigorously assessed, as encouraged by a 2023 ISAPP paper focused on emerging issues in probiotic safety (see here). However, the claims of harm made – although generated from studies in humans – are not based on clinical endpoints. Instead they are based on either microbiome endpoints (Suez et al. 2018) or on post hoc analysis of biomarker outcomes (Wastyk et al. 2023). The limitations of the Suez et al. 2018 study were discussed in more detail previously (See: Clinical evidence and not microbiota outcomes drive value of probiotics). This paper evaluated the effect of one multi-strain probiotic product and is the only paper I am aware of that shows that probiotics inhibit microbiome recovery after antibiotic treatment. The paucity of supporting evidence for the harm supposedly documented in this paper is not mentioned in the stories. It is noteworthy that in the Wastyk et al. 2023 paper the authors acknowledge that the study did not achieve its primary objectives, and in referring to their post hoc analysis (including the ‘evidence’ for harm), they specifically acknowledge that such analysis is not conclusive evidence: “We next leveraged aspects of our study design … in a discovery analysis process to reveal trends that could inform possible … hypotheses for future studies.” These studies are best used for generating hypotheses requiring further study.

Another criticism that was leveraged as evidence that probiotics cause harm is that probiotics reduce microbiota diversity. Any probiotic-induced reduction in diversity of fecal microbiota has not been shown to be associated with harm. Further, most studies show no significant overall changes in microbiome composition in response to traditional probiotic administration. However, it should be understood that the value of diversity as a marker of health remains unproven. The evidence is from observational studies and only shows associations, not causality.

You can’t both object to criticisms based only on microbiome data but then promote probiotics based on it.

As stated, relying on microbiota endpoints to advance the idea that probiotics cause harm is not justified. But I cannot escape the fact that probiotic proponents in part contribute to this thinking. When probiotics are marketed as being able to ‘balance the microbiota’, without clinical data to substantiate a benefit, aren’t they promoting the same limited science?

Adherence to definitions of biotics needed

ISAPP has rigorously considered and offered definitions for probiotics, prebiotics, synbiotics, postbiotics and fermented foods (see here for a summary), which have been presented in highly cited reviews in Nature Reviews Gastroenterology and Hepatology (see here, here, here, here and here). These efforts were undertaken to advance a common understanding of these terms, so that precision can be attained in communications on them.

This objective has been far from realized. Misuse of these terms continues on product labels, in scientific publications, and in popular press communications.

The articles cited above compelled me to offer some take home messages for those responsible for accurately communicating science:

“Prebiotics + probiotics = postbiotics”, a heading in the National Geographic article, is completely wrong.

Probiotics are: Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host

Prebiotic is: A substrate that is selectively utilized by host microorganisms conferring a health benefit on the host

Postbiotic is: Preparation of inanimate microorganisms and/or their components that confers a health benefit on the host

Fermented foods are not the same as probiotics. Most fermented foods have not been proven to improve health (associative studies have suggested, but in most cases not proven, health benefits), many do not retain live microbes, and most are not made with microbes characterized to the strain level. All these are requirements to meet the definition of a probiotic. See here, here and here for clear discussions of this issue.
Fermented foods are not the magic bullet that many portray them as. Yes – for that subset of fermented foods that retain live microbes – they may contribute a diversity of live microbes to the diet. ISAPP has recently researched this area (see recent ISAPP publications here and here). And yes, they are tasty. However, the evidence level for benefits of traditional fermented foods is nowhere near the level for probiotics. Still, healthcare professionals critical of evidence supporting probiotic benefits commonly recommend fermented foods.
Postbiotics do not refer to ‘metabolites from probiotics’. See here for why ISAPP focused the definition of postbiotic on inactivated microbes with or without their metabolites.
In simplistic language, prebiotics can be viewed as food for beneficial microbes, but, typically, prebiotics target the normal microbes in the gut, not probiotics. See here.

Conclusion

Both the positive and negative effects of probiotics based on microbiome assembly can be misrepresented in the press, by some marketing claims, and sometimes in scientific literature. The field will benefit from communications that acknowledge the limitations of available science. Further, it’s important for clarity in communication that the field coalesces around established definitions and honor the criteria needed to meet those definitions. Additionally, scientists and medical professionals should apply the same scrutiny and critical thinking to fermented foods as they do to probiotics.

ISAPP encourages healthy debate, critical review of new studies and innovative research. Since ISAPP’s mission is focused on promoting the science of these substances, journalists are invited to reach out as needed to ISAPP for an evidence-based perspective on this evolving field (www.ISAPPscience.org).

Episode 18: The definition of postbiotics

March 6, 2023/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 definition of postbiotics, with Dr. Gabriel Vinderola and Prof. Seppo Salminen

Episode summary:

In this episode, the ISAPP podcast hosts join guests Gabriel Vinderola, PhD, Principal Researcher at the
National Scientific and Technical Research Council (CONICET) and Associate Professor at University of Litoral in Argentina, and Seppo Salminen, PhD, Professor at University of Turku in Finland, to discuss the relatively recent definition of postbiotics and what kinds of substances are included in this category. They talk about the criteria for something to qualify as a postbiotic, common mechanisms of action for postbiotics, and how postbiotic science has brought new perspectives on the study of probiotics.

Key topics from this episode:

What are postbiotics? Dr. Vinderola and Prof. Salminen dive deep into the definition of postbiotics created in 2021 and what it entails.
Postbiotics, similar to probiotics, prebiotics, and synbiotics, must provide health benefits to the host.
The nature of the postbiotic preparation is important for its health benefits. When the inactivation process is changed, this can lead to altered health benefits, and clinical studies must be repeated to ensure the desired health benefits are maintained.
They explain why “inanimate” was chosen to describe the microorganisms / components in a postbiotic preparation.
What is the mode of action, or how do postbiotics work?
- Postbiotics show similar mechanisms of action to probiotics, except for ones requiring viability, since postbiotics will not grow and produce metabolic byproducts in the host.
- Postbiotics can benefit the host via physical interaction with the host epithelial and immune cells.
- A primary mechanism of action is likely to be through activation of the immune system, through which postbiotics can affect inflammation and some disease conditions.
- Postbiotics may also affect the microbiome composition and ability to inhibit pathogens.
From a regulatory point of view, inanimate microorganisms may represent an easier category to prove safe for users. For industry, postbiotics may be more convenient with a longer shelf life.
Some controversy still exists around the ISAPP-led postbiotic definition, and this has led to valuable discussions that are crucial to scientific progress. So far the authors of the definition have defended their stance.

