International Journal of Food Microbiology 140 (2010) 93–101

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Review

Functional intestinal microbiome, new frontiers in prebiotic design

Marco Candela a, Simone Maccaferri a, Silvia Turroni a, Paola Carnevali b, Patrizia Brigidi a,⁎
a
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b
R&D Food Microbiology & Bioprocess Research, Barilla G&R f.lli SpA, 44133 Parma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In this review we focus on the revision of the prebiotic concept in the context of the new metagenomic era.
Received 2 March 2010 Functional metagenomic data provided by the Human Microbiome Project are revolutionizing the view of
Received in revised form 13 April 2010 the symbiotic relationship between the intestinal microbiota and the human host. A deeper knowledge of
Accepted 16 April 2010
the mechanisms that govern the dynamic interplay between diet, intestinal microbiota and host nutrition
opens the way to better information on the prebiotic structure–function relationships, tailoring prebiotic
Keywords:
Intestinal microbiota
formula into specific health attributes. On the other hand, functional genomic studies of the sourdough
Microbiome microbial communities allow to scan the environmental variability to identify novel metabolic traits for the
Functional genomics biosynthesis of new potential prebiotic molecules. The integration of the functional analyses provided by the
Prebiotics massive sequencing of bacterial genomes and metagenomes will allow the rational production of a desired
prebiotic molecule with specific functional properties.
© 2010 Elsevier B.V. All rights reserved.

Contents

1. The human intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

2. Functional genomics of the human intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3. Intestinal microbiota metabolism of dietary compounds, impact on host nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4. Microbiota adaptation to changes in diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
5. Disease-associated unbalances of the human intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6. Diet-dependent manipulation of the human intestinal microbiota with prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7. Cereal sourdough fermentation, new formula from the past . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

1. The human intestinal microbiota
Human beings are “metaorganisms” as a result of millennia of co-
evolution with an incredible number of symbiont microbial cells
living in our body. The vast majority of these bacterial mutualists
(from 10 to 100 trillions) inhabits the gastrointestinal tract (GIT) and
constitutes the human intestinal microbiota (Eckburg et al., 2005;
Turnbaugh et al., 2007). With ≥100 times as many genes as our
2.85 billion base pair human genome, the collective genome of our
symbiont intestinal microorganisms (microbiome) endows us with
crucial physiological traits that we had not evolved on our own (Gill
⁎ Corresponding author. Tel.: + 39 051 2099743; fax: + 39 051 2099 736.
E-mail address: patrizia.brigidi@unibo.it (P. Brigidi).
et al., 2006; Neish, 2009; O'Hara and Shanahan, 2006; Xu et al., 2007).
Throughout the extension of the host metabolic capacity to indigest-
ible polysaccharides, the regulation of fat storage and the biosynthesis
of essential vitamins, the intestinal microbiota exerts a key contribu-
tion to the human energy balance and nutrition. Moreover, the fine
and dynamic cross-talk between intestinal microorganisms and GIT
immune system is crucial for its development and homeostasis
(Sansonetti and Medzhitov, 2009). Intestinal microorganisms educate
the host immune system to tolerance against harmless antigens while,
at the same time, concur in the maintenance of a fast responsiveness
towards harmful pathogens. In addition, precluding the colonization
from allochthonous microorganisms, the gut microbiota exerts a
“barrier effect”, defending the host from colonization by opportunistic
pathogens. Finally, studies carried out by means of gnotobiotic mice
revealed that the structural and functional development of the human
gastrointestinal epithelium is intimately connected with the presence

