Abstract diet. Prebiotics significantly rely on the


The standpoint of prebiotic now is already well
reported for managing gut dysbiosis (microbiota imbalance) in many clinical
studies tested on animals and human. A
regular consumption of prebiotic is generally accepted as required component of
a healthy diet. Prebiotics significantly rely on the fermentation in the colon
to give beneficial effects inside human body. Principally along the large
intestine or colon, inhabit the numerous species of bacteria. The word ‘colon’
itself resembles colony or the colonization of microbiota of which engage in
the fermentation of prebiotics. Studies also revealed the complexity of the gut
microbiome establishing a causal link between bacterial shifts and host health are
not straightforward.

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Metabolism; inulin; enterocytes; colon; carbohydrates


Numerous definitions of prebiotics have been proposed
over the past decades. It was first properly define by Gibson in 1995 then
eventually modified and rephrased by other well-known researchers such as Reid
(2003) and Roberfroid (2007). Recently the term prebiotics redefine as a
selectively fermented ingredient that results in changes(species specific) in
the composition and/or activity of the gut microbiota, thus conferring
benefit(s) upon host health (Gibson et al. 2010). The colon is heavily colonized by diverse
microorganisms dominated by several phyla such as Firmicutes and Bacteriodetes (Walker et al. 2011). At present, it is widely accepted that the
microbial within the colon expressed a complex ecosystem that interacts with multiple
host metabolic pathways (Tremaroli and Bäckhed
2012). Recent advances in microbial community analysis
confirmed the link between diet and gut bacterial composition (Ríos-Covián et al.
2016, David et al. 2014). High prevalence of proteolytic bacteria (e.g.,
species of Clostridium, Peptococcus, Peptostreptococcus,
and Fusobacteria) was considered detrimental because some typical
metabolites of protein breakdown, including ammonia and phenolic compounds, may
induce direct toxic effects or can act as (co)carcinogens(Davila et al. 2013). But the presumed negative effect of protein
fermentation is mainly based on in vitro studies and, so far, cannot be
demonstrated in humans (Windey et al. 2012). There is therefore the need of an exhaustive
review on the current knowledge of the health effects of prebiotic is presented
in line with ideas of the mechanisms that possibly explained how these intestinal
bacteria counteract the imbalance condition in individual. The first part of this review will deliberate about
major prebiotics that have been studied and how prebiotic could modulate gut
microbiota composition in general relationship between prebiotics and gut
microbiota with some diseases reported on studies in which prebiotics have been
used. The second part describes the structure and functions of
carbohydrate-based prebiotic (inulin, fructooligosaccharides and
galactooloigosaccharides) and the mechanisms involve during the fermentation by
gut microbiota. The last part of the review explores structure and functions of
various other potential non-carbohydrate prebiotics (alkaloid, alcohol, plant
sterols and drugs) and also illustrate how carbohydrates and non-carbohydrates
prebiotics showed dissimilar metabolism upon being fermented. Finally, the
paper will conclude with a discussion on many other emerging potential
prebiotics yet to be discover.



Prebiotic potential of a complex carbohydrate depends
on many factors: monomeric composition, linkage type, degree of polymerization
(DP), crystallinity, solubility and association with other substrates (Hamaker and Tuncil
2014). Inulin,
inulin-type fructooligosaccharides (FOS), galactooligosaccharides and lactulose
have been already approved as prebiotics (Kothari, Patel, and
Goyal 2014). Degradation of carbohydrates prebiotics yields
mainly short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate),
which are not harmful at normal physiological concentrations and are
acknowledged to provide energy for enterocytes
and thereby improve energy harvest
and defense mechanism on the intestinal epithelial cells (Lee and Hase 2014).


and Fructooligosacharide (FOS)