Episode abbreviations and links:

ISAPP scientific consensus definition of postbiotics, published in Nature Reviews Gastroenterology & Hepatology: The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics
Study on a postbiotic for addressing chronic stress in students: Health Benefits of Lactobacillus gasseri CP2305 Tablets in Young Adults Exposed to Chronic Stress: A Randomized, Double-Blind, Placebo-Controlled Study
Study using live bifidobacteria to address irritable bowel syndrome: Randomised clinical trial: Bifidobacterium bifidum MIMBb75 significantly alleviates irritable bowel syndrome and improves quality of life–a double-blind, placebo-controlled study
Study using dead bifidobacteria (same strain as above) to address irritable bowel syndrome: Heat-inactivated Bifidobacterium bifidum MIMBb75 (SYN-HI-001) in the treatment of irritable bowel syndrome: a multicentre, randomised, double-blind, placebo-controlled clinical trial
Opinion from the European Food Safety Authority (EFSA) Panel on Nutrition, Novel Foods and Food Allergens (NDA) on pasteurized Akkermansia muciniphila as a novel food (NF) pursuant to Regulation (EU) 2015/2283: Safety of pasteurised Akkermansia muciniphila as a novel food pursuant to Regulation (EU) 2015/2283

Additional Resources:

Postbiotics. ISAPP infographic (also available in Japanese and Spanish).

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

Definition of postbiotics: A panel debate in Amsterdam. ISAPP blog post.

About Dr. Gabriel Vinderola:

Gabriel Vinderola graduated at the Faculty of Chemical Engineering from the National University of Litoral (Santa Fe, Argentina) in 1997. He obtained his Ph.D. in Chemistry in 2002 at the same University. He collaborated with several research teams in Canada, Spain, France, Italy, Germany, Brazil and Finland. He is presently Principal Researcher of the National Scientific and Technical Research Council (CONICET) and Associate Professor at the Food Engineering Department of his home Faculty. He participated in 1999 in the development of the first commercial cheese carrying probiotic bacteria in Latin America. In 2011, he was awarded the prize in Food Technology for young scientists, by the National Academy of Natural, Physic and Exact Sciences from Argentina. He published more than 120 original scientific publications in international refereed journals and book chapters. From 2020 to present, he serves as a member of the board of directors of the International Scientific Association for Probiotics and Prebiotcis (ISAPP). He is engaged in science communication to the general public through Instagram (@gvinde).

About Prof. Seppo Salminen:

Seppo Salminen, MSc, MS, PhD, is a Senior Advisor, Functional Foods Forum (FFF) at the University of Turku. His areas of expertise are gut microbiota, probiotics and prebiotics, nutrition and food safety, and EU regulations. Seppo teaches the topics of lactic acid biotechnology, functional foods and EU legislation and conducts research into food and health, intestinal microbiota, probiotics, prebiotics, functional foods, food legislation, health claims, and novel foods.

Definition of postbiotics: A panel debate in Amsterdam

December 5, 2022/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola, PhD, Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at the Dairy Products Institute (CONICET-UNL), Santa Fe, Argentina.

A panel debate titled “Postbiotics, definition and scopes” was convened at the 9^th Beneficial Microbes conference in Amsterdam on November 14, 2022. The aim of this panel was to advance the discussion about postbiotics in the aftermath of some published disagreement (see here and here) about the definition of postbiotics produced and published by ISAPP: “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. The debaters included Prof. Seppo Salminen and myself (Dr. Gabriel Vinderola), both members of the board of directors of ISAPP and co-authors of the ISAPP postbiotics definition, supporting the ISAPP definition, and Prof. Lorenzo Morelli (in attendance virtually) and Dr. Guus Roeselers challenging the ISAPP definition. The debate was attended by around 150 persons, and consisted of 15-minute opening arguments on both sides, followed by a 30 min open discussion guided by the conference chair, Dr. Koen Venema.

I introduced ISAPP as a non-profit organization dedicated to advancing the science on probiotics, prebiotics and related substances. Among many other activities, ISAPP has produced 5 different consensus definitions: probiotics, prebiotics, synbiotics, postbiotics and fermented foods. Each consensus panel was composed of academic scientists with different backgrounds, expertise and perspectives, comprising at least 11 authors from 4 – 10 countries, who came together to incorporate broad perspectives and engage in thoughtful debate. To date, all 5 consensus papers have had almost half a millon accesses at Nature Reviews Gastroentetology and Hepatology, the journal where all of the definitions are published.

The discussion within ISAPP about the need for a postbiotic definition dates back to our 2019 annual meeting. Emerging research on the health benefits conferred by non-viable microbes, their fragments and metabolites was discussed at the meeting, and this planted the seed for a definition that would cover this area. Many different terms such as heat-killed probiotics, heat-treated probiotics, heat-inactivated probiotics, tyndallized probiotics, paraprobiotics, ghost probiotics, cell fragments, cell lysates and postbiotics had been used to encompass these substances.

The panel discussed these different terms and previously published definitions. Those opposed to the ISAPP definition preferred the Tsilingiri and Rescigno (2013)¹ definition of postbiotics, which focuses on metabolites produced by probiotics. I reviewed the limitations of that definition, which were outlined in Salminen et al. (2021)². One concern is that requiring a postbiotic to be derived from a probiotic creates an unnecessary burden of first meeting the criteria for a probiotic before developing a postbiotic.

Morelli emphasized the importance of definitions for regulatory bodies and stated that researchers should provide guidance on criteria to meet a definition. He quoted the first published definition of postbiotic by Tsilingiri and Rescigno in 2013¹: “any factor resulting from the metabolic activity of a probiotic or any released molecule capable of conferring beneficial effects to the host in a direct or indirect way”. Morelli stated that one value of this definition was that it was clear to regulators; metabolites are measurable and produced by microbes already accepted as food components with a long history of safe use. He considered this of paramount relevance as otherwise, the novel foods path would be required. He challenged the ISAPP approach as defining a substance that was unclear how to measure. Morelli showed pictures depicting the deterioration of the biomass of freeze-dried cultures during storage, to underscore the challenges of controlling the quality of products based on biomass of non-viable microbes. He added, “If we don´t know which are the components responsible for the health benefits, then it is challenging to determine what to measure.” He questioned the ability to establish the shelf life of such a product. The need to be precise in terms of how to quantify the active components of non-viable cells was essential to his criticism of ISAPP’s definition of postbiotics. Prof. Morelli concluded that researchers must address this issue of quantification methods, both to advance research and to provide regulatory bodies needed approaches to regulating non-viable microbes.

Conclusions from the debate were that the flaws of definitions previous to the ISAPP definition are apparent, and that the substance defined by ISAPP was useful to delineate, but that clear approaches to measurement of the active component(s) of non-viable microbes are needed to make the ISAPP definition workable in scientific and regulatory circles. The debate was very worthwhile, since science advances through respectful debates such as this.

It is clear that characterization of postbiotic products may be challenging, especially with increased complexity that arises by use of multiple inanimate strains, inclusion of metabolic endproducts, and the presence of whole and fragmented cells. But these challenges are not unique to postbiotics. Probiotic products can comprise complex mixtures of multiple strains as well as metabolic products (as the biomass during industrial production is harvested for freeze-drying, but not washed), along with significant amounts of non-viable microbes, which all may contribute to the overall health benefit. These facts are usually overlooked when relying just on viable cells for quantification.