0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
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94 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101
of intestinal bacteria (Round and Mazmanian, 2009). Enhancing the
epithelial cell turnover and angiogenesis, and reducing the transe-
pithelial permeability, the intestinal microbiota favors the renewal
and barrier function of the gastrointestinal epithelium (Neish, 2009).
Moreover, intestinal microorganisms exert a role of primary impor-
tance in the proper structuring of inductive and effector sites of the
host GIT immune system (Garrett et al., 2010).
Even if the human intestinal microbiota is one of the most densely
populated microbial ecosystems in nature, it is characterized by a
relatively low level of phylogenetic diversity (Xu et al., 2007). 16S
rRNA gene sequence-based surveys of the intestinal microbial
ecosystem indicated that it is populated by only 8 of the 70 known
bacterial divisions and, of these, 5 are less abundant (Rajilić-
Stojanović et al., 2007) (Fig. 1). With a relative abundance of 25%
and 65%, respectively, the two dominant phyla Bacteroidetes and
Firmicutes represent together up to 90% of the total microbiota,
whereas Actinobacteria, Proteobacteria and Fusobacteria are the
subdominant phyla with a relative abundance of about 5, 8, and 1%,
respectively. However, it is at the lower taxonomic levels that we
assist a real bloom of bacterial biodiversity. At least 1800 genera
[≥90% of sequence identity (ID)] and 16,000 phylotypes at the species
level (≥97% ID) have been so far identified, and an even greater
diversity at the species level has been predicted (Peterson et al., 2008;
Zoetendal et al., 2008). Interestingly, 70% of these phylotypes are
subject-specific, and no phylotype is present at more than about 0.5%
abundance in all subjects (Turnbaugh et al., 2009). Even if the
intestinal microbiota of each individual is characterized by a peculiar
individual pattern consisting in a subject-specific complement of
hundreds of genera and thousands of species, the host-driven “top-
down” selection for the functional stability of the intestinal ecosystem
forced the development of a high level of functional redundancy
among members of the intestinal microbial community. As a
consequence, differences in the microorganism lineage among
individuals are greater than the differences in the representation of
the gene networks embedded in the bacterial genomes (Peterson
et al., 2008), and the existence of a core microbiome at a functional
level shared by all individual has been recently hypothesized (Turn-
baugh et al., 2009). Coding for genes involved in important metabolic
functions, this core functional microbiome is fundamental to support
the mutualistic relationship with the human host.
Fig. 1. Relative proportion of the phylotypes belonging to 8 of the 70 known bacterial
divisions which have been found in the human gut microbiota.
2. Functional genomics of the human intestinal microbiota
The mutualistic co-evolution between intestinal microbes and
human host resulted in the biochemical specialization of symbiotic
microorganisms to efficiently utilize the energy sources provided by
the host and, at the same time, in the host adaptation to utilize the
nutritional input provided by the novel metabolic processes conferred
by bacteria (Bäckhed et al., 2005; Martin et al., 2007). The best
example of this fine and dynamic microbiota-host metabolome
network is the extraction of energy from non-digestible plant
polysaccharides. Introduced with diet, plant polysaccharides reach
unchanged the colon where they are degraded by the microbial
consortia living in the human GIT. The principal end products of
bacterial fermentation processes are short chain fatty acids (SCFAs):
acetate, propionate, and butyrate. Adsorbed by the host enterocytes,
butyrate constitutes an important energy source for the colonic
epithelium (Neish, 2009). On the other hand, acetate and propionate
access the portal circulation and impact on the host lipid metabolism
(Laparra and Sanz, 2009). In particular, while acetate contributes to
lipid and cholesterol synthesis in the liver, propionate can inhibit the
effect of acetate.
The human microbiome project (HMP), an interdisciplinary effort
consisting in multiple projects for sequencing the human intestinal
microbiome, is currently launched around the entire globe with the
aim to explore the entire metabolic potential of the human intestinal
microbiota (Turnbaugh et al., 2007). At present, the microbiome of 20
Caucasian subjects has already been sequenced. Compared to the
previously sequenced bacterial genomes, the human intestinal
microbiota is generally enriched for COG (Clusters of Orthologous
Groups) (Tatusov et al., 2001) and KEGG (Kyoto Encyclopedia of
Genes and Genomes) (Kanehisa et al., 2004) categories involved in
metabolism (Gill et al., 2006; Turnbaugh et al., 2009). Indeed,
pathways involved in carbohydrate metabolism, energy metabolism,
generation of SCFAs, amino acid metabolism, biosynthesis of second-
ary metabolites, and metabolism of cofactors and vitamins are highly
represented in the collective genomes of intestinal symbionts (Fig. 2).
In particular, the human intestinal microbiota is endowed with a real
arsenal of carbohydrate-active enzymes (CAZymes), many of which
are not present in the human glycobiome. The percentage of
sequences in the gut microbiome that have been assigned to CAZymes
is greater than all the other KEGG pathways, indicating the abundance
and diversity of microbiome genes directed towards the metabolism
of a wide range of polysaccharides. A total of 156 CAZy families has
been found within at least one human gut microbiome, including 77
glycoside hydrolase, 21 carbohydrate-binding module, 35 glycosyl-
transferase, 12 polysaccharide lyase and 11 carbohydrate-esterase
families (Turnbaugh et al., 2009). This peculiar glycobiome confers to
the human microbiota the capacity to degrade several different
glycans which the human host cannot metabolize, ranging from the
host glycans enclosed in mucus to the array of glycans contained in
plant polysaccharides, such as xylan-, pectin- and arabinose-contain-
ing carbohydrate structures.
