The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics — TEI-tagged view

Kelly S. SwansonpersName, Glenn R. GibsonpersName, Robert HutkinspersName, Raylene A. ReimerpersName, Gregor ReidpersName, Kristin VerbekepersName, Karen P. ScottpersName, Hannah D. HolscherpersName, Meghan B. AzadpersName, Nathalie M. DelzennepersName, Mary Ellen SanderspersName (2020date). “The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics.” Nature Reviews Gastroenterology & Hepatologytitle·j.

Object of study — the term synbioticterm·dom: its definition, its two categories, the evidence needed to use it, and its use by stakeholders

Research question — what a synbioticterm·dom is, stated as a clear, concise and testable definition, and what trials and safety evidence are needed to use the term

Contribution — an updated consensus definition of synbioticterm·dom, with complementary and synergistic categories and minimum evidence criteria for each

Full text — tagged

The International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom (ISAPP) consensus statement on the definition and scope of synbioticsterm·dom Abstract In May 2019, the International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom (ISAPP) convened a panel of nutritionists, physiologists and microbiologists to review the definition and scope of synbioticsterm·dom. The panel updated the definition of a synbioticterm·dom to “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefitterm·key on the host”. The panel concluded that defining synbioticsterm·dom as simply a mixture of probioticsterm·dom and prebioticsterm·dom could suppress the innovation of synbioticsterm·dom that are designed to function cooperatively. Requiring that each component must meet the evidence and dose requirements for probioticsterm·dom and prebioticsterm·dom individually could also present an obstacle. Rather, the panel clarified that a complementary synbioticterm·dom, which has not been designed so that its component parts function cooperatively, must be composed of a probioticterm·dom plus a prebioticterm·dom, whereas a synergistic synbioticterm·dom does not need to be so. A synergistic synbioticterm·dom is a synbioticterm·dom for which the substrate is designed to be selectively utilized by the co-administered microorganisms. This Consensus Statement further explores the levels of evidence (existing and required), safety, effects upon targets and implications for stakeholders of the synbioticterm·dom concept.

Subject terms: Nutritional supplements, Microbiota, Nutrition therapy Introduction Notable properties of the gut microbiotaterm·key include its functionality and resilience 1 . A stable gut community protects the host against invading microorganisms and helps maintain homeostasis, including immune regulation. Nonetheless, disruptions occur owing to dietary shifts, antibiotic use, age or infection, leading to a gut microbiotaterm·key that can contribute to a range of inflammatory, pathogenic and metabolic conditions such as inflammatory bowel diseases, colorectal cancer, metabolic syndrome and atopy 2 . Several strategies have been proposed to modulate the composition and/or function of the gut microbiotaterm·key, including faecal microbiota transplants, the application of probioticsterm·dom and other live microorganisms, and the use of non-digestible dietary substrates such as prebioticsterm·dom 3 , 4 .

When the synbioticterm·dom concept was first described 25 years ago, the notion that selectively fermentable non-digestible food ingredients (prebioticsterm·dom) could be combined with probioticsterm·dom was envisioned 5 . Thus, synbioticsterm·dom were loosely defined as mixtures of “probioticsterm·dom and prebioticsterm·dom that beneficially affect the host” 5 . The term itself was formed from the Greek prefix ‘syn’, meaning ‘together’ and the suffix ‘biotic’, meaning ‘pertaining to life’. Despite the availability of similarly worded definitions, confusion exists among stakeholders, including scientists, about what constitutes a synbioticterm·dom 6 – 9 . A general misunderstanding might have been, in part, because the original definition itself — that is, “mixtures of probioticsterm·dom and prebioticsterm·dom that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract, by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of health-promoting bacteria, thus improving host welfare” — was too wordy and lacked precision 5 . In addition, the expansion of the entire ‘–biotics’ category, including terms such as postbioticterm·dom 10 and pharmabioticterm·dom 11 , almost certainly further contributes to confusion. To provide clarity and guidance regarding appropriate use of the term ‘synbioticterm·dom’, in May 2019, the International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom (ISAPP) convened an expert panel of academic scientists to address the current status of synbioticsterm·dom, including its definition. The outcomes of that meeting and subsequent discussions comprise this Consensus Statement. A summary of key conclusions is shown in Box 1 . Herein, we consider applicable efficacy and mechanistic evidence on combination probioticterm·dom plus prebioticterm·dom products, we recommend the research needed to establish a ‘synbioticterm·dom’ formulation, discuss the safety considerations, and reflect on implications for stakeholders of the synbioticterm·dom concept.

The definition of synbioticterm·dom has been updated to “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefitterm·key on the host”.

Within this definition, ‘host’ microorganisms comprise both autochthonousterm·dom (resident or colonizing the host) and allochthonousterm·dom (externally applied, such as probioticsterm·dom) microorganisms, either of which can be targets for the substrate contained in the synbioticterm·dom.

Two subsets of synbioticsterm·dom were defined: complementary and synergistic. A ‘synergistic synbioticterm·dom’ is a synbioticterm·dom in which the substrate is designed to be selectively utilized by the co-administered microorganism(s). A ‘complementary synbioticterm·dom’ is a synbioticterm·dom composed of a probioticterm·dom combined with a prebioticterm·dom, which is designed to target autochthonousterm·dom microorganisms. Minimum criteria for the existing probioticterm·dom and prebioticterm·dom must be met for both components of a complementary synbioticterm·dom.

Beneficial effect(s) of a synbioticterm·dom on health must be confirmed in the target hostterm·key, which might include humans, companion animals, or agricultural species or a subpopulation (such as different age or developmental stage, health status, sex or living situation) thereof.

For a synergistic synbioticterm·dom, evidence of selective utilizationterm·dom of the substrate must be demonstrated in the same study establishing the health benefitterm·key. The aim is to demonstrate that the combined effect is better than the estimated effects of each component separately. This step is not required for a complementary synbioticterm·dom, as it contains a prebioticterm·dom for which selective utilizationterm·dom has already been established.

A synbioticterm·dom can be applied to intestinal or extra-intestinal microbial ecosystems and might be formulated into products fitting an array of regulatory categories (such as foods, non-foods, feeds, drugs or nutritional supplements).

