Ann Microbiol (2012) 62:15–30
DOI 10.1007/s13213-011-0306-6
REVIEW ARTICLE
Role of intestinal microbiota in colon cancer prevention
Loredana Baffoni & Francesca Gaggìa & Diana Di Gioia &
Bruno Biavati
Received: 18 February 2011 / Accepted: 21 June 2011 / Published online: 20 July 2011
# Springer-Verlag and the University of Milan 2011
Abstract Environmental and hereditary factors, together
with lifestyle, are important factors in colon cancer
development. Considering the increasing incidence of this
disease, especially in the developed western world, the last
decade has seen much attention directed towards under-
standing possible prevention strategies. Efforts to study the
intestinal microbiota and its interaction with the host have
underlined that disbiosis in colonic bacterial composition is
a risk factor for colon cancer. Modulation of the composi-
tion of intestinal microbiota through the use of probiotic,
prebiotic and synbiotic products could therefore represent a
strategy for prevention of cancer development. The mech-
anisms underlying the probiotic-prebiotic anticarcinogenic
effect involve a combination of events: e.g. binding of
mutagens, suppression of bacteria that convert pro-
carcinogens into carcinogens, immune system stimulation,
and a reduction in the level of certain intestinal bacterial
enzymes that promote carcinogen formation.
Keywords Colon cancer . Mechanism of prevention .
Probiotic . Prebiotic . Synbiotic
Introduction
The intestine constitutes the largest site of interaction
between an individual and the surrounding environment.
L. Baffoni (*) : F. Gaggìa : D. Di Gioia : B. Biavati
Department of Agroenvironmental Sciences and Technologies,
University of Bologna,
viale Fanin 42,
40127 Bologna, Italy
e-mail: loredana.baffoni@unibo.it
Intestinal homeostasis relies on the equilibrium between
absorption (nutrients, ions), secretion (ions, IgA), and the
barrier capacity of the digestive epithelium towards
pathogens and macromolecules. These functions are con-
trolled through multiple interactions with the endocrine,
neurocrine, stromal and immune cells and with the resident
bacterial microbiota that regulates epithelial functions. The
development and maintenance of a healthy microbiota is
extremely important for the correct maturation of the gut
associated lymphoid tissue (GALT) and the intestinal
mucosa (Macpherson and Harris 2002; Schiffrin and Blum
2002; McCracken and Lorenz 2001).
The human microbiota is a complex ecosystem harbour-
ing more than 1,000 species (Qin et al. 2010) although its
true size and diversity remain largely unknown (Marchesi
2010; Hattori and Taylor 2009; Ley et al. 2006). Microbial
colonisation begins immediately after birth with a succes-
sion of different species colonising each gastrointestinal
(GI) site until an equilibrium is reached (Guarner and
Malagelada 2003; O’Hara and Shanahan 2006). Because
intestinal motility is slow, and oxidation-reduction poten-
tials are very low, the large intestine is undoubtedly the
richest colonisation site in the human body, harbouring a
great number of bacteria (1010–1011 cfu/g intestinal content)
(Hao and Lee 2004), constituted mainly by anaerobes or
facultative anaerobes, including Bacteroides, Peptostrepto-
coccus, Eubacterium, Bifidobacterium, Ruminococcus,
Fusobacterium, Clostridium, Lactobacillus, Enterococcus,
Enterobacter, Escherichia coli.
The colonic microbiota influences a variety of intestinal
functions and plays a key role in nutrition, in maintaining
the integrity of the epithelial barrier, and in the development
of mucosal immunity (Shanahan 2002). Unabsorbed dietary
sugars, such as lactose, alcohols and undigested polysac-
charides are salvaged by bacterial enzymes, and fermented
16
into short-chain fatty acids (SCFAs) that are used as an
energy source by the colonic mucosa. SCFAs promote
the growth of intestinal epithelial cells and control their
proliferation and differentiation. Moreover, enteric bac-
teria produce valuable vitamins, such as folate, vitamins
of B group (B1, B2, B6, B12) and vitamin K
(Ballongue 2004). The relationship between the host
immune system and the indigenous microbiota is impor-
tant in protecting the host from pathogen colonisation. In
this regard, intestinal bacteria produce a variety of
substances, ranging from relatively nonspecific acids,
fatty acids and peroxides, to highly specific bacteriocins
that can inhibit or kill potentially pathogenic bacteria
(Servin 2004).
Importance of prevention in colon cancer development
Cancer is a combination of various metabolic and physio-
logic disturbances in the cell that are directly or indirectly
related to the influence of genetic makeup. Generally, all
cancers involve the malfunction of genes that control cell
growth and division. The process by which cancer develops
is called carcinogenesis. Generally, the carcinogenesis
process starts when DNA is damaged by chemicals or
radiation (carcinogen). Viruses are also potent inducers of
cancer, and usually induce carcinogenesis by introducing
new DNA sequences. Normal cells have DNA repair
machinery, so that, most of the time, when DNA is
damaged the cell is able to repair it. Genetic instability
provides a mechanism through which normal cells can
accumulate sufficient mutations to become malignant.
