Trends in

Microbiology OPEN ACCESS

Special Issue: Regulation of Human Health by the Microbiota
Feature Review

Shaping the Future of Probiotics and Prebiotics

Marla Cunningham ,1,* M. Andrea Azcarate-Peril,2 Alan Barnard,3 Valerie Benoit,4 Roberta Grimaldi,5
Denis Guyonnet,6 Hannah D. Holscher,7 Kirsty Hunter,8 Sarmauli Manurung,9 David Obis,10
Mariya I. Petrova,11 Robert E. Steinert,12,13 Kelly S. Swanson,14 Douwe van Sinderen,15
Jelena Vulevic,16,17 and Glenn R. Gibson17

Recent and ongoing developments in microbiome science are enabling new Highlights
frontiers of research for probiotics and prebiotics. Novel types, mechanisms, An expanding range of candidate
and applications currently under study have the potential to change scientific probiotic species and prebiotic
substrates is emerging to address
understanding as well as nutritional and healthcare applications of these newly elucidated data-driven micro-
interventions. The expansion of related fields of microbiome-targeted interven- bial niches and host targets.
tions, and an evolving landscape for implementation across regulatory, policy,
prescriber, and consumer spheres, portends an era of significant change. In this Overlapping with, and adjacent to,
the probiotic and prebiotic fields,
review we examine recent, emerging, and anticipated trends in probiotic and new variants of microbiome-modulating
prebiotic science, and create a vision for broad areas of developing influence in interventions are developing, including
the field. synbiotics, postbiotics, microbial con-
sortia, live biotherapeutic products, and
genetically modified organisms, with
Background and Current State renewed interest in polyphenols, fibres,
Probiotics (see Glossary) and prebiotics have received escalating attention in recent years in and fermented foods.

the scientific, healthcare, and public arenas. Publicity around microbiome research has also
Personalised nutrition and precision
broadened the public perception of microorganisms, beyond disease-causing agents that should medicine are beginning to influence the
be avoided, to a more rational view integrating an understanding of the beneficial roles of micro- application of probiotics and prebiotics,
organisms in human health. In line with these advances, public awareness and acceptance of with growing interest in modulation of mi-
crobial signatures of health and disease.
probiotics and prebiotics continues to expand [1], with probiotic industry growth estimated at
7% annually [2], and prebiotic growth forecast at 12.7% over the next 8 years [3]. Demand for probiotics and prebiotics
across divergent product formats is driv-
While there is a general consumer view that probiotics and prebiotics are beneficial, there is still a ing innovation in quality assurance tech-
niques to measure dose, viability, and
gap in understanding on definitions of the terms 'probiotics' and 'prebiotics', their benefits to structural and functional integrity.
health, how they function, and where to find the best sources in food and healthcare products
[1,4]. Both probiotics and prebiotics are increasingly incorporated into a wide range of foods,
beverages, and topical products (even toilet paper), in some cases with questionable or no scien-
1
tific validation of any health benefit to the host, as is the requirement of existing consensus Department of Science and Innovation,
Metagenics, PO Box 675, Virginia BC,
definitions. QLD, 4014, Australia
2
UNC Departments of Medicine and
In this scientific field, definitions for both are clearly established, with the International Scien- Nutrition, Microbiome Core Facility,
University of North Carolina, Chapel Hill,
tific Association for Probiotics and Prebiotics (ISAPP) having convened consensus panels NC, USA
whereby experts reviewed and published the science behind probiotics [5] and prebiotics 3
Campaign It! Ltd, London, UK
4
[6]. The conclusions of these panels highlighted that they play an integral role in health sta- Bell Institute of Health and Nutrition,
General Mills, Minneapolis, MN, USA
tus. Some key mechanisms have been elucidated (Box 1) and both have been used in a va- 5
Clinical Development, GSK Consumer
riety of health states, prophylactically and therapeutically. Healthcare, Weybridge, UK
6
Diana Nova, Symrise Nutrition,
Clichy-la-Garenne, France
Currently, multiple spheres of influence are acting on the probiotic and prebiotic fields (Figure 1). 7
Department of Food Science and
Broad technological advances in data collection and analytical tools are enabling the exploration Human Nutrition and Division of
of new candidate probiotics and prebiotics as well as providing deeper insights into their interac- Nutritional Sciences, University of
Illinois, Urbana, IL, USA
tions with the microbiome and host. Interest continues to grow into new applications of probiotics 8
Department of Sport Science,
and prebiotics across health conditions, body sites, population subgroups, and delivery formats. Nottingham Trent University, UK

Trends in Microbiology, August 2021, Vol. 29, No. 8 https://doi.org/10.1016/j.tim.2021.01.003 667

© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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9
Box 1. Mechanisms of Action of Probiotics and Prebiotics Emerging Sciences Research, Reckitt
Benckiser, Nijmegen, The Netherlands
Mechanisms of action of probiotics and prebiotics are complex, diverse, heterogeneous, and often strain- and compound- 10
Danone Nutricia Research, Palaiseau
specific. While many have been described, there remain calls for increased understanding, especially structure–function
Cedex, France
explanations of observed health effects and long-term influences [134,152,153]. 11
Winclove Probiotics, Amsterdam, The
Netherlands
Probiotics interact with both the host and the microbiome via molecular effectors present on the cell structure or secreted 12
R&D Human Nutrition and Health,
as metabolic products. Probiotic metabolites can act on the microbiota by crossfeeding interactions, changes in the gas- DSM Nutritional Products Ltd, Basel,
trointestinal microenvironment (e.g., pH lowering), competition for nutrients and binding sites, and inhibition of growth via Switzerland
the production of strain-specific antibacterial compounds including bacteriocins [133,152,153]. Such microbiota-directed 13
Department of Surgery, Division of
effects contribute to the ability of probiotics to mediate health benefits in pathogen overgrowth states such as vaginal and Visceral and Transplantation Surgery,
oral dysbioses [153]. University Hospital Zürich, Switzerland
14
Department of Animal Sciences,
With regard to host cells, probiotic effector molecules can interact directly with receptors in intestinal epithelial, University of Illinois at Urbana-
enteroendocrine, and immune cells as well as vagal afferent fibres. These interactions produce local gut effects, such as Champaign, Urbana, IL, USA
15
enhancement of intestinal barrier integrity and inflammation (e.g., via Toll-like receptors), as well as systemic effects via host Microbiology BioSciences Institute,
immune, endocrine, and nervous system mediators [133,152,153]. Probiotics can also perform enzymatic metabolism of University College Cork, Cork, Ireland
16
host compounds such as bile salts and ingested xenobiotics [152]. Specific probiotic surface-associated effector veMico Ltd, Reading, UK
17
molecules include pili, lipoteichoic acids, exopolysaccharides, and various surface-layer proteins, many of which are Department of Food and Nutritional
strain-specific and therefore mediate the delivery of strain-specific effects [133,153]. Sciences, University of Reading,
Reading, UK
Classical prebiotic effects are mediated through consumption of the substrate by specific groups within the microbiota,
promoting their growth and metabolic activity. Provision of substrate to select group/s of bacteria can also indirectly
influence other bacterial groups within the microbiome – promoting growth through crossfeeding interactions as well as
inhibitory effects via pathogen displacement. Resulting changes in microbial composition and metabolite concentrations
from prebiotic administration impact host epithelial, immune, nervous, and endocrine signalling and mediate health bene- *Correspondence:
fits such as improvements in bowel function, immune response, glucose and lipid metabolism, bone health, and regulation [email protected]
of appetite and satiety [6]. Chief by-products of bacterial prebiotic metabolism are the SCFAs acetate, butyrate, and (M. Cunningham).
propionate, which are well recognised to interact with these host systems and facilitate many prebiotic effects [10].