Implied in the definition is that safety of the synbioticterm·dom for the intended use is established.

‘Symbioticterm·dom’ is not a synonym of synbioticterm·dom and is incorrect in this context.

Methods ISAPP is a non-profit collaboration of scientists dedicated to advancing scientific excellence and providing objective, science-based information on probioticsterm·dom and prebioticsterm·dom. The organization’s activities are funded by companies involved in the sale of probioticsterm·dom and prebioticsterm·dom, but ISAPP is guided by an international, all-volunteer academic board that functions independently. This ISAPP-organized panel was composed of experts in microbiology, nutrition and gastrointestinal physiology, including many who were involved in the latest updates of the probioticterm·dom 12 and prebioticterm·dom 13 definitions according to ISAPP. Panellists were charged with accomplishing the following goals: consider what a synbioticterm·dom is and provide a clear, concise and testable definition; suggest the appropriate experimental conditions necessary to establish synbioticterm·dom activity; describe the evidence required to demonstrate the health benefitsterm·key and establish safety; and provide guidance for stakeholders, including researchers, industry, public health professionals and regulatory agencies.

Prior to the meeting, panellists developed a discussion outline and target questions. During the meeting, panellists presented the perspectives and evidence regarding the core issues involved. Debate ensued until a consensus was achieved. After the meeting, individual panellists wrote sections of the summary, which were compiled by K.S.S., G.R.G., R.H. and M.E.S. into a draft report. This document was edited and agreed upon by all panel members. The authors would like to thank members of the ISAPP board of directors who did not directly participate in this consensus panel but who reviewed, criticized and approved this manuscript: D. Merenstein, H. Szajewska, M. Marco, E. Quigley, S. Lebeer and S. Salminen.

An updated definition The panel updated the definition of a synbioticterm·dom to “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefitterm·key on the host”. ‘Host’ microorganisms in this context include both autochthonousterm·dom microorganisms (resident or colonizing the host) and allochthonousterm·dom microorganisms (externally applied, such as probioticsterm·dom), which, even if transiently present, do constitute a component of the host microbiota.

The panel considered defining synbioticsterm·dom as simply a mixture of probioticsterm·dom and prebioticsterm·dom. Common to the definition of both probioticsterm·dom and prebioticsterm·dom is the requirement that each independently provides a health benefitterm·key and the dose of each must be adequate to independently achieve those benefit(s). However, the panel recognized the possibility that a functional synbioticterm·dom could be formulated at doses below those at which the probioticterm·dom or prebioticterm·dom could independently exert health benefitsterm·key. Alternatively, a particular microorganism might lack probioticterm·dom functions even at high dosages owing to competition or other ecological effects but, in the presence of a suitable substrate, could provide a health benefitterm·key. Likewise, a novel substrate, again even at high doses, might not by itself provide benefits but could do so when combined with a selected live microorganism(s) that it can enhance. Such formulations comprise a live microorganism and a substrate that depend on the presence of one another and function in concert. Simply put, the microbial component does not necessarily have to be a standalone probioticterm·dom and the non-digestible substrate does not necessarily have to be a standalone prebioticterm·dom, but, if together they provide a health benefitterm·key, then the mixture can be called a synbioticterm·dom. This proposed definition of a synbioticterm·dom should encourage innovation in formulations by not requiring that component parts meet the strict definitions of either a probioticterm·dom or a prebioticterm·dom.

However, the panel also recognized that a current common usage of the term synbioticterm·dom includes products that combine a probioticterm·dom and a prebioticterm·dom. Such a combination product might not have any evidence of co-dependent function but is instead designed for the components to work independently to promote an observed health benefitterm·key(s). The panel agreed that such a formulation could be considered a synbioticterm·dom, provided that components meet the respective probioticterm·dom 12 and prebioticterm·dom 13 definitions (Fig. 1 ). Thus, the probioticterm·dom strain(s) is chosen based on the benefits that it provides to the host, while the prebioticterm·dom is designed to promote the growth and activities of beneficial members of the indigenous microbiota and provide a health benefitterm·key 14 . Furthermore, a combination of probioticterm·dom plus prebioticterm·dom must be tested to confirm that a health benefitterm·key is conferred by the combined formulation compared with a placebo. Otherwise, the product should not be labelled a synbioticterm·dom. Based on these qualifiers, the panel retained the definition that a combination of a probioticterm·dom and a prebioticterm·dom be denoted as a complementary synbioticterm·dom (discussed later) 14 .

Synbioticterm·dom categories. Synbioticsterm·dom can be formulated using two approaches. A complementary synbioticterm·dom comprises a probioticterm·dom plus a prebioticterm·dom (more than one of each can be used), working independently to achieve one or more health benefitsterm·key. Probioticterm·dom and prebioticterm·dom components of the complementary synbioticterm·dom must meet the minimum criteria, as stipulated previously 12 , 13 . A synergistic synbioticterm·dom is composed of a live microorganism and a selectively utilized substrate but neither needs to meet the minimum criteria stipulated previously for probioticsterm·dom and prebioticsterm·dom. Instead, these components are designed to work together, with the substrate being selectively utilized by the co-administered microorganism. The panel considered whether all synbioticsterm·dom should be synergistic. However, the absence of such substances today speaks to the difficulty of achieving the required evidence. The panel judged that it was more important for the definition to be useful rather than hypothetical.

The other type of synbioticterm·dom previously envisioned is a synergistic synbioticterm·dom, which is a synbioticterm·dom in which the substrate is designed to be selectively utilized by the co-administered microorganisms. To be a synergistic synbioticterm·dom, the live microorganism is selected based on its ability to provide a health benefitterm·key and the substrate is chosen to primarily support the growth or activity of that selected microorganism 14 . Although the substrate might also enrich other beneficial members of the gastrointestinal microbiota, its main target is the ingested microorganism.

Designing and demonstrating the efficacy of a synergistic synbioticterm·dom is experimentally challenging. To our knowledge, nearly all synbioticsterm·dom used in published clinical trials or available commercially are of the complementary form 15 , irrespective of whether they have been deliberately designed or named as such. Approaches to designing complementary and synergistic synbioticsterm·dom are shown in Fig. 2 .

