Biomedicine & Pharmacotherapy 130 (2020) 110625

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Immune boosting functional foods and their mechanisms: A critical

evaluation of probiotics and prebiotics
Tolulope Joshua Ashaolu a, b, *
a
Smart Agriculture Research and Application Team, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
b
Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Viet Nam

A R T I C L E I N F O A B S T R A C T

Keywords: Comprehensive studies conducted on the link between the gut microbiome and immunity in recent decades have
Functional foods correspondingly led to ever increasing interests in functional foods, especially probiotics and prebiotics. Pro­
Probiotics biotics and prebiotics play crucial roles in managing the intestinal microbiota in order to improve host health,
Prebiotics
even though their influence on other body sites are being investigated. Different colonic bacteria metabolize
Microbiota
dietary prebiotics to produce beneficial metabolites, especially short chain fatty acids (SCFAs) that improve
Short chain fatty acids
luminal contents and intestinal performance, while positively affecting overall host physiology. Thus, this review
provides a general perspective of the immune system, the gut immune system and its microbiota. The review also
evaluates functional foods with critical but comprehensive perspectives into probiotics and prebiotics, their
immune boosting and mechanisms of action. It is recommended that further mechanistic and translational
studies are conducted to promote health, social life and also empower poverty-stricken communities.

1. Introduction though it is not their only pharmacologically important zone of the

There is a trendy demand for various food components that can
improve immune response. Functional foods can regulate the immune
system through immune response enhancement or inhibition, which
provide host defenses against infection, and suppress allergies and
inflammation [1]. The production of these food components such as
prebiotics and probiotics, is ever increasing with the intention of
fighting chronic health problems like non-alcoholic fatty liver disease
(NAFLD), cancer, obesity, diabetes and cardiovascular diseases. Pres­
ently, the most known functional foods are the probiotics and prebiotics
based on their health promoting properties.
Several investigations and in-depth studies have established pro­
biotics with both clear and unclear mechanisms, and thus it is suggested
that they be incorporated into health management as alternative ther­
apy or complements to pharmaceutical agents, foods and lifestyle [2].
Prebiotics were known to be selectively degraded by host microorgan­
isms to release beneficial metabolites. Prebiotics are not just enhancers
of bifidobacteria and lactobacilli anymore, they have gained recognition
regarding their physiological and systemic functions [3].
Usually, the gut has been implicated in immune cells activity and
overall immunity based on probiotics and prebiotics interventions, even
body. This review provides an overview of the gut immune system and
their microbiota. The definitions and trending knowledge of both pro­
biotic and prebiotic action mechanisms with regards to immunity were
critically evaluated, while capping it all with prospective outlook, con­
cerns, challenges, and conclusive remarks.
2. A brief overview of the immune system
The immune system is broadly categorized into innate (non-specific)
or adaptive (specific). The specific immunity is linked to the immuno­
globulins (Ig), produced by B cells in response to antigenic attacks, such
as pathogens and allergens. The Igs are further divided into IgA, IgG and
IgE classes - all produced via isotype switching upon activation. Of them
all, the IgAs are more relevant to the present review, as they can be
found in saliva, tears, sweat, breast milk and mucosal secretions. The
IgAs also help in controlling commensal bacteria at mucosal sites as well
as protecting the body from infection [1]. T-cells are another group of
specific/adaptive immune cells found in the small and large intestines.
They have receptors that consist of α- and β-chains, and are further
classified based on their surface expression of CD4 or CD8 molecules.
CD4+ T cells are helper T cells (Th) assisting with other immune cells

* Correspondence to: Smart Agriculture Research and Application Team, Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Viet Nam.
E-mail address: tolulopeashaolu@tdtu.edu.vn.