In addition to nutritive effects on microbes, prebiotic molecules are also recognised to interact directly with host receptors,
modulating immune and gut epithelial cell signalling with local effects on inflammation and barrier function [154].

Furthermore, evolution in regulatory frameworks, clinical guidelines and industry trends is

influencing the implementation of probiotics and prebiotics into nutrition and healthcare. As our
knowledge continues to expand in each of these fields, a broad and integrated review of trends
shaping the future of probiotics and prebiotics is timely.

Probiotics – Novel Species, Health Targets, and Evaluation Frameworks

Traditionally, lactobacilli, bifidobacteria, and other lactic acid-producing bacteria (LAB) have been
used as probiotics, primarily isolated from fermented dairy products and the faecal microbiome. As
knowledge of the breadth of the human microbiome and its functions has expanded, the future
holds a range of potential new discovery approaches [7] as well as new potential probiotic taxa. De-
velopments in affordable complete genome sequencing and powerful cultivation methods have
allowed isolation and characterisation of a new range of microorganisms from human microbiomes
with potential health benefits and the opportunity to be developed as next-generation probiotics
[8] (Figure 2). Various bacteria, such as Roseburia intestinalis, Faecalibacterium prausnitzii, Eubacte-
rium spp., Bacteroides spp. and Akkermansia muciniphila, have been isolated from the human gut
with growing interest in their probiotic potential [8,9]. These candidates represent a significant propor-
tion of the currently cultivable human gut microbiome and offer physiological functions that are not
always directly conferred by bifidobacteria or lactobacilli, such as the production of butyrate, propio-
nate, and other bioactives [10]. Converting these species into industrially viable probiotics presents
challenges as their requirement for rich growth media and anaerobic conditions adds cost and com-
plexity, as well as investment in determining optimal fermentation and manufacturing processes over
time. Despite these difficulties, A. muciniphila is one of the more promising candidates. Isolated in
2004 [11], it has been tested in preclinical animal models and shown to prevent development of obe-
sity, with bacterial pasteurisation increasing stability and efficacy of the species. Initial proof-of-

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concept studies have taken place in humans and shown that both live and pasteurised A. muciniphila Glossary
is safe to use in humans and improves several metabolic parameters [12]. Live A. muciniphila is al- Bioinformatics: computational
ready on the market in a multispecies synbiotic preparation, containing inulin, Bifidobacterium analyses of biological data.
longum subsp. infantis and other anaerobic bacteria (Clostridium beijerinckii, Clostridium butyricum, Candidate prebiotic
oligosaccharides: oligomers that may
and Anaerobutyricum hallii) and was shown to improve glucose levels in type 2 diabetics [13].
fulfil the current criteria for prebiotic but
lack (in vivo) confirmation currently.
The gut microbiome will not be the only source of new candidate probiotic strains (Figure 1). Niches of Crossfeeding: whereby one or more
strong interest for discovery of new species, and as targets for intervention, include the female metabolic products from
microorganism(s) can serve as growth
urogenital tract, oral cavity, nasopharyngeal tract, and skin [14–16]. Species or genera associated substrates for others.
with health in these regions are being investigated as potential interventions to restore microbial Faecal microbial transplantation:
populations and therefore physiological homeostasis in disease states. Examples include the skin the transfer (through a processed
commensal isolate Staphylococcus hominis for eczema and atopic dermatitis [17], and Lactobacillus mixture of liquid stool) of healthy bacteria
from a donor into the intestines of the
crispatus for vaginal dysbiosis [18]. Fermented foods are the most common natural source of patient (recipient).
potentially probiotic strains of LAB, and consumption has been associated with significant health Fatty acids: carboxylic acids, with
benefits, including reduced risk of type 2 diabetes and cardiovascular diseases [19] as well as a aliphatic chains, that can be either
saturated or unsaturated.
putatively beneficial metabolomic profile [20]. These foods are most likely the major source of LAB
Fermentable fibres: dietary fibres
in the human gut microbiome [21] and show potential for future probiotic development. Fermented broken down by microbial growth in the
and unfermented food sources of future probiotics may include fruits, vegetables, grains/cereals, gut.
dairy, meat and fish products, and honey, as well as environmental sources such as soil [22]. Fermented foods: foods and
beverages that have involved microbial
growth and activities.
In addition to the core heartlands of gut and immune health, emerging target conditions for pro- Generally recognised as safe
biotic therapy include subfertility [23], liver disease [24], mood disorders [25], cognition [26], oral (GRAS): a notification to the FDA stating
health [27], asthma [28], metabolic disease [29], hypercholesterolaemia [30], and obesity [31]. that a substance is generally recognised,
among qualified experts, as having been
adequately shown to be safe under the
Significant emphasis will be placed on investigating the safety of novel species and genera conditions of its intended use.
considered for the development of new probiotic products [8,9]. Many commonly exploited Genetically modified organisms:
and currently available probiotic strains benefit from a generally recognized as safe (GRAS) organisms whose genetic material has
been altered using genetic engineering
status in the USA or belong to species with qualified presumption of safety (QPS) status techniques.
with the European Food Safety Authority (EFSA), yet this is not yet the case for other candidate Gut-simulation models: in vitro
novel probiotic species that have no history of use. Submission through GRAS, QPS, and systems that mimic the human or animal
novel food frameworks may enable a path to commercialisation, and for pharmaceutical gut.
Live biotherapeutic products:
applications, novel regulatory frameworks are emerging, for example, the live biotherapeutic biological products that contains live
products category being defined by the Food and Drug Administration (FDA)i and the organisms and are applicable to the
European Directorate for the Quality of Medicines [32]. A complete characterisation of strains prevention, treatment, or cure of a
disease.
from these new species will likely be required [33], comprising retrospective analysis of possible
Metabolome: the combined metabolic
human disease linked with the taxa considered, full genome sequence, antibiotic resistance outputs of the host and its microbial
genes, toxin genes, transferrable genetic elements, virulence factors, proven safety in animal content.
models, pharmacokinetics, pharmacodynamics, and Phase I–III trials. Many live biotherapeutic Metagenomic: the study of the
collective genome of microorganisms
products with appropriate clinical evidence will fall within the current scientific definition of
from an environmental sample.
probiotics [5] (Figure 2), albeit attracting specific regulatory attention. Microbial consortia: a mixture of
microbial species with symbiotic
The discovery of defined therapeutic microbial consortia with network interactions and syner- interactions. It can include well-defined
consortia with fully characterised
gistic effects [34] will augment the development of single-strain organisms in the future and re-
members or undefined mixtures.
main in the scope of the current probiotic definition, if well characterised [5] (Figure 2). Adjacent Microbiome signature: a
to probiotics, postbiotics1 – microbial fragments and metabolites [35] – have been shown to characteristic pattern of
share many, though not all, mechanisms of their live probiotic counterparts. Some new promising microorganisms.
Next-generation probiotics:
gut isolates will also most likely be commercialised under the postbiotic category, such as the probiotics from genera with no history of
use as probiotics, and which are likely to
be delivered under drug regulatory
1
frameworks.
An updated expert consensus panel definition, convened by ISAPP, is currently in press.