Since the structure of inulin and FOS only differs
from its polymer chain length, we took an approach to discuss both prebiotics
together.  Both are linear fructopolysaccharides
comprised of -(glucose-fructose)-subunits. Inulin is commercially manufactured
from plants, mostly from chicory (Buclaw 2016). Structurally, inulin and FOS is a made up of ?-D
(2?1) linked fructosyl units having a glucose moiety at the terminal reducing
end joined by ?-D (1?2) linkage(Di Bartolomeo,
Startek, and Van den Ende 2013).
Inulin has a unique structure among polysaccharides as no bond of the furanose
ring is part of the macromolecular backbone (Singh, Singh, and
Kennedy 2016). As
studied by Niness the chicory-derived inulin can be enzymatically hydrolysed by
endoinulinases (Sarbini and Rastall
2011) to
produce prebiotic FOS known as FFn-type FOS where F stands for fructose and ‘n’
is the number of fructosyl units(Goh and Klaenhammer
2015). Aside
of plant sources, inulin-type FOS are enzymatically produced from sucrose via
transfructosylation using microbial fructosyl-transferases (EC of Aspergillus as well as ?-fructofuranosidases
(EC or ?-D-fructosyltransferases (Niness 1999) reacted under transfer-favoring conditions. FOS
molecules 1-ketose (GF2), nystose (GF3) and 1F-?- fructofuranosylnystose (GF4) (Oku, Tokunaga, and
Hosoya 1984). They
are naturally found in various food items like raw Jerusalem artichoke tubers which shown to give beneficial effects on
immunity, blood metabolites, intestinal morphometry and hindgut fermentation of
rats (Samal et al. 2015).?The chain length or degree of polymerization (DP)
plays an important role in the gut fermentation of these prebiotics. Generally,
inulin has a DP ranging from 2-60 fructose units. But a few reports have
mentioned the DP as high as 100 while FOS characterized by a DP of 2 to 10,
with an average DP of 4 (Niness 1999).


Inulin and FOS derived from it are recognized and
already widely used as prebiotics (Visnapuu, Mardo, and
Alamae 2015). The
short chain FOSs are fermented by the bacteria present in the proximal colon, while
the long chain FOSs are fermented in distal colon (Meyer and
Stasse-Wolthuis 2009). A Q-PCR assay of gut microbiota of healthy
volunteers indicated that 16 days of inulin administration significantly
increased the populations of Bifidobacterium adolescentis and B. bifidum (Ramirez-Farias et al.
This effect is expected, because bifidobacteria possess ?-fructofuranosidases
specifically cleaving ?-2,1 linkages(Ryan, Fitzgerald, and
van Sinderen 2005). Extracellular
hydrolysis of FOS and inulin has been previously reported in another strain of L.
paracasei, Lactobacillus pentosus, and other species such as Streptococcus
mutans and Streptomyces exfoliates (Goh, Lee, and Hutkins 2007). The Bacteroides strains tested
showed some growth on peptides available in basal medium in the presence of
carbohydrates (Scott et al. 2014). As studied by Scott, the butyrate-producing
strains Firmicutes families Lachnospiraceae and Ruminococcaceae exhibited
different growth profiles on the substrates, which included starch, inulin,
fructooligosaccharides (FOS), galactooligosaccharides (GOS) and
xylooligosaccharides (XOS). As the fructan chain length increased, the ability
of some other strain to utilize it is decreased. Long-chain inulin was utilised
by Roseburia inulinivorans, but by none of the Bifidobacterium species examined
here. There are only six Firmicutes strains from 11 tested  strains that are sble to grow in XOS showing more
selective growth than FOS. These results illustrate the selectivity of
different prebiotics and help to explain why some are butyrogenic (Scott et al. 2014). Two isolates of L. plantarum (P14 and P76) from fermented fish were able to utilize
inulin. ?-fructosidase, the inulin-degrading enzyme, was detected in both
supernatant and cell wall extract of both strains. No activity was observed in
the cytoplasmic fraction, indicating that this key enzyme was either
membrane-bound or extracellularly secreted. The proteogenomic data revealed inulin degrading and
uptaking routes widely expressed among L. plantarum strains, which related to
fosRABCDXE operon and pstBCA genes (Buntin et al. 2016). ?