https://doi.org/10.1016/j.biopha.2020.110625
Received 3 July 2020; Received in revised form 2 August 2020; Accepted 5 August 2020
Available online 11 August 2020
0753-3322/© 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
T.J. Ashaolu
functions. These helper T cells assist with the production of multifarious
cytokines such as interferon γ (IFN-γ), interleukin-4 (IL-4), and
interleukin-7 (IL-17).
3. The gut immune system and its microbiota
The gut is divided into the upper and the lower regions. The upper
region comprises of the mouth, pharynx, esophagus, stomach and duo­
denum while the lower part mainly consists of the small and large in­
testines. The gut microbiota are predominantly present in the lower
region, and this makes the intestinal immune system quite important.
This immune system is composed of gut-associated lymphoid tissue
(GALT) and other cells, meant to protect the gut from several types of
antigens readily supplied from foods, commensal and pathogenic bac­
teria. Also, many and diverse intestinal cells are responsible for stimu­
lating IgA isotypes production, which further buffer up the gut
immunity. The helper T-cells 17 (Th17 cells) are also part of the gut
immune system that keep the mucosal barrier intact while stimulating
the intestinal epithelial cells to produce antimicrobial peptides [4].
The human gut is predominantly inoculated at birth, and the diverse
microbiome develop following certain feeding and dietary patterns until
around 3–5 years of age, which then look similar to the adults [5]. These
organisms interfere with the production of several cells and tissues
within the gut in order to confer immunity on the system. Aging, diet,
infection or indiscriminate use of medications may pose some challenges
to upset the make-up of the microbes as well as their fermentation end
products. In fact, a perturbation of the gut microbiota will lead to certain
acute and chronic disorders such as inflammatory bowel disease (IBD),
irritable bowel syndrome (IBS) and obesity [6,7]. The fact that gut
fermentation and microbial functions largely depend on diet imply that
the consumption of functional foods like probiotics and prebiotics will
be a good dietary approach to improve the human gut microbiota for
increased health benefits. Bacteria like Bifidobacteria possess special
transport mechanisms, cellular and extracellular glycosidases, which
actively digest low molecular weight prebiotic carbohydrates, while
Bacteroides break down high molecular weight prebiotic carbohydrates
[8,9]. However, Ruminococcus spp. will degrade resistant starch into low
molecular weight dextrins, capable of being utilized by other colonic
organisms [121]. These gut microbes are continuously being engineered
into marketable probiotics, which alongside prebiotics are considered
among functional foods demanding health claims and other authenti­
cations from relevant regulatory bodies.
4. Functional foods
The definitions ascribed to functional foods are many, and include
foods marketed with health claims; food sources with positive physio­
logical properties beyond their nutritional uses of providing essential
nutrients; and natural substances that can be consumed daily meant to
regulate or affect a system of the body upon ingestion [10,11]. The
European Consensus provided the commonest and most recent defini­
tion, which states that ‘a food can be regarded as functional if it is
satisfactorily demonstrated to affect beneficially one or more target
functions in the body, beyond adequate nutritional effects, in a way that
is relevant to either improved stage of health and well-being and/or
reduction of risk of disease’ [12]. A functional food can be natural,
components-added, or components-eliminated foods via biotechnolog­
ical processes [13]. The ingredients in functional foods based on tech­
nological factors can help in preventing some diseases or improve
performance and well-being of consumers beyond its nutritional role,
either whole or specific groups of the population defined by age or ge­
netic differences [14,15].
In fact, functional foods have been classified based on food type or
active components added or naturally present in the foods i.e. fibre,
flavonoids, vitamins, minerals, fatty acids, carotenoids, and so on [16].
Some functional foods are regarded as microbial medicines, i.e.
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Biomedicine & Pharmacotherapy 130 (2020) 110625
prebiotics and probiotics meant to target obesity and metabolic syn­
drome [17], as well as high blood pressure, oxidation, cellular damage
and other diseases from sources/raw materials like vegetables, cereals,
diary and meat products [15,18,19]. Host health benefits which include
reduction of macular degeneration and prostate cancer risks were
associated with certain lutein and lycopene, while flavones, catechins
and anthocyanidins have been shown to possess antioxidative properties
by chelating free radicals and reducing the incidence of certain chronic
diseases [15]. Essential minerals and dietary elements were previously
included in the production of functional foods such as calcium, iron, zinc
and vitamins [20]; and now, among the most prominent and well
researched functional foods are the probiotics and prebiotics, which
have the capacity of boosting consumers immune status [21]. Each of
them has its own immunomodulatory and immune boosting benefits and
they can beneficially interact in a “symbiotic” process to boost the im­
munity of the human host.
5. Probiotics
The intention of consuming probiotics is to harness their benefits in a
symbiotic or commensal relationship with the intestinal microbes.
Initially, probiotics were defined as contributors to host’s intestinal
microbial balance [2]. The modern consensus definition of probiotics is
that they are living microorganisms, which when administered in
adequate amounts can confer health and well-being to the host [22].
Thus, probiotics benefits are beyond the mediation of gut microbiota
based on associated mechanisms. A paramount criterion for obtaining
effective probiotics is their ability to adhere to the gastrointestinal tract
(intestinal mucosa and epithelial cells), in order to avoid being passed
out via gut motility, and thus multiply, colonize and modulate the im­
mune system of the whole body by competitively suppressing pathogens
[21,23].
The commonest probiotics used in functional foods and other fer­
mented products include Lactobacilli, Enterococci, Bifidobacteria and
Leuconostoc spp. Other than these lactic acid bacteria group, yeasts such
as Saccharomyces spp. are also used as probiotics [24]. In fact, the
market for probiotics is ever increasing in size, and consumers’ demands
have risen spontaneously based on the immune-enhancing benefits of
probiotics. They are already being sold as conventional foods, dietary
supplements, medical foods and drugs in the United States, and are often
administrated in capsules, liquid or powder forms [24]. The immune
boosting effects and mechanisms of some probiotics are listed in Table 1.
5.1. Immune boosting and mechanisms of action
The action mechanisms of probiotics interfere with the gut epithelial
and immune cells composition and functions. Several insights into the
mechanisms of probiotics are deduced from in vitro, animal, cell culture
or ex vivo human studies, and not all mechanisms have been confirmed
in every probiotic strain or humans because a probiotic cell may exhibit
several mechanisms simultaneously, further complexed by diverse fac­
tors [2]. There is a probiotic inherent immunomodulatory property
which varies from strain to strain. For instance, a probiotic strain
significantly reduced sepsis in a study, but upon reformulation allowed
enterocolitis in infants [43,44]. There is a colonization resistance
concept, which is possibly the sum outcome of the functioning of many
of the different immune-boosting mechanisms of probiotics action in
concert; it is described as the occupation of host tissues by the native gut
microbiota in order to exclude potential pathogenic infections [2,45].
The validated and proposed action mechanisms of probiotics are
represented in Fig. 1. Probiotics are well known to boost the immunity of
humans by protecting against gastrointestinal pathogens, thus the
mechanisms of action by which they exert their beneficial effects on the
host include secretion of antimicrobial substances, competitive exclu­
sion for adhesion sites and nutritional sources, enhancement of intesti­
nal barrier function, and immunomodulation [24].
T.J. Ashaolu Biomedicine & Pharmacotherapy 130 (2020) 110625