Many commercial products carrying inanimate microbes and metabolic fermentation products, that potentially fit the ISAPP definition of postbiotics, are already available in the market. These are diverse products such as a mixture of two lactobacilli aimed at treating infant and adult diarrhea³ or a fermented infant formula to support pediatric growth⁴. Similar products also target animal nutrition⁵. A tightly controlled manufacturing process may be the path forward to warrant reproducibility of health benefits. Suitable characterization methodologies such as flow cytometry for non-viable microbes and mass spectrometry for metabolites seem to be relevant to sufficient postbiotic product characterization.

In brief, the ISAPP definition itself seemed well accepted by the meeting participants, but concerns were raised about how to quantify postbiotics according to the definition. We intend to address this point through consultations with experts, proposing scientific paths to help conceptualize factors that need to be considered for postbiotic quantification.

Panel debate about ISAPP’s definition of postbiotics held at Beneficial Microbes conference in Amsterdam on November 14th, 2022. On the stage, from left to right: Koen Venema (conference chair), Gabriel Vinderola, Seppo Salminen, Guus Roeselers and Lorenzo Morelli (on screen).

References

Tsilingiri, K. & Rescigno, M. Postbiotics: What else? Benef. Microbes (2013) doi:10.3920/BM2012.0046.
Salminen, S. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. (2021) doi:10.1038/s41575-021-00440-6.
Malagón-Rojas, J. N., Mantziari, A., Salminen, S. & Szajewska, H. Postbiotics for Preventing and Treating Common Infectious Diseases in Children: A Systematic Review. Nutrients 12, (2020).
Béghin, L. et al. Fermented infant formula (with Bifidobacterium breve C50 and Streptococcus thermophilus O65) with prebiotic oligosaccharides is safe and modulates the gut microbiota towards a microbiota closer to that of breastfed infants. Clin. Nutr. 40, 778–787 (2021).
Kaufman, J. D. et al. A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms. Sci. Rep. 11, 6407 (2021).

Additional reading:

Follow up from ISAPP webinar – Probiotics, prebiotics, synbiotics, postbiotics and fermented foods: how to implement ISAPP consensus definitions

Postbiotics: The concept and their use in healthy populations

Watch / listen to the debate here: https://youtu.be/pATNfhQY4P4

Episode 12: Postbiotics and probiotics in Japan: A researcher’s perspective

October 6, 2022/in Podcast, Season One /by KC

Podcast: Play in new window | Download

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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 Prebiotic (ISAPP), a nonprofit scientific organization dedicated to advancing the science of these fields.

Postbiotics and probiotics in Japan: A researcher’s perspective, with Prof. Akihito Endo

Episode summary:

In this episode, ISAPP podcast host Dan Tancredi talks with food microbiologist Prof. Akihito Endo from Tokyo University of Agriculture in Japan, who was an author on the published ISAPP scientific consensus definition of postbiotics. Endo describes the unique properties of fructophilic lactic acid bacteria, and talks about the landscape of probiotic and postbiotic products in Japan.

Key topics from this episode:

Prof. Tomotari Mitsuoka originally introduced the concept of probiotics, prebiotics, and “biogenics” in Japan – the latter are similar to postbiotics.
Japan is a leading country in postbiotic applications, with many companies actively producing postbiotic products with killed bacterial cells.
Endo has done research on fructophilic lactic acid bacteria (FLAB), which favor fructose instead of glucose; they are found in flowers, fruits, fermented foods, and honeybee guts.
Novel species of FLAB have been discovered recently, and Endo found novel bacteriocin-producing FLAB. The bacteroicins may be active against pathogens.
Dead cells of FLAB are present in fermented foods so they have a history of safe consumption. There is one postbiotic product with FLAB in Japan at present.
Endo tested fresh honey and found it has viable FLAB. After 2 weeks they die because of antimicrobials present in honey. But there’s a safe consumption history even of the viable cells, albeit at low levels. He is interested in exploring them as probiotics in food products.
The Japanese regulatory environment has two health claim systems for ‘biotics’: FOSHU, FFC. FOSHU is more restricted, while FFC can have more diverse health claims.
A large variety of postbiotic products are available in Japan.
One mechanism by which FLAB confer health benefits is through IgA induction (i.e. influencing immune activity).

Episode abbreviations and links:

The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics

Mitsuoka T. 1998. Functional Food: Probiotics, Prebiotics, Biogenics. Intestinal Flora and Probiotics.

Mitsuoka. 2011. History and Evolution of Probiotics. Japanese Journal of Lactic Acid Bacteria.

Background on fructophilic lactic acid bacteria: Are fructophilic lactic acid bacteria (FLAB) beneficial to humans?

Viable fructophilic lactic acid bacteria present in honeybee-based food products

Japanese categories for health benefit claims on foods (for more details, see ISAPP consensus statement):

FOSHU – Food for Specialized Health Use
FFC – Food with Functional Claims

On Kikunae Ikeda’s discovery of umami: Glutamate: from discovery as a food flavor to role as a basic taste (umami)

Additional resources:

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

Postbiotics. ISAPP infographic

What is a postbiotic? ISAPP video

About Prof. Aki Endo:

Akihito Endo is a research professor at Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, Japan. He obtained a PhD degree of Fermentation Science with a topic on Lactic Acid Bacterial Diversity during Shochu Fermentation at Tokyo University of Agriculture in 2005. After he studied as a postdoc in Stellenbosch University (South Africa) and in University of Turku (Finland), he started to work in Tokyo University of Agriculture in 2013. His research themes are ecology and food application of fructophilic lactic acid bacteria and oligosaccharide metabolism in human gut anaerobes. He is a member of Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms, International Committee on Systematics of Prokaryotes since 2011 and a board member of Japanese Society for Lactic Acid Bacteria since 2021.

ISAPP’s 2021 year in review

December 14, 2021/in ISAPP Science Blog /by KC

By Mary Ellen Sanders, PhD, ISAPP Executive Science Officer

The upcoming year-end naturally leads us to reflect about what has transpired over the past 12 months. From my perspective working with ISAPP, I witnessed ISAPP board members and the broader ISAPP community working creatively and diligently to find solutions to scientific challenges in probiotics, prebiotics and related fields. Let’s look back together at some of the key developments of 2021.

ISAPP published outcomes from two consensus panels this year, one on fermented foods and one on postbiotics. The popularity of these articles astounds me, with 49K and 29K accesses respectively, as of this writing. I think this reflects recognition on the part of the scientific community of the value – for all stakeholders – of concise, well-considered scientific definitions of terms that we deal with on a daily basis. If we can all agree on what we mean when we use a term, confusion is abated and progress is facilitated. The postbiotics definition was greeted with some resistance, however, and it will remain to be seen how this is resolved. But I think ISAPP’s response about this objection makes it clear that productive definitions are difficult to generate. Even if the field ultimately embraces another definition, it is heartening to engage in scientific debate about ideas and try to find alignment.