Design and mechanisms of action of complementary and synergistic synbioticsterm·dom. Two approaches to designing synbioticsterm·dom are represented here. The complementary approach combines a prebioticterm·dom and a probioticterm·dom that work independently to elicit one or more health benefitsterm·key. The prebioticterm·dom and probioticterm·dom must each meet applicable criteria (Table 3 ). The prebioticterm·dom functions by modulating the resident microbiota in a manner associated with an improved health outcome. The synergistic approach selects a substrate that is utilized by the co-administered live microorganism, enhancing its functionality. Synergistic synbioticsterm·dom work together (not independently) to bring about the resulting health benefitsterm·key.

It is important to clarify that, although synbioticsterm·dom can be formulated to provide synergistic activities, the ‘syn’ prefix in the word ‘synbioticterm·dom’ is not intended to imply synergy; it means ‘together’. Of note, the term ‘symbioticterm·dom’ is often misused in this context and is not the same as synbioticterm·dom. Symbioticterm·dom, as used in biology 16 , refers to an ecological relationship in which one organism (the symbiont) lives in a long-term relationship in a natural ecosystem with another organism (the host), that is, in symbiosis.

Characterization needed for synbioticsterm·dom A synbioticterm·dom should be characterized to the extent needed to ensure safety and a consistent performance. Live microbial component(s) of the synbioticterm·dom should have a publicly available genome sequence and annotation, be assessed for any genes of safety concern (for example, toxin production or transferrable antibiotic resistance), named using current taxonomic nomenclature and carry a traceable strain designation. The strain(s) should also be deposited into a recognized international culture collection that permits access by scientists to conduct research. In short, the safety, identity, purity and potency of the live microorganism should be clearly and accurately described according to the best available methods that meet applicable regulatory standards for the product category.

The structure and purity of the substrate should be stated and characterized by appropriate chemical analyses. This process includes testing for microbial and other contaminants as per regulatory standards for the country of sale. The level of purity required will depend on what is needed to ensure a consistent performance and safety of the product. The level of active substrate in commercial preparations of prebioticsterm·dom available worldwide ranges considerably, often from 35% to 99% 17 – 19 . The monosaccharides and disaccharides carried over from the production process that are present in prebioticterm·dom preparations are typically digested and absorbed by the host in the upper gastrointestinal tract after oral ingestion. A relevant issue is whether the material used in the formulation is sufficient to deliver a consistent dose of the active component and result in reproducible selective utilizationterm·dom by microorganisms and beneficial health effect(s) in the target hostterm·key. This issue highlights that studies should communicate the content of the active ingredient being tested in addition to the quantity of the overall product. For example, a 6 g dosage of 50% pure galacto-oligosaccharides would provide 3 g of the active substrate and should be reported as such.

The active ingredients of a synbioticterm·dom must be sufficiently stable. Ensuring stability of the live microbial component of a synbioticterm·dom can be challenging 20 . When live microorganisms are combined with a substrate(s) in a matrix (for example, liquid, dried or ointment), the burden is on manufacturers to ensure that the dosage of the live microorganism required to confer the stated health benefitterm·key is delivered throughout the shelf-life. Live microorganism viability is highly dependent on the matrix, storage temperature, pH and oxygen level of the product: for liquid products, the shelf-life might be as short as 1–2 weeks, for lyophilized or encapsulated products, the shelf-life might be as long as 2 years. Packaging and storage conditions must control critical factors, such as water activity and temperature, through production to distribution and usage. Furthermore, a synbioticterm·dom must undergo assessment of safety for the intended use, as described later.

Current levels of evidence Numerous randomized controlled trials (RCTs) in humans across a range of populations, from healthy individuals to those with acute and chronic diseases, have been conducted to examine the health benefitsterm·key of putative synbioticsterm·dom. Many trials have been conducted in adults with metabolic diseases, including overweight and obesity 21 , 22 , type 2 diabetes mellitus 23 , 24 and non-alcoholic fatty liver disease 25 , 26 . Other outcomes, such as irritable bowel syndrome 27 , surgical infections 28 , 29 , chronic kidney disease 30 , 31 and atopic dermatitis 32 , have also been investigated. Consequently, many systematic reviews and meta-analyses of RCTs evaluating the effect of putative synbioticsterm·dom on disease targets have been published 22 , 23 , 32 – 35 and this approach is an accepted means of evaluating the evidence for health benefitsterm·key 36 – 38 . Examples are given in Table 1 , which focuses on RCTs demonstrating benefits. Null trials have also been published across a variety of outcomes, including, for example, some studies aimed at the prevention of surgical infections 39 , 40 , treatment of obesity 41 , management of gestational diabetes mellitus 42 and eradication of Helicobacter pylori infection 43 , 44 . However, evidence of a health benefitterm·key is not in itself sufficient to call a formulation of live microorganism(s) plus selectively utilized substrate(s) a ‘synbioticterm·dom’. Concomitant evidence of selective utilizationterm·dom by either the endogenous microbiota (complementary synbioticterm·dom) or the co-administered live microorganism (synergistic synbioticterm·dom) must also be generated.

Human trials of orally administered combinations of live microorganisms and a substrate reporting health outcomes Across studies, species from the genera Lactobacillus , Bifidobacterium and Streptococcus are the most commonly used live microorganisms within the formulations tested. The substrate components are usually galacto-oligosaccharides, inulin or fructo-oligosaccharides but doses vary considerably, from as low as 100 mg to as much as 10–15 g per day. For example, in a double-blind RCT, twice daily consumption of a synbioticterm·dom composed of Lactobacillus acidophilus 10 (10 9 CFU), Lactobacillus rhamnosus HS111 (10 9 CFU), Lactobacillus casei 10 (10 9 CFU), Bifidobacterium bifidum (10 9 CFU) and fructo-oligosaccharides (100 mg) 4 days before and 10 days after surgery for periampullary neoplasms resulted in a lower number of postoperative infections, a shorter duration of antibiotic therapy, fewer non-infectious complications, shorter length of hospital stay and reduced mortality than in patients receiving a placebo (sucrose) 45 . Although such low doses would not be expected to provide a prebioticterm·dom effect in complementary synbioticsterm·dom, they could, in theory, be sufficient to stimulate a cognate microorganism in a synergistic synbioticterm·dom formulation. In one RCT 46 of 225 adults with overweight and obesity, a combination of Bifidobacterium animalis subsp. lactis 420 and polydextrose (12 g per day) resulted in a relative reduction in body fat massterm·var of 4.5%, whereas the individual treatments had no effect. However, selective utilizationterm·dom was not established in this study. Thus, by itself, this study does not provide sufficient evidence that the tested combination was either a complementary or a synergistic synbioticterm·dom.