are derived from milk lactose of bovine by ?-galactosidase to produce several oligomers of
different chain lengths (Prenosil et al. 1987). They usually with 2 to 10 dp
chain with a terminal glucose. The main raw material for its production for
commercial products is usually whey-derived lactose (Yanahira et al. 1995). GOS
is produced by ?-galactosidase that
have transgalactosylation activities, which results in the formation of 4′- or
6′-galactosylactose, longer oligosaccharides, transgalactosylated disaccharides
and nonreducing oligosaccharides (Angus et al. 2005). For GOS production, the
enzymes and conditions used determine the various glycosidic linkages in the
final products. ?-galactosidase from
various fungi, yeasts and bacteria are usually immobilized on microparticle carriers
such as ion exchange resins, chitosan, cellulose and agarose beads, or fibrous
supports such as cotton cloths, which leads to the formation of different GOS
products. ?(1,2), ? (1,3) and ?(1,4) linkages
and branched glucose residues occur, while (1,4) and (1,6) linkages are present
in the galactan fragment. Variability of GOS glycosidic linkages may be one of
the reasons to acid digestion (Tomomatsu 1994). The amount of GOS produced from
lactose has also been shown to depend on the initial concentrations of lactose
present in the reaction mixture, and not on the concentration of ?-galactosidase.







Pathway 1

Utilisation of fructan-type
oligosaccharides in Lactobacillus

The capability to utilize carbohydrates depends on the
presence of a functional transport system and intracellular metabolic pathways (Buntin et al. 2016). The ability to hydrolized the prebiotics come
from the ?-fructosidase
and ?-fructofuranosidase, belongs to glycosyl hydrolase
family 32 and contains conserved amino acid residues essential for its
catalytic activity (Pons et al. 1998). In L. acidophilus and Bifidobacterium breve,
ABC transporters have been observed to be involved in FOS uptake (Ryan, Fitzgerald, and van Sinderen 2005).The inulin-degrading enzyme by the bacteria is
quite rare among lactobacilli, which mainly limited to some strains of L.
paracasei, L. casei, L. acidophilus and L. delbrueckii (Takemura et al. 2010, Tsujikawa, Nomoto, and Osawa
2013, Velikova et al. 2014, Barrangou et al. 2003). Lactobacillus species that
metabolize oligofructose, FOS utilization appears to occur via one of two
catabolic pathway (a)The substrate is transported intact and hydrolyzed
by a cytoplasmic ?-fructofuranosidase,
(b) Extracellular hydrolysis of substrates is catalyzed by a cell
surface–associated ?-fructofuranosidase,
followed by subsequent uptake of the hydrolytic products (i.e., fructose,
sucrose and glucose) via one or more transporters(Goh and Klaenhammer 2015). Three pathways have been documented to be
associated including fos operon(Goh, Lee, and Hutkins 2007, Goh et al. 2006), msm operon (Barrangou et al. 2003),and Pts1BCA operon(Saulnier et al. 2007)  to
degrade the prebiotics in Lactobacillus. The Pts1BCA operon
encoded a sucrose phosphoenolpyruvate
(PEP)-dependent phosphotransferase system (PTS) to transport short-chain FOS
(scFOS) into the cytosol of Lactobacillus
plantarum WCSF1 catalyzed by an
cytoplasmic ?-fructofuranosidase, encoded by sacA to degrade scFOS (Saulnier et al. 2007). The fosABCDXE
operon, involved in the FOS utilization pathway of Lactobacillus
paracasei 1195 has adopted a rather different strategy by encodes components
of a putative fructose/mannose PEP-dependent PTS and a ?-fructosidase precursor
(FosE). The FosE contains an N-terminal signal peptide sequence and an LPQAG
cell-wall anchor motif in the C-terminal region, indicating its location
outside the cells. A cell wall–anchored ?-FFase (FosE) mediates extracellular hydrolysis
of FOS substrates (Goh, Lee, and Hutkins 2007). Another system for FOS utilization that has
been genetically characterized in Lactobacillus acidophilus NCFM was multiple
sugar metabolism (msm) operon. This msm operon coded for an adenosine
triphosphate (ATP)-dependent binding cassette (ABC)-type transporter (msmEFGK) and a
putative intracellular ?-fructosidase found to ?hydrolyse sucrose, inulin-type fructans and transport
and hydrolysis of FOS (Barrangou et al. 2003). Lactobacillus rhamnosus GG,
which possesses functional fructose PTS transporters, enabled the recombinant
strain to grow efficiently on GFn- and FFn-type FOS, sucrose, inulin, and levan
(Goh et al. 2007).