With the attempt to reveal the strategies for habitat and niche
adaptation for the different members of the human gut microbial
community, several comparative functional analyses of the genome
sequences of species belonging to the gut microbiota have been
recently carried out. Xu et al. (2003, 2007) sequenced the genome of
three gut Bacteroides: B. thetaiotaomicron, B. vulgatus, and B. distasonis.
Compared to non-gut microorganisms, the gut Bacteroides contain a
large repertory of genes involved in acquisition and metabolism of
polysaccharides, such as glycoside hydrolases (GHs), polysaccharide
lyases (PLs), paralogous of SusC and SusD (outer membrane proteins
involved in recognition and import of specific carbohydrate struc-
tures), and a large array of environmental sensors and regulators.
Interestingly, all these genes are organized in different selectively
regulated polysaccharide utilization loci (PULs), each encoding for
 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101
Fig. 2. Comparison of the number of KEGG genes involved in metabolic pathways within all the assembled data of human, bacterial and archaeal genomes and human intestinal
metagenome. KEGG genes are organized in functional categories (glycan, amino acid, nucleotide, lipid, carbohydrate, and xenobiotic metabolism).
functions necessary to detect, bind, degrade and import a particular
carbohydrate encountered in the gut habitat. The comparison among
the three Bacteroides genomes revealed diverse ecological strategies
and niche adaptations. Endowed with a proteome particularly
enriched in GHs for degrading plant and host glycans, the generalist
B. thetaiotaomicron can opportunistically shift between different
glycan sources. Differently, B. distasonis has the smallest repertory of
genes associated with carbon source degradation. It lacks several
hemicellulases, pectinases and other polysaccharidases, such as
chitinases that target non-plant carbohydrates. On the other hand,
B. distasonis has two classes of carbohydrate-processing enzymes
which are more represented than in other gut Bacteroidetes: α-
amylase-related proteins and N-acetylhexosaminidases. In concert
with polysaccharide deacetylases, these enzymes allow the bacteria to
make deacetylated products from host glycans available for itself and
other microorganisms. Finally, the specialist B. vulgatus has the largest
and most complete complement of enzymes that target pectin, a
common fruit-associated class of glycans.
Recently, a comparative study of functional genomics has been
carried out on the probiotic genera Bifidobacterium and Lactobacillus,
two common members of the human intestinal microbiota (Ventura
et al., 2009). More than 8% of B. longum and B. adolescentis genes
encode for enzymes involved in carbohydrate metabolism. This
percentage is analogous to the ones reported for the gut Bacteroidetes
and is 30% higher with respect to that of other GIT-resident bacteria,
such as Escherichia coli, and the non-GIT Lactococcus lactis. Among the
bifidobacterial enzymes involved in carbohydrate metabolism, there
are several GHs specific for the degradation of plant-derived dietary
fibers, the complex sialic acid-containing carbohydrates of mucin, and
the glycosphingolipids of human milk (Keenan and Patton, 1995).
Differently from B. longum and B. adolescentis, B. breve, one of the
dominant members of the infant microbiota, also possesses several
enzymes involved in the breakdown of complex polysaccharides, such
as starch, amylopectin and pullulan. Interestingly, bifidobacterial GHs
are organized in different genetic loci, each containing genes specific
for the uptake of a GH sugar substrate. These uptake systems involve
the concerted action of ATP-binding cassette (ABC) transporters,
permeases and proton symporters that mediate the capture of high
molecular weight carbohydrate substrates on the cell surface. Proving
95
their considerable degree of auxotrophy, bifidobacteria possess
several enzymes involved in the breakdown and internalization of
peptides. Similarly, the functional genomic analysis of intestinal
Lactobacillus species revealed their auxotrophy for amino acids and
carbohydrates (Kleerebezem and Vaughan, 2009). Lactobacilli sustain
their auxotrophy with a large gene array for transport functions, such
as sugar internalization systems. Lactobacilli also contain genes
encoding for metabolism of complex polysaccharides and mucus-
binding cell surface proteins (Ventura et al., 2009).
Very recently, the genome sequences of the two prominent human
gut derived Firmicutes, Eubacterium rectale and E. eligens, have been
provided and a comparative functional genomic analysis has been
carried out (Mahowald et al., 2009). Firmicutes have significantly
fewer polysaccharide-degrading enzymes and more ABC transporters
than Bacteroidetes.
Considering the complexity of the human intestinal microbiota,
the number of sequenced bacterial genomes is still small. However,
the comparative functional genomic analysis of these intestinal
microorganisms provided pioneer information regarding their adap-
tation to the gastrointestinal environment and their niche specializa-
tion. All the available genomes from GIT bacterial species showed a
common pattern of KEGG pathways, which may reflect common
features in adaptation to the human gut ecosystem. On the other
hand, differences in the number of genes within the functional
categories as well as in their specific sequences have been identified.
These differences are sufficient to define a niche specialization that
allows avoiding a direct competition between autochthonous species,
without renouncing the basal level of functional redundancy
necessary to confer stability to the symbiotic relationship.
3. Intestinal microbiota metabolism of dietary compounds, impact
on host nutrition
The metabolism of intestinal symbiont microorganisms is mainly
supported by two kinds of energy sources: non-digestible xylan-,
pectin- and arabinose-containing dietary carbohydrates, such as plant-
derived pectin, cellulose, hemicellulose and resistant starches, and
endogenous mucus glycans, which constitute a reservoir to mitigate the
effects of changes in availability of dietary polysaccharides (Flint et al.,
96 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101