However, cells possess important mechanisms to combat
genomic instability, which is driven by the loss of cell-cycle
checkpoints, persistent DNA damage or telomere dysfunc-
tion. Central to this mechanism is the tumour-suppressor
protein p53. The p53 protein is important in transducing
diverse signals such as a range of stresses (i.e. DNA
damage, hypoxia or proliferative signals) into tumour-
suppressive apoptotic or growth-arresting responses; this
implies that there is strong selection for tumour cells to lose
p53 function (Brown and Attardi 2005; Evan and Vousden
2001). The exact mechanisms whereby the development of
cancer is mediated through mutations are still obscure;
since carcinogenesis is a multistep process, just how many
mutations in DNA are required for the development of the
complete carcinogenesis process remains unknown. As
many as ten distinct mutations may have to accumulate in
a cell before the cell becomes cancerous (Jain et al. 2009).
The increasing prevalence of human colorectal cancer
is attracting the attention of health professionals and
researchers seeking more efficacious therapeutic and
prevention strategies.
Ann Microbiol (2012) 62:15–30
Early detection and surgery have significantly reduced
both mortality and morbidity in patients affected by
colorectal cancer, but survival after surgical treatment for
advanced tumour has not seen significant improvements in
recent years. Hence, prevention of the development of
colorectal cancer appears to be a more rational and effective
strategy. The multistep nature of colorectal cancer, together
with the concept of carcinogenesis (i.e. the phenomenon by
which independent premalignant foci may progress concur-
rently and at different rates to give rise to multiple primary
tumours), make the colon a peculiarly suitable target organ
for any given chemoprevention study. Indeed, chemo-
prevention of colorectal cancer in humans has been the
focus of a number of studies where fibre, vitamins, calcium,
low-fat diet, and non-steroidal anti-inflammatory drugs
have all been shown to affect the incidence of this disease.
Approximately 70% of colorectal cancer is associated with
environmental factors, mainly diet (Saikali et al. 2004).
Thus, much attention has focused on decreasing cancer risk
through changes in dietary habits, consumption of pro-
biotics and increasing intake of dietary fibres (prebiotics). It
has been reported that ingestion of probiotics, prebiotics, or
a combination of both (synbiotics) plays an important role
in the prevention of colorectal cancer, and represents a
novel new therapeutic option (Jain et al. 2009; Le Leu et al.
2010; Choi et al. 2006).
Probiotic, prebiotic and synbiotic: a definition
Many definitions have been proposed for the term probiotic.
As originally defined by Fuller, a probiotic is “a live microbial
feed supplement which beneficially affects the host health by
improving its intestinal microbial balance” (Fuller 1989). The
most recent definition provided by FAO-WHO is: “live
microorganisms which, when administered in adequate
amounts, confer a health benefit on the host” (FAO/WHO
2002). Probiotics belong mainly to the genera Lactobacillus
and Bifidobacterium (Macfarlane and Cummings 1999;
Biavati et al. 2000; Biavati and Mattarelli 2006). Potential
health benefits deriving from probiotics may vary depending
on the type of probiotic consumed. Probiotic species that can
be used in food or pharmaceutical preparations need to have
Qualified Presumption of Safety (QPS) status. The QPS
concept provides a generic assessment system for use within
the European Food Safety Authority (EFSA) that in principle
can be applied to all requests received for the safety
assessments of microorganisms deliberately introduced into
the food chain. In essence, this proposed that a safety
assessment of a defined taxonomic group (e.g. genus or
group of related species) could be made based on four
pillars: establishing identity, body of knowledge, possible
pathogenicity and end use. If the taxonomic group did not
Ann Microbiol (2012) 62:15–30 17

raise safety concerns or, if safety concerns existed but could
be excluded through targeted characterisation studies, QPS
status could be granted. QPS granted microorganisms are
listed in Table 1 (EFSA 2008). The United States Food and
Drug Administration (FDA)—the US equivalent of EFSA—
on the other hand applies the Generally Recognized As Safe
(GRAS) concept. Both QPS and GRAS share the same core
values; however, considering the different social and regula-
tory climate present in Europe, issues of importance to
Europe would not necessary influence a GRAS listing.

