The Concept of Postbiotics. — TEI-tagged view

Gabriel VinderolapersName, Mary Ellen SanderspersName, Seppo SalminenpersName (2022date). “The Concept of Postbiotics..” Foodstitle·j.

Object of study — the definitions of postbioticsterm·dom

Research question — what the consensus definition of postbioticterm·dom means, why it was chosen, and what it implies for foods

Contribution — a defence of the ISAPPterm definition, which centres the inanimate microbe rather than its metabolites

Full text — tagged

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The Concept of Postbioticsterm·dom.

Abstract The scientific community has proposed terms such as non-viable probioticsterm·dom, paraprobioticsterm·key, ghostbiotics, heat-inactivated probioticsterm·dom or, most commonly, postbioticsterm·dom, to refer to inanimate microorganismsterm·key and/or their components that confer health benefitsterm·key. This article addresses the various characteristics of different definitions of ‘postbioticsterm·dom’ that have emerged over past years. In 2021, the International Scientific Association for Probioticsterm·dom and Prebiotics (ISAPPterm) defined a postbioticterm·dom as “a preparation of inanimate microorganismsterm·key and/or their components that confers a health benefitterm·key on the host”. This definition of postbioticterm·dom requires that the whole or components of inactivated microbes be present, with or without metabolic end products. The definition proposed by ISAPPterm is comprehensive enough to allow the development of postbioticsterm·dom from different microorganisms, to be applied in different body sites, encouraging innovation in a promising area for any regulatory category and for companion or production animals, and plant or human health. From a technological perspective, probioticterm·dom products may contain inanimate microorganismsterm·key, which have the potential to impart a health benefitterm·key. However, their contribution to health in most cases has not been established, even if at least one probioticterm·dom has been shown to confer the same health benefitterm·key by live or inanimate cells.

1. Introduction Cell viability has long been regarded as important for a probioticterm·dom to confer a health benefitterm·key. However, it has been long recognized that non-viable microbes, their cell components, and their metabolites also can impact health. A number of different terms can be found in the published research to address these preparations: non-viable probioticsterm·dom, heat-killed probioticsterm·dom, tyndallized probioticsterm·dom, cell lysates, paraprobioticsterm·key, ghostbiotics and postbioticsterm·dom [ 1 ]. In mid-2021, a definition of postbioticsterm·dom was proposed by the International Scientific Association of Probioticsterm·dom and Prebiotics (ISAPPterm), stating that a postbioticterm·dom is a “preparation of inanimate microorganismsterm·key and/or their components that confers a health benefitterm·key on the host” [ 1 ]. This definition was debated for more than one year by a consensus panel composed of scientists with different backgrounds and perspectives. Usage of this term has grown over recent years, but until 2021, no consensus definition had been published, and indeed, many different conceptualizations of this term were in use. This precise, well-considered definition provides a basis for clarity and accuracy in communications about postbioticsterm·dom, to the benefit of scientists, industry and regulators. This paper discusses the consensus term ‘postbioticterm·dom’, including the rationale behind the definition, and explores their technological development and future use in foods.

2. Postbioticterm·dom Definition Postbioticterm·dom is a term derived from the Greek for ‘post’, meaning after, and ‘bios’, meaning life. Further, the ‘biotic’ family of terms (probioticsterm·dom, prebiotics, synbiotics and postbioticsterm·dom) coalesces around microbes (or their substrates) ( Table 1 ). Therefore, the term postbioticterm·dom appropriately refers to substances derived after the microorganisms are no longer alive, or, in other words, inanimate, dead or inactivated. The microbes comprising a postbioticterm·dom may be inanimate, intact cells or may be structural fragments of microbes, such as cell walls. Many preparations of postbioticsterm·dom also retain microbe-produced substances, such as metabolites, proteins, or peptides, which may contribute to the overall health effect conferred by a postbioticterm·dom, but such components are not essential to a postbioticterm·dom. A postbioticterm·dom must be derived from a well-defined microorganism or combination of microorganisms for which genomic sequences are known and prepared using a delineated technological process of biomass production and inactivationterm·key, which can be reliably reproduced.