In general, the appropriate dose, duration and composition of a synbioticterm·dom needed to confer a health benefitterm·key are likely to be specific to the context, including outcome and baseline host target site microbiota 47 , as well as coexisting environmental factors such as medication, habitual diet and, perhaps, yet-to-be-identified host genetic factors.

Necessary evidence for synbioticsterm·dom A synbioticterm·dom must contain a live microorganism and a selectively utilized substrate. For complementary synbioticsterm·dom, the respective components must fulfil the evidence and dose requirements for both a probioticterm·dom and prebioticterm·dom. Furthermore, the combination must be shown in an appropriately designed trial to confer a health benefitterm·key in the target hostterm·key. A product containing a probioticterm·dom and a prebioticterm·dom that only has evidence for each component individually, and not as a combination product, should not be called a synbioticterm·dom. A key evidentiary requirement for a synergistic synbioticterm·dom is that there be at least one appropriately designed study of the synbioticterm·dom in the target hostterm·key that demonstrates both selective utilizationterm·dom of the substrate and a health benefitterm·key (Box 2 ).

Generating this evidence requires appropriately designed, adequately powered experimental trials conducted on the target hostterm·key. These studies should follow standard human trial design and reporting guidelines 48 and consider best practices for diet–microbiota research 49 . The study should also meet the criteria outlined in CONSORTterm·key 48 and should be registered, including a description of all outcomes, prior to recruitment. These CONSORTterm·key criteria include guiding principles for microorganisms, microbiota-related compliance and outcome measures, relevant subgroups, and statistical considerations for evaluating the microbiota as a mediator of clinical effects.

Ideally, the health benefitterm·key of a synbioticterm·dom would be superadditiveterm·dom, that is, it would exceed the benefit observed for the sum of the individual components. However, owing to the difficulties in demonstrating different levels of health benefitterm·key in an efficacy trial, the panel did not insist upon this aspect. Instead, we advocate requiring a measurable, confirmed health benefitterm·key of the synbioticterm·dom, which is conferred, at least in part, through selective utilizationterm·dom of the substrate(s) provided in the synbioticterm·dom. Although in vitro and animal models are used frequently to test the effects of probioticsterm·dom, prebioticsterm·dom and synbioticsterm·dom 50 – 53 , it is our position that these have not been fully validated as predictive and that definitive tests of these interventions must be performed in the target hostterm·key. Of course, model experiments can add mechanistic insights, in particular in cases for which available samples from studies in the target hostterm·key have limited usefulness (for example, faeces, which are not reflective of upstream intestinal microbial or metabolic activities).

As noted, it is required that the health benefitterm·key of the synbioticterm·dom mixture be confirmed, even when established probioticterm·dom(s) and prebioticterm·dom(s) (that is, ones that meet criteria stipulated in respective definitions and are used at efficacious doses) are used to formulate components of the synbioticterm·dom. This requirement would account for any potential antagonistic effect of the combination that might diminish the health benefitsterm·key of each component independently. Although unlikely, such antagonism is theoretically possible, as shown in in vitro studies whereby some carbohydrate substrates increased the production of antimicrobial compounds by probioticsterm·dom 54 . Depending on which microorganism is able to utilize the substrate, which antimicrobial factors are produced, and which taxa are neutralized or killed by them, such a scenario could lead to positive or negative outcomes. Therefore, in the absence of evidence that the combination product provides a health benefitterm·key, a product should simply be labelled as “contains probioticsterm·dom and prebioticsterm·dom”. SanderspersName et al. 55 provide a perspective on determining whether evidence from studies using one formulation (for example, a prebioticterm·dom or a probioticterm·dom alone) can be extrapolated to different formulations (for example, when combining a prebioticterm·dom and a probioticterm·dom).

The qualifier ‘established’ refers to a probioticterm·dom or prebioticterm·dom that meets the requirements of the globally accepted definitions from International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom consensus 12 , 13 . Studies are done in the target hostterm·key.

Example 1 In a blinded four-arm study, 41 healthy volunteers received either Bifidobacterium animalis subsp. lactis Bi-07 (10 9 CFU per day), 8 g per day xylo-oligosaccharides (XOS), the combination of both or an inert maltodextrin control for 21 days 128 . XOS enhanced bifidobacteria counts in faeces in both the synbioticterm·dom and prebioticterm·dom groups compared with the control and improved plasma lipid profiles and modulated markers of immune function in healthy adults. The lowest reported use of analgesics was observed during combination supplementation along with a reduced expression of CD19 on B cells (as markers of immune function). Thus, the combination exerted certain benefits that were not afforded by the probioticterm·dom or prebioticterm·dom alone. This study shows a prebioticterm·dom status for XOS used at 8 g per day. Previous evidence supports that Bifidobacterium animalis subsp. lactis Bi-07 at 10 9 CFU per day met criteria for a probioticterm·dom 112 , 129 . Taken together, this tested product meets our definition of a complementary synbioticterm·dom. However, because an increase of Bifidobacterium was not observed in individuals fed the combination product, it does not meet our definition of a synergistic synbioticterm·dom.