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abovementioned pasteurised A. muciniphila or bioactive proteins from this species that have Non-carbohydrate substrates:
microbial growth factors that are
shown beneficial effects [36]. independent of saccharolytic growth.
Omics: comprehensive analysis of
Prebiotics – Novel Substances, Sources, and Applications complete genetic or molecular profiles of
organisms, including genomics,
At the instigation of the prebiotic concept for gut microbiota management [37], and for many transcriptomics, proteomics, or
years since, the primary premise of prebiotics has been selectively fermented carbohydrates act- metabolomics.
ing in the colon and modulating levels of resident lactobacilli and bifidobacteria said to elicit health Polyphenols: naturally occurring plant
compounds containing phenol groups.
effects. In recent years, omics techniques have improved mechanistic in vitro and in vivo re-
Postbiotics: 1bacterial fragments with
search, as well as human clinical trials, to determine a fuller extent of prebiotic impacts. Current or without bioactive products of
targets for prebiotics have now expanded, beyond LAB, to a wider range of microbial responders microbial growth that are of benefit for
[6]. Similar to probiotics, these include candidate health-promoting genera such as Roseburia the host.
Prebiotic: a substrate that is selectively
spp., Eubacterium spp., Akkermansia spp., Christensensella spp., Propionibacterium spp., and
utilised by host microorganisms
Faecalibacterium spp. Prebiotics may be used to stimulate the growth of these and other conferring a health benefit.
bacterial groups directly or indirectly through crossfeeding interactions. One of the key health- Probiotics: live microorganisms that,
promoting benefits of such genera is their production of short-chain fatty acids (SCFAs) that when administered in adequate
amounts, confer a health benefit on the
regulate a range of gut and ex-gut functions, including gut epithelial and mucus barrier function,
host.
immunity, inflammation, glucose and lipid metabolism, energy expenditure, and satiety [10]. Qualified presumption of safety
(QPS): a status granted to genera,
Currently, a narrow range of confirmed prebiotic substances exists, with galactans and fructans species, or subspecies of
microorganisms by the EFSA after an
(e.g., inulin) dominating the market. The desire to stimulate a wider group of commensal organisms application is received and an
has allowed the development of novel candidate prebiotic compounds (Figure 2). These will likely assessment of available evidence on
include carbohydrate-based substances derived from plants – the source of traditional prebiotics characterisation, safety, and intended
such as inulin – but may also include those that mimic animal-derived substrates use is conducted.
Synbiotics: a mixture, comprising live
(oligosaccharides present in milk; O-linked glycans present in mucins), yeast-based substances, microorganisms and substrate(s)
and many non-carbohydrate substances including polyphenolics, fatty acids, herbs, and selectively utilised by host
other micronutrients. Over 8000 known polyphenols exist in plants, vegetables, and fruits, and microorganisms, that confers a health
benefit on the host.
many reach the colon intact, to be utilised by resident microorganisms [38]. Some polyphenols
Vitamins: organic molecules that are
have been shown to have prebiotic potential, such as cranberry-rich extracts stimulating essential micronutrients to support an
A. muciniphila [39], or to provide antimicrobial action against pathogens [40]. organism’s metabolism.

In the future, prebiotics will likely be isolated from novel sources (Figure 1) as focus on sustainability,
cost, and scale emerges [3]. The 1.3 billion tons of food waste generated annually in the food
chainii represents a rich and sustainable source of natural bioactive ingredients. Many side
streams from fruit, vegetable, and grain processing contain potential prebiotics, such as pectin
from orange peel [41] and arabinoxylans from distillery and brewing waste [42]. Future prebiotic
compounds may also be chemically or structurally modified by the application of sonication,
high pressure, acid, enzyme and oxidation treatments, in order to modify functionality. Further,
unique combinations of prebiotics in optimised mixtures may provide the ability to create new
profiles of benefits [43].

There is also growing interest in the use of prebiotics to affect other microbiomes within the host,
such as the female urogenital tract, oral cavity, and skin. As an example, prebiotic glucomannan
hydrolysates have been shown to modulate the skin microbiome and reduce acne when admin-
istrated topically [44]. There is also interest in more targeted prebiotic delivery within the distal
colon for treatment or prevention of colorectal cancer and ulcerative colitis. A mixture of different
chain length prebiotics, or specific delivery technologies, may allow delivery of intact prebiotics
towards the distal colon and selective stimulation of carbohydrate-metabolising genera therein,
reducing local proteolysis and the concomitant production of undesirable metabolites. Such
modulation of the colonic microbial metabolome to a healthier profile is likely to become a key
target for prebiotics, beyond simple microbial growth promotion [45]. The ability of prebiotics to

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Sources of probiotics Spheres of influence on Sources of prebiotics

Human development and implementation Plants and
microbiota fungi

Discovery
Gut Skin Health-associated microbes (top-down) Food
waste Plants
Target-based discovery (bottom-up)
Humanistic screening models
Urogenital Others Fungi
Evaluation
Genomic and structural characterisation, safety requirements Animals and
Fermented Integrated data – metagenomics, metabolomics microbes
foods Novel health benefits and disease targets

Microbial Mammalian
Manufacturing and formulation synthesis milk
Yogurt Vegetable
Novel production and extraction techniques
Quality assurance developments
Novel delivery technologies and formats Gut
Cheese glycans

Regulatory and policy

Nutrition, supplement, and drug category evolution Chemical and physical
Environment modification
Regulatory claim evaluation requirements
Policy and practice guidelines

Plants Ultrasound Acid

Soil
Implementation
Personalised healthcare and nutrition
Consumer and prescriber recognition and awareness
Animals Market growth and innovation demand Enzymes

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Figure 1. Influences on the Future of Prebiotics and Probiotics. The figure shows current and emerging influences on probiotics and prebiotics, including novel
sources, new discovery and evaluation techniques, manufacturing and formulation advances, regulatory and policy changes, and influences on implementation in
nutrition and healthcare. It is important to note that developments in one area influence growth in others – for example, the discovery of novel probiotic genera
necessitates evolution in manufacturing techniques and regulatory frameworks.

control detrimental bacterial growth via pathogen exclusion and virulence attenuation is an area of
interest and may extend beyond bacteria into viral pathogens [46]. Prebiotics, such as human
milk oligosaccharides (HMOs), may also act as decoy receptors that prevent the attachment
of pathogenic microorganisms, or via immunomodulatory interactions with host gut epithelial or
immune cells [47].

As of December 2020, there were 245 registered clinical trials (ClinicalTrials.gov) which have
completed evaluation of prebiotics (alone or in combination with probiotics) on ageing, autism,
bariatric surgery, colic, colon cancer, atopic dermatitis, constipation, diarrhoea, infant growth,
irritable bowel syndrome (IBS), obesity, and other conditions. The number of studies and
investigational targets are suggestive of significant investment in the development of prebiotics
as bioactive ingredients or supplements for a range of potential applications.

Stretching the Boundaries of Prebiotics

Polyphenols and other intestinal microbiome-modulating carbohydrates (including resistant
starch, polydextrose, xylo-oligosaccharides, pectin, and HMOs) are considered prebiotic candi-
dates but have not yet met the ISAPP consensus definition [6,48]. Also, overlapping and adjacent
to the current boundaries of prebiotics (Figure 2) are fermentable fibres and other substrates
such as vitamins, minerals, and fatty acids [6]. While all require further determination through
in vivo studies, such compounds are emerging to be utilised by the indigenous microbiome
and may have the potential to impart beneficial health effects upon the host.