Prior to settling on the definition of postbioticterm·dom, the ISAPPterm panel considered other definitions that had been previously published ( Table 2 ), as well as the Greek derivation of the word. Most published definitions of postbioticterm·dom focused on metabolites or factors produced by microbes. Some also included dead or inactivated microbes. Other stipulations in some definitions were problematic. For example, the requirement that a postbioticterm·dom should be produced from a probioticterm·dom places capricious restrictions on a postbioticterm·dom. A probioticterm·dom by definition must meet specific criteria, including a documented health benefitterm·key [ 9 ]. It is not clear what the value is of stipulating that the starting material for a postbioticterm·dom must in itself be proven to be a probioticterm·dom, when a postbioticterm·dom is so different from a probioticterm·dom and an established health benefitterm·key for a live microbe does not predict a benefit in an inactivated form. Such a definition would also be a barrier to innovation as the research path should first establish that a microbe is a probioticterm·dom, or restrict the starting microbe to already established probioticsterm·dom, and then go on to meet the criteria for a postbioticterm·dom when the latter should be sufficient. Further, definitions that focused on metabolites from probioticsterm·dom were problematic as, by that definition, a metabolite produced by a probioticterm·dom could be a postbioticterm·dom, whereas the chemically identical metabolite produced by a microbe not established to be a probioticterm·dom would not be. Another concern about some previous definitions is the absence of a clear requirement for a health benefitterm·key. If no health benefitterm·key is specified, then it is uncertain what value is added by the use of a postbioticterm·dom. Some definitions were unclear if the postbioticterm·dom was to be administered to a target host or could instead be produced in situ via the normal activities of resident or administered microorganisms. Definitions that do not distinguish between a product that is administered and substances that are produced in situ have an unclear path to translation into foods, feeds and other final products. Finally, other definitions stipulated that postbioticsterm·dom target only the gut lumen [ 10 ], leaving out the possibility of applying postbioticsterm·dom to other surfaces, such as the vagina or the skin. Taken together, the previously published definitions of postbioticterm·dom were deemed to be vague and lacking consideration of important, practical factors. Further, as discussed later, exciting new research in the area of potential health benefitsterm·key of inactivated microbes has accumulated. Hence, the ISAPPterm consensus panel focused on the inanimate microbe, not on their metabolic outputs. Opposition to the ISAPPterm definition was levied shortly after its publication [ 11 ], and the corresponding reply refuting the criticism was also published [ 12 ].

3. How Postbioticsterm·dom May Impact Our Vision about Probioticsterm·dom for Future Research Probioticterm·dom foods and food supplements should be formulated to confer the expected health benefitterm·key. This requires the product to retain efficacious levels of live microbes in the product through the end of its shelf lifeterm·key. However, live cells coexist with non-viable cells, even in a fresh, overnight culture of the strain ( Figure 1 ), but potentially to an even greater extent at the end of shelf lifeterm·key.