Table 1 List of microorganisms granted Qualified Presumption of Safety (QPS) status (EFSA 2008) (kindly provided by Gaggìa et al. 2010)

Gram-positive non-sporulating bacteria

Bifidobacterium adolescentis
Bifidobacterium animalis Bifidobacterium breve
Bifidobacterium bifidum Bifidobacterium longum
Corynebacterium glutamicum
Lactobacillus acidophilus Lactobacillus delbrueckii Lactobacillus panis
Lactobacillus amylolyticus Lactobacillus farciminis Lactobacillus paracasei
Lactobacillus amylovorus Lactobacillus fermentum Lactobacillus paraplantarum
Lactobacillus alimentarius Lactobacillus gallinarum Lactobacillus pentosus
Lactobacillus aviaries Lactobacillus gasseri Lactobacillus plantarum
Lactobacillus brevis Lactobacillus helveticus Lactobacillus pontis
Lactobacillus buchneri Lactobacillus hilgardii Lactobacillus reuteri
Lactobacillus casei Lactobacillus johnsonii Lactobacillus rhamnosus
Lactobacillus coryniformis Lactobacillus kefiranofaciens Lactobacillus sakei
Lactobacillus crispatus Lactobacillus kefiri Lactobacillus salivarius
Lactobacillus curvatus Lactobacillus mucosae Lactobacillus sanfranciscensis
Lactococcus lactis
Leuconostoc citreum
Leuconostoc lactis
Leuconostoc mesenteroides
Pediococcus acidilactici
Pediococcus dextrinicus
Pediococcus pentosaceus
Propionibacterium freudenreichii
Streptococcus thermophilus

Gram-positive sporulating bacteria

Bacillus amyloliquefaciens
Bacillus atrophaeus Bacillus lentus Bacillus pumilus
Bacillus clausii Bacillus licheniformis Bacillus subtilis
Bacillus coagulans Bacillus megaterium Bacillus vallismortis
Bacillus fusiformis Bacillus mojavensis Geobacillus stearothermophillus

Yeast
Debaryomyces hansenii
Hanseniaspora uvarum
Kluyveromyces lactis
Kluyveromyces marxianus
Pichia angusta Pichia jadinii
Pichia anomala Pichia pastoris
Saccharomyces bayanus Saccharomyces pastorianus (synonym
Saccharomyces cerevisiae Saccharomyces carlsbergensis)
Schizosaccharomyces pombe
Xanthophyllomyces dendrorhous
18
Prebiotic compounds are defined as “non-digestible food
ingredients that beneficially affect the host by selectively
stimulating the growth and/or activity of one or a limited
number of bacteria in the colon” (Gibson and Roberfroid
1995). For a dietary substrate to be classified as a prebiotic,
at least three criteria are required: (1) the substrate must not
be hydrolyzed or absorbed in the stomach or small
intestine; (2) it must be selective for beneficial commensal
bacteria, such as the bifidobacteria, in the large intestine; (3)
fermentation of the substrate should induce beneficial luminal/
systemic effects within the host (Scantlebuy-Manning and
Gibson 2004). Dietary modulation of the human gut micro-
biota with prebiotics has been investigated for many years to
improve gut microbial balance (Hord 2008; Roberfroid
2007; Rastall et al. 2005).
Synbiotics may be defined as mixtures of probiotics and
prebiotics that beneficially affect the host by improving the
survival and implantation of live microbial dietary supple-
ments in the gastrointestinal tract (Gibson and Roberfroid
1995). Roberfroid (1998) suggested that a combination of
prebiotics and probiotics might be more active on the colon
than the individual components alone. The effectiveness of
a synbiotic formula is, however, strictly dependent on the
probiotic strain properties and its fermentation ability
towards the selected prebiotic compound.
Mechanisms underlying cancer prevention
The anti-carcinogenic properties of probiotic bacteria are
probably linked to multiple activities: (1) carcinogen
binding, (2) modulation of the other intestinal bacteria
enzymes, (3) effects on oxidative stress, (4) production of
beneficial compounds, (5) immune modulation, and (6)
apoptotic deletion.
Binding/absorption of carcinogens
Studies confirm that some strains of lactic acid bacteria (LAB)
are able to bind food carcinogens (e.g. mycotoxins or
heterocyclic aromatic amines present in cooked meat and
fish), excluding them from the human gut and reducing
thereby the rate of exposure to these highly toxic compounds.
Aflatoxins are naturally occurring mycotoxins and are
among the most carcinogenic substances known, being
metabolized by the liver to a reactive epoxide intermediate.
Aflatoxin B1 (AFB1) is considered the most toxic and is
produced by Aspergillus flavus and Aspergillus parasiticus,
which also produce aflatoxin B2 (AFB2). Aflatoxins G1 and
G2 (AFG1 and AFG2) are produced by Aspergillus para-
siticus. Aflatoxin M1 (AFM1) was discovered in the milk of
cows fed with mouldy grain and is produced following a
conversion process of aflatoxin B1 in the animal’s liver.
Ann Microbiol (2012) 62:15–30
Ochratoxin A (OTA), a toxin produced by Aspergillus
ochraceus and Penicillium verrucosum, is one of the most
abundant food-contaminating mycotoxins in the world.