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Fermentable
fibres
Genetically
modified
Candidate prebiotic
organisms
oligosaccharides
Probiotics Prebiotics
Next-
generation Synbiotics
probiotics
Live biotherapeutic
products

Faecal Polyphenols
microbial
transplantation
Microbial Fermented
consortia foods Fatty acids Non-carbohydrate
substrates

Vitamins
Postbiotics

(A) Microbes and microbe-derived preparations (B) Microbial substrates

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Figure 2. Probiotics, Prebiotics, and Adjacent Fields. Many areas of new discovery intersect and are adjacent to the currently defined fields of probiotics and
prebiotics. The figure depicts a conceptual map of these established and emerging concepts, with (A) microbes and microbe-derived preparations represented in
shades of blue; (B) microbial substrates represented in shades of yellow; and preparations containing both represented in green. Current and potential relationships
between fields are depicted by the overlap of shapes (note: the size and shape representing each field is for visual purposes only). Refer to the Glossary for definitions
of each field.

During the most recent decade, several papers have proposed alternative definitions of prebiotics,
with broader scope in order to better integrate emerging microbiome-modulating compounds
[49–51]. In particular, the requirement for ‘selective utilisation’ by a limited number of species or
genera has been questioned (despite an overall consensus in the literature to the contrary). As
our capacity has grown for detailed resolution and deeper understanding of the members and in-
teractions within the microbiome, one assertion is that differentiating in a binary manner between
growth effects on beneficial and nonbeneficial growth targets has become increasingly difficult.
Proponents also note that some of the health benefits of currently recognised prebiotics may be
ascribed to non-species-specific ecological or functional modulation of the microbiome, such as
increases in SCFA production or broad compositional modulation [51]. It is important to recognise
that blurring the boundaries between prebiotics and fermentable fibres in such a way would be in
conflict with the premise upon which prebiotics were built, that is, as a method for selectively
enhancing the growth of specific microorganisms with associated health benefits.

Other nonfermentable modulators of the microbiome also lie adjacent to the prebiotic scope
(Figure 2) and will play a role in the future of microbiome modulation. While vitamins are normally
absorbed in the small intestine, their administration in large amounts or in colon-targeted formula-
tions can exert modulation of the colonic microbiome, as has been demonstrated with both
riboflavin and niacin [52,53]. Furthermore, genomic studies have suggested that B vitamin

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exchange may be a component of normal symbiotic relationships among gut microbial species
[54], and several lines of evidence suggest a potential corrective role for colonic vitamin administra-
tion in some disease states [55].

Synbiotics and Complex Mixtures

Combining the effects of fermentable substrates and live microorganisms are blends known as
synbiotics. Synbiotics may be complementary or synergistic in nature. Complementary synbiotics
are comprised of a combination of an accepted prebiotic and probiotic, as their mechanisms of
action can be independent of each other, and both the prebiotic and the probiotic must have
their own demonstrated health benefits. Alternatively, synergistic synbiotics contain a fermentable
substrate for the coadministered live microbe, where the substrate and the microbe may or may
not be able to elicit a health benefit independently of the other. In this case the individual compo-
nents do not necessarily need to be confirmed probiotics or prebiotics (Figure 2); however, they
must have a demonstrated health benefit in combination [56]. Similar to the probiotic and
prebiotic fields, the future of synbiotics will be influenced by the development of novel strains
and substrates, informed by and targeted to vacant microbiome niches in individuals and
subgroups, with potential applications in both gastrointestinal and ex-gut sites.

Other possible mixtures, which sit outside of the synbiotic definition, will likely be developed in the
future, as food industry trends around gut health show no sign of slowing. Such mixtures may in-
clude combinations of live or killed microorganisms, coupled with potential microbiome-modulating
compounds such as fermentable substrates, vitamins, and minerals, phytochemicals, and other
plant-based materials. Fermented foods can be considered to be naturally occurring
microbiome-targeted mixtures (Figure 2), delivering microbes and microbial substrates together,
with a range of bioactive fermentation metabolites [19]. The increasing popularity of such foods
will likely drive everyday consumer recognition of prebiotics, probiotics, and synbiotics moving
forward.

Personalised Probiotics and Prebiotics

The search for individual and group microbiome signatures to predict disease incidence, pro-
gression, and response to treatment is a key area of microbiome research, enabled by efficient
and powerful processing of large data sets [57]. Unique taxonomic profiles and specific genera
and species have been associated with health and disease status [58] as well as host biomarkers,
dietary and lifestyle characteristics [59] in large cross-sectional studies. Based on these data,
there is significant interest in targeted strategies to modulate microbial composition within
hosts on a personalised or population subgroup level. Probiotics and prebiotics present as
promising candidate interventions with the potential to ‘redirect’ these signatures towards health
[7,57,60], utilising multiple potential modes of action (Table 1).

One approach is to replace under-represented microbes in vacant taxonomic niches; however,

LAB show only limited potential for direct microbial ‘replenishment’ strategies as they represent
only a small proportion of the adult gut microbiome. While probiotics have demonstrated little
impact or disruption to microbiome composition in healthy states [61], some lactobacilli and
Saccharomyces species have demonstrated partial restoration of commensal microbiome
groups in various manifestations and models of dysbiosis, including antibiotic administration,
alcohol-induced disruption, and in children with cystic fibrosis [62–64]. Substantially modifying
the adult microbiome in the future through replenishment strategies may be a possibility through
the development of novel microbiome-derived probiotics (see earlier), with the majority of current
candidates identified through top-down approaches as health-associated microbes [7]. How-
ever, it is also important to note that probiotics can exert independent health benefits outside

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Table 1. Precision Application of Probiotics and Prebiotics – Potential Strategies

Target Target component Precision application strategy
Host Health state Target unique host health states
• Targeted treatment of symptoms and disease states utilising
specific clinical trialled probiotic strain/s or prebiotic compound/s
Genotype, phenotype, Tailor for unique host characteristics
environment • Match suitability of specific probiotics and prebiotics for particular
diet, lifestyle, demographic, genetic factors
Microbiome Composition Fill vacant compositional niches (‘missing’ microbes)
• Reintroduce health-associated bacteria with microbiome-derived
probiotics
• Promote growth of under-represented species through targeted
prebiotics or crossfeeding interactions from probiotics
Reduce over-represented microbes
• Inhibit growth of detrimental microbes by creating unfavourable
environments, e.g., production of antimicrobial compounds
Function Fill vacant functional niches (‘missing’ functions)
• Augment beneficial metabolite levels through provision of relevant
prebiotic substrates and/or probiotics with specific metabolic
capacity
Reduce deleterious microbial metabolites
• Inhibit the production, or support the catabolism, of detrimental
compounds

of colonisation or microbiome modulation, and that probiotic ability to modulate the microbiome
should not be considered a prerequisite for its utility.