Table 1 Regarding antimicrobials production, probiotics produce diverse

The immune boosting effects and mechanisms of selected probiotics. substances such as organic acids, hydrogen peroxide and bacteriocins,
Probiotic organism Immune boosting Mechanisms Citation which are capable of inhibiting both Gram-positive and Gram-negative
functions bacteria. Several strains of Lactobacillus spp. secrete low molecular
Lactobacillus Detoxify toxins Binding via surface Jebali et al. weight bacteriocins (LMWB) and high molecular weight bacteriocins
plantarum structures [25] (class III), which are less than, and greater than 1000 Da, respectively.
MON03 The LMWB are antimicrobial in activity. The mechanism of action in­
Lactobacillus kefiri Detoxify toxins Adsorption and Taheur et al. volves the destruction of target pathogenic cells via pore formation or
KFLM3 biotransformation [26]
Saccharomyces
cell wall synthesis inhibition [46], as found with nisin, which binds the
cerevisiae precursor lipid II of spore-forming bacilli cell wall in order to prevent its
KFGY7 biosynthesis. This later forms an aggregation to invaginate the antimi­
Acetobacter crobial peptides for pore formation within the bacterial cell membrane,
syzygii KFGM1
thereby making the low molecular weight compounds to leak, weaken
Lactobacillus Detoxify toxins Binding via surface Ismail et al.
helveticus ATCC structures [27] the proton motive force, and consequently, cause cell death [47,48].
12046 Another mechanism under this category of antimicrobial substances
Bacillus Detoxify toxins Enzymic degradation Rao et al. secretion involves the production of acetic and lactic acids for acidifi­
licheniformis [28] cation in order to inhibit the growth of pathogens such as Salmonella spp.
CFR1
Saccharomyces Detoxify toxins Binding via surface Ismail et al.
The intracellular pH become reduced to acidify the cytoplasm, while the
cerevisiae HR structures [27] proton motive force become collapsed. The cumulation of these actions
125a leads to inhibition of nutrients transport and consequently, bactericidal
Lactococcus lactis Detoxify toxins Binding via surface Ismail et al. effects [49,50]. In addition, probiotic bacteria secrete microcins, which
JF 3102, structures [27]
act by binding iron siderophore receptors for cell entry or produce
Lactobacillus
plantarum NRRL harmful substances after gaining entrance into the cell. These processes
B-4496 lead to the inhibition of intracellular enzymes like ATP-synthase, DNA
Streptomyces cacaoi Detoxify toxins Enzymic degradation Harkai et al. gyrase and RNA polymerase, as well as their functions such as mRNA
subsp. Asoensis [29] translation, and then cause pathogen cell death.
K234,
Competitive exclusion mechanism involves adhesion sites and nu­
Streptomyces
luteogriseus trients competition between a probiotic organism and a pathogenic or­
K144, ganism. This enables the probiotic to inhibit the pathogen’s growth. The
Streptomyces probiotics may utilize their surface protein structures to achieve this like
rimosus K145
the case with mucins, or make their environment unfavorable for the
Stimulate dendritic Kikuchi et al.
Secrete IgA
cells to produce IL-6 [30] pathogens via secretion of antimicrobials including organic acids [24].
Stimulate dendritic Gut pathogens and LAB members have similar binding sites to the host
Sakai et al.
Produce IgA cells to produce TGF-
[31]
receptors [51,52], while microbe-associated molecular patterns
β (MAMPs) can bind the host pattern recognition receptors (PRRs) like
Improve natural
Stimulate secretion Takeda et al. toll-like receptors (TLRs) with the aim of out-competing the pathogens
killer (NK) cell
of IL-12 [32] [53]. Other than these means, pathogenic adhesion may be inhibited by
activity
Stimulate secretion Fujiwara probiotics via receptor disruption by probiotic enzymes, biosurfactants
Inhibit Th2 activity
of IL-12 et al. [33] secretion, and receptor analog production [24,54].
Stimulate activated T Kanzato
Several other Inhibit Th2 activity Intestinal epithelial barriers are well maintained by probiotic or­
cells death et al. [34]
Lactobacillus
Aoki-
ganisms to protect against certain intestinal hyper-acidity and diseases
strains Improve oral like obesity, inflammatory bowel diseases, and irritable bowel syn­
Induce Tregs Yoshida
tolerance
et al. [35] drome. Some studies demonstrated that probiotics increase gene
Weaken pro- expression associated with tight junction signaling, thus keeping intes­
inflammatory
tinal barrier integrity intact [24]. Examples include the modulation of
Reduce cytokines and Shimazu
inflammation chemokines by et al. [36] genes that encode adherence junction proteins like E-cadherin and
down-regulation of β-catenin, as well as their phosphorylation and the abundance of protein
TLR-signals kinase C (PKC) isoforms such as PKCδ by probiotic lactobacilli [55].
Chlebicz and
Binding via surface Probiotics do not only maintain the intestinal barriers, they also
Detoxify toxins Śliżewska
structures
[120]
potentiate their reparative process after damage. For instance, Staphy­
Reduce Kawashima lococcus thermophilus and Lactobacillus acidophilus markedly inhibited
Induce of IFN-β
inflammation et al. [37] enteroinvasive Escherichia coli from adherence to, invasion, mainte­
Up-regulate Nakamura nance and enhancement of HT29, and Caco-2 cells up-regulation of
Produce IgA
expression of pIgR et al. [38]
transepithelial resistance, cytoskeletal and tight junction protein phos­
Reduce A20 and
Modulate anti-viral
improve IRF-3, IFN-
Ishizuka phorylation [24]. Over-expression of mucin glycoproteins by probiotics
activity et al. [39]
Several other b, MxA, RNasel also confer immunity on host’s gut by keeping the intestinal barrier
Bifidobacterium
Protect from
Produce acetate and intact. The mucins are found in the epithelial mucus layer above the
strains improve intestinal Fukuda et al. intestinal epithelium and are associated with immune boosting. Mucin
enteropathogenic
defence with [40]
infection
epithelial cells
secretion may be enhanced by probiotics as a mechanistic improvement
Suppress Th2 Iwabuchi to intestinal barrier physiology in order to curb pathogenic adhesion and
Inhibit allergy
chemokines et al. [41] survival [56,57]. Some lactobacilli do increase mucin secretion in
Reduce Miyauchi human intestinal cell lines via adhesion to their monolayers, as found in
Inhibit IL-17
inflammation et al. [42]
the expression of MUC2, MUC3 and MUC5AC in HT29 cells [58].
The defensins, which are small peptides that are active against bac­
teria, fungi and viruses, and can also be produced by probiotic-
stimulated epithelial cells for barrier integrity, function and stability