Fumonisins are a family of toxins produced by several species
of Fusarium moulds, which occur mainly in maize, wheat
and other cereals. Aflatoxins, OTA and fumonisins are
considered carcinogens by the World Health Organization
International Agency for Research on Cancer (IARC 1993).
Many studies have investigated the binding of aflatoxins
to lactic acid and probiotic bacteria (Fazeli et al. 2009;
Bueno et al. 2007; Haskard et al. 2001). The binding of the
toxin is strain-dependent and is reversible in nature (Fazeli
et al. 2009; Haskard et al. 2001). Some researchers have
suggested that aflatoxin molecules bind cell wall compo-
nents of the bacteria (Hernandez-Mendoza et al. 2009;
Haskard et al. 2001), but cell surface hydrophobicity may
also play an important role (Oatley et al. 2000). Lahtinen et
al. (2004) stressed that cell wall polysaccharides and
peptidoglycan may be the most important elements respon-
sible for the binding mechanism. Some experiments also
reveal that heat treatment of bacterial cells improves their
ability to remove aflatoxins AFM1 and AFB1, possibly due
to the formation of Maillard reaction products between
polysaccharides, peptides, and proteins (El-Nezami et al.
2002; Pierides et al. 2000). In contrast, research by El-
Nezami and coworkers (1998a) showed that heat-treated
dairy strains of LAB seem to have the same ability to
remove AFB1 as viable bacteria, indicating that viability
may not be an essential prerequisite. Polarity of the toxins
also plays an important role in the binding mechanism. The
percentage of aflatoxin removed by dairy strains of LAB
and bifidobacteria decreases in the order: AFB1 > AFB2 >
AFG1 > AFG2, which is correlated with the decreasing
polarity of these toxins and is consistent with hydrophobic
interactions, suggesting the involvement of the latter in the
binding mechanism (Haskard et al. 2000). Another critical
factor, as reported by El-Nezami et al. (1998b), is the size
of the bacterial population involved in aflatoxin binding; it is
estimated that a minimum of approximately 2 x 109 CFU/ml
is required for significant AFB1 removal. The same amount
seems to be necessary for binding of OTA (Fuchs et al.
2008) by strains of Lactobacillus, Propionibacterium and
Bifidobacterium (Kabak and Dobson 2009).
Studies also indicate that probiotic bacteria can bind
other toxic compounds, e.g. toxic secondary bile acids
produced by the metabolic activity of some intestinal
bacteria (see next section).
Modulation of intestinal bacteria enzymes
In addition to the aforementioned binding mechanisms,
probiotic microorganisms could play a role in the preven-
tion of the initiation phase of carcinogenesis by decreasing
Ann Microbiol (2012) 62:15–30
exposure to activated faecal carcinogens. Animal and human
studies with Lactobacillus and Bifidobacterium supplements
show a drop in detrimental faecal enzyme concentrations
such as nitroreductase, β-glucuronidase, azoreductase, 7α-
dehydroxylase and urease (Kumar et al. 2010). It is well
known that these enzymes, which are present in some
intestinal bacteria, are able to convert pro-carcinogen
compounds into carcinogens in the human colon.
Nitroreductases from bacteria are oxygen-insensitive and
catalyse the reduction of nitro-compounds, producing
nitroso, hydroxylamine, and/or amino derivatives. These
nitroreductases have been studied in a number of bacteria
including enterobacteria (e.g. Escherichia coli, Salmonella
typhimurium, Enterobacter cloacae), and exhibit varying
substrate specificities, being able to reduce a wide range of
nitroaromatics such as nitrophenols, nitrobenzenes, and
nitrobenzoates. A large number of nitroaromatics are
present in the environment because of their use in
manufacturing processes and as antimicrobial agents, and
they are also generated as by-products of combustion
processes. Nitroaromatic compounds have caused consid-
erable health concern because their metabolism through
reductive pathways may lead to potent genotoxic and/or
mutagenic metabolites. Thus, enzymatic reduction by
nitroreductases gives rise to reactive intermediates that can
undergo nucleophilic additions with DNA and other macro-
molecules, suggesting a possible mechanism for their
cytotoxicity (Guillén et al. 2009). Some species of the
following genera encode nitroreductases: Clostridium
spp. (e.g. C. leptum, C. paraputrificum, C. clostridiiforme,
C. perfringens, C. septicum, C. sporogenes and C.
butyricum), Bacteroides spp. (B. fragilis, B. thetaiotaomi-
cron, B. ovatus and B. vulgatus) (McBain and Macfarlane
1998).