Prebiotics may also assist to correct compositional imbalance through promoting the growth of
under-represented species. While conventional prebiotics are primarily known for their
bifidogenic effects, crossfeeding interactions have shown the potential of inulin to modify some
other limited intestinal microbiome groups, including Faecalibacterium spp. and Anaerostipes
spp. [65]. In the future, novel and emerging prebiotic compounds may be able to be used in
targeted ways to manipulate the microbiome and its metabolic outputs. Many of the structural
characteristics of prebiotics are known to influence which microbes can utilise the substrate, in-
cluding monosaccharide structure, degree of polymerisation, branching, linkages, and addition of
functional groups or other modifications [43]. Clinical intervention studies utilising multiple sources
of either type II [66] or type IV [67] resistant starch, each with distinctive structural properties, have
demonstrated modulation of specific taxa and SCFAs unique to each compound. To aggregate
data from disparate studies, Lam and Cheung [43] proposed the creation of a multidimensional
prebiotic structure–microbiome matrix, sequentially testing and collating data from prebiotic inter-
ventions and mapping the resulting microbiome impact from each structural variation. Such infor-
mation could be teamed with machine learning to predict structural characteristics of a prebiotic
required for the modulation of specific microbiome profiles, and lead to custom prebiotic and
synbiotic production based on these characteristics [43]. Data collection and predictive modelling
could also capture microbial metabolic interactions [57], layering in the potentially complex eco-
system effects of designer prebiotic administration and mixtures thereof.

Taxonomic microbiome characterisation is increasingly being combined with metagenomic or

metabolomic data to understand what functions microbes might be performing. Integrated data
sets may assist to identify loss of microbiome functions, or vacant ‘functional niches’, important
to host health and providing further potential for precision medicine intervention [68]. A recent
longitudinal study [69], employing metagenomic and metabolomic analyses in samples from IBS

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patients and healthy controls, found differences in the levels of certain faecal metabolites and
relative abundance of taxa between the IBS constipation and diarrhoea subtypes and healthy con-
trols, as well as targets for potential probiotic intervention being proposed. For example, elevated
levels of primary bile acids could be addressed by the introduction of bile acid-metabolising micro-
bial consortia; suboptimal hypoxanthine production could be augmented through local microbial
production or inhibition of enzymatic breakdown; and SCFA and tryptamine production could be
increased for motility benefits in constipation [69]. Prebiotics and synbiotics may also assist to
address over- or under-represented metabolic microbiome pathways in the future. Direct application
of postbiotics to replenish missing compounds [60] is also a promising area of future investigation.

Other opportunities for precision application include targeting therapies based on the prediction
of responders and nonresponders. It has been shown that specific prebiotics may be more able
to confer a health benefit when they are given to an individual with appropriate baseline commen-
sal microbes to utilise the substrate. Microbial composition at baseline has been demonstrated to
predict microbiome response, fermentation rates, and butyrate production resulting from resis-
tant starch [66,70] and hydrolysed guar gum [71] supplementation. Interindividual intestinal
microbiome differences have also been linked to differential clinical response to prebiotics with re-
gard to stool consistency [71] in healthy adults and hepatic lipid metabolism in hepatic steatosis
patients [72]. This provides the possibility for more optimal matching of prebiotics to individual
microbiome characteristics. Metabotypes will also likely play a key role in personalised prebiotic
innovation into the future. The ability of individuals to metabolise polyphenols into smaller bioavail-
able metabolites through gut microbiota is one example. Specific bioactive molecules may only
be produced from polyphenols in the presence of specific gut microbiota species [73], and pro-
duction of these metabolites may serve as a useful surrogate marker of the ability of a subject to
benefit from a given polyphenol.

Nonresponse is also an issue for probiotics [74], which may be overcome in the future with pre-
cision application. For probiotics, the baseline microbiome provides a degree of colonisation re-
sistance that can impact the duration of residence as well as the penetration into the mucosal
microbiome, and potentially impact biological activity [7]. Several studies have demonstrated
variability in probiotic colonisation between individuals [75,76], either persistently or transiently
during supplementation; however, whether there is a resulting effect on clinical outcomes is cur-
rently unknown. Zmora and colleagues [76] identified multiple host immune factors (as well as
microbiome composition) as determinants of mucosal colonisation of administered probiotics.
Transcriptomics from mucosal biopsies revealed caecal host immune activity against Gram-
positive bacteria in colonisation-resistant individuals, inviting speculation of a personalised
predisposition to the creation of an inhospitable environment to probiotics.

Vacant functional niches identified by metabolomic stool features may also be predictive of pro-
biotic response. In children with gastroenteritis, a meaningful reduction in diarrhoea and lower in-
testinal inflammation in response to Limosilactobacillus reuteri DSM 17938 intervention was
predicted by lower baseline levels of faecal metabolites, including lactate [77]. Authors suggested
that a vacant metabolic niche may have existed in the responders that was amenable to the ac-
tivity of lactobacilli. In healthy individuals administered Lacticaseibacillus paracasei DG, baseline
levels of butyrate were found to predict the directional butyrate response to therapy [78]. These
results provide the potential for faecal metabolomics to be incorporated into future baseline mea-
sures to observe and predict individual response to probiotic therapy.

Future research could support the realisation of possibilities in this field in a number of ways.
Enhanced characterisation of the biological response to probiotics and prebiotics in clinical trials

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would enable deeper understanding of these interventions and their potential for precision
application. Calls have been made for increased use of integrated, multi-omic approaches to
characterise probiotic and prebiotic effects, including metagenomic, metatranscriptomic, and
metabolomic technologies [79,80]. Recognition of the relevance of effects within the mucosal
microbiome will likely predict inclusion of more invasive sampling in future trials as well as the de-
velopment of noninvasive techniques for sampling different regions of the gut [60]. Such deeper
characterisation would be coupled with analysis and reporting of clinical and biomarker response
at subgroup and individual levels. Improved reporting of responders and nonresponders in inter-
vention trials would enable aggregation of individual or small-subgroup data into meta-analyses
to better develop predictive modelling of microbial and host responses to prebiotics [79] and
probiotics. Further, the inclusion of multiple prebiotics or probiotics in head-to-head comparative
studies [7] will enable the detection of unique directional effects through direct comparison, and
facilitate identification of optimal interventions for groups, subgroups, or individuals.

Hypotheses developed from these avenues of research could be tested prospectively in future
clinical trials, with cohort selection or group stratification based on microbiome and other host
factors, such as diet or genetics. Subject selection may be possible on baseline factors, or in
the case of identifying metabotypes, a run-in period may be utilised to identify the response of
a specific microbial species and/or the production of specific metabolites after a short period of
intervention. The exclusion of a large portion of nonresponders in the screening phase of clinical
trials would increase predicted effect sizes and effectively test personalisation hypotheses.

As effective biomarkers of probiotic and prebiotic response are identified, implementation into
practice may warrant the conduct of extensive testing at the individual level [7], a potentially costly
and burdensome activity. While microbiome testing services for the general public are commer-
cially available, accessibility constraints such as cost and service delivery exist, and use in nutrition
and healthcare is not yet widespread [81]. Furthermore, extensive validation and refinement of
putative host or microbiome biomarkers will be required for clinical implementation. Ease of use
through the integration of data sets into digitalised decision-making tools [7] will facilitate con-
sumer nutrition and healthcare implementation alike, and effective dissemination through educa-
tion and practice guidelines will influence readiness and acceptance from healthcare
professionals to adopt a personalised approach in their practices [82].

Global Healthcare Challenges

Probiotics and prebiotics may also play a role in the future in addressing current and emerging
healthcare challenges, including those caused by microbes.