3
T.J. Ashaolu
Fig. 1. Action mechanisms of probiotics.
Caption: Probiotics offer several means of boosting host immunity, ranging from influencing immune and other host cells directly, production of antimicrobial
components and cross-feeding other microbes, to a combination of many mechanisms of action.
[59]. These defensins include antimicrobial proteins (AMPs), which act
as enzymes by degrading cell wall structures or non-enzymatic rupturing
of the pathogen’s cell membrane via cationic binding, and thus promote
pathogen death with defensin pores incorporated in its membrane.
Probiotics are living organisms with immunomodulatory benefits,
and some of these immunomodulatory effects are produced when
attenuated or dead, implying that probiotic effects are not necessarily
obtained from live organisms [1]. The enhancement of the antibodies
such as IgG and IgA by probiotics like LAB and bifidobacteria may be
useful in preventing invasion of the mucosal barriers as LAB can act on
intestinal dendritic and epithelial cells or improve the expression of
polymeric immunoglobulin receptor (pIgR) [30,31,60]. Some LAB
members can use TLR2 to stimulate IL-6 or TGF-β production from
dendritic cells [38]. Lactic acid bacteria also stimulate IL-12 production
mediated by polysaccharides in order to augment NK activity, which
then enhances host defense [1].
Some probiotics also demonstrate their ability to increase antibody
production for the purpose of improving defences against pathogenic
invasions and thereby serve as adjuvant vaccines [61,62]. The amounts
of anti-inflammatory cytokines such as tumor necrotic factor (TNF) can
be increased by some probiotic strains, which then aid with colon cancer
and colitis inhibition or therapy [63]. Probiotic organisms do confer
immune boosting benefits to the gut while providing some counterbal­
ance to inflammation caused by an invading foreign body [64]. They act
with monocytes and lymphocytes, which play important roles in innate
and adaptive immunity, as well as epithelial and dendritic cells.
Dendritic cells and intestinal epithelial cells (IECs) use their pattern
recognition receptors (PPRs) such as the TLRs to communicate with
probiotics and other gut microbes, which then play significant roles in
immune regulation. The TLRs are transmembrane proteins located
within endocytic vesicular membranes and other intracellular cell
membranes [65]. The TLRs cytoplasm have conservative
4
Biomedicine & Pharmacotherapy 130 (2020) 110625
Toll/interleukin-1 (IL-1) receptor (TIR), responsible for several adaptor
molecules interactions like myeloid differentiation protein (MyD88)
that leads to the activation of both mitogen-activated protein kinase
(MAPK) and NF-κB [53], essential in cytokines and other immune cells
production.
In addition to these, probiotics can induce anti-inflammatory cyto­
kines such as IL-10 and TGF-β, by down-regulating the expression of pro-
inflammatory cytokines, such as TNF and IFN-γ in order to enhance
mucosal immune responses [64,66]. However, certain probiotic bacteria
alter cytokine production via modulation of the signals transduced by
the cells. They may inhibit I-κB via ubiquitination by blockade of the
proteasome activity or interfere with RelA gene localization in a
γ-dependent signal cascade, which is stimulated by proliferative
peroxisome [67,68]. Upon activation by probiotic bacteria, Th0 are
differentiated into Tregs by DCs to inhibit Th1, Th2 and Th17 responsive
actions capable of inflammation [24].
Moreover, cell-surface structures like capsule and fimbriae are used
by certain probiotic organisms as mechanistic drivers for their immune
boosting activities [2].
Indeed, some studies also indicate that the immunoregulatory effects
of probiotics may be beneficial in NAFLD treatment as they modulate the
intestinal microbiota; improve epithelial barrier function and strengthen
the intestinal wall by decreasing its permeability; reduce bacterial
translocation and endotoxemia; improve intestinal inflammation; and
reduce oxidative and inflammatory liver damage [69]. Although pro­
biotics and prebiotics have been proposed in the treatment and pre­
vention of patients with obesity-related NAFLD, their therapeutic use is
not supported by high-quality clinical studies, even though an effective
short-term treatment for NAFLD, as part of a multidisciplinary approach,
has been claimed [69]. Being the intestinal mucosal barrier of immune
nature, the microbiota may be viewed as part of this system because of
the mutual influence occurring between the host and the luminal
T.J. Ashaolu Biomedicine & Pharmacotherapy 130 (2020) 110625