Intestinal β-glucuronidase may be involved in the
development of large bowel cancer. The activity of β-
glucuronidase in faecal samples or colon contents is
increased by high fat diets. Genotoxic and carcinogenic
heterocyclic aromatic amines (HHAs), e.g. 2-amino-3-
methylimidazo[4,5-f]quinoline (IQ), are formed in meat
and fish during cooking. Following absorption in the upper
part of the gastrointestinal tract, IQ is metabolized mainly
in the liver by xenobiotic-metabolising enzymes. Among
them, UDP-glucuronosyl transferases lead to harmless
glucuronidated derivatives that are partly excreted via the
bile into the digestive lumen, where they come into contact
with the resident microbiota. β-glucuronidase could con-
tribute to IQ genotoxicity by releasing reactive intermedi-
ates from IQ glucuronides (Humblot et al. 2007). This
enzyme activity is reported to be present in some intestinal
bacteria such as E. coli, Peptostreptococcus, Bacteroides
(e.g. B. fragilis, B. thetaiotaomicron) and C. perfringens.
Faecal bile acid excretion is also increased by a fatty diet;
19
bile acids are secreted into the duodenum in the form of
conjugates, which are degraded by bacterial enzymes in
the large intestine and transformed to deconjugated or
secondary bile acids that are toxic. A high dietary intake
of fat and red meat results in a significantly higher
excretion of faecal secondary bile acids (Reddy et al.
1980; Bernstein et al. 2009) and conjugated bile acids
seem to enhance excretion of β-glucuronidase from, for
example, E. coli and C. perfringens.
Azo dye compounds represent a large group of chem-
icals that are used extensively in the textile, pharmaceutical,
food, and cosmetic industries. Although the commonly
used azo dyes are not mutagenic in the standard Ames plate
assay, they are reduced by azoreductases from intestinal
bacteria and, to a lesser extent, by enzymes of the cytosolic
and microsomal fractions of the liver. Some intestinal
bacteria that contain azoreductase belong to the following
genera: Bacteroides spp. (B. fragilis, B. thetaiotaomicron,
B. ovatus and B. vulgatus), Eubacterium spp. (e.g. E.
hadrum) (Rafii et al. 1990), Clostridium spp. (e.g. C.
leptum, C. paraputrificum, C. clostridiiforme, C. perfrin-
gens, C. sporogenes, C. septicum, and C. butyricum)
(McBain and Macfarlane 1998), Butyrivibrio. Azo dye
compounds are linked to bladder cancer in humans and to
hepatocarcinoma and nuclear anomalies in intestinal epi-
thelial cells in mice. Thus, a number of azo dyes are
classified as carcinogenic (Rafii et al. 1990).
7α-Dehydroxylase is a detrimental enzyme responsible
for producing harmful secondary bile acids that exert a
cytotoxic effect towards epithelial cells. This enzyme
removes the 7-alpha hydroxyl group in bile acids. The
dehydroxylation of chenodeoxycholic acid leads to the
formation of lithocholic acid (litho = stone), which is
poorly water-soluble and toxic to cells, producing DNA
breaks (Bernstein et al. 2005). Bile acids formed by
synthesis in the liver are termed “primary” bile acids,
and those made by bacteria are termed “secondary” bile
acids (Commane et al. 2005).
Urease is en enzyme that breaks the C–N linkage of urea
to form CO2, NH3 and H2O. Urease-producing bacteria
inhabiting the GI tract are important in both their nutritional
and pathological aspects because they are involved in
nitrogen recycling, and the resulting product, ammonia, can
be harmful to host health. The following species of
anaerobes include urease-producing strains: Bacteroides
multiacidus (now: Mitsuokella multiacida), Clostridium
symbiosum, Eubacterium aerofaciens (now: Collinsella
aerofaciens), Eubacterium lentum (now: Eggerthella lenta),
Fusobacterium necrophorum, Fusobacterium varium, Pep-
tococcus asaccharolyticus (now: Peptoniphilus asacchar-
olyticus), Peptococcus prevotii (now: Anaerococcus
prevotii), and Peptostreptococcus productus (now: Blautia
producta) (Suzuki et al. 1979).
20
Effects on oxidative stress
Oxidative stress results when the balance between the
production of ROS (reactive oxygen species) overrides the
antioxidant capability of the target cell. ROS may interact
with and modify cellular protein, lipid, and DNA, which
results in altered target cell function. The accumulation of
oxidative damage has been implicated in both acute and
chronic cell injury, including participation in the formation
of cancer (Halliwell 2007). ROS can be produced both
endogenously and exogenously. Endogenous oxidative
stress can be the result of normal cellular metabolism and
oxidative phosphorylation. The metabolism of substances
by the P450 enzyme system generates oxygen free radicals
through normal or futile cycling mechanisms (Parke and
Ioannides 1990). Exogenous sources of ROS can also
impact on the overall oxidative status of a cell. Drugs,
hormones, and other xenobiotic chemicals can produce
ROS by either direct or indirect mechanisms (Trush and
Kensler 1991; Halliwell 1996). Alternatively, oxidative
stress can also occur when a decrease in the antioxidant
capacity of a cell occurs. Non-enzymatic antioxidant levels
[vitamin E, vitamin C, glutathione (GSH), etc.] and
enzymatic antioxidant levels (superoxide dismutase, GSH
peroxidase, and catalase) in the cell can be decreased
through modification in gene expression, reduced uptake in
the diet, or can be overloaded in ROS production, which
creates a net increase in the amount of oxygen free radicals
present in the cell (Vuillaume 1987; Barber and Harris
1994). Several human chronic disease states (e.g. cirrhosis,
atherosclerosis etc.), including cancer, are associated with
oxidative stress (Valko et al. 2007). Some lactobacilli
possess antioxidative activity, and are able to decrease the
risk of accumulation of ROS during the ingestion of food.