2007). The degradation of this heterogeneous set of complex oligosac-
charides involves a strong metabolic syntrophy among the diverse
microorganisms which constitute the intestinal microbiota. Indigestible
polysaccharides that reach the large intestine are the first substrate of
specialized primary degraders. Different primary degraders specifically
colonize a particular dietary substrate, with a strong substrate specificity
conferred by the expression of the appropriate system for substrate
attachment, degradation and uptake. The metabolic activity of primary
degraders results in the release of a wide range of polysaccharides that
become available for secondary colonizers (Fig. 3). These microorgan-
isms can live in biofilm association with the primary degraders or less
closely adherent to substrates or planktonic.
The comparison between germ-free (GF) and conventionally
raised (CONV-R) mice has led to the discovery of the dramatic impact
that indigenous microbes exert on the host nutrition (Bäckhed et al.,
2004, 2005). According to the Authors, young adult CONV-R mice
have 40% more total body fat than the GF counterparts fed with the
same polysaccharide-rich diet. This strong impact that intestinal
symbiont microorganisms exert on the host energy balance depends
on their capability to affect both energy yield and storage from diet.
As previously discussed, the gut microbiota enhances the energy
yield by processing indigestible dietary polysaccharides to SCFAs,
which constitute a fundamental energy source for human colonic
epithelium and provide from 5 to 15% of the total energy requirement
(Neish, 2009). Differently, intestinal microorganisms exert a strong
impact on energy storage by interacting with the host lipoprotein
lipase (LPL)-mediated process for triglyceride storage in adipocytes.
In particular, throughout the suppression of the intestinal epithelium
expression of the LPL-inhibitor fasting-induced adipose factor (Fiaf),
the intestinal microorganisms promote the absorption of polysac-
charides from the gut lumen (Bäckhed et al., 2004). Moreover,
intestinal microorganisms increase the glucose uptake in the host
intestine and produce a substantial elevation in serum glucose and
insulin, stimulating the hepatic lipogenesis.
4. Microbiota adaptation to changes in diet
Pioneer whole bacterial genome transcriptional studies have been
recently carried out on mono- and bi-associated mice allowing, for the
first time, to shed some light on the dynamics that govern the syntrophic
bacterial metabolism in the GIT and the bacterial adaptation to different
diets. Gnotobiotic mice colonized exclusively with B. thetaiotaomicron
have been utilized to study the highly adaptive carbohydrate metab-
olism of this intestinal anaerobe (Bäckhed et al., 2005). In mice
maintained under a standard high plant polysaccharide chow diet
containing xylose, galactose, arabinose and glucose as monosaccharide
components, B. thetaiotaomicron directly colonizes small nutrient
platforms composed of undigested foods (dietary fibers), shed epithelial
cells and mucus fragments. Transcriptional profiling of B. thetaiotaomi-
cron revealed the up-regulation of the appropriate subset of SusC and
SusD paralogs and GHs for plant polysaccharide binding and hydrolysis.
On the other hand, when mice are fed with a simple sugar diet (glucose
and sucrose), B. thetaiotaomicron increases the expression of SusC and
SusD paralogs and GHs involved in capturing and retrieving carbohy-
drates from mucus glycans. These findings proved the B. thetaiotaomi-
cron capability to turn to host polysaccharides in the absence of the
dietary ones, demonstrating the eclectic nature of this microorganism
capable to maximize the energy harvest in response to changes in diet.
Very recently, an elegant reductionist approach has been applied to
investigate the syntrophic interaction between two members of the two
major divisions of the human intestinal microbiota: the GIT-Bacter-
oidetes B. thetaiotaomicron and the GIT-Firmicutes E. rectale (Mahowald
et al., 2009). Syntrophism has been characterized by comparing the
whole genome transcriptional profiling of each species in mono- and bi-
associated gnotobiotic mice. Interestingly, according to Mahowald et al.
(2009), B. thetaiotaomicron adapts to the presence of E. rectale by the up-
regulation of PULs that confer to the microorganism the capacity to
increase the variety of glycan substrates utilized, including those
derived from the host and that E. rectale is unable to access. Differently,