Keeping with the idea of postbiotics, a noteworthy development this year was the opinion from the European Food Safety Authority that the postbiotic made from heat-treated Akkermansia muciniphila is safe for use as a novel food in the EU. Undoubtedly, this development is a bellwether for likely future developments in this emerging area as some technological advantages to postbiotics will make these substances an attractive alternative to probiotics IF the scientific evidence for health benefits becomes available.

Recognizing the existing need for translational information for clinicians, ISAPP developed a continuing education course for dietitians. Published in March, it has currently reached close to 6000 dietitians. This course focused on probiotics, prebiotics and fermented foods: what they and how they might be applied in dietetic practice. It is a freely available, self-study course and completion provides two continuing education credits for dietitians.

On a sad note, in March of this year, ISAPP suffered the loss of Prof. Todd Klaenhammer. Todd was a founding ISAPP board member, and directed many of our activities over the course of his 18-year term on the board. He was also my dear friend and major advisor for my graduate degrees at NC State many years ago. As one former collaborator put it, “I was not prepared to finish enjoying his friendship and mentorship.” See here for a tribute to Prof. Klaenhammer on the ISAPP blog: In Memoriam: Todd Klaenhammer.

So where will 2022 lead ISAPP? The organization has now published five consensus definitions: probiotics, prebiotics, synbiotics, postbiotics and fermented foods – extending its purview beyond where it started, with probiotics and prebiotics. Through the year ahead, ISAPP is committed to providing science-based information on the whole ‘biotics’ family of substances as well as fermented foods. Our Students and Fellows Association is growing, supported by the opportunity for young scientists to compete for the Glenn Gibson Early Career Researcher Prize. We continue to see our industry membership expand. Through our new Instagram account and other online platforms, our overall community is increasing. The ISAPP board of directors continues to evolve as well, with several long-term members leaving the board to make room for younger leaders in the field who will direct the future of the organization. This applies to me as well, as I have made the difficult decision to depart ISAPP in June of 2023. Thus, hiring a new executive director/executive science officer is an important priority for ISAPP in 2022. My 20 years with ISAPP have seen the organization evolve tremendously, through the hard work of incredible board members as well as many external contributors. We will strive to make 2022 – our 20^th anniversary – ISAPP’s best year yet.

Should the concept of postbiotics make us see probiotics from a new perspective?

October 5, 2021/in ISAPP Science Blog, Uncategorized /by KC

By Dr. Gabriel Vinderola, PhD, 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), Santa Fe, Argentina

In early May 2021 an ISAPP consensus panel defined postbiotics as “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host“. The fact that non-viable microbes may still deliver health benefits is not new for the scientific community and was reviewed more than 20 years ago. More recent studies demonstrating health effects of non-viable microbes spurred interest in this topic, leading ISAPP to carefully consider the emerging use of the term ‘postbiotic’ and provide a clear, modern, concise definition.

Postbiotics can be contrasted with probiotics: live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In practice, probiotics have likely always coexisted with inanimate microbes, as live microbes will die at all phases of production of a product. In the past, it seems the presence of inanimate microbes as part of probiotic products was not really considered. We all knew they were there, but made a default assumption that they had limited significance. As we consider postbiotics, though, we should perhaps look again at how to address the inanimate components of probiotic products.

The presence of inanimate cells in probiotic preparations: from lab to product

A loop of a fresh, overnight, live culture of a probiotic strain may still contain non-viable cells (Fig. 1). During the biomass production of a probiotic culture, an abundance of live cells can be observed in the exponential growth phase, but as the culture enters the stationary phase, a significant increase in the proportion of non-viable cells occurs (Fig. 2). Yet the culture may display a satisfactory high number of viable cells as verified by traditional plating on agar media. Some years ago, it was reported that a fresh culture of a lactobacilli strain may display a live/dead cells ratio of ca. 100/1. However, this ratio may change to 1:1 after freeze-drying, as studied using flow cytometry, a technology that allows the quantification of both live and dead cells in a culture-independent way. Therefore, a recently freeze-dried culture of a probiotic strain may contain 10¹⁰ log CFU/g of live cells, but also the same amount of non-viable cells.

Food supplements may have a shelf life between 12 and 24 months at room temperature and over this time, a proportion of cells will likely lose viability along the shelf life. This depends on the intrinsic resistance of the strain, the nature of the matrix used for freeze-drying, the water activity remaining after lyophilization, the package and the storage conditions. Taking this into consideration, the probiotic supplements industry overfills probiotic capsules or sachets with 1.5 to 4 times more live cells, in order to warrant the delivery at the end of shelf life of the minimum amount of live cells to be able to deliver the expected health benefit. Considering that both freeze-drying and long-term storage may significantly increase the proportion of inanimate microorganisms in a probiotic supplement, a probiotic supplement could easily consist of more inanimate microorganisms than live ones. Yet if the products delivers the minimum amount of live cells to confer a health benefit, this makes the product fit the definition of probiotics so it must be considered a probiotic product. The probiotic focus has been prevalent during previous clinical trials and also during the shelf life of a probiotic product. Maybe we were just overlooking what was going on beyond the information obtained by CFU. These new insights do not change the status of a probiotic, but with due attention given to postbiotic components, offers the possibility to have better and better characterized products in the future.

Figure 1, above – Fluorescence microscopy images of an overnight (18h) culture of bifidobacteria (left) and lactobacilli (right) showing live (green) and non-viable cells (red). The Live/dead BackLight Invitrogen® kit was used for staining cells.

Figure 2, above – Fluorescence microscopy images of a culture of lactobacilli in the exponential (left) and late stationary (right) growth phase showing live (green) and non-viable cells (red). The Live/dead BackLight Invitrogen® kit was used for staining cells.

Are dead probiotics ‘postbiotics’?

What is the contribution of these inanimate cells to the overall health benefit observed for the probiotic culture? In most cases, no evidence exists documenting health benefits of inanimate probiotics. But we may have reason to suspect it may be relevant. For example, a live culture of Bifidobacterium bifidum MIMBb75 significantly alleviated irritable bowel syndrome symptoms and improved quality of life in a double-blind, placebo-controlled study when delivered at 10⁹ CFU, but also the same strain performed equally well for the same end-point when delivered as a heat-inactivated culture. Also, a novel next-generation probiotic strain of Akkermansia muciniphila performed equally well in its live and pasteurized form for improving several metabolic parameters in overweight and obese volunteers. In these cases, it can be said that both strains fit simultaneously the probiotic and the postbiotic definitions.

However, does this mean that as the strain gradually loses viability during storage it gradually becomes a postbiotic? No! This is because method of strain inactivation may play an important role in the health benefit observed. For example, the health benefit delivered by a strain that underwent a heat inactivation can not be assumed to have the same functionality if it is left to die on its own on the shelves. A heat treatment may, for instance, modifiy the spatial display of surface proteins and this may lead to a different immunomodulating capacity of the strain when compared to spontaneous and gradual cell viability lost along storage.