microorganisms. Alteration of the mucosal barrier function with
accompanying increased permeability and/or bacterial translocation
has been linked to many conditions [70]. Further investigations into
several other factors that may relieve or aid a damaged mucosal barrier
healing process for intestinal and systemic immune maintenance, are
required.
6. Prebiotics
From the onset, prebiotics were referred to indigestible food in­
gredients capable of providing health benefits to the host through se­
lective stimulation of the activity of colonic bacteria [71]. Subsequent
comprehension of the gut microbiota and technological advancements
such as metagenomics, nutrigenetics and metatranscriptomics have
further elucidated on the intricacies and delicateness of this definition.
For instance, the International Scientific Association of Probiotics and
Prebiotics (ISAPP) in London (United Kingdom), has defined a prebiotic
as “a substrate that is selectively utilized by host microorganisms
conferring a health benefit”. This occurred in 2016, and is the most
recent definition [72].
Therefore, prebiotics may not necessarily be carbohydrates, and may
also be applied to other atypical “substrates” in the human body apart
from the gut such as the skin or vaginal tract, capable of being colonized
by the probiotics. This process have to follow stringent microbial
selectivity. Other than the established prebiotics that are
oligosaccharides [e.g. fructooligosaccharides (FOS), gal­
actooligosaccharides (GOS), inulin and xylooligosaccharides (XOS)],
which cannot be digested by enzymic activities, rather used up by gut
microbes like Saccharomyces, bifidobacteria, eubacteria and lactobacilli;
certain compounds like proteins or peptides, unsaturated fatty acids,
flavonoids, micronutrients and macronutrients also demonstrated pre­
biotic properties [73]. Critical observations show that prebiotics im­
mune boosting effects are associated with probiotics stimulation and
their metabolites produced such as short chain and branched chain fatty
acids; and may also be derived from other uncommon microbial taxa
such as faecalibacteria, eubacteria or roseburia [21,74].
Speculated ecosystemic metabolism of polysaccharides have been
repeatedly presented but their modus operandi within the gastrointes­
tinal tract is not clear yet [75]. Functional redundancy exists despite the
presence of diverse taxa in gut microbiota. The ecological functions of
bacteria are specific in different individuals [76], and less vivid
comprehension of the functional ecology of the gut microbiota poses a
challenge of describing prebiotics action mechanisms [2]. However,
probable mechanisms of prebiotics leading to human health benefits can
be postulated as shown in Fig. 2.
6.1. Immune boosting and mechanisms of action
Dietary prebiotics are usually those indigestible fibrous compounds
passed through the upper gut in order to stimulate probiotics growth. A

Fig. 2. Action mechanisms of prebiotics.

Caption: Upon selective utilization of prebiotics in the gut, the microbiota grow to exert various immune boosting functions at species and strain multi-levels. The
bacterial cell wall and other biomass increase immunomodulation and fecal bulking for healthy bowel movement. Metabolites like organic acids reduce luminal pH,
harmful to pathogens but aid solubility and absorbability of minerals such as calcium, as well as positively regulate hormones and epithelial integrity. GLP1:
glucagon like peptide1; M cell: microfold cell; NK: natural killer; PYY: peptide YY; TGFβ: transforming growth factor-β; TH1, TH2: type 1 T helper, type 2 T helper;
Treg: regulatory T; ZO1: zonula occludens 1. Adapted from Sanders et al. [2].