The rise of antimicrobial-resistant pathogens is a World Health Organisation priority, and the use
of antibiotics as growth promoters and for infection prophylaxis in animal husbandry has led to
large-scale antimicrobial resistance [83]. While bans on their use as feed additives now exist in
many Western nationsiii,iv, in middle-income countries antimicrobial use [84] and resistance [83]
in livestock continues to increase. In the future, probiotics and prebiotics may be increasingly
used as alternative growth-promoting and health-enhancing feed additives, due to their modulat-
ing effects on animal immunity, gut microbiota, feed intake, and productivity [85,86]. Certain pro-
biotic strains have shown results in the decolonisation of antimicrobial-resistant pathogens from
the human gut [87,88], and both probiotics and prebiotics may provide protective effects against
multidrug-resistant infections via effects within the microbiome, epithelial barrier, and immune
system [89], suggesting potential for a future role in reducing the burden of antimicrobial resis-
tance in hospitals and healthcare. Furthermore, probiotics and their antimicrobial by-products
are being investigated as novel future alternatives to antibiotics [90,91].

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Over the last decade, the challenges of influenza and coronaviruses, including H1N1 (swine flu),
severe acute respiratory syndrome coronavirus [SARS-CoV (SARS)], and SARS-CoV-2 [corona-
virus disease 2019 (COVID-19)], have created massive healthcare, societal, and economic bur-
dens. In the period between the emergence of a new mutant virus and the development and
testing of an effective vaccine, safe and low-cost prophylactic agents with nonspecific immune,
anti-inflammatory, and antiviral effects could provide an avenue for intervention. Probiotics and
prebiotics have been suggested as candidate components of preventative and acute care
strategies for COVID-19 infection [92–94]. Known for their ability to regulate multiple aspects of
the immune response [95], a body of evidence has previously demonstrated the effectiveness
of specific probiotics in preventing upper respiratory tract viral infections [96] and reducing the
risk of ventilator-associated pneumonia [92]. One report on a small group of hospitalised
COVID-19 patients found reduced morbidity and mortality with the addition of a multistrain
probiotic formulation to standard care [97]. While widespread clinical data do not yet support
their use for prevention or treatment of serious viral infections, this represents a potential future
area of investigation for probiotic and prebiotic research to investigate their safety and effective-
ness as adjunctive therapies.

New Discovery and Research Methodologies

Significant developments in technologies and associated methodologies have enabled many ad-
vances in the prebiotic and probiotic field in recent years and will continue to play a key role in the
future. As the breadth of technologies and bioinformatic techniques expands, increased emphasis
is being placed on the reproducibility of microbiome results and removal of bias introduced by pro-
tocols. Interpretative variations can enter at many stages, including collection and processing of
samples, gene sequencing, database use, and data analyses, any of which may cloud biologically
relevant signatures including interindividual variations [98]. Furthermore, sample size and statistical
power will become increasingly important when conducting microbiome studies [99].

Microbiome-derived leads have generated many current candidate probiotic genera of interest.
However, target-based approaches are also being widely utilised for the development of
probiotics, where libraries of bacteria are screened in silico, in vitro, or ex vivo for mechanistic ac-
tivity on host- or microbiome-related pathways of interest [7]. Furthermore, there are increasing
regulatory and scientific demands to support clinically proven beneficial activities of prebiotics
and probiotics with detailed mechanistic insights. These challenges are beginning to be met
through the development, improvement, and implementation of existing and new discovery
and research methodologies. An expanding group of ‘omics’ technologies, responsible for
many recent advances, are now moving from stand-alone data-generating vehicles to fully inte-
grated, systems biology-oriented ‘meta’-technologies [100]. Future and ongoing efforts will no
doubt improve current bioinformatics tools for data interrogation, integration and processing,
with holistic predictions being facilitated through machine learning and artificial intelligence
[101–103].

The cost of molecular methodologies has been decreasing, being powered by microfluidics and
nanofluidics, which generate an astonishing amount of data. The miniaturisation of enzymatic re-
actions has also allowed for absolute quantification of molecules in droplet quantitative PCR re-
actions, including high-throughput qPCR and digital PCR [104], which rely on robotics to
dispense volumes in the range of 10 nanolitres. Such technologies have permitted, for example,
the absolute quantification of 12 Bifidobacterium species in response to prebiotic galacto-
oligosaccharides (GOS), utilising volumes as low as 3 μl of total DNA [105]. Likewise, improve-
ments in sequencing quality beyond traditional short-read next-generation sequencing, as well
as longer-read generating platforms, including single-molecule real-time (SMRT) sequencing,

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which has been developed based on the nonclonal amplification and sequencing of single DNA
molecules [106], will also expand our ability to better characterise microorganisms to species
and even strain levels.

Likewise, novel high-throughput cultivation approaches, such as single-cell encapsulation in

droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (EMD) [107,108],
the ichip [104], and employment of multiple culture conditions associated with long incubation
periods, have permitted the identification of new microbial species [109]. Such novel isolates,
for example antibiotic-producing bacteria with potential beneficial effects [107,110], can now
be rapidly and comprehensively characterised through next-generation sequencing and liquid
chromatography–mass spectrometry analysis. Recently developed instruments like the Prospector
System (GALT, CA, USA), which allow the simultaneous cultivation and screening of isolates
based on specific phenotypes, the ability to use substrates of interest like prebiotics, or the
generation of metabolites, will rapidly advance the collection, characterisation, and application
of novel strains.

Advances in bioinformatic platforms and parallel-computing workflows have enhanced our ability
to convert the terabytes of sequence data into useable data in a fraction of the time, allowing for
generation of probiotic databases and large-scale analyses of probiotic strains [111,112]. Simi-
larly, movement of some software and wrapper programs into more user-friendly graphical
user interfaces, rather than only being available through cumbersome command lines, has also
helped to remove analysis barriers for probiotic and prebiotic effects.

Nonetheless, it should be borne in mind that many such technologies deliver in vitro information
on potential prebiotics and probiotics that may not represent in vivo behaviour. A lack of transla-
tion of in vitro mechanisms to clinical effects can occur in probiotic and prebiotic development.
Moreover, while in vitro fermentation models are currently used in development to simulate im-
pacts of prebiotics and probiotics on the microbiome, such models do not replicate the host
physiological interactions. Current efforts to improve these translation challenges include the de-
velopment of humanistic models for screening, including humanised microbiome animal models,
organoids from human biopsy samples, coculture experiments of epithelia and microbes, ‘organ
on a chip’ models, and ‘in human’ discovery models. Such models will increasingly be used not
only for discovery and prediction purposes but also to elucidate the mechanisms of action of pre-
viously demonstrated clinical effects. None will replace the necessity of in vivo assessments,
which remain definitive tests for probiotic and prebiotic efficacy.

Quality Assurance Developments

There is demand for incorporation of temperature- and moisture-stable probiotics and prebiotics
into novel foods and supplements, in increasing levels and complex combinations. This trend has
placed a burden on the evolution of quality assurance methodologies to ensure stability, shelf life,
batch consistency, and functional integrity of such bioactive ingredients [113–115]. Over the
years, numerous studies have called into question the quality and purity of commercial probiotic
products [116], and calls have been made for standardised assessment and certification tools to
improve trust from end users and other stakeholders [2]. The same should be applied to
prebiotics.

Appropriate identification of a probiotic strain indicates to which genus, species, and subspecies
or strain it belongs [117], according to valid (and for lactobacilli, recently updated [118]) nomen-
clature. Conventionally, phenotypic methods of probiotic identification are used in commercial
quality control laboratories, which provide low resolution, often to the genus level only [119].