Characterizing probiotic products with an eye to the presence of non-viable cells

By definition, probiotics must be quantified. In the past, this quantification has been limited to numbers of viable cells, typically using a colony count method. This is wholly appropriate, as probiotics must be alive. Yet for the future, will it become necessary to quantify the numbers of non-viable microbial cells as well? With evidence emerging that these non-viable cells may be functional components, then a reasonable argument can be made that this component of a probiotic product should also be quantified. This has implications for characterizing products for use in intervention trials and for the marketplace. The challenge for the marketplace is that probiotic products should deliver the functionality observed in intervention trials.

Reports of trials typically indicate a viable count of the probiotic being tested, but these can be reported in different ways. For example, the statement may indicate delivery of 1.9 × 10⁷ CFU/day of the strain XXX, or delivery of > 1.9 × 10⁷ CFU/day of the strain XXX. These are very different and neither gives any indication of the level of non-viable microbes. The first expression is a specific measure of the viable count at a particular point in time. The second indicates a target minimum and the actual count of viable cells could be much greater. Counts all along the intervention are rarely reported, even though that count could change substantively over time. Papers rarely report if the same batch or different batches of the probiotic preparation were used. The potentially increasing proportion of inanimate microbes is never reported.

In light of postbiotics, future studies should report quantifications of both live and inanimate microbes. Although it is not clear at this time what role inanimate microbes may play in probiotic efficacy, a first step is understanding the composition of probiotic products.

ISAPP members can access Dr. Vinderola’s webinar on this topic here. Email info@nullisappscience.org if you require the password.

Bacterial vesicles: Emerging potential postbiotics

July 22, 2021/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola, PhD, 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), Santa Fe, Argentina

The recently published ISAPP consensus paper defines a postbiotic as “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host“. Such a definition quickly brings to mind that a postbiotic is not equivalent to microbial metabolites. A postbiotic should also contain inanimate microbial cells or cell fragments. Metabolites or fermentation products may be present, but they are not required.

Because microbes are complex entities, we must be open to innovative understandings of what a postbiotic might entail. Indeed, although not explicitly mentioned in the ISAPP consensus paper, extracellular membrane vesicles may comprise an innovative conceptualization of a postbiotic, falling within the ‘cell component’ part of the postbiotic definition.

Bacterial vesicles

Extracellular membrane vesicles (EMV) are universal carriers of biological information produced in all domains of life. Bacterial EMV are small, spheroidal, membrane-derived proteoliposomal nanostructures, typically ranging from 25 – 250 nm in diameter, containing proteins, lipids, nucleic acids, metabolites, numerous surface molecules and many other biomolecules derived from their progenitor bacteria (Figure 1). Bacterial vesicles have been known for more than 50 years as structures able to carry cellular material (Ñahui Palomino et al. 2021). However, studies on membrane vesicles derived from Gram-positive bacteria are more recent as it was for a time believed they were incapable of producing vesicles due to their thick and complex cell walls, and the lack of an outer membrane. Today, EMVs have been isolated from Gram-positive probiotic bacteria, including those belonging to the Lactobacillaceae family (under which Lactobacillus was recently split into many new genera) and the Bifidobacterium genus. In probiotic bacteria, vesicles may mediate quorum sensing and material exchange. Perhaps even more important, they can act as mediators of bacteria-to-cell and bacteria-to-bacteria interactions. As bacterial EMV are inanimate structures that cannot replicate, they fit the postbiotic definiton as cell components as long as other criteria stipulated by the definition are met.

Figure 1. Membrane vesicles budding on the surface of L. reuteri DSM 17938 and released into the surrounding medium. These vesicles were described in by Grande et al. 2017. Photo used with permission of BioGaia.

Functions of bacterial vesicles related to potential health benefits

Underlying mechanisms and corresponding molecules driving health effects of bacterial vesicles are not well understood, in part due to reliance on in vitro models. Bacterial EMV derived from Lactobacillaceae spp., Bifidobacterium spp., and Akkermansia spp. have been reported to alleviate metabolic syndrome and allergy symptoms, promote T-cell activation and IgA production, strengthen gut barrier function, and exhibit anti-viral and immunomodulatory properties (Kim et al. 2016; Tan et al. 2018; Ashrafian et al. 2019; Molina-Tijeras et al. 2019; Palomino et al. 2019; Shehata et al. 2019; Bäuerl et al. 2020). Interestingly, vesicles from Limosilactobacillus reuteri DSM 17938 (West et al. 2020) and Lacticaseibacillus casei BL23 (Domínguez Rubio et al. 2017) may accomplish some of the the effects of these probiotic bacteria. In fact it is not unreasonable to think that EMVs may be already present and active in probiotic products.

Challenges for bacterial vesicle production

To develop a postbiotic from microbial EMVs, many challenges need to be overcome. Defining optimal cultivation conditions, and methods for vesicle release, isolation and scaling up are some of the challenges of bacterial vesicle production. There are several studies showing that altering the cultivation parameters can impact vesicle production. Examples of treatments shown to increase vesicle release include exposure to UV radiation and antibiotic pressure (Gamalier et al. 2017; Gill et al. 2019). Exposure to glycine has also been shown to increase vesicle production (Hirayama & Nakao 2020). Interventions during culture, for example by introducing agitation and varying pH, can possibly be ways to potentiate vesicle release and increase their bioactivity (Müller et al. 2021). A recent report also revealed that B. longum NCC2705 released a myriad of vesicles when cultured in human fecal fermentation broth, but not in basal GAM anaerobic medium (Figure 1). Moreover, the B. longum vesicle production pattern differed among individual fecal samples suggesting that metabolites derived from symbiotic microbiota stimulate the active production of vesicles in a different manner (Nishiyama et al. 2020). Whether any of these treatments and culture conditions are general or strain specific remains to be elucidated. Large differences in the number of vesicles that may be obtained by different extraction methods can occur (Tian et al. 2020). Tangential flow filtration or the use of antibodies targeting specific epitopes of the vesicles are some of the options proposed for the large scale isolation of EMV (Klimentová & Stulík 2015).

Figure 2. Left: Bifidobacterium longum NCC2705 grown on GAM broth. Right: secretion of membrane vesicles by Bifidobacterium longum NCC2705: the strain was cultured in bacterial-free human fecal fermentation broth and secreted a myriad of membrane vesicles. Reported and adapted from Nishiyama et al. 2020.

Progress has been made on the production of bacterial vesicles in recent years, yet several issues remain to be clarified including how vesicles are generated from the progenitor microbe, how the composition of vesicles changes according to the culture conditions, how to target specific bacterial vesicle purification from a pool of vesicles derived from other organisms (for example, bacterial vesicles produced in milky media can be accompanied by vesicles from eukaryotic cells present in the milk), safety aspects, quantification methods and the regulation of their use by the corresponding authority.