5
T.J. Ashaolu Biomedicine & Pharmacotherapy 130 (2020) 110625

simple case study is indigestible FOS, made of 1-kestose (GF2) and
nystose (GF3), metabolized specifically by bifidobacteria and bacter­
oides amongst others, as energy sources for replication in the gastroin­
testinal tract [1]. This process alters the gut ecosystem based on series of
the bacterial dynamics and movement of their metabolites, which then
stimulate IgA secretion, an important intestinal immune system
component [77]. In the same process, the larger GF3 molecule can in­
crease intestinal bacteroides in order to stimulate IgA production [1,77].
Prebiotics may also interfere with type 2 T-helper reactions in order
to alleviate allergy according to some studies in infants. Arslanoglu et al.
[78] found out that feeding healthy term infants who are at atopy risk
with prebiotic-supplemented hypoallergenic formula for 6 months could
lead to a significant reduction in allergies prevalence even after 5 years
of the feeding programme. In a similar study, there were reduced
wheezing, atopic dermatitis and urticaria in infants fed with long-chain
FOS and GOS formula, administered in a double-blind, randomized,
placebo-controlled trial as compared to infants fed with non-prebiotic
formula [79,80]. In these studies, the exact mechanisms remain unclear.
SCFAs produced via prebiotic degradation by the intestinal micro­
biota also act in defense of the immune system. Tarantino & Finelli [69]
reported that prebiotic treatment had some varied impacts among
NAFLD and obese individuals who had undergone gastric bypass surgery
with intriguing hypothesis based on molecular and biochemical mech­
anisms. Processes like minor conversion of indigestible carbohydrates
into SCFAs or decreased local alcohol production may reduce the risk of
worsening NAFLD by ameliorating insulin resistance. However, it is
currently still unclear as to which bacterial groups play a role in the
development of obesity in humans [69]. The production of acetic acids
as bifidobacteria metabolites in the intestinal epithelial cells can prevent
intestinal enterohemorrhagic Escherichia coli O157 infection [40]. There
is a strong relationship between SCFAs and G-protein coupled receptors
(GPRs) because SCFAs activate GPR41 and GPR43 on intestinal
epithelial cells in order to stimulate immune responses via chemokines
and cytokines swift secretion, reactions already confirmed to help in
tissue inflammation and immunity in mice studies [81].
In addition to these mechanisms, certain fatty acid receptors
(FFARs), including FFAR2 and FFAR3 may react with gut fermented
metabolites like SCFAs for lipolysis enhancement and incretin glucagon-
like peptide-1 (GLP-1) secretion [82]. Several tissues possess these re­
ceptors indicative of possible action mechanism linkage between
immunomodulation and prebiotic fermentation.
Regarding appetite regulation, many mechanisms have been impli­
cated, such as the interaction between SCFAs and colonic cells to pro­
duce anorexigenic hormones like peptide YY (PYY) and GLP-1; or
metabolism escape of SCFAs through the colonic epithelial cells to the
liver via the hepatic portal vein in order to create a satiety signal stim­
ulated by propionate-mediated gluconeogenesis [83]. Leptin induce­
ment can also result from fatty acid receptors interaction with SCFAs in
the blood stream, or else cross the blood–brain barrier and enter the
hypothalamus to promote anorectic signals [84]. A detailed mechanistic
illustration of SCFAs production and their consequential pathways upon
prebiotics fermentation and bacterial cross-feeding is shown in Fig. 3.
From another outlook, model systems have been used to make in vitro
probe into prebiotics defence against pathogens by the inhibition of
pathogen growth via decreased intestinal pH upon organic acids pro­
duction [85,86].
Age-related immune boosting efficacy of prebiotics was investigated
in a human study for ten weeks, whereby some elderly individuals were
fed daily with GOS, and it was found out that their immune cells func­
tions in the likes of natural killer cells were more proactive, and their

Fig. 3. SCFAs production and their consequential pathways upon prebiotics fermentation and bacterial cross-feeding.
Caption: Organic acids (e.g. CO2 and H2) and short chain fatty acids (SCFAs: lactate, acetate, propionate and butyrate) are the major metabolites produced by gut
microbial fermentation of prebiotics. Acetate is the largest amount of SCFAs produced, and often released by Methanobrevibacter smithii and Blautia hydrogenotrophica
via reductive acetogenesis or pyruvate acetyl-CoA pathways. Firmicutes are involved in butyrate production from two acetate-derived acetyl-CoA molecules, con­
verted via the intermediates to butyryl CoA. This subsequently synthesize butyrate via butyryl CoA: acetate CoA transferase or butyrate kinase pathways. Propionate
has multi-production pathways such as the acrylate pathway, where Firmicutes reduces pyruvate to lactate by the enzymic action of lactoyl-CoA dehydratase to
produce propionate. In succinate pathway, propionate is produced by Firmicutes and Bacteriodetes through succinate decarboxylation. The third pathway involves
the conversion of fucose and rhamnose from propionaldehyde to propionyl-CoA by CoA-dependent propionaldehyde dehydrogenase. Other metabolites such as
lactate are produced in fewer amounts. Then, all these SCFAs modulate transepithelial fluid transport for membrane integrity maintenance, mucosa improvement and
disease relief. Adapted from Louis et al. [119] and Neri-Numa and Pastore [74].