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The availability of whole-genome sequencing (WGS) and next-generation sequencing (NGS) have
changed studies of microbial communities and are routinely used in food quality and safety prac-
tices [120]. However, these techniques are not currently widely adopted in quality control labora-
tories, despite their potential to enable accurate identification of probiotics in multistrain mixes, as
well as the presence of potential contaminants [121,122]. Increased utilisation will depend on
time-to-results reduction, cost effectiveness, and curation of databasesv [123].

Microbial viability is classically measured through culturability, that is, colony-forming units (CFUs);
however, this method has significant limitations, including an inability to enumerate organisms in a
viable but nonculturable state [124]. Laboratories currently seek faster, cheaper, less labour-
intensive, and more reliable methods, with applicability across various matrices and emerging
new genera. Viability measurement by flow cytometry is available for certain species of LAB,
and the combination with polyclonal antibodies shows promise for enumerating multistrain
mixes [125]. Flow cytometry can also be applied to assess damage from oxidative stress [126].
Other emerging alternatives to plating methods include chip-based digital PCR, droplet digital
PCR, or electrochemical magnetic bead-based immunosensor techniques [127–129]. Impor-
tantly, as the majority of probiotic efficacy trials report doses in CFUs, a rationale for equivalence
of new quantification results to classical colony-count techniques will be needed for use in quality
assurance of commercial products. Furthermore, widespread industry adoption of these ap-
proaches will require scaling to the routine laboratory, standardisation, and methodological vali-
dation [130]. Application of these new methods to test products during human intervention
trials will also help to ensure implementation.

Improvements in the stability of live cells in products throughout shelf life across different product
formats will continue to be a future focus. Understanding strain phenomics and fluxomics with big
data and machine learning may also help mastering product efficacy and stability in various appli-
cations [131]. Furthermore, beyond simple probiotic viability measures, the integrity of extracellu-
lar structures, enzymatic activities, or other effector molecules may help to assess probiotic
functionality in the final application [132], provided such candidate markers are able to demon-
strate a robust role in probiotic efficacy [132–134].

For prebiotics, it is well established that they may be degraded by food-processing methods,
including pH changes and heat [135,136], and therefore widespread adoption of standardised
quality assurance methods for prebiotic products is an important step in the future of effective,
reliable, and diverse products. Prebiotic quality assurance can take the form of chemical and struc-
tural analyses, which determine dose and maintenance of molecular integrity; or functional assays,
to measure retention of activity (i.e., microbial metabolism of the prebiotic and the associated health
benefit in final products). Relatively simple functional assays enable the quantification of prebiotic
effects on bacterial growth [137] and have been successfully used to determine the effect of
food-processing techniques on prebiotic properties [138]. These culture media-based assays
which use single organisms, coupled with culture enumeration of the target microbes, may be su-
perseded in routine use by more complex models in the future. Gut-simulation models capture
the complex interaction of digestive processes and mixed microbial fermentation and are often
combined with DNA-based methods to ensure comprehensive determination of microbial re-
sponse [139]. Such techniques can be applied to prebiotic food products, overcoming the need
for purification steps required for chemical analysis techniques.

Direct prebiotic compound analysis can take the form of chromatographic and electrophoretic
methods for quantitative information, while spectroscopic techniques provide more detailed struc-
tural characterisation [140]. Gas chromatography and high-performance liquid chromatography

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are frequently used for prebiotic determination [140]; however, their application in commercial food
matrices is complex and might not always be accessible for routine quality control. To address this,
a method of high-performance anion-exchange chromatography with pulsed amperometric de-
tection has been coupled with characterisation by size-exclusion chromatography with multiangle
light scattering and refractive index detection. This method provides a sensitive and reliable ap-
proach for accurate quantification of prebiotic compounds in complex dietary matrices without
the need for fractionation [141]. Such methods may become increasingly important in the future
as prebiotics become incorporated into an ever-expanding variety of foods, beverages, and
other consumer products. Microstructural morphological examination via environmental scanning
electron microscopy and detailed structural analysis via spectroscopic techniques can provide
detailed information on new candidate prebiotics [142]; however, they are unlikely to be used in
quality assurance laboratories on a routine basis. For novel polymeric prebiotics with highly com-
plex structures, an approach similar to the use of trypsin for the study of complex proteins has
been proposed, whereby structure-specific glycosidic hydrolases can be used to cleave oligomers
more amendable to mass spectrometry approaches [43].

Implementation into Policy and Practice

While a convincing body of evidence [143,144] exists on the effectiveness of certain probiotics
and prebiotics for a broad range of health applications, their consistent implementation into nutri-
tion and healthcare remains limited. Sentiments from both healthcare practitioners and con-
sumers commonly contain a degree of scepticism about the potential usefulness of probiotics
and their level of clinical evidence [145].

The ability to communicate about probiotics and prebiotics to key stakeholders is heavily
influenced by the regulatory environment. Probiotics and prebiotics hold different regulatory
status across countries and regions, further differing in considerations for use in product formu-
lations, health claims, labelling, or other end-user communication [146]. While many countries
have successfully implemented specific regulatory frameworks and authorised evidence-based
probiotic and prebiotic claims, in other jurisdictions approved claims are scarce and current com-
munication about health benefits in the market is therefore limited. The European framework in
particular (Box 2) is an important influence on the future of industry-driven probiotic and prebiotic
research, given its stringent requirements and significant market size. The scientific and technical
requirements for bringing products to market and for claims are therefore very diverse around the
world, just considering food and dietary supplements. Stakeholders operating under distinct ju-
risdictions need to comply with these divergent frameworks, sometimes with different levels of
scientific requirements for the same concepts [146]. Several evidence-based categorisations
and straightforward criteria might be considered for probiotics [5,117]. Existing and new initiatives
for further international harmonisation should be encouragedvi. Working towards convergent
frameworks, and at the same time adapting these to upcoming scientific and technological dis-
coveries will be one of the major implementation challenges for future prebiotics and probiotics.
It is likely that a reconciliation between scientific recognition and regulatory acceptance is the
way forward and further harmonisation of regulatory approaches will improve the uptake of
prebiotic and probiotic products into mainstream nutrition and health care.