Utilisation of FOS in

The ability of
bifidobacteria to ferment FOS, specifically shorter-chain oligofructose, is a
universal phenoty.  FOS fermentation relies
on intracellular ?-FFase and dedicated permeases or ABC transporters. Most Bifidobacterium
species grow poorly on inulin, and extracellular enzymes with specificities
for long-chain fructans are rare among bifidobacteria, prefer for shorter chain
FOS substrates. The operon is apparently induced by FOS of the GFn type but not
the FFn type. CscA hydrolyzed the ?-2,1 linkage between the glucose and
fructose moieties of oligofructose but not the ?-2,1 linkage between two
fructose moieties within the same substrate, leaving behind chains of fructose
molecules as residual hydrolytic products. Studies of FOS and inulin
fermentation with mixed fecal cultures demonstrated that other fecal bacterial
species served as primary degraders of inulin; they provided cross-feeding of
monosaccharides and short-chain oligofructose for bifidobacteria (Rossi et al. 2005).


Utilisation of GOS in Lactobacillus

The molecular mechanism of GOS utilization by
probiotic microbes was first described in L. acidophilus NCFM, when
researchers identified the lactose permease (LacS) as the key player in GOS
metabolism (Andersen et al. 2011). GOS specifically induces the gal-lac operon
that encodes LacS, a galactoside-pentose-hexuronide (GPH) family permease, two
cytoplasmic ?-galactosidases (GH42 LacA and GH2 LacLM), and enzymes of the
Leloir pathway for galactose metabolism. GOS is transported via the GPH-type
LacS permease and hydrolyzed by LacA and LacLM into glucose and galactose,
which are subsequently metabolized via the glycolytic and Leloir pathways,
respectively. The lac operon was also inducible in lactose-grown cells
(Barrangou et al. 2006), indicating that the lac operon in L.
acidophilus was responsible for the metabolism of lactose, GOS, and
potentially other galactosides. Inactivation of lacS abolished growth on
GOS, lactose, or lactitol as sole carbon source. These indicate that LacS as
the sole transporter for GOS and suggested that LacS has broad substrate specificity
for ?-galactosides. The gene cluster was also upregulated by bile exposure
(Pfeiler et al. 2007), revealing an adaptive combination of gut-evolved traits
for bile tolerance and utilization of carbohydrates common in mammals.
Additionally, the adoption of an MFS-type transporter allows energy-efficient,
rapid adaptive transport of GOS and enables scavenging of the substrates in a
nutrient-competitive niche. L. bulgaricus possesses a lacZ GH2
?-galactosidase, and its lac operon shares high similarity to that of Streptococcus
thermophiles. Differences in gene arrangement and the types of encoded
?-galactosidases reflect specific adaptation among these LAB toward the
metabolism of a variety of ?-galactoside substrates.

Researchers predicted that, similar to L.
acidophilus, the transport and hydrolysis of GOS and other ?-galactosides
such as lactose and lactulose by L. ruminus ATCC 26544 are mediated by
GPH family lactose permeases (LacY) and ?-galactosidases (LacZ) (O’Donnell et
al. 2011). Two operons of lacIZY are present in the human strain ATCC
26544, whereas genes associated with ?-galactoside metabolism are completely
absent in the bovine strain ATCC 27782.