In fact, ROS are produced during passage of nutrients
through the GI tract, and the natural production of host
antioxidants decreases rostrally (Blau et al. 1999, Bruce et
al., 2000). LAB are able to degrade the superoxide anion
and hydrogen peroxide. However, the type of superoxide
dismutase (SOD) expressed in antioxidative strains has not
been assessed. It remains an open question if the anti-
oxidative potency of strains is associated with their survival
in stressful environments. Kullisaar et al. (2002) reported
that two strains of Lactobacillus fermentum with substantial
antioxidative activity, express Mn-SOD (manganese-super-
oxide dismutase) and have significantly increased resistance
to several ROS such as hydrogen peroxide, superoxide and
hydroxyl radicals. LGG and LGG-fermented milk are
demonstrated to be potent scavengers of superoxide anion
and inhibitors of lipid peroxidation reactions in vitro
(Ahotupa et al. 1996). Lin and Chang (2000) show that a
strain of Bifidobacterium longum ATCC 15708 and a strain
of Lactobacillus acidophilus ATCC 4356 display antiox-
Ann Microbiol (2012) 62:15–30
idative activity, inhibiting linoleic acid peroxidation by
28–48% and also show the ability to scavenge the α-
diphenyl-β-picrylhydrazyl free radical, scavenging 21–
52%. The intact cells of these two intestinal bacteria
demonstrate a high inhibitory effect on the cytotoxicity of
4-nitroquinoline-N-oxide (4NQO, a quinoline derivative
and tumorigenic compound). The cytotoxicity of 4NQO is
reduced by approximately one-half by L. acidophilus and
by almost 90% by B. longum. Nevertheless, no inhibition of
cytoxicity is observed for intracellular cell-free extracts of
109 cells of B. longum and L. acidophilus. Pediococcus
pentosaceus 16:1 and Lactobacillus plantarum 2592 pro-
duce antioxidants after 18 h growth corresponding to
100 μg vitamin C, Lactobacillus paracasei F19 a slightly
lower amount, and yet another, L. paracasei, does not exert
any antioxidative activity, again emphasizing that these
characteristics are strain-dependent (Kruszewska et al.
2002). In another study, obligatory homofermentative
lactobacilli display high antioxidant activity whereas this
property is highly strain-dependent among facultative and
obligate heterofermentative lactobacilli (Annuk et al. 2003;
Kumar et al. 2010).
Production of beneficial compounds
Bacterial fermentation in the colonic lumen produces a
variety of short chain fatty acid (SCFA) metabolites (e.g.
acetate, butyrate, propionate) that are potential anti-
carcinogenic agents within the gut. Butyrate has been
implicated in cellular homeostasis of the normal colonic
mucosa, and this is thought to be one of the mechanisms of
the chemoprotective effect of fibre. In vitro studies indicate
that butyrate causes cell cycle arrest, differentiation or
apoptosis in a number of transformed cell lines (Yu et al.
2010; Bingham et al. 2003). Femia et al. (2002) stress the
importance of oligofructose administration, alone or to-
gether with probiotic microorganisms, to increase SCFA in
the colon, and decreases the incidence of tumour develop-
ment in a rat model.
Moreover, the production of SCFA and other organic
acids by LAB and probiotics reduces intestinal pH,
preventing thereby the growth of putrefactive bacteria and
pathogens and modulating detrimental enzymes that acti-
vate toxic metabolites (see section above on Modulation of
intestinal bacteria enzymes) (Servin 2004). A prebiotic/
probiotic-induced decrease in luminal colonic pH may
function to improve mineral solubility and uptake, namely,
calcium, magnesium, and iron. The availability of minerals
is also enhanced through bacterial fermentation of sub-
stances such as phytate (myoinositol hexaphosphate),
which remove divalent cations. In particular, calcium is
suggested to be beneficial toward colorectal cancer, with
increasing evidence that it inhibits proliferation and
Ann Microbiol (2012) 62:15–30
enhances differentiation and apoptosis of mucosal cells
(Lamprecht and Lipkins 2003; Jain et al. 2009).
Some probiotic strains also produce molecules with
antagonistic activity against intestinal pathogens called
bacteriocins. Bacteriocins are a heterogeneous group of
ribosomally synthesized peptides or proteins displaying
antimicrobial activity against other bacteria. LAB bacter-
iocins seem to be targeted primarily to other LAB, which
are likely to be the most prominent competitor in the
(acidic) ecological niche in which these bacteria reside.