In fact, cells that go unseen by traditional agar plating used to enumerate viable cells in probioticterm·dom products, such as viable but not culturable cellsterm·dom (VBNCterm·dom) or dead cells, may constitute a major proportion of a probioticterm·dom culture, and this proportion likely varies according to many factors, including the pH and phase of growth of the culture prior to harvesting. Cultures harvested in the late stationary phase typically contain a larger proportion of non-viable cells than in the early stationary phase or in the exponential phase, and cultures grown at pH close to neutrality contain a larger proportion of non-viable cells than those grown at a lower pH [ 18 ]. Non-viable cells can be observed through fluorescence microscopy or flow cytometry [ 19 ]. Using flow cytometry, researchers demonstrated that a fresh culture of Lacticaseibacillus rhamnosus R001 contained a 100:1 ratio of live:dead cells, where just after freeze drying, the number of dead cells significantly increased to render almost a 1:1 ratio [ 20 ]. Taking into account that some loss of cell viability occurs during the manufacture of probioticterm·dom cultures and during the shelf lifeterm·key of the product, manufacturers usually overfill the products with live cells beyond the declared dose to assure adequate levels of live cells at the end of the product shelf-life. Overfilling can range from 1.5 to 4 times the number of live cells expected by the end of the shelf lifeterm·key, depending on the strain, dosage form and packaging [ 21 ]. However, there is a scarcity of published data about industrial overfilling practices [ 21 ]. In a completely theoretical scenario, a probioticterm·dom food supplement that is expected to deliver, for example, 60 billion CFU, and which was verified during development to lose 75% viability by the end of shelf lifeterm·key, should be overfilled to 240 billion CFU at manufacture. By the end of its shelf lifeterm·key, it would still be able to deliver 60 billion live cells, but also 180 billion inanimate cells. The implications of this high level of non-viable cells in commercial products have not been addressed in the published literature. Can we consider the 180 billion non-viable cells delivered in such a product to be postbioticsterm·dom? The answer is ‘no’, primarily because in order to be a postbioticterm·dom, those non-viable cells themselves must be shown to confer a health benefitterm·key. Further, the product as described remains a probioticterm·dom as it is able to deliver an efficacious dose of live microorganisms.

However, one may still hypothesize that inanimate cells can participate in the overall health benefitterm·key expected for a probioticterm·dom product. This hypothesis can be made on the basis that some strains, and for some endpoints, deliver similar benefits as viable and non-viable microbes [ 8 , 22 , 23 ]. Since the probioticterm·dom definition does not exclude inanimate microbes, and since it is technologically infeasible to rid a probioticterm·dom of inanimate cells, the significance of their presence in probioticterm·dom products deserves investigation. Such research is complicated by the fact that a probioticterm·dom product may have variable and significant amounts of inanimate cells. The challenge is to understand what would be the potential contribution of inanimate cells to the health benefitterm·key as, for instance, they can still display the surface antigens of a live cell, which could be involved in the mechanism of action that supports the health benefitterm·key.

The accumulation of inanimate cells over the shelf lifeterm·key of a probioticterm·dom food supplement may be interesting to consider in future efficacy trials where long-term administration of the probioticterm·dom takes place. For example, in studies of 12 months’ duration, it is rarely reported if the same batch of the probioticterm·dom supplement was used, or if different fresh batches were supplied and with what frequency [ 24 ]. The level of non-viable cells these products contain over the duration of the trial are mostly not reported. The design of future clinical trials to collect and report these data will help us to better understand the contribution of inanimate microbes to the health effects conferred by probioticsterm·dom.

4. Characteristics of Postbioticsterm·dom as They Relate to Foods Probioticsterm·dom are live microorganisms that, when administered in adequate amounts, confer a health benefitterm·key on the host [ 3 ]. Probioticsterm·dom have been included in a variety of products: fermented (yoghurt, cheese) or non-fermented (cereal or chocolate bars, fruit juices, smoothies) foods, which can be of dairy origin or plant-based, with yoghurt (or fermented milks in general) perhaps the food most commonly used for the delivery of probioticsterm·dom [ 25 ]. However, certain food characteristics (acidity, water activity, specific chemical compounds) or storage conditions (moisture, temperature, package permeability to oxygen, time) may impose challenges to the proper survival of probioticsterm·dom in foods during production and storage [ 16 ]. In this context, the unsatisfactory survival of certain probioticterm·dom strains in some foods has been systematically reported [ 26 ]. Therefore, inanimate microbes are also likely components of many food products.