Example 2 Krumbeck et al. 21 conducted a parallel multi-arm, double-blinded randomized control trial in people with obesity (17–19 per group) to assess the effects of a synbioticterm·dom containing 5 g of galacto-oligosaccharide (GOS) and 10 9 Bifidobacterium adolescentis IVS-1 on gut barrier function. The test strain had been obtained by an in vivo selection strategy intended to select for strains that would be expected to provide synergistic outcomes. In addition to the synbioticterm·dom, GOS, B. adolescentis IVS-1 and placebo (lactose) controls were also included. After 3 weeks of consumption, genus-specific and strain-specific quantitative real-time PCR were performed to assess changes in absolute abundances of bifidobacteria. To assess intestinal permeabilityterm·var, non-metabolizable sugars were measured in urine following the consumption of a sugar mixture. Although the results showed that all three treatments (GOS only, B. adolescentis IVS-1 only and the combination) markedly improved gut barrier function, there was no statistically significant difference among the three groups. The B. adolescentis IVS-1 group was significantly enriched but the addition of GOS as a synbioticterm·dom did not further increase strain abundance. Likewise, all three treatments significantly increased the absolute abundance of faecal bifidobacteria compared to baseline, but there was no statistically significant difference among the three groups. Although the combination, probioticterm·dom and prebioticterm·dom arms all improved the markers of colonic permeability and all increased faecal bifidobacteria levels, the study did not support our definition of a synergistic synbioticterm·dom.

Example 3 In a hypothetical study, an established prebioticterm·dom substrate (for example, GOS or inulin) at the dose shown to be both selectively utilized and have a health benefitterm·key (primary outcome showing improved probably of response) is combined with an established probioticterm·dom at the dose shown to have a health benefitterm·key in the same target hostterm·key. This combination product is tested and shown to confer a health benefitterm·key compared with the control (not necessarily the same benefit as previously tested for the probioticterm·dom and prebioticterm·dom). This product would meet our definition of a complementary synbioticterm·dom.

Example 4 In a hypothetical study, a substrate (not an established prebioticterm·dom) at 1 g per dose is combined with 10 6 CFU of a live microorganism (not an established probioticterm·dom). Preclinical testing suggests that the live microorganism selectively utilizes the substrate. A study tracking both health and microbiota end points in the target hostterm·key comprises 10 6 CFU per dose of live microorganism alone, 1 g per dose of substrate, 10 6 CFU per dose of the live microorganism plus 1 g per dose of the substrate, and an inert control. Microbiota analysis supports selective utilizationterm·dom by the combination. Concomitantly, a health or therapeutic end point is improved by the combination. The combined effect is better than the estimated effects of each component separately. This product would meet our definition of a synergistic synbioticterm·dom.

Example 5 A substrate (not an established prebioticterm·dom) plus a live microorganism (not an established probioticterm·dom) is tested against the control. It is found to confer a health benefitterm·key and increase levels of bifidobacteria in faeces. This result does not provide evidence for either a synergistic or complementary synbioticterm·dom. To fulfil criteria for a synergistic synbioticterm·dom, it must be demonstrated that the health benefitterm·key and selective utilizationterm·dom of the substrate exceed those observed for the control and for each individual component. As the materials comprising the mixture, at the dose used, were not established probioticsterm·dom or prebioticsterm·dom, it does not meet our definition of a complementary synbioticterm·dom.

Guiding principles for synbioticterm·dom research Design A randomized, double-blind, placebo-controlled trial is recommended for synbioticterm·dom research. The study should be registered with accepted protocol and results systems such as ClinicalTrials.gov. Study quality factors include, but are not limited to, appropriate blinding, randomization, the allocation concealment mechanism, reporting of inclusion and/or exclusion criteria and adverse events (AEs), completion of intention-to-treat analyses, and adequate statistical power. Crossover designs might be preferred to account for the individualized nature of the gut microbiotaterm·key. The wash-outterm·var period length should be based on the primary outcome with consideration for secondary outcomes; a 2-week wash-outterm·var between conditions is generally adequate for gut microbiotaterm·key outcomes, although longer times might be necessary for some populations (for example, the elderly or for those with constipation or functional bowel disorders). Parallel-arm designs might be required for long-term outcomes (such as weight loss or glycaemia). The number of study groups required depends on whether the researchers intend to demonstrate synergism (Table 2 ). Health outcomes and selective utilizationterm·dom of the substrate by host microbiota must be demonstrated in the same study. When experimental trials are not feasible or ethical, observational trials, including prospective longitudinal studies, are useful; these studies must accurately capture synbioticterm·dom exposure and control for relevant confounders (such as diet or antibiotics).

Evidence required for synbioticsterm·dom using doses delivered in product Population or participants When choosing the study population, many parameters need to be defined, including the target hostterm·key (including non-human species), target life stage (for example, pregnant women, infants, children, adults or elderly) and health status (for example, healthy, at-risk, or with a diagnosed disease or condition). Furthermore, microbiome-related factors, such as recent probioticterm·dom, prebioticterm·dom, synbioticterm·dom or antibiotic use, microbiota-disrupting medications and diet can be considered as (in)eligibility criteria, depending on primary health and microbiota outcomes. A 2-week wash-in period is generally adequate for probioticsterm·dom; however, certain populations might require longer wash-in periods (such as 4 weeks for those with slow transit time) 56 . The time since last antibiotic exposure will vary depending on a range of factors, including type of antibiotic, dose, duration and whether antibiotic use has occurred on multiple occasions in fairly short succession 57 , 58 . A general recommendation is difficult to make, although a common exclusion is individuals who have taken antibiotics 3 months prior to a study. However, at a minimum, 4 weeks since cessation of antibiotics is recommended. Non-antibiotic prescription medications should also be considered if they can influence bowel function and the microbiome 59 .

The population subgroups of interest should be specified. Clinical factors (such as sex or BMI), baseline microbiome features (such as presence of specific microbial taxa) and other microbiome-relevant factors (such as dietary fibre intake, maternal secretor status for studies on human milk oligosaccharides or birth mode) might be relevant to analyse. For studies on the elderly, consider age-associated differences in transit time, gastric acidity and diet diversity. For mother–infant dyads, consider possible vertical transmission of microorganisms from mother to infant in breast milk, microorganisms and substrates naturally present in breast milk, and the unique characteristics of immature infant microbiome.

Intervention The intervention should be fully described to enable replication of the study. This entails an adequate description of substrate and live microorganism dosages, specification of the microbial strain, the structure and purity of the substrate, and the timing and route of administration of the intervention. Subject compliance with an oral intervention can include measurement of the administered microorganisms in stool.