6
T.J. Ashaolu Biomedicine & Pharmacotherapy 130 (2020) 110625

phagocytic activities increased [87]. Once there is a probiotic compe­
tition for prebiotic nutrients, stabilized by the growing population of
commensal microbes, lesser available nutrients will be left for patho­
genic organisms, and thus force them into redundancy, suppression and
inhibition.
Moreover, on the basis of mineral nutrients route, absorption occur
in the small intestine via active transport mechanisms. The drop in
luminal pH due to the production of SCFAs among other acidic metab­
olites can lead to increased calcium solubility, readily available for
passive uptake and consequent immune boosting via immune cells
activation or bone strength enhancement among other benefits. How­
ever, the solubility of calcium salts present in many food supplements
are pH-dependent and are less available. So, calcium solubility may
indeed increase with increased pH based on the starting pH [88]. In
some studies whereby young adolescents consumed a mix of FOS and
inulin or GOS, absorption and mineralization of calcium into bone was
significantly increased, thus reducing the risks associated with osteo­
porosis in later life [89,90]. Although previous studies in animal models
supported these research [91], but clinically validated long-term studies
are not available [2].
Finally, prebiotics play important roles in the metabolic processes
associated with immunomodulation. Gut barrier dysfunction allows
movement of various inflammatory mediators like the bacterial lipo­
polysaccharide (LPS) from the gut into the blood stream, a process
known as metabolic endotoxaemia, and shown to be an important factor
in the development of obesity and diabetes in mice models [92]. The gut
barrier integrity associated with immunity, can be enhanced when
prebiotics and subsequent SCFAs release act. GOS has proved this point

Table 2
Prebiotics influences on immune boosting recently demonstrated in in vitro, animal and human studies.
Prebiotics source Experimental design In vitro, Analysed metabolites/bacteria Resultant influences Citation
animal or
human
studies

Orange juice enriched with
hesperidin and naringin
Galactooligosaccharides (GOS)
and sialyllactose
Blackcurrant enriched with
prebiotic polyphenols
Grapeseed flour enriched with
prebiotic polyphenols and kefir-
derived lactic acid bacteria
Aronia juice enriched with
prebiotic polyphenols
MSPrebiotic®
Inulin
Hesperidin
B-GOS®
Vivinal-GOS®
Blueberry polyphenols extract
scGOS/lcFOS plus fermented milk
Orafti® inulin-type fructans
Ten healthy children and
women in a controlled non-
randomized clinical study for
sixty days.
Caco-2, colon and gingival
epithelial cells lines.
Sixten Sprague-Dawley rats fed
for 6 weeks, randomly divided
into 8 groups.
Ten Mice in random groups of
six fed for 9 weeks.
A Simulator of the human
intestinal microbial ecosystem,
Caco-2 and endothelial cells
Coculture were used for 2
weeks.
Eighty-four mid-age and elderly
adults consumed the prebiotic in
a prospective, randomized,
blinded, placebo-controlled trial
for 12 weeks.
Caco-2 cells plus fecal batch
fermentations.
Eighteen Lewis rats in random
groups of three fed for 4 weeks.
Forty-one children with autism
spectrum disorder were
randomly divided into 2 groups
and fed for 6 weeks.
Eighteen crayfish in random
groups of twelve fed for 97 days.
Ten mice in random groups of
six fed for 12 weeks.
Twenty-one Lewis rats in
random groups of three fed with
a mixture of FOS,GOS and
fermented Milk for 21 days.
Two hundred and nine healthy
children, randomized, placebo-
controlled, double- blind trial,
fed for 24 weeks.
Human Acetate, butyrate, lactobacilli and
bifidobacteria
In vitro Lactate, propionate, butyrate,
bifidobacteria and Bacteroides.
Animal Butyrate and Bacteroides-
Prevotella-Prophyromonas group
increased while bifidobacteria and
C. perfringens decreased.
Animal Propionic acid.
In vitro Increased propionate, butyrate,
akkermansia and Firmicutes.
Decreased bifidobacteria and
acetate.
Human Butyrate, bifidobacteria and
proteobacteria
In vitro Acetate, propionate, butyrate,
bifidobacteria, actinobacteria and
members of Bacteroides and
Lentisphaerae.
Animal Coated caecal bacteria coated with
increased lactobacilli, enterococci
and staphylococci. Decreased
Clostridium and Eubacterium spp.
Human Veillonellaceae, Lachnospiraceae,
Faecalibacterium, Bacteroides and
Bifidobacterium spp.
Animal Fewer lactic acid bacteria, and
higher number of intestinal
heterotrophic bacteria.
Animal Deferribacteres, Actinobacteria,
Proteobacteria, Bifidobacterium,
Desulfovibrio, Prevotella,
Adlercreutzia, Helicobacter and
Flexispira spp.
Animal Streptococcus thermophilus strain
065 and Bifidobacterium breve strain
C50
Human Bifidobacteria
Up-regulation of insulin, glucose, Lima et al.
triglycerides and total cholesterol. [100]
Regulation and modulation of Perdijk
genes associated with cell cycle et al. [101]
stages and cyclin-dependent
kinases.
Influences on food intake and body Paturi et al.
weight and disrupted large intestine [102]
biomarkers.
Adipogenesis, permeable intestine, Cho et al.
systemic inflammation, glucose and [103]
insulin resistance.
Pro-inflammatory markers such as Wu et al.
interleukin-8 and monocytes were [104]
down-regulated.
Lowered insulin and glucose levels. Alfa et al.
[105]
Improved and enhanced gut barrier Van Den
and immune function (nuclear Abbeele
factor kappa b, transepithelial et al. [106]
electrical resistance, and
interleukins-10 and -6.
Immune modulation and up- Estruel-
regulation of Immunoglobulin A Amades
and lymphocytes. et al. [107]
Urine and faecal metabolites Grimaldi
changed, reduced bowel movement et al. [108]
and abdominal pain, and improved
anti-social behavior.
Increased innate immunity, stress Nedaei et al.
resistance and digestion. [109]
Modulated leptin levels, reduced Jiao et al.
cholesterols and body weight. [110]
Improved lipid metabolism by
modulation of lipid genes.
Reduced diarrhea symptoms and Rigo-
inhibited rotavirus replication. Adrover
et al. [111]
Had immune boosting effects and Soldi et al.
reduced antibiotic-induced [112]
intestinal microbiota perturbations