Their future as potential postbiotics

Membrane vesicles are an exciting opportunity for the development of postbiotics. A potential benefit of vesicles is that their small size compared to whole cells may enable them to more readily migrate to host tissues that could not be otherwise reached by a whole cell (Kulp & Kuehn 2010). Their nanostructure enables them to penetrate through the gut barrier and to be delivered to previously unreachable sites through the bloodstream or lymphatic vessels, and to interact with different cell types (Jones et al. 2020). For example, bacterial rRNA and rDNA found in the bloodstream and the brain of Alzheimer’s patients were postulated to have originated from bacteria vesicles (Park et al. 2017). Safety of EMVs must be carefully considered and assessed, even if they are derived from microbes generally recognized as safe, as their small size may increase penetration capacity with potential and yet unknown systemic effects. Novel postbiotics derived from microbial membrane vesicles is an intriguing area for future research to better understand production parameters, safety and functionality.

Thanks to Cheng Chung Yong, postdoctoral researcher at Morinaga Milk Industry Co., LTD (Japan) and Ludwig Lundqvist, industrial PhD student at BioGaia AB (Sweden) for their contributions to this blog, and Mary Ellen Sanders and Sarah Lebeer from ISAPP for fruitful discussions.

References

Ashrafian, F., Shahriary, A., Behrouzi, A., Moradi, H.R., Keshavarz Azizi Raftar, S., Lari, A., Hadifar, S., Yaghoubfar, R., Ahmadi Badi, S., Khatami, S. and Vaziri, F., 2019. Akkermansia muciniphila-derived extracellular vesicles as a mucosal delivery vector for amelioration of obesity in mice. Frontiers in microbiology, 10, p.2155.

Bäuerl, C., Coll-Marqués, J.M., Tarazona-González, C. and Pérez-Martínez, G., 2020. Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface. Scientific reports, 10(1), pp.1-12.

Domínguez Rubio, A.P., Martínez, J.H., Martínez Casillas, D.C., Coluccio Leskow, F., Piuri, M. and Pérez, O.E., 2017. Lactobacillus casei BL23 produces microvesicles carrying proteins that have been associated with its probiotic effect. Frontiers in microbiology, 8, p.1783.

Gamalier, J.P., Silva, T.P., Zarantonello, V., Dias, F.F. and Melo, R.C., 2017. Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation. Microbiological research, 194, pp.38-46.

Grande, R., Celia, C., Mincione, G., Stringaro, A., Di Marzio, L., Colone, M., Di Marcantonio, M.C., Savino, L., Puca, V., Santoliquido, R. and Locatelli, M., 2017. Detection and physicochemical characterization of membrane vesicles (MVs) of Lactobacillus reuteri DSM 17938. Frontiers in microbiology, 8, p.1040.

Gill, S., Catchpole, R. & Forterre, P., 2019. Extracellular membrane vesicles in the three domains of life and beyond. FEMS microbiology reviews, 43(3), pp.273–303.

Hirayama, S. & Nakao, R., 2020. Glycine significantly enhances bacterial membrane vesicle production: a powerful approach for isolation of LPS-reduced membrane vesicles of probiotic Escherichia coli. Microbial biotechnology, 13(4), pp.1162–1178.

Jones, E.J., Booth, C., Fonseca, S., Parker, A., Cross, K., Miquel-Clopés, A., Hautefort, I., Mayer, U., Wileman, T., Stentz, R. and Carding, S.R., 2020. The uptake, trafficking, and biodistribution of Bacteroides thetaiotaomicron generated outer membrane vesicles. Frontiers in microbiology, 11, p.57.

Kim, J.H., Jeun, E.J., Hong, C.P., Kim, S.H., Jang, M.S., Lee, E.J., Moon, S.J., Yun, C.H., Im, S.H., Jeong, S.G. and Park, B.Y., 2016. Extracellular vesicle–derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression. Journal of Allergy and Clinical Immunology, 137(2), pp.507-516.

Kulp, A. & Kuehn, M.J., 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annual review of microbiology, 64, pp.163–184.

Molina-Tijeras, J.A., Gálvez, J. & Rodríguez-Cabezas, M.E., 2019. The immunomodulatory properties of extracellular vesicles derived from probiotics: a novel approach for the management of gastrointestinal diseases. Nutrients, 11(5), p.1038.

Müller, L., Kuhn, T., Koch, M. and Fuhrmann, G., 2021. Stimulation of probiotic bacteria induces release of membrane vesicles with augmented anti-inflammatory activity. ACS Applied Bio Materials, 4(5), pp.3739-3748.

Ñahui Palomino, R.A., Vanpouille, C., Costantini, P.E. and Margolis, L., 2021. Microbiota–host communications: Bacterial extracellular vesicles as a common language. PLoS Pathogens, 17(5), p.e1009508.

Nishiyama, K., Takaki, T., Sugiyama, M., Fukuda, I., Aiso, M., Mukai, T., Odamaki, T., Xiao, J. Z., Osawa, R., & Okada, N. 2020. Extracellular vesicles produced by Bifidobacterium longum export mucin-binding proteins. Applied and Environmental Microbiology, 86(19), e01464-20.

Palomino, R.A.Ñ., Vanpouille, C., Laghi, L., Parolin, C., Melikov, K., Backlund, P., Vitali, B. and Margolis, L., 2019. Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues. Nature communications, 10(1), pp.1-14.

Park, J.Y., Choi, J., Lee, Y., Lee, J.E., Lee, E.H., Kwon, H.J., Yang, J., Jeong, B.R., Kim, Y.K. and Han, P.L., 2017. Metagenome analysis of bodily microbiota in a mouse model of Alzheimer disease using bacteria-derived membrane vesicles in blood. Experimental neurobiology, 26(6), p.369.

Shehata, M.M., Mostafa, A., Teubner, L., Mahmoud, S.H., Kandeil, A., Elshesheny, R., Boubak, T.A., Frantz, R., Pietra, L.L., Pleschka, S. and Osman, A., 2019. Bacterial outer membrane vesicles (omvs)-based dual vaccine for influenza a h1n1 virus and mers-cov. Vaccines, 7(2), p.46.

Tan, K., Li, R., Huang, X. and Liu, Q., 2018. Outer membrane vesicles: current status and future direction of these novel vaccine adjuvants. Frontiers in microbiology, 9, p.783.

Tian, Y., Gong, M., Hu, Y., Liu, H., Zhang, W., Zhang, M., Hu, X., Aubert, D., Zhu, S., Wu, L. and Yan, X., 2020. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. Journal of extracellular vesicles, 9(1), p.1697028.