However, LAB bacteriocins also show activity towards a
number of potential Gram-positive food spoilage and/or
pathogenic bacteria, for example towards Listeria. LAB are
GRAS and so are their bacteriocins, which do not affect
humans or other eukaryotes. Bacteriocins are thus receiving
great attention, since applications as ‘natural’ food preser-
vatives, and maybe even as antibiotics, may be envisaged.
Since about 1990, this field has grown dramatically and this
has led to the discovery and characterisation of a large
number of bacteriocins (García et al. 2010; De Vuyst and
Leroy 2007).
Conjugated linoleic acids (CLA) are a family of
positional and geometric isomers of linoleic acid that have
been associated with several health benefits. Different
bacteria have been identified as capable of synthesizing
CLA. These include both probiotic strains and dairy starter
cultures as well as strains of human intestinal origin (Mills
et al. 2009; Alonso et al. 2003; Barrett et al. 2007; Coakley
et al. 2006; Ewaschuk et al. 2006, Coakley et al. 2003).
Several works report the ability of Bifidobacterium spp.
to convert LA into CLA (Van Nieuwenhove et al. 2007;
Barrett et al. 2007; Ewaschuk et al. 2006; Coakley et al.
2009; Rodríguez-Alcalá et al. 2011). B. breve is considered
the most efficient CLA-producing species, with conversion
rates up to 65% following 48 h fermentation (Coakley et al.
2003, 2009). However, some strains of B. breve display a
low conversion rate (Barrett et al. 2007), thus suggesting a
strain-specific behaviour. B. breve and B. longum from
human faecal samples demonstrate conversion rates of up
to 75% following 48 h fermentation of linoleic acid to c9,
t11 CLA (Barrett et al. 2007). The dairy starters Lacto-
coccus lactis and Streptococcus thermophilus also exhibit
CLA-producing activity (Lin et al. 1999) as well as the
strain Pediococcus acidilactici (Kishino et al. 2002). The
isomers cis-9, trans-11 (c9, t11) and trans-10, cis-12 (t10,
c12), have demonstrated exceptional abilities in vitro, and
in some cases in vivo, to prevent various types of cancer
(Belury 2002; Bhattacharya et al. 2006; Kelley et al.
2007), hypertension, atherosclerosis, obesity, and diabetes,
as well as an ability to improve immune function, bone
formation-promoting properties and to reduce total body
fat composition (Bhattacharya et al. 2006; Nagao and
Yanagita 2005).
21
Numerous researchers report that LAB also have the
ability to synthesise folate, for example industrial starter
bacteria Lactococcus lactis, Streptococcus thermophilus,
and Leuconostoc species and some probiotic strains (e.g.
bifidobacteria, lactobacilli, propionibacteria, and Saccharo-
myces cerevisiae) (Iyer and Tomar 2009; Pompei et al.
2007). Folic acid, one of the B vitamins (B9), acts as
cofactor in numerous biochemical reactions through its
ability to donate or accept one-carbon units. Mammals are
unable to synthesise folic acid de novo and so they must
obtain it either from the diet or from microbial breakdown
in the gut. Folate deficiency induces cytogenetic damage
and mutations both in vivo and in vitro, with increases in
DNA strand breakage, chromosomal aberrations and micro-
nuclei formation (Duthie 1999). A number of reviews have
focussed on the health benefits associated with increased
folate intake, and many countries possess mandatory folate
enrichment programs. Lately, a number of studies have
shown that high intakes of chemically synthesized folic
acid, but not natural folates, can cause adverse effects in
some individuals, such as the masking of the haematolog-
ical manifestations of vitamin B12 deficiency, leukaemia,
arthritis, bowel cancer, and ectopic pregnancies. Fermented
milk products are reported to contain high amounts of folate
produced by food-grade bacteria, primarily LAB. The focus
has therefore shifted toward natural folate, i.e. folate
produced by LAB, and levels of folate present in foods
fermented by, or containing, these valuable microorganisms.
The proper selection and use of folate-producing micro-
organisms is an interesting strategy to increase “natural”
folate levels in foods (Iyer and Tomar 2009).
Dietary flavonoids, a class of semi-essential food
components, have long been believed to exert protective
effects against many diseases, in particular cardiovascular
disease and cancer. This has been well supported by myriad
studies examining potential sites and modes of action.
There has been a proliferation of mechanistic studies,
concerning mainly effects on cell signalling pathways.
However, these studies are focused entirely on the
flavonoid aglycones, not the glycosides—the flavonoid
forms present in the human diet. Studies have confirmed
that glycoside hydrolysis is necessary prior to absorption
(Piskula 2000), and that this is accomplished by the β-
glucosidase enzyme. This enzyme is produced by different
strains of probiotic bacteria and this could be an advantage
since it may help the release of flavonoids in the large
bowel (Kumar et al. 2010; Marotti et al. 2007).