Fig. 3. (A) Establishment of syntrophic consortia in the human colonic ecosystem. Insoluble complex polysaccharides are converted in SCFAs and soluble oligosaccharides by the
primary degrader bacteria, which are characterized by the presence of specific enzyme not shared with other commensal bacteria. The primary fermentation end products fuel
different cross-feeding mechanisms by which secondary degrader bacteria originate a plethora of SCFAs. (B) Example of major bacterial metabolic pathways of oligosaccharides
fermentation and metabolic cross-feeding reported in literature.
 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101
E. rectale responds to B. thetaiotaomicron with the down-regulation of
GHs and the up-regulation of three simple sugar transport systems for
cellobiose, galactoside and arabinose/lactose, as well as peptide and
amino acid transporters. Moreover, the E. rectale enzymes involved in
the production of butyrate are the most highly expressed in bi-
associated mice. Taken together, these observations indicate that E.
rectale is better able to access nutrients in the presence of B.
thetaiotaomicron and utilizes the B. thetaiotaomicron-derived acetate
to generate increasing amounts of butyrate in mouse ceca. The impact of
two diverse dietary conditions on the syntrophic interaction between B.
thetaiotaomicron and E. rectale has been also investigated. In response to
a standard polysaccharide-rich chow diet, B. thetaiotaomicron up-
regulates several PULs involved in the degradation of dietary plant
polysaccharides and E. rectale responds with the concomitant up-
regulation of sugar transporters and GHs. The final result is a balanced
syntrophic metabolism where B. thetaiotaomicron processes complex
plant polysaccharides as primary degrader and distributes the products
of digestion to E. rectale, which, in turn, synthesizes butyrate. This
nutrient interchange between Bacteroidetes and Firmicutes is dramat-
ically interrupted when mice were fed with a high-fat and high-sugar
Western-type diet. The highly adaptive B. thetaiotaomicron responds to
this change in diet by the up-regulation of PULs specific for the
degradation of the host mucus polysaccharides. Completely devoid of
GHs that can process host glycans, E. rectale responds to the high-fat and
high-sugar Western-type diet with the down-regulation of several GHs
and sugar transporters, with an overall marked reduction in the GIT
colonization levels and a lower butyrate production. These data are in
agreement with the observations that human subjects fed with diets
deficient in complex polysaccharides harbor low levels of butyrate
producer GIT-Firmicutes, such as E. rectale (Duncan et al., 2007).
One of the most fascinating discoveries concerning the diet
response of the human intestinal microbial community has been
obtained by the characterization of the intestinal microbiota of obese
people (Turnbaugh et al., 2009). The abnormal diet energy input in
obese people favors a microbiota enriched in Firmicutes and
Actinobacteria with a low relative abundance of Bacteroidetes.
Functional microbiome analysis revealed that this peculiar transition
of the intestinal microbiota is associated with a lower level of
functional diversity in comparison with a Bacteroidetes-enriched
bacterial community, which is typical of lean people. The Authors
concluded that, like a fertilizer runoff rather than a rainforest, the diet
of obese people favors the bloom of a microbial community with a
reduced functional diversity.
5. Disease-associated unbalances of the human
intestinal microbiota
Although considered generally stable, numerous host-related
factors can shape the intestinal microbiota composition (Zoetendal
et al., 2008). Indeed, host health, antibiotic therapies, gastrointestinal
inflammation, age as well as environmental factors, such as
geographical location, ethnic and diet can impact on the intestinal
microbiota in terms of bacterial groups represented and their relative
abundance. In particular, there is an increasing evidence of a shift in
species composition in the gut microbiota in response to changes in
diet (Flint et al., 2007). For instance, a high-fat Western-type diet has
been associated with a significant decrease in the overall diversity of
the intestinal microbiota and an increase in the relative abundance of
Actinobacteria and Firmicutes respect to Bacteroidetes.
Since the intestinal microbiota impacts on a wide range of host
biological processes (Gill et al., 2006), it is widely expected that
several human diseases will be characterized by peculiar transitions in
the intestinal microbiota composition (McKenna et al., 2008).
Perturbations of the microbiota composition have been hypothesized
to be involved in diverse diseases, such as inflammatory bowel
diseases (IBD), type II diabetes, non-alcoholic fatty liver disease, and
97
sporadic colorectal cancer (CRC). Moreover, intestinal microbiota
unbalances may have a role in several allergic and atopic disorders,
which are showing a dramatic increase in incidence within the
Western population, such as immune dysregulation, asthma, and
atopy affecting skin, respiratory tract and gut (Jia et al., 2008; Rawls,
2007; Round and Mazmanian, 2009). 16S rRNA gene surveys of
disease-associated unbalances of the human intestinal microbiota
have been recently carried out. In particular, IBD is characterized by a
marked reduction of bacterial diversity in the Clostridium cluster IV
and XIVa belonging to Firmicutes, a decline in Bacteroidetes
biodiversity and a corresponding increase in Proteobacteria and
Bacillus (Frank et al., 2007; Sokol et al., 2008). Analogously, intestinal
inflammation has been generally related with a marked increase in
Enterobacteriaceae and a corresponding decrease in members of the
resident colonic bacteria (Pédron and Sansonetti, 2008; Stecher et al.,
2007). Finally, a potential role of intestinal symbiont microorganisms
in CRC development and protection has been hypothesized (Davis and
Milner, 2009; Hope et al., 2005). Certain intestinal microorganisms
could impact on CRC by the metabolism of dietary compounds, their
overall immunostimulatory activity and, eventually, the production of
toxins that perturb the regulation of cell growth.
6. Diet-dependent manipulation of the human intestinal
microbiota with prebiotics
Human beings and their intestinal symbiont microbial communities
coevolved in response to alterations of the host diet. With the
introduction of plant polysaccharides in diet, humans acquired an
adaptable and rapidly evolving bacterial metagenome (Ley et al., 2008).
Harboring an immense diversity of saccharolytic enzymes, the symbiont
microbiota provides humans with an arsenal of GHs which is necessary
to harvest energy from the enormous structural diversity of plant
polysaccharides. Thus, that diet has the potential to manipulate the
intestinal microbiota is a matter of fact and, at present, prebiotics
represent a useful dietary method for influencing the composition of the
human gut microbial community. Prebiotics formula can be included in
a wide range of foods, such as bakery, dairy and beverage. Prebiotics
were originally defined as “a non-digestible food ingredient that
beneficially affects the host by selectively stimulating the growth and/
or activity of one or a limited number of bacteria in the colon that can
improve the host health” (Gibson and Roberfroid, 1995). Generally,
prebiotics are oligosaccharides or more complex saccharides that are
selectively metabolized by some commensal groups, including species
considered to be beneficial for the host. The concept of prebiotics has
been recently formalized by the establishment of three scientific criteria
that a food ingredient must satisfy to be considered as prebiotic (Gibson
et al., 2004): (i) resistance to gastric acidity and hydrolysis by
mammalian enzymes and gastrointestinal absorption; (ii) substrate of
fermentation by intestinal microorganisms belonging to the human
microbiota; and (iii) selective stimulation of the growth and/or activity
of intestinal bacteria associated with health and wellbeing. Several
complex oligosaccharides fulfill the three criteria and can be effectively
considered as prebiotics (Table 1). Most promising prebiotics are non-
digestible fructo-oligosaccharides (FOS), such as inulin and oligofruc-
tose. Inulins are common plant storage carbohydrates which are
nutritionally classified as dietary fibers. Inulin-type fructans are present
in a range of different plants including wheat, onion, banana, garlic, leek
and Agave tequilana (Gomez et al., 2009). Numerous in vitro and in vivo
studies proved that inulin and oligofructose selectively stimulate the
health-promoting groups of the human intestinal microbiota, bifido-
bacteria and Lactobacilli (Gibson et al., 1995). Transgalacto-oligosac-
charides (GOS) are a mixture of oligosaccharides derived from the
enzymatic transglycosylation of lactose. Even if the data on their non-
digestibility do not fully match the criteria (Tomomatsu, 1994), studies
in gnotobiotic mice inoculated with the human faecal microbiota and in
human volunteers indicated that GOS induce a significant increase in
98 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101