7
T.J. Ashaolu
in an in vivo study as it improved the barrier function [93]. There is a
common ground for the acceptance of prebiotics metabolic effects that
they are positive regulators of glucose balance, inflammatory response
and blood lipid profile in humans following similar hypotheses
described earlier, even though the results have never been the same
[94–96]. Mice models indicated that glycemic index could be improved
by inulin because of its direct inhibitory effect on the intestinal iso­
maltase–sucrase enzyme complex [97]. In epithelial cell lines, the in
vitro fermentation of GOS induced tight junction proteins expression and
decreased transepithelial flux [98,99], a mechanism not yet clear in vivo
and a bit daunting as a task. Table 2 shows prebiotics influences on
immune boosting recently demonstrated in in vitro, animal and human
studies.
7. Further considerations
Most consumers embrace the idea of health promoting foods and
food ingredients. Inclusion of probiotics and prebiotics in their foods or
direct consumption of these functional food products as dietary sup­
plements have their own challenges too. Upon consumption, probiotic
organisms should be stable and remain viable when passed through the
digestive tract in order to perform the expected activity and confer the
needed benefits. And once in the colon, they should be able to metab­
olize their counterpart prebiotics for synergistic and robust beneficial
outcomes.
It appears that safety is the most paramount measure required when
developing probiotic microorganisms, but interestingly most of them are
safe [113]. Newer biotechnological developments and better compre­
hension of the organisms’ action mechanisms have led to engineering
newer strains with much better strengths than their progenitors, thus
manufacturing an entirely different probiotics [114]. Nevertheless,
presuming that the newer species and strains of probiotics are equally
safe as their progenitors will be preposterous. In fact, at the molecular
level, the genes may be mutated and may be unsafe. Therefore, how safe
and efficacious the novel probiotic organisms will be before commer­
cialization is a question to be meticulously pondered on.
Another consideration is the neutral, antagonistic or synergistic ef­
fects of probiotic bacteria on several other substances and products like
the antibiotics. The concern that some probiotics may transfer resistance
genes to pathogenic bacteria require strain resistance testing to a wide
variety of common classes of antibiotics such as tetracyclines, quino­
lones and macrolides. The transmission of drug resistance genes to the
pathogenic bacteria should also be ascertained [24]. Next generation
“omics”-based technologies will ease the necessary tests needed to do
this, and thus help ascertain how safe and quality the identified pro­
biotic strain is.
Prebiotics are largely carbohydrates but some are non-carbohydrates
and serve as novel food ingredients with health promoting functions.
How these ingredients interact with the gut microorganisms or
consumed probiotics, with their widely diversified structures are how­
ever yet to be completely comprehended [74].
Finally, the availability of probiotic organisms in less developed re­
gions of the world cannot be compared with the developed regions. The
potentials of beneficial organisms and local food sources to improve
consumers overall well-being in developing countries cannot be over­
emphasized just as their affordability and accessibility to many con­
sumers in developed nations should not be undermined. However, in a
recent programme, probiotics L. rhamnosus (GR-1 or Yoba 2012) and
plus S. thermophilus C106 were introduced in sachets for the production
of multifarious fermented foods such as yoghurt, millet, cereals and
juices, which help to bolster consumers’ health and improve the social
status and well-being of poverty-stricken societies [2,115].
8. Conclusions
Functional foods such as probiotics and prebiotics are the future of
8
Biomedicine & Pharmacotherapy 130 (2020) 110625
health-promoting foods. The consumption of probiotics such as lacto­
bacilli and bidobacteria may proffer an option to antibiotics for pre­
vention and treatment of microbial infections based on certain
mechanisms including antimicrobial toxins production, maintenance of
intestinal barrier integrity, competing for nutrients and adhesion, as
well as modulation of the immune system. This is not dissimilar to ef­
fects and mechanisms of prebiotics, which are entangled in synergistic
processes with the beneficial probiotic organisms. Thus, better
comprehension of the mechanisms involved are required, as well as
extensive human studies to validate excellent potent probiotic species/
strains and effective amount of doses meant for particular diseases.
Generally, the results of these interventions may be valuable for target
populations. Researchers are bestowed with the responsibility of
considering the importance of their findings to consumers, as well as
their fundamentality to overall conclusions on products’ efficacy.
Moreover, more investigations and translational research on probiotics
and prebiotics meant to help developing countries’ consumers are
recommended.
Author contributions
TJA conceptualized, researched, compiled and analyzed data, as well
as drafted and revised the manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The work was self-supported. The author also expresses his profound
gratitude to the Smart Agriculture Research and Application Team,
Faculty of Applied Sciences, Ton Duc Thang University, HCMC,
Vietnam.
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