West, C.L., Stanisz, A.M., Mao, Y.K., Champagne-Jorgensen, K., Bienenstock, J. and Kunze, W.A., 2020. Microvesicles from Lactobacillus reuteri (DSM-17938) completely reproduce modulation of gut motility by bacteria in mice. PloS one, 15(1

Follow up from ISAPP webinar – Probiotics, prebiotics, synbiotics, postbiotics and fermented foods: how to implement ISAPP consensus definitions

June 7, 2021/in ISAPP Science Blog /by KC

By Mary Ellen Sanders PhD, Executive Science Officer, ISAPP

On the heels of the most recent ISAPP consensus paper – this one on postbiotics – ISAPP sponsored a webinar for industry members titled Probiotics, prebiotics, synbiotics, postbiotics and fermented foods: how to implement ISAPP consensus definitions. This webinar featured short presentations outlining definitions and key attributes of these five substances. Ample time remained for the 10 ISAPP board members to field questions from attendees.

When considering the definitions, it’s important to remember that the definition is a starting point – not all criteria can be included. Using the probiotic definition as an example, Prof. Colin Hill noted that the definition is only 15 words – Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. This is a useful definition, stipulating the core characteristics of a probiotic. However, important criteria such as safety and identity are not specified in the definition yet are clearly delineated in the full paper on probiotics.

Several interesting topics emerged from this discussion, which will be explored in future blog posts. These include:

What is meant by host health? Microbe mediated benefits are numerous. But not all benefits are a benefit to host health. Benefits for user appearance, pets and potentially livestock may be measurable, economically important and desirable, but may not encompass ‘host health’.
What types of endpoints are appropriate for studies to meet the requirement of a health benefit? Endpoints that indicate improved health (such as symptom alleviation, reduced incidence of infections or quality of life measures) are targeted. Some physiological measures that may be linked to health (such as increased fecal short chain fatty acids or changes in microbiota composition) may not be sufficient.
What are the regulatory implications from these definitions? As suggested by the National Law Review article on the ISAPP consensus definitions, attorneys are interested in the scientific positions on how these terms are defined and characterized. Further, some regulatory actions – such as by Codex Alimentarius in defining probiotics – are underway. ISAPP is open to suggestions about the best way to communicate these definitions to regulators.
Is any follow-up by ISAPP to these papers anticipated in order to clarify criteria and provide simple guidance to their implementation?

Simple guidance to these substances can be found in the infographics: probiotics, probiotic criteria, prebiotics, fermented foods, how are probiotic foods and fermented foods different, synbiotics, and postbiotics. As mentioned above, watch for blog updates on implementation of the definitions for different stakeholder groups.

The recording of this webinar is available here under password protection for ISAPP industry members only.

Related information:

Consensus panel papers, all published in Nature Reviews Gastroenterology and Hepatology:

A roundup of the ISAPP consensus definitions: probiotics, prebiotics, synbiotics, postbiotics and fermented foods

May 11, 2021/in ISAPP Science Blog /by KC

By Dr. Mary Ellen Sanders, PhD, ISAPP Executive Science Officer

ISAPP has long recognized the importance of precise definitions of the ‘biotic’ family of terms. As a scientific organization working to advance global knowledge about probiotics, prebiotics, synbiotics, postbiotics and fermented foods, we believe carrying out rigorous scientific studies—and comparing one result to another—is more difficult if we do not start with a clear definition of what we are studying.

Over the past 8 years, ISAPP has endeavored to bring clarity to these definitions for scientists and other stakeholders. ISAPP board members have met with other top experts representing multiple perspectives and specialties in the field to develop precise, useful and appropriate definitions of the terms probiotics, prebiotics, synbiotics, postbiotics and fermented foods. The definitions of these first four terms have all entailed the requirement that the substance be shown to confer a health benefit in the target host. Fermented foods have multitudes of sensorial, nutritional and technological benefits, which drive their utility. A health benefit is not required.

The problem with health benefits

The definitions provide significant advantages for the scientific community in terms of clarity but complexity arises when the same definitions are accepted by regulatory agencies. This requirement for a health benefit as part of the probiotic definition has been rigorously implemented in the European Union. Currently, with the exception of a few member states, the term probiotic is prohibited. The logic is that since a health benefit is inherent to the term probiotic and since there are no approved health claims for probiotics in the EU*, the term ‘probiotic’ is seen to be acting as a proxy for a health claim. This has frustrated probiotic product companies who believe they have met the scientific criteria for probiotics, yet cannot identify their product as a probiotic in the marketplace because they have not received endorsement of their claims by the EU. This is not an issue resulting from an unclear definition, since probiotics surely should provide a health benefit, but rather from a lack of agreement as to what level of evidence is sufficient to substantiate a health benefit.

ISAPP remains committed to the importance of requiring a health benefit for the ‘biotic’ family of terms (outlined in the table below). It’s clear that all of these definitions are meaningless unless the requirement that they confer a health benefit is considered as essential by all stakeholders. One could reasonably discuss whether the required levels of evidence for foods and supplements are too high in some regulatory jurisdictions, but the value of these substances collapses in the absence of a health benefit.

Summary of ISAPP consensus definitions

With the publication of the most recent ISAPP consensus paper, this one on postbiotics, I offer a summary below of the five consensus definitions published by ISAPP. Each definition is part of a comprehensive paper resulting from focused discussions among experts in the field and published in Nature Reviews Gastroenterology and Hepatology (NRGH). These papers are among the top most viewed of all time on the NRGH website and are increasingly cited by scientists and regulators.

Table. Summary of ISAPP Consensus Definitions of the ‘Biotics’ Family of Substances. Probiotics, prebiotics, synbiotics and postbiotics have in common the requirement for a health benefit. They may apply to any target host, any regulatory category and must be safe for their intended use. Fermented foods fall only under the foods category and no health benefit is required.

	Definition	Key features of the definition	Reference
Probiotics	Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host	Grammatical correction of the 2001 FAO/WHO definition. No mechanism is stipulated by the definition.	Hill et al. 2014
Prebiotics	A substrate that is selectively utilized by host microorganisms conferring a health benefit	Prebiotics are distinct from fiber. Beneficial impact on resident microbiota and demonstration of health benefit required in same study.	Gibson et al. 2017
Synbiotics	A mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host	Two types of synbiotics defined: complementary and synergistic. Complementary synbiotics comprise probiotic(s) plus prebiotic(s), meeting requirements for criteria for each. Synergistic synbiotics comprise substrate(s) selectively utilized by co-administered live microbe(s), but independently, the components do not have to meet criteria for prebiotic or probiotic.	Swanson et al. 2020
Postbiotics	Preparation of inanimate microorganisms and/or their components that confers a health benefit on the host	Postbiotics are prepared from live microbes that undergo inactivation and the cells or cellular structures must be retained. Filtrates or isolated components from the growth of live microbes are not postbiotics. A probiotic that is killed is not automatically a postbiotic; the preparation must be shown to confer a health benefit, as well as meet all other criteria for a postbiotic.	Salminen et al. 2021
Fermented Foods	Foods made through desired microbial growth and enzymatic conversions of food components	Fermented foods are not the same as probiotics. They are not required to have live microbes characterized to the strain level nor have evidence of a health benefit. Types of fermented foods are many and are specific to geographic regions. Compared to the raw foods they are made from, they may have improved taste, digestibility, safety, and nutritional value.	Marco et al. 2021

*Actually, there is one approved health claim in the EU for a probiotic: Scientific Opinion on the substantiation of health claims related to live yoghurt cultures and improved lactose digestion (ID 1143, 2976) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

Further reading: Defining emerging ‘biotics’

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

May 5, 2021/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.