Table 1
Comparison of several candidate prebiotics in terms of satisfaction of the three criteria defined by Gibson and Roberfroid (1995).

Name of the candidate prebiotics Criterion 1: resistance to gastric Criteria 2 and 3: fermentation by the Classification of the
acidity, hydrolysis by mammalian intestinal microbiota and selective candidate prebiotics
enzymes and gastrointestinal stimulation of the bacterial growth
absorption

Inulin
β(2 ↔ 1)fructosyl-glucose linkages
β(1 → 2)fructosyl-fructose linkages
Resistant to digestive processes
(Cherbut, 2002)
Selective growth stimulation of: Inulin fulfills all the three
– Bifidobacterium (Gibson et al., 1995) criteria and is classified as
Significant decrease of the concentration of: prebiotic
– Bacteroides (Gibson et al., 1995)
– Eubacterium rectale–Clostridium cluster
XIVa group (Harmsen et al., 2002; Rao, 2001)

GOS
Transgalacto-oligosaccharides, mixture
of oligosaccharides derived from lactose
by enzymatic transglycosylation
Tri- to penta-saccharides with β(1 → 6),
β(1 → 3) and β(1 → 4) linkages
The data of non-digestibility do
not fully match the criterion, even
if there are suggestions that GOS
reach the colon intact
(Tomomatsu, 1994)
Selective growth stimulation of: Even though the first criterion
– Bifidobacterium (Boehm et al., 2002; for prebiotic classification
Bouhnik et al., 1997; Ito et al., 1993; is not totally fulfilled, GOS
Moro et al., 2002; Rowland and Tanaka, 1993) can be classified as prebiotics
– Lactobacillus (Ito et al., 1993;
Rowland and Tanaka, 1993)
Significant decrease of the concentration of:−
– Bacteroides and Candida (Ito et al., 1993)
– Enterobacteria (Rowland and Tanaka, 1993)

Lactulose
Disaccharide galactosyl β(1 → 4) fructose
derived from isomerization of lactose
Human and calf intestinal
β-galactosidases do not degrade
lactulose (Gibson and Angus, 2000) Terada et al., 1993; Tuohy et al., 2002)
Selective growth stimulation of: Lactulose fulfills all the three
– Bifidobacterium (Ballongue et al., 1997; criteria and is classified as
prebiotic
–Lactobacillus and Streptococcus
(Ballongue et al., 1997)
Significant decrease of the concentration of:
– C. perfrigens and Bacteroides
(Ballongue et al., 1997; Terada et al., 1993)
–Lactobacillus and Streptococcus (Terada et al., 1993)
–Enterobacteria and Eubacterium (Ballongue et al., 1997)

IMO
Isomalto-oligosaccharides derived from
starch by action of α-amylases,
pullulanases and α-glucosidases
α(1 → 6) linkages
No human data are available.
Studies in rats demonstrated
that IMO are slowly digested
in the jejunum (Kaneko et al.,
1995)
In vitro studies demonstrated that IMO are Some of the evidences for the
metabolized by members of Bifidobacterium, prebiotic status of IMO appear
Enterococcus faecalis, Bacteroides and Clostridium to be promising, but still not
genera (Kohmoto et al., 1988). sufficient.
No selective stimulation of bacterial growth has IMO cannot be presently
been demonstrated in vitro. classified as prebiotics.
In vivo studies demonstrated that the concentration
of bifidobacteria significantly increases after IMO
administration (Kaneko et al., 1994; Kohmoto et al., 1991).

Lactosucrose
Produced from a mixture of lactose
and sucrose using the enzyme
β-fructofuranosidase
No data are available about
digestibility of lactosucrose
Selective growth stimulation of: Bifidobacterium The evidences for the prebiotic
(Kumemura et al., 1992, Ohkusa et al., 1995) status of lactosucrose are still
Significant decrease of the concentration of: not sufficient.
– Clostridium (Kumemura et al., 1992) Lactosucrose cannot be
– Bacteroides (Ohkusa et al., 1995) presently classified as
prebiotic.

XOS
Xylo-oligosaccharides derived
by enzymatic hydrolysis of xylan
The parent molecule (xylan) is
recognized as a dietary fiber,
indicating that XOS may reach
the colon intact.
No data are available about
digestibility of XOS.
In vitro studies demonstrated that XOS are metabolized The evidences for the prebiotic
by members of Bifidobacterium, Bacteroides and status of XOS are still not
Clostridium genera, but not Lactobacillus sufficient.
(Jaskari et al., 1998). XOS cannot be presently
In vivo studies demonstrated a selective growth classified as prebiotics.
stimulation of Bifidobacterium and Megasphaera
(Okazaki et al., 1990).