The translation of scientific findings into changes to healthcare practice has repeatedly been
shown to be a lengthy and inconsistent process [147]. Strategies aimed at microbiome modula-
tion for health maintenance and disease prevention are currently notably absent from most health
care systems, although professional guidelines have made some recommendations [148]. While
the growing field of implementation science aims to address common issues across many
healthcare fields [149], a novel approach being carried out in the UK is direct political influence

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Box 2. Challenges and Opportunities in a European Framework Outstanding Questions

The EU-wide framework for nutritional and health claims is a challenging case for probiotics and prebiotics in foods and
Can we facilitate the reintroduction of
food supplements. In this market, probiotic and prebiotic effects have been largely rejected by assessment panels for
‘missing’ desirable microbial species
health claims, with notable exceptions [155,156], despite being based upon otherwise peer-reviewed scientific evidence,
into vacant niches in a microbiome?
exploiting up-to-date technologies and being, in many cases, mechanistically driven [155–157].
Will novel probiotic strains prove to
The EFSA operates an ingredient-specific pre-market approval process for any health claim, with a rigorous evaluation be safe and achieve relevant health
process. Regulations specify rules for the authorisation of nutritional and health claims in Member States, clarified by outcomes?
the Commission in specific guidance. Interested companies must follow a specific application process. To prepare dos-
siers, applicants can rely on extensive guidance, revised according to experience in a process open to public consultation. Will the most promising probiotics of
The EFSA is responsible for evaluating the scientific basis of applications; it then publishes evaluations as Scientific Opin- the future be derived from human,
ions in the open-access EFSA Journalvii. Finally, claims are authorised according to the final decision by the Commission animal, or plant hosts, foods or other
and Member States, including actual claim wording and conditions of use. A public EU register, with over 2300 entries as sources?
of 2020, is available, including nonauthorised claims. Member States are responsible for implementing the regulations and
the authorised claims. Can we achieve better translation of
candidate microbes and substrates
As highlighted in specific reviews, few applications for prebiotics and probiotics have been successful, lacking either suf- to proven probiotics and prebiotics
ficient characterisation, a beneficial effect for human health, establishment of a cause-and-effect relationship, or biological with health benefits, through the
plausibility [146,155–158]. This limited positive track record creates a high uncertainty and unpredictability, especially for development of in situ and in-human
future applicants in the always-evolving field of human health and nutrition. The EFSA follows technical and scientific de- discovery models?
velopments and reviews its procedures and methodologies regularly, as illustrated by guidance on weight of evidence
[159], biological relevance [160], on whole-genome sequencing dataviii, or the uptake of Lactobacillus genus taxonomy How do we move beyond binary
changes in QPS [161]. However, for future applications to be successful, further evolution in scientific standards will be methods of microbial classification
necessary to accommodate future scientific and technological developments such as those described in this review, from ‘health-associated/beneficial’ or
and especially cope with long-standing, wide-ranging trends such as systems biology and omics methods, personalised ‘disease-associated/detrimental’, to
nutrition, or the role of the gut microbiome in human health. more sophisticated understanding of
ecosystem interactions, functionality,
and therapeutic targets?
via the translation of key microbiome science into emotive and relevant benefits for policy makers.
In the All-Party Parliamentary Group (APPG) on the Human Gut Microbiome, a group of political What are the host-related or
microbiome-related factors that
campaigners and microbiome scientists aim to educate and convince key decision-makers
determine individual response or nonre-
(including politicians) to incorporate the role of the gut microbiome into policy. In the complex sponse to probiotics and prebiotics?
field of prebiotic and probiotic science, a fact-based ‘storytelling’ or narrative approach ‘decodes’
the science, making it accessible, relevant, comprehensible, and increases the chances of How can we continue to elucidate a
deeper mechanistic understanding of
engagement and action [150,151]. Realising the full potential of probiotics and prebiotics in
probiotic and prebiotic mechanisms
healthcare will require increased recognition at all levels, from consumers, prescribers, and of actions?
governments, and subsequent integration into policy, practice, and lifestyle.
Will the putative effects of prebiotics
on pathogen exclusion and virulence
Concluding Remarks and Future Perspectives attenuation provide a useful pathogen-
The wealth of research into microbiome-targeted nutrition and therapeutics has expanded the control strategy for the microbiome?
fields of probiotics and prebiotics as well as many related interventions. Both within and outside
Will synbiotics provide significantly
of the current definitions, new probiotics and prebiotics will emerge, challenging scientific as well
enhanced health benefits compared
as regulatory definitions. Many substances will be derived from novel sources that meet eco- with individual probiotic and prebiotic
nomic and environmental needs to target a growing range of compositional and functional niches application, due to cooperative
within the microbiome. Industry trends and consumer preferences will continue to drive demand interactions in the intestine?

for integration of probiotic, prebiotic, and other bioactive substances into a plethora of formats, Will the advent of live biotherapeutic
supported by advancements in delivery technologies and quality assurance. While the gut will products and genetically modified
likely remain as the heartland of these therapies, clinically proven applications will continue to organisms alter the public perception
and utilisation of probiotics?
expand in the respiratory system, immune system, urogenital tract, skin, nervous system, oral
cavity, cardiometabolic system, and weight-management field. Emerging healthcare challenges What other metabolic pathways in
will drive research into new areas of global health importance, and a growing body of evidence microbes (such as vitamin utilisation in
for key applications will guide increased implementation in healthcare policy and practice. the colonic microbiome) can be
exploited for microbiome modulation?

Accelerating advances in biotechnology and bioinformatics show no sign of slowing and will pro- Which structural or functional features
vide detailed mechanistic insights into the action of prebiotics and probiotics as well as leads to of prebiotics and probiotics will be
identify new candidate organisms and substrates. Discovery and validation techniques will

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continue to undergo refinement, increasing reliability and reproducibility of study findings. This will most informative for verification in
quality assurance assays for food and
further enable the comparability of data sets and larger aggregate insights from multiple research supplement products?
streams. These insights, as well as continued investment into large intervention and population-
based studies, will uncover new ways to improve dietary relevance and clinical efficacy as well as How can effective messages about
probiotic and prebiotic effects
target these interventions and tailor them to individuals’ biology and microbiome. Such a vision is
be translated to consumers, health
our predicted future of probiotics and prebiotics (see also Outstanding Questions). agencies, and key opinion leaders?

Acknowledgments
The authors gratefully acknowledge the International Scientific Association for Probiotics and Prebiotics (ISAPP) for providing the
forum for collaboration and discussion at their 2019 Annual Meeting and supporting the open access publication of this manu-
script. We thank all the participants of the discussion group ‘The Future of Probiotics and Prebiotics’ at the 2019 ISAPP meeting.

Declaration of Interests
M.C., V.B., R.G., D.G., S.M., D.O., M.I.P., and R.E.S. are involved in research and development projects as employees
of companies that produce, sell, and distribute food and/or healthcare products, including probiotics and prebiotics
(M.C., Metagenics; V.B., General Mills; R.G., GSK Consumer Healthcare; D.G., Symrise Nutrition; S.M., Reckitt Benckiser;
D.O., Danone Nutricia Research; M.I.P., Winclove Probiotics; R.E.S., DSM Nutritional Products).

H.D.H has received grant funding from the Hass Avocado Board, the Almond Board of California, and the National Honey
Board; presentation honoraria from Kellogg’s Company, PepsiCo, Nutricia North America, Tate & Lyle; and serves on the
Grains Food Foundation scientific advisory board.

M.A.A., A.B., K.H., K.S.S., D.S., J.V., and G.G. have no conflicts of interest to declare.

Resources
i
www.fda.gov/files/vaccines,%20blood%20&%20biologics/published/Early-Clinical-Trials-With-Live-Biotherapeutic-
Products–Chemistry–Manufacturing–and-Control-Information–Guidance-for-Industry.pdf
ii
www.fao.org/food-loss-and-food-waste/en
iii
https://ec.europa.eu/commission/presscorner/detail/en/IP_05_1687
iv
www.accessscience.com/content/u-s-bans-antibiotics-use-for-enhancing-growth-in-livestock/BR0125171
v
www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Costs-Data
vi
www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%
252Fsites%252Fcodex%252FMeetings%252FCX-720-39%252FCRDs%252FCRD03.pdf
vii
https://efsa.onlinelibrary.wiley.com/journal/18314732
viii
www.efsa.europa.eu/en/consultations/call/public-consultation-efsa-statement-requirements-whole-genome

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