A postbioticterm·dom was defined in this work. Figure 2 depicts various ways in which an inanimate culture could be prepared before assessing it as a candidate postbioticterm·dom. Without a cautious consideration of both definitions and scopes, probioticsterm·dom and postbioticsterm·dom could be regarded as two sides of the same coin, with the loss of viability being the path to go from one category (probioticterm·dom) to the other (postbioticterm·dom). However, a dead probioticterm·dom in a food does not make it a postbioticterm·dom food even if some strains may be used in both categories. For instance, Bifidobacterium bifidum MIMBb75 significantly alleviates irritable bowel syndrome in its viable form [ 22 ] and also as a heat-inactivated culture [ 23 ]. A viable culture of Lactobacillus gasseri CP2305 was able to enhance quality of life and clinical symptoms in patients with irritable bowel syndrome [ 27 ], and as an inactivated culture was able to positively regulate gut environment and function [ 28 ]. In a similar fashion, Akkermansia muciniphila ATCC BAA-835 improved several metabolic parameters in obese and overweight volunteers when administered alive or in its pasteurized, inanimate form. Despite these examples, a microbe can only be regarded as a postbioticterm·dom if it is properly characterized, deliberately prepared with a reproducible method for inactivationterm·key, and shown to confer a health benefitterm·key. A probioticterm·dom microorganism that gradually loses cell viability over the shelf lifeterm·key of the food does not gradually become a postbioticterm·dom; it is simply a probioticterm·dom food that, if formulated properly, will deliver an efficacious dose of live cells until the end of its shelf lifeterm·key.

Although yoghurt is defined by the Codex Alimentarius [ 29 ] to include live starter cultures, a product known as ambient yoghurtterm·dom has emerged. Ambient yoghurtterm·dom is conventional yoghurt that is subjected to thermal treatment in order to inactivate starter cultures. As such, ambient yoghurtterm·dom is more correctly named “yoghurt-based product for ambient distribution”. These products do not need to be stored or distributed in cold conditions. Ambient yoghurtterm·dom is experiencing growth in popularity, spreading across Asia and into Africa and Latin America. In China, ambient drinking yoghurt first appeared on the market in 2010, and this fast-growing market amounts to almost 2.5 billion liters and now accounts for almost 50% of total yoghurt sales ( https://www.tetrapak.com/insights/cases-articles/the-rise-of-ambient-yoghurt , accessed on 22 February 2022). The viability of starter bacteria is emphasized in food standards and regulations for yoghurt and fermented milks. Cultured milks that do not deliver viable cultures have longer shelf lives and easier storage, but beneficial health effects conferred by non-viable microbes, beyond those conferred by the micro- and macro-nutrients, therein remain to be demonstrated. Most health benefitsterm·key reported for fermented milks have been documented using viable bacteria, and data on non-viable preparations are often limited. One study showed that the treatment of acute gastro-enteritis demonstrated clinical efficacy in shortening the duration of diarrheaterm using fermented milks with both viable and non-viable forms [ 30 ]. In this sense, ambient yoghurtterm·dom may fit the definition of a postbioticterm·dom food once health benefitsterm·key are properly demonstrated through efficacy trials.

Infant formulasterm·key are another example of foods used to deliver probioticsterm·dom, prebiotics, synbiotics and also postbioticsterm·dom [ 31 ]. In particular, Bifidobacterium breve C50 and Streptococcus thermophilus 065 have been used to produce a fermented infant formulaterm·key whose microbes are inactivated by spray drying after fermentation. The infant formulaterm·key carries inanimate microbes and fermentation products. A series of clinical studies in children demonstrated its safety and postbioticterm·dom properties, such as modulation of the gut microbiota to be closer to that of breastfed infants [ 32 ], reduction in the severity of acute diarrhea [ 33 ], improved inflammatory and immune markers, which might be related to some features of gastrointestinal tolerance [ 34 ], reduction in digestive and respiratory events in infants at high risk of allergy [ 35 ] and the induction of positive effects on thymus size and stool pH in healthy term infants [ 36 ]. In a broader approach, postbioticsterm·dom were systematically reviewed in relation to the prevention and treatment of common infectious diseases among children younger than 5 years. Seven RCTs involving 1740 children met the inclusion criteria. For therapeutic trials, supplementation with heat-killed Lactobacillus acidophilus LBterm reduced the duration of diarrheaterm. For preventive trials, the pooled results from two RCTs showed that heat-inactivated L. paracasei CBA L74 reduced the risk of diarrhea, pharyngitis and laryngitis [ 37 ].