Utilisation of GOS in

B. longum NCC2705 and B. breve UCC2003
possess analogous extracellular GH53 cell membrane–bound endogalactanases
(GalA) capable of degrading plant-derived galactan. The enzyme liberates
galactotriose from galactan polymers with ?(1–4) and ?(1–3) linkages as well as
GOS in an exotype fashion toward the reducing end of the polymers (Hinz et al.
2005). The galactotriose is imported by the cells via an ABC transporter
(GalCDE) and further hydrolyzed by an intracellular GH42 ?-galactosidase (GalG)
encoded in the same locus (galCDEGRA) (O’Connell Motherway et al. 2010).
Among strains of B. breve, the presence of GalA was also an important
determinant for efficient GOS utilization (O’Connell Motherway et al. 2013).
The endogalactanase specifically targets GOS with DP > 3. The partially
degraded GOS were primarily imported via the aforementioned GalCDE ABC
transporter and hydrolyzed by GalG. In addition to the galCDEGRA cluster,
growth on pGOS also induced two additional gene loci that each encode a LacS
permease and the GosDEC ABC transporter, both associated with a
?-galactosidase, LacZ (GH2) and GosG (GH42), respectively. Only inactivation of
the galA, galC (ABC transporter substrate-binding protein), and galG
genes resulted in impaired growth on pGOS, indicating that the LacS-LacZ
and GosDEC-GosG systems play complementary roles in GOS utilization. On the
other hand, LacS in L. acidophilus is the sole transporter for GOS,
lactose, and lactulose (Andersen et al. 2011), whereas in B. breve, LacS
is the sole uptake system for only the latter two substrates (O’Connell
Motherway et al. 2013). In B. lactis Bl-04, GOS specifically induced the
expression of two operons encoding (a) a putative MFS lactose permease
and a GH2 ?-galactosidase and (b) an ABC transporter and a GH42
?-galactosidase, respectively (Andersen et al. 2013). The genetic of these two
operons was similar to that of the lacSZ and gosRDEGC operons in B.
breve, respectively, although the proteins shared only moderate sequence


Pathway 2

of organic acids



Colonic acetate is coming from
acetogenic bacteria, which are able to synthesize it from hydrogen and carbon
dioxide or formic acid through the Wood–Ljungdahl pathway (Louis et al., 2014).



Three different
pathways are used by colonic bacteria for?propionate
formation: succinate pathway, acrylate pathway,?and propanodiol
pathway (Reichardt et al., 2014). The succinate route(dominant) utilizes
succinate as a substrate for?propionate
formation and involves the descarboxylation of?methylmalonyl-CoA
to propionyl-CoA. This pathway is present?in several Firmicutes,
belonging to the Negativicutes class,?and in Bacteroidetes.
Acrylate pathway lactate is?converted to
propionate through the activity of the lactoyl-?CoA dehydratase
and downstream enzymatic reactions; this?route appears to
be limited to a few members of the?families Veillonellaceae
and Lachnospiraceae (Flint et al.,?2015). Propanodiol
pathway, characterized by the?conversion of
deoxysugars to propionate, the CoA-dependent?propionaldehyde
dehydrogenase, that converts propionaldehyde?to propionyl-CoA,
has been suggested as a marker for?this route. This
metabolic pathway is present in bacteria?which are
phylogenetically distant, including proteobacteria?and members of the Lachnospiraceae family (Louis et al.,?2014; Reichardt et al., 2014).?


The butyrate
kinase pathway employs phosphotransbutyrylase and butyrate kinase enzymes to
convert butyryl-CoA into butyrate (Louis et al., 2004). This route is not
common among members of the gut microbiota and is mainly limited to some Coprococcus
species (Flint et al., 2015). In contrast, the butyryl-CoA: acetate
CoA-transferase pathway, in which butyryl-CoA is converted to butyrate in a
single step enzymatic reaction, is used by the majority of gut
butyrate-producers (Louis et al., 2010), including some of the most abundant
genera of the intestinal microbiota, such as Faecalibacterium, Eubacterium,
and Roseburia. Remarkably, the production of butyrate and propionate
by the same bacterium is not common and only a few anaerobes, such as Roseburia
inulinivorans and Coprococcus catus, are able to produce both (Louis
et al., 2014).