The duration of the intervention will be determined based on outcome; microbiota changes can be rapid but health outcomes will vary from days to weeks (such as constipation, stool frequency) to months (such as reduced fat mass, glycaemia). The background diet must be considered and, if possible, monitored as diet could provide a source of prebioticterm·dom substrates that are intrinsic and intact within certain foods (such as onions, wheat) as well as live microorganisms (such as fermented foods). Diet monitoring should include the use of validated methods such as the National Cancer Institute Dietary History Questionnaire (DHQ), Automated Self-Administered 24-hour dietary assessment tool (ASA24) and nutrient analysis software (such as Nutrition Data System for Research).

Placebo or control Design of a placebo or control is driven by the comparisons of interest. Often, a fully inert control is preferred. For formulations within pills or sachets, a low dose of highly digestible ingredients (such as maltodextrin or corn starch) or slowly fermentable fibre (such as microcrystalline cellulose) are acceptable placebos, which enables double blinding. For interventions composed of a food or beverage, the control group must be carefully considered, including differences in flavour, texture and nutrient content. These factors will also determine whether the study can be considered as a single-blinded or double-blinded design.

Outcome A synbioticterm·dom requires both a health outcome and a microbiota outcome in the same study. Primary and secondary health outcome(s) must be clearly specified. The microbiota outcome should be hypothesis-driven and provide insight into the microbiota-mediated mechanisms underlying the health outcome. Outcomes may include the abundance and viability of the administered live microorganism, changes in overall microbiota and/or microbiome composition, abundance of specific taxa and/or strains, microbial-derived metabolites, or others. Metabolomic analysis could also be performed using liquid chromatography–mass spectrometry, nuclear magnetic resonance or other suitable methods.

Statistics A statistician’s involvement in the design phase of a study will provide confidence that the sample size is sufficient for the defined outcomes using intention-to-treat analysis. Mediation analyses might be useful to assess whether microbiota effects are contributing to the health benefitterm·key. Ancillary analyses, which could include population subgroups, responders versus non-respondersterm·dom or exploratory microbiota analyses should be pre-specified.

Finally, as with all trials, funding sources should be transparently reported, AEs must be carefully recorded and reported, and study design, execution and reporting should comply with CONSORTterm·key guidelines 48 to minimize study bias.

Designing a synbioticterm·dom trial There are multiple ways to design a synbioticterm·dom trial intended to examine health effects in the target hostterm·key. Here, we provide some recommendations based on the criteria established herein to demonstrate complementary or synergistic synbioticsterm·dom. Pre-specified subgroup analyses should be considered based on the relevant clinical factors (such as sex and age), baseline gut microbiotaterm·key factors, dietary factors (such as typical dietary fibre intake) or other relevant factors (such as responder analysis). The number of experimental groups or ‘study arms’ required to prove efficacy and support mechanistic understanding will depend on the existing level of evidence for the component live microorganism(s) and selectively utilized substrate(s) as well as the researchers’ intent to determine whether components of the test mixture are complementary or acting as synergistic synbioticsterm·dom (Table 2 ).

When selecting the study design and determining the duration of the intervention and (if applicable) of the wash-outterm·var period, general recommendations are difficult to make. Factors that will impact these decisions include the live microorganism dose and its ability to persist in situ as well as the characteristics of study subjects; for example, in elderly individuals or persons with slow intestinal transit time, a longer wash-outterm·var period could be required compared with a study conducted in younger individuals 56 . Parallel arm designs would be appropriate for studies that aim to demonstrate improvements in metabolic health measures, such as of adiposity or glycaemic control, as these outcomes typically require longer-term interventions (that is, 12 weeks or more). Gut microbiotaterm·key changes can be rapid (within days) 60 . Currently, changes in the gut microbiotaterm·key and microbial metabolites, such as short-chain fatty acids, are not accepted by regulatory bodies as measures of a health outcome 61 .

Responders and non-respondersterm·dom among study participants in intervention studies with probioticsterm·dom and prebioticsterm·dom are commonly observed, which could justify including the probability of a positive response as a study end point. In the context of synbioticsterm·dom, non-respondersterm·dom include individuals for whom little or no change in gut microbiotaterm·key composition and/or clinical end point occurs compared with placebo. Various reasons for the non-responderterm·dom phenotype include limited available niches for desired gut microorganisms (either native or introduced as probioticsterm·dom) 47 , limited substrates to support microbial growth 62 , host immune system expressing intolerance for specific microorganisms 63 or a lack of specific microorganisms in the host microbiota 3 . Although many scenarios of formulations for synbioticsterm·dom can be conceived, we provide both actual and hypothetical examples to illustrate the concept (Box 2 ).

Trials for synergistic synbioticsterm·dom Studies on a synergistic synbioticterm·dom that compare the synbioticterm·dom to the control can provide supportive evidence but do not constitute the primary evidence needed to confirm a synergistic synbioticterm·dom. Instead, a study including the combination, the substrate alone, the live microorganisms alone and a control should be conducted. Using an appropriate statistical model, the aim is to demonstrate that the combined effect is better than the estimated effects of each component separately. Evidence of selective utilizationterm·dom of the substrate by the co-administered live microorganism must be obtained from the same trial demonstrating the health benefitterm·key.

Trials for complementary synbioticsterm·dom A two-arm parallel or crossover study would be sufficient to test a complementary synbioticterm·dom. The aim is to demonstrate that the combination is better than the placebo group with a relevant health end point. As a demonstrated prebioticterm·dom is used to formulate a complementary synbioticterm·dom, we do not require that selective utilizationterm·dom by the indigenous microbiota be reconfirmed in this clinical trial.

Measurement of selective utilizationterm·dom Measurement of selective utilizationterm·dom by the microbiota might involve different approaches, including both in vitro model systems and in vivo studies in the target hostterm·key. For example, selective utilizationterm·dom of a substrate could be demonstrated experimentally using well-established in vitro gut models 64 that include measurement of substrates and products during a prescribed time course. Ultimately, however, studies must be done in the target hostterm·key to show that specific microorganisms or taxa had been enriched or their activity enhanced by a particular substrate. For synergistic synbioticsterm·dom, methods that quantify the live microorganism should be used to show that the target strain has indeed been enriched 21 and other suitable methods can be used to show that its function has been enhanced. Microbiota features that are useful to measure can include characterization of the overall gut microbial community and microbiota diversity 65 , abundance and/or activity of specific taxa 66 , microorganism–microorganism or microorganism–host proximity 67 , the presence and/or abundance of specific microbial genes or gene clusters of interest, and/or metabolite concentrations 68 .