Food supplements form a promising category for the development of new postbioticterm·dom products as the lack of viability may offer longer stability to postbioticterm·dom products compared to probioticterm·dom food supplements. Tablets containing heat-inactivated L. gasseri CP2305 were administered for 4 weeks to patients with irritable bowel syndrome, resulting in improved subjective and objective symptoms [ 27 ]. The same tablets were used in a longer-term study (24 weeks), which improved chronic stress in healthy young adults preparing themselves for the national examination for medical practitioners [ 38 ]. Capsules containing 10 9 cells of non-viable B. bifidum MIMBb75 were administered for 8 weeks to patients diagnosed with irritable bowel symptoms according to Rome III criteria. The inanimate culture was able to substantially alleviate symptoms in a real-life setting [ 23 ].

The concept of postbioticsterm·dom will likely broaden the spectrum of microbes used for functional purposes. Species other than those belonging to the traditionally safe genus Bifidobacterium or the family Lactobacillaceae, which could not be administered live due to concerns about their safety, have been explored as potential postbioticsterm·dom. For instance, the safety of heat-killed Mycobacterium manresensis was assessed as a novel food pursuant to EU regulation 2015/228330, and this inanimate culture showed potential against the development of active tuberculosis in a pilot human study [ 39 ].

5. Conclusions Evidence is accumulating that inanimate microorganismsterm·key and/or their components are able to confer health benefitsterm·key when administered in adequate amounts to a host. There is also research to support the concept that microbe-derived metabolites administered to a host may drive some beneficial physiological effects. Postbioticsterm·dom pull together these two aspects of microbial influence on health into products that can be developed as foods, therapeutics and other product types to be administered for health outcomes. Mechanistic research points to important microbe-derived small molecules such as neurochemicals, short-chain fatty acids, defensins, bacteriocins and others that are produced in situ that likely mediate the many roles colonizing microbes exert on physiological function. However, this microbial activity is distinct from postbioticsterm·dom, which must be administered to a host. Different terms have been used over the years to address this evolving area of research. To facilitate communication to health professionals, the industry, regulators and the general public, uniting under one, well-defined, consensus definition, make clear what fits and what does not fit within the postbioticterm·dom category. Further, it will allow better tracking of scientific papers for future systematic reviewsterm·mth and meta-analyses on the topic.

It is clear that most probioticterm·dom products contain inanimate microorganismsterm·key, but this does not make them ‘postbioticsterm·dom’. Probioticterm·dom products are correctly considered to be probioticsterm·dom as long as they are able to deliver the necessary dose of live cells to confer the expected health benefitterm·key. Inanimate cells in probioticterm·dom products may contribute to the health benefitterm·key delivered, but few have been studied, and their contribution is not clear so far. There is a need for future research to understand: (1) Which inanimate microbes, with or without associated metabolites, are able to confer a health benefitterm·key; (2) What mechanisms are driving the benefits; (3) What role inanimate microbes contained in probioticterm·dom products may play in driving health benefitsterm·key.

The ISAPPterm definition for postbioticsterm·dom is not restricted to microbes that produce metabolites in the gut, is comprehensive enough to allow the development of postbioticsterm·dom from different microorganisms and encompasses products targeting non-gut anatomical sites. Further, this definition encourages innovation in a promising area for companion or production animals and plant or human health.