Bifidobacterium and Lactobacillus and a corresponding decrease in
Enterobacteria (Boehm et al., 2002; Bouhnik et al., 1997; Ito et al., 1993;
Moro et al., 2002; Rowland and Tanaka, 1993). Lactulose is one of the
best characterized prebiotic components. It is manufactured by the
isomerization of lactose to generate the disaccharide galactosyl β-(1-4)
fructose. Completely resistant to the human intestinal β-galactosidases,
lactulose has a well documented prebiotic effect (Gibson and Angus,
2000). As reported in a double-blind placebo-controlled trial, lactulose
induces a significant increase in Bifidobacterium, Lactobacillus and
Streptococcus concomitantly with a significant decrease in Bacteroi-
detes, Clostridium, coliforms and Eubacterium (Ballongue et al., 1997).
Besides FOS, GOS and lactulose, several other potential prebiotic
compounds have been identified, such as isomalto-oligosaccharides
(IMO) (Kaneko et al., 1994; Kohmoto et al., 1991), lactosucrose
(Kumemura et al., 1992; Ohkusa et al., 1995), xylo-oligosaccharides
(XOS) (Jaskari et al., 1998; Okazaki et al., 1990), soyabean oligosacchar-
ides (Hopkins et al., 1998) and gluco-oligosaccharides (Wichienchot
et al., 2006). However, evidences are still not sufficient for the
 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101
classification of these oligosaccharides as prebiotics in accordance with
the three rules reported above. Recently, the prebiotic potential of oat
has been also investigated (Kedia et al., 2009). According to the Authors,
the oat bran fraction shows a decrease in cultivable anaerobes and
clostridia, and an increase in bifidobacteria and Lactobacilli population.
Very recently, fermentation properties of panose, a new potential IMO-
type prebiotic compound, have been described (Mäkeläinen et al.,
2009). In a complex in vitro model simulating the human colon, panose
enhanced the growth of Bifidobacterium and reduced significantly the
growth of Bacteroides and Clostridium.
With an impact on gut microbiota composition and metabolism,
prebiotics and their derived metabolites can exert different func-
tional properties, such as: the prevention of pathogen adhesion and
colonization; the modulation of bowel habits; the regulation of lipid
and glucose metabolism (Laparra and Sanz, 2009; Sherman et al.,
2009). It has been recently reported that oligofructose supplemen-
tation lowers hunger, promotes weight loss and improves glucor-
egulation in obese and healthy adults (Parnell and Reimer, 2009). The
mechanism by which the nutritional modulation of gut microbiota by
prebiotics impacts on the appetite sensation is poorly understood,
however a fascinating hypothesis has been advanced (Cani et al.,
2009a). The increase in SCFA production due to the oligofructose
fermentation by intestinal microorganisms stimulated the biosyn-
thesis of the satiety inducing hormones, such as glucagon-like
peptide 1 (GLP-1), peptide YY (PYY) and ghrelin. According to the
Authors, it is the higher concentration of these gut peptides in plasma
that concurs in the induction of a decrease in subjective hunger
ratings and a correspondent increase in satiety. Even if further
investigations are surely needed to understand how prebiotics can
modulate satiety in humans, they can be regarded as a new potential
tool for controlling food intake and glucose homeostasis, and to
provide a possible candidate strategy in managing obesity and its
associated comorbidities. A significant step forward in this direction
has been provided by Cani et al. (2009b). Alterations of the intestinal
microbiota following a high-fat diet strongly increase the intestinal
permeability resulting in a higher plasmatic lipopolysaccharide (LPS)
level. Plasma LPS significantly contributes to the development of
obesity-related inflammatory liver diseases, such as non-alcoholic
fatty liver and non-alcoholic steatohepatitis. The Authors showed
experimental evidences that the modulation of the intestinal
microbiota by using prebiotics in obese mice acts favorably on the
intestinal barrier, lowering the high-fat diet-induced LPS endotox-
aemia and systemic and liver inflammation. Microbiota modulations
in response to prebiotic carbohydrates are associated with an
enhanced glucagon-like peptide-2 (GLP-2) production in the colon.
Positively correlated with the higher expression of ZO-1 and occludin
mRNA, the GLP-2 production improves the mucosal barrier function
leading to a decrease of plasma LPS. The influence of prebiotics on
host immunity, defense and inflammatory processes has been
recently reviewed (Lomax and Calder, 2009). According to the
Authors, FOS are effective in the improvement of both innate and
adaptive host gastrointestinal immune defenses, and significantly
decrease the host susceptibility to gut infections and atopic
disorders. Pouillart et al. (2009) investigated the impact of Nutriose,
a fermentable low-digestible carbohydrate mixture of different
glucose polymers, on IBD symptoms by using piglet model of
human IBD. Nutriose supplementation of piglets treated with
trinitrobenzene sulfonic acid for colitis induction promoted a
beneficial shift in the bacterial microbiota profile to butyrogenic
genera, such as Peptostreptococcus, Fusobacterium and Bifidobacterium.
This effect was associated with a down-regulation of some proinflam-
matory factors and a concomitant increase in the expression of the
immunomodulatory cytokine IL-10 and the up-regulation of the
secretory IgA. Enhancing a butyrogenic microbiota, Nutriose can thus
regulate IBD through an immunomodulatory pathway and alleviate
symptoms like body weight loss and bloody stools.
99
7. Cereal sourdough fermentation, new formula from the past
The market of prebiotics in foods is rapidly growing worldwide,
and there is increasing interest in developing new formulas that
benefit the human gastrointestinal health. Most of the market
prebiotics are inulin-based FOS or GOS. Even if the prebiotic status
of these compounds has been already established, according to the
literature their activity is limited to the stimulation of Bifidobacterium
and Lactobacillus, which represent only a minimal fraction of the
human microbiota biodiversity. At the light of the recent advances in
understanding the biodiversity and the enormous metabolic com-
plexity of the human intestinal microbiota, and in view of the
importance of the metabolic cross-feeding, the selective stimulation
of only few members of the intestinal microbiota can be considered a
limited vision of the potential of prebiotic compounds to manipulate
the intestinal microbial community. It is in this frame that Gibson
(2008) suggested an update of the prebiotic concept: “a prebiotic is a