Safety measures for synbioticsterm·dom Prebioticsterm·dom and probioticsterm·dom tested to date have a strong safety record 69 – 74 , and synbioticsterm·dom formulated with them might also be presumed safe for the same intended uses. However, novel formulations must be suitably assessed for safety. Unfortunately, historically, many probioticterm·dom and prebioticterm·dom intervention trials have not adequately reported the types and frequency of AEs or serious AEs, perhaps owing to expectations that, as food ingredients, these products were inherently safe or that AEs could be due to failure to comply with norms for reporting harms in RCTs. Nonetheless, clear guidance for reporting AEs and serious AEs is provided by CONSORTterm·key 75 and these standards should be followed. Describing such events as “unrelated to the study product”, without justification for this statement, is unacceptable.

A systematic review of 384 interventions involving prebioticsterm·dom, probioticsterm·dom and synbioticsterm·dom found that no safety-related data were reported in 37% of the RCTs and 89 studies only used generic statements to describe AEs 76 . Up to 98% of studies did not provide a definition of AEs or serious AEs, the number of participant withdrawals due to AEs, or the number of AEs and serious AEs per study group. Taken together, these results are evidence that some studies inadequately collect or report data on AEs. van den Nieuwboer and colleagues 72 , 77 – 79 reviewed the AEs reported in studies with probioticterm·dom and synbioticterm·dom interventions and classified them according to the Common Terminology Criteria for Adverse Events system 80 ; they found that the incidence of AEs in each category was either no different from or often lower in the control groups than in the treatment group.

Thorough safety assessment of a synergistic synbioticterm·dom requires consideration that the added microorganism will express enhanced functionality in the presence of a targeted substrate such as improved growth or altered metabolic or physiological activity in vivo. This aspect suggests that safety assessments conducted on the live microorganism in isolation might not be sufficient to enable a conclusion about its safety when paired with a substrate that alters physiology or might in effect alter the dose delivered in vivo. Implications of such an interaction should be considered when assessing the safety of synergistic synbioticsterm·dom.

Implications of this consensus The panel recognizes the importance of developing a scientifically valid definition of the term synbioticterm·dom. The intention is to provide clarity for a diverse range of stakeholders, including consumers, regulators, health-care providers, researchers, industry and scientific organizations communicating about synbioticsterm·dom. Table 3 is designed to clearly lay out the criteria for synbioticsterm·dom in comparison to probioticsterm·dom and prebioticsterm·dom. Within the commercial supply chain, many different industry sectors, including ingredient suppliers, end-product manufacturers and retailers are very interested in how synbioticsterm·dom are defined. As synbioticterm·dom products are being used more frequently in clinical trials, it is incumbent upon scientists to clarify the appropriate use of this term as has been done for the terms ‘probioticterm·dom’ 12 and ‘prebioticterm·dom’ 13 .

Minimum criteria to appropriately use the terms ‘probioticterm·dom’, ‘prebioticterm·dom’ and ‘synbioticterm·dom’

Although the term ‘synbioticterm·dom’ might not be as well recognized as probioticsterm·dom and prebioticsterm·dom, it is nonetheless found on product labels, in popular press articles and in the scientific literature. The first mention of a synbioticterm·dom was in 1995 yet, according to PubMed searches, in 2019, 269 papers were published using the term. Consumer exposure is expected to increase. Thus, it is hoped that the definition herein is clear and widely accepted and will counter misuse of the term, including by scientists 8 . Table 3 provides the minimum criteria for the correct use of terms associated with synbioticsterm·dom.

The importance of context The panel urges stakeholders to carefully consider context for communications to consumers on the health benefitsterm·key of synbioticsterm·dom. The overall impression of any communication should lead consumers to understand the claim in a manner that is consistent with the evidence; any misleading use of the term constitutes misuse. Communication of the tested health benefitterm·key must accurately reflect what was reported in the clinical trial; results should not be extrapolated to health conditions, populations or synbioticsterm·dom that have not been studied.

Regulators Regulatory authorities are primarily focused on two issues: product safety and product labelling. These encompass both truthfulness and compliance with regulatory statutes. Even if the term synbioticterm·dom is not included in governmental guidelines or regulations, the use of our proposed scientific definition of the term will aid regulatory oversight of products labelled as ‘synbioticterm·dom’.

Regulatory statutes will differ with regard to geographical regions, regulatory categories, types of allowable claims and premarket approval. Furthermore, different standards exist for manufacturing, efficacy and safety depending on geographical region and product category. The term synbioticterm·dom does not stipulate a regulatory category, so (simply stated), regulatory requirements for a synbioticterm·dom would need to meet those that apply to the category (for example, drug, food or supplement) of the marketed product.

Regulatory complications can result in regions that impose probiotic-specific regulations. For example, Canada 81 , 82 , Italy 83 , Argentina 84 , Chile 85 , Colombia 86 and Brazil 87 have requirements specific to probioticterm·dom foods or supplements. Furthermore, there is a proposal 88 under consideration by Codex Alimentariusterm that could result in probiotic-specific global standards 89 . The Codex Alimentariusterm is a collection of standards, guidelines and codes of practice adopted under the auspices of the Food and Agricultural Organization of the United Nations and World Health Organization to protect consumer health and promote fair practices in food trade. Codex Alimentariusterm standards could affect probioticterm·dom products traded globally. Could product manufacturers potentially avoid these regulations by labelling a product as a synbioticterm·dom? Without the use of the term probioticterm·dom or prebioticterm·dom, relevant specific regulations would not be triggered. Perhaps this approach would not provide a marketing advantage, as consumer recognition and demand for synbioticterm·dom products is currently not as advanced as for probioticsterm·dom and prebioticsterm·dom. However, regions supporting probiotic-specific (or prebiotic-specific) regulations will need to address how the concept of synbioticsterm·dom fits into those regulations. Our stipulation is that manufacturers of complementary synbioticsterm·dom should meet current probioticterm·dom or prebioticterm·dom regulations.