What’s the evidence on ‘biotics’ for health? A summary from five ISAPP board members

March 9, 2021/in Consumer Blog, ISAPP Science Blog /by KC

Evidence on the health benefits of gut-targeted ‘biotics’ – probiotics, prebiotics, synbiotics, and postbiotics – has greatly increased over the past two decades, but it can be difficult to sort through the thousands of studies that exist today to learn which of these ingredients are appropriate in which situations. At a recent World of Microbiome virtual conference, ISAPP board members participated in a panel that provided an overview of what we currently know about the health benefits of ‘biotics’ and how they are best used.

Here’s a summary of what the board members had to say:

Dr. Mary Ellen Sanders: Probiotics and fermented foods

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”.
Unfortunately, published assessments of probiotic products available on the market show that these products often fall short of required evidence. For example, their labels may not adequately describe the contents (including genus / species / strain in the product); they may not guarantee the efficacious dose through the end of the shelf life.
Contrary to common belief, probiotics do not need to colonize in the target site (e.g. the gut), impact gut microbiota composition, be derived from humans, or be resistant to stomach acid and other gut secretions such as bile.
Fermented foods are those made “through desired microbial growth and enzymatic conversions of food components”. The recent increased interest in fermented foods may come from people’s increased awareness of the role of gut microbes in overall health, but it is important to note that we have little direct evidence that the transient effects of fermented food microbes on the gut microbiota actually lead to health benefits. With that said, observational studies suggest that consuming some traditional fermented foods is associated with improved health outcomes.

Prof. Dan Merenstein, MD: Probiotics – How do I know what to prescribe for adult health?

A (limited) survey showed that most dietary supplement probiotic products cannot be linked to evidence because they do not provide enough information to determine what evidence exists to support their use – especially strains in the product. However, there are some probiotic products that have robust evidence.
Should every adult take a probiotic? The best evidence supports probiotics for improved lactose digestion and for prevention of difficile infection. Probiotics have also been shown to prevent common illnesses; reduce the duration of gut symptoms; and perhaps even reduce antibiotic consumption.
Studies will reveal more about the microbiome and about how probiotics work, for whom and for what indications. As with diet, the answer will most likely not be same for each person.

Prof. Glenn Gibson: Prebiotics and Synbiotics

A prebiotic is “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Researchers can test these substances’ activity in various ways: batch cultures, micro batch cultures, metabolite analysis, molecular microbiology methods, CF gut models, with in vivo (e.g. human) studies being required. Prebiotics appear to have particular utility in elderly populations, and may be helpful in repressing infections, inflammation and allergies. They have also been researched in clinical states such as IBS, IBD, autism and obesity related issues (Gibson et al., 2017).
A synbiotic is “a mixture, comprising live microorganisms and substrate(s) selectively utilized by host microorganisms, that confers a health benefit on the host.” While more studies are needed to say precisely which are useful in which situations, synbiotics have shown promise for several aspects of health in adults (Swanson et al. 2020): surgical infections and complications, metabolic disorders (including T2DM and glycaemia), irritable bowel syndrome, Helicobacter pylori infection and atopic dermatitis.

Prof. Hania Szajewska, MD: Biotics for pediatric use

Beneficial effects of ‘biotics’ are possible in pediatrics, but each ‘biotic’ needs to be evaluated separately. High-quality research is essential.
It is important that we view the use of ‘biotics’ in the context of other things in a child’s life and other interventions.
Breast milk is the best option for feeding infants
If breastfeeding is not an option, infant formulae supplemented with probiotics and/or prebiotics and/or postbiotics are available on the market.
Pro-/pre-/synbiotic supplemented formulae evaluated so far seem safe with some favorable clinical effects possible, but the evidence is not robust enough overall to be able to recommend routine use of these formulae.
Evidence is convincing on probiotics for prevention of necrotizing enterocolitis in preterm infants.
Medical societies differ in their recommendations for probiotics to treat acute gastroenteritis in children – they appear beneficial but not essential.
Synbiotics are less studied, but early evidence indicates they may be useful for preventing sepsis in infants and preventing / treating allergy and atopic dermatitis in children.

Prof. Gabriel Vinderola: Postbiotics

The concept of non-viable microbes exerting a health benefit has been around for a while, but different terms were used for these ingredients. Creating a scientific consensus definition will improve communication with health professionals, industry, regulators, and the general public. It will allow clear criteria for what qualifies as a postbiotic, and allow better tracking of scientific papers for future systematic reviews and meta-analyses.
The ISAPP consensus definition (in press) of a postbiotic is: “A preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”.
Postbiotics are stable, so no cold-chain is needed to deliver them to the consumer. Safety is of less concern because the microbes are not alive and thus cannot cause bacteraemia.
Research in the coming years will reveal more about postbiotics and the ways in which they can promote human health.

See here for the entire presentation on Biotics for Health.

Probiotics and fermented foods, by Dr. Mary Ellen Sanders (@1:15)

Postbiotics, by Prof. Gabriel Vinderola (@18:22)

Prebiotics and synbiotics, by Prof. Glenn Gibson (@33:24)

‘Biotics’ for pediatric use, by Prof. Hania Szajewska (@47:55 )

Probiotics: How do I know what to prescribe for adult health? by Prof. Dan Merenstein (@1:04:51)

Q&A (@1:20:00)

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

August 26, 2020/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.”

Posts

Increased recognition of biotics as a category

Steps forward and steps back in the regulation of live microbial interventions

Wider awareness of the postbiotic concept and definition

Advances in technologies for analyzing different sites in the digestive tract

First convincing evidence linking intake of live microbes with health benefits

Increased interest in candidate prebiotics

Sharper focus on evidence for the health and sustainability benefits of fermented foods

Novel findings related to lactic acid bacteria

Progress on the benefits and mechanisms of action for probiotics to improve the effectiveness of cancer immunotherapies

Updated resource available on probiotics and prebiotics in gastroenterology

Two competing terms

Questions about the ISAPP definition of postbiotic

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

Conclusions

References

The Science, Microbes & Health Podcast

The definition of postbiotics, with Dr. Gabriel Vinderola and Prof. Seppo Salminen

The Science, Microbes & Health Podcast

Postbiotics and probiotics in Japan: A researcher’s perspective, with Prof. Akihito Endo

Related information:

The problem with health benefits

Summary of ISAPP consensus definitions

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