selectively fermented ingredient that allows specific changes, both in
the composition and/or activity in the gastrointestinal microbiota that
confers benefits upon host wellbeing and health”. Thus, a complete
view of the impact of a prebiotic product on the intestinal microbiota
cannot be assessed regardless to the usage of 16S rRNA based
community profiling techniques. On the other hand, it is mandatory to
increase the arsenal of prebiotic molecules, extending the limits of
prebiotic activity over the major components of the human intestinal
microbiota, such as certain Bacteroidetes and members of the
Clostridium cluster IV and XIV. In this perspective, the sourdough
fermentation can represent a source of novel prebiotic compounds.
Dating back to Egypt where bread was commonly produced by lactic
acid bacteria (LAB), cereal sourdough fermentation is one of the oldest
biotechnological processes (Poutanen et al., 2009). Spontaneous
fermentation was used as leavening agent just activating the natural
occurring microbes in milled grains. Only with the development of
industrial baking, white wheat flour and baker's yeast have become
the major practice for making bread.
Sourdough is basically a mixture of water and flour that is
fermented by a heterogeneous microbial community, which includes
LAB and yeasts (Zannini et al., 2009). The sourdough microbiota
typically consists of acid-tolerant Lactobacilli, such as L. sanfrancis-
censis, L. pontis, L. panis, L. reuteri, L. amylovorus and L. frumenti, as well
as members of the genera Leuconostoc and Weissella (Katina et al.,
2009). Microorganisms are maintained active through daily additions
of water and flour, leading to the establishment of a dynamic
microbial community highly adaptable to changing in the type of
flour, nutrient availability and fermentation parameters. During the
cereal sourdough fermentation, typically up to 24 h at moderate
temperature, the metabolic activity of the microorganisms interact
with the grain constituents, mainly starch and non-starch poly-
saccharides, such as β-glucan, polyfructan, xylose and arabinoxylan
(Korakli et al., 2002). The sourdough fermentation process leads to the
production of oligosaccharides and exopolysaccharides (EPS), which
represent important prebiotic compounds. Not digested in the small
intestine, EPS stimulate colonic carbohydrate fermentation to SCFAs
and increase the cell count of beneficial members of the intestinal
microbiota. Interestingly, besides their importance as a resource of
new prebiotic compounds, the promising application of EPS as anti-
adhesion factors for the prevention of enteropathogen colonization is
currently emerging (Kitov et al., 2000; Shoaf et al., 2006).
Generally composed of fructan, glucan and FOS, the vast array of
the EPS produced in the diverse sourdough fermentations can
represent a resource of new, and partially unexplored, potential
prebiotic compounds (Korakli et al., 2002; Tieking et al., 2003). Very
recently, with the attempt to combine the biodiversity of the
sourdough microbial communities with the possibility to identify
novel potential prebiotic formula, a functional genomic analysis of the
cereal microbial communities has been carried out (Gänzle et al.,
100 M. Candela et al. / International Journal of Food Microbiology 140 (2010) 93–101
2009). The functional genomic analysis of the cereal-associated LAB
allowed to describe the bacterial metabolic pathways that lead to the
formation of oligosaccharides and EPS in the cereal fermentation
processes. The first metabolic step involves the action of extracellular
glycansucrases, GHs or glucoside phosphorylases, which drive the
formation of maltose from sucrose and glucose, maltose and glucose,
or glucose-1-phosphate and glucose, respectively. Subsequently,
maltose is metabolized by disaccharide-converting enzymes that
drive the production of a remarkable variety of oligosaccharides. In
particular, the biosynthesis of EPS involves glucansucrase or fructan-
sucrase enzymes that utilize oligosaccharides as well as mono-, di-
and oligosaccharides, such as sucrose, lactose and galactose, as final
acceptors. The presence of sucrose as acceptor carbohydrate results in
formation of kestose and higher inulin-type FOS, whereas lactosu-
crose or lactulose are formed when lactose and galactose are present
as acceptors, respectively. The use of maltose, malto-oligosaccharides,
arabinose and xylose as acceptor carbohydrates for levansucrases has
been also described (Tieking et al., 2005).
These functional genomic analyses showed the complexity and the
heterogeneity of the metabolic pathways that lead to EPS formation,
allowing to perceive the importance of the microbial biodiversity in
the sourdough fermentation as a possible source of an arsenal of novel
prebiotic products with peculiar activities towards the diverse
components of the human intestinal microbiota.
8. Conclusions
The functional analysis of the genetic information provided by the
massive sequencing of bacterial genomes and metagenomes implied a
complete revision of the relationship between human beings and the
surrounding microbial environments. Metagenome sequencing of the
human intestinal microbiota is revealing the complexity and the vast
metabolic potential of our symbiont microbial community. The
integration of the metagenome data provided by the world wide HMP
project with metabolomic studies, will open the way to a system view of
the human microbiota-host co-metabolism. This system approach will
drive to a deeper comprehension of the biological mechanisms on the
basis of the dynamics of the microbiota-host mutualisms, allowing to
understand the role of intestinal microorganisms in health and disease.
In the context of this new omics era, prebiotics can represent a potential
tool for the rational manipulation of the human intestinal microbiota.
The knowledge of the individual metabolic profiles of the intestinal
microorganisms, and the integration of a microbe-specific profile in the
community metabolome network, will allow a rational manipulation of
the human intestinal microbiota by the selection of a specific prebiotic
component. At the same time, functional genomics studies of the
sourdough microbial community metagenomes will let to scan the
microbial variability of this ecosystem to identify novel metabolic traits
for the biosynthesis of new prebiotic molecules. Finally, better
information on the prebiotic structure–function relationships, together
with knowledge of the role of intestinal microorganisms in health and
disease, will make possible to tailor prebiotics into specific health
attributes.
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