One regulatory consequence of probioticterm·dom and prebioticterm·dom definitions is that the European Union has determined that labelling a food product as such amounts to an implied health claim 90 . As health claims in the European Union must be approved, the use of ‘probioticterm·dom’ or ‘prebioticterm·dom’ on a food label is subject to a health claim approval process. Despite controversy surrounding this situation, we can expect that the European Union might adopt a similar position for the use of the term ‘synbioticterm·dom’ because it also requires evidence of a health benefitterm·key.

Scientists Synbioticsterm·dom present challenges for researchers, including deciphering mechanisms underlying the health benefitterm·key and proving that the microbiota are modulated via complementary or synergistic means. Unless appropriate experimental methods of verification are used, the product under assessment should not be referred to as ‘synbioticterm·dom’.

As for probioticsterm·dom, prebioticsterm·dom and synbioticsterm·dom, it might be possible a priori to stratify study participants to enhance either the magnitude of the response or the number of responders to a given synbioticterm·dom treatment. The baseline composition of gut microbiotaterm·key of study participants could be one useful stratification factor 91 , 92 . This stratification might improve mechanistic understanding of observed effects and could be useful for characterizing responders and non-respondersterm·dom. Ultimately, this approach could enable better translation of clinical trial outcomes to those who are likely to benefit.

Scientists involved in the publication process (authors, editors, reviewers) should use the term synbioticterm·dom correctly and reject erroneous or unsupported use of the term. Even if a given treatment fails to demonstrate an effect (that is, acceptance of the null hypothesis) in a well-conducted study, such results still warrant publication 93 .

Industry The business community plays a vital part in translating the outcomes of fundamental and clinical research into commercial products that can benefit consumers. To the extent that industry funds research, it is essential that studies adhere to well-established guidelines to manage conflicts of interest and minimize bias 94 , 95 . Further, industry bears the duty of responsible manufacture, quality control and marketing of synbioticterm·dom products. It is incumbent on industry to follow good manufacturing practices, engage in truthful labelling and product promotion as well as adhere to appropriate use of the term ‘synbioticsterm·dom’.

Media The media (TV, radio, newspapers, magazines, websites and social media) have an increasingly central role in communicating science. These efforts require commitment and skill to craft clear, simple messages that are true to the science. Too often, scientists or their institutions do not effectively translate complex research findings so that they can be understood by the lay public. At times, overextended or inflated representations of research findings can be communicated through official academic or journal press releases. Communicators are therefore encouraged to distinguish between association studies and those presenting causal evidence, to describe single studies in the context of the totality of evidence, to avoid overgeneralizing results (for example, from model organisms such as mice) and to note study limitations. This requirement is true for null studies as well as those suggesting benefits from the interventions being tested. In this context, the media should adopt the scientific definition of synbioticsterm·dom herein.

Conclusions This Consensus Statement provides a new definition for a ‘synbioticterm·dom’ based on review by a panel of experts. To summarize (Box 1 ), a synbioticterm·dom is a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefitterm·key on the host. Two categories of synbioticsterm·dom are recognized. A complementary synbioticterm·dom is composed of a probioticterm·dom and a prebioticterm·dom that together confer one or more health benefitsterm·key but do not require co-dependent function; the components must be used at doses that have been shown to be effective for the components alone. A synergistic synbioticterm·dom contains a substrate that is selectively utilized by the co-administered live microorganism(s). Synbioticterm·dom products are not confined to human applications but could also include companion animals and livestock. They might also be directed to specific subpopulations (age, sex, health status) of the target species. The definition can also be applied to intestinal or extra-intestinal microbial ecosystems. The hope is that, going forward, this updated definition will be utilized and that it will aid in advancing synbioticterm·dom research, improve stakeholder understanding and enable better communication to consumers.

Acknowledgements This panel was supported by the International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom (ISAPP), a not-for-profit organization dedicated to advancing the science of probioticsterm·dom and prebioticsterm·dom. ISAPP’s activities are determined by its volunteer board of directors, comprising academic scientists. Through its Industry Advisory Committee, ISAPP incorporates industry scientists in its activities and raises funds. ISAPP strives to be an objective, science-based voice for the probioticterm·dom and prebioticterm·dom fields. The content of this paper was reviewed and approved by the ISAPP board of directors. The Rowett Institute (K.P.S.) receives financial support from the Scottish Government (RESAS).

Author contributions All authors participated in the panel from which the ideas for this paper were generated. K.S.S, G.R.G, R.H., R.A.R., G.R., K.V., K.P.S., H.D.H., M.B.A. and M.E.S. contributed written sections for the paper. K.S.S. and M.E.S. compiled the paper, with invaluable support from G.R.G. and R.H. All authors reviewed, commented on and approved the final version of the paper.

Competing interests K.S.S. has received funding from Ingredion, Beneo GmbH and Gnubiotics Sciences. G.R.G. currently receives research funding from GlaxoSmithKline, Pukka Herbs, and consulted for Clasado and Pepsico. G.R. has consulted for Seed, KGK Science, Acerus Pharmaceuticals, Danone, Chris Hansen, Altmann, Kimberly Clark and Metagenics. R.H. has received grants or honoraria from Mead Johnson Nutrition, Pharmavite, Danone, Beachbody, and PepsiCo and is a co-owner of Synbioticterm·dom Health. R.A.R. has received presentation honoraria from Beneo and research funding from General Mills and Sensus. K.V. received presentation honoraria from Beneo and Yakult, consultancy fees from Comet Bio and research funding from Nestlé. K.P.S. currently receives research funding from Probi and has received presentation honoraria from Yakult. H.D.H. has received presentation honoraria from Kellogg’s, PepsiCo and Nutricia North America. M.B.A. has received grants and presentation honoraria from Prolacta Biosciences and presentation honoraria from Medela. M.E.S. has been compensated for consulting, serving on advisory boards or for speaking engagements from California Dairy Research Foundation, Church & Dwight, Clorox, Danone North America, Danone Research, General Mills, Georgetown, GlaxoSmithKline, International Scientific Association for Probioticsterm·dom and Prebioticsterm·dom, JHeimbach LLC, Kellogg, Kerry, Mead Johnson, PepsiCo, Probi, Trouw, Visalia Dairy Company, Winclove, Yakult and Yakult Foundation of India. N.M.D. declares no competing interests.

Footnotes References