Immune system stimulation
The influence of the resident microbiota on mucosal
immune function and gut health has become an area of
scientific and clinical importance (Fuller 1991; Cebra 1999;
22
Lee 2009). There is an active dialogue between the
commensal microbiota and the host mucosal immune
system (Macpherson and Harris 2002). This cross-talk
elicits different host responses to commensal and patho-
genic bacteria. The healthy host is able to elicit a good
mucosal immune response against luminal antigens and to
maintain a “physiological state of inflammation” in the gut,
but is also able to face up to invading commensal
organisms and pathogens. In the healthy host, penetration
of the commensal bacteria is usually prevented by the
barrier afforded by the intestinal epithelium and the
immune cells associated with the mucosa, which are highly
adapted to the presence of the normal microbiota. The
functioning of the gut mucosal immune system requires a
complex network of signals, with multiple interactions
between commensals, foreign antigens and eukaryotic cells.
These include epithelial cells, macrophages, dendritic cells,
and cells belonging to the non-specific barriers: mucus-
producing cells such as goblet cells, and Paneth cells,
which secrete antimicrobial peptides (Rook and Brunet
2005). Three different routes exist for the uptake of luminal
antigens: dendritic cells, specialized M cells from the
Peyer’s patches, and individual M cells found in the villous
epithelium (Mowat 2003; Neutra et al. 1996). The
anatomical location of the immune cells from the innate
response (macrophages and dendritic cells), and the way in
which these cells acquire antigens, are crucial in determining
the nature of the subsequent responses. In the gut immune
response induced by commensal bacteria, the antigen
presentation from the luminal microbiota leads to the
generation of large quantities of local immunoglobulin A
(IgA) without induction of systemic immunity (Macpherson
and Slack 2007).
In this complex cross-talk between commensal micro-
biota and the intestinal immune system, how can probiotics
affect gut mucosal immunity? It is obvious that these non-
pathogenic bacteria must interact with the epithelial cells
and with the immune cells associated with the gut in order
to trigger the network of immune signals. The increase in
the number of IgA-producing cells is the most remarkable
property induced by probiotic microorganisms or by milk
fermented with LAB (Park et al. 2002; LeBlanc et al.
2002). IgAs play a critical role in mucosal immunity. In its
secretory form, IgA is the main immunoglobulin found in
mucous secretions, including tears, saliva, colostrum and
secretions from the genito-urinary tract, gastrointestinal
tract, prostate and respiratory epithelium. Within the two
IgA subclasses (IgA1 and IgA2) in humans, IgA1 is
produced preferentially in nasal and bronchial mucosa,
and IgA2 is produced predominately in the gut. IgA2 is
more resistant to pathogen proteases and is present at the
highest levels in the distal intestine, which has the heaviest
bacterial load. Commensal bacteria promote IgA2 class
Ann Microbiol (2012) 62:15–30
switching in human B cells (He et al. 2007). The IgA+ B
cells induced in Peyer’s patches circulate through the
mesenteric lymphatic nodes to enter the blood via the
thoracic duct and return to the intestinal mucosa, repopulat-
ing distant mucosal sites such as the bronchus. Some
probiotic microorganisms are also able to increase the IgA
cycle, in a dose-dependent manner (Villena et al. 2008;
Perdigon et al. 1999). T-independent IgA induction is also
demonstrated; cytokines, including transforming growth
factor β (TGF- β), interleukin-4 (IL-4), and IL-2, IL-6,
and IL-10, work in a synergistic way with immune cells
other than T cells and can promote the switch from IgM to
IgA expression (He et al. 2007). Stimulation with probiotic
bacteria induces signals on epithelial and immune cells that
evoke different patterns of cytokines in the intestine
(Perdigón et al. 2002; O’Flaherty et al. 2010), depending
on the dose and the strain administered. Maldonado
Galdeano et al. (2007), analysing the profile of cytokines
induced by some LAB, observe that the most remarkable
effect for all the probiotic strains tested is the increase in
tumour necrosis factor-alpha (TNF-α), gamma-interferon
(IFN-γ) and of the regulatory cytokine IL-10. This effect is
obtained without increasing the inflammatory response.
Induction of TNF-α by probiotic bacteria would be
necessary to initiate cross-talk between the immune cells
associated with the lamina propria and the intestinal
epithelial cells, while IFN- γ would play a physiological
role. It has been demonstrated that this cytokine is
necessary for the maturation of some immune cells, such
as dendritic cells, and also controls their cellular prolifer-
ation at the intestinal level (Rumbo et al. 2004). It could be
postulated that probiotics (as whole cells or as antigenic
fragments) interact with the M cells in Peyer’s patches, with
gut epithelial cells, and with the associated immune cells.
Following interaction with these cells, the release of
cytokines is induced to up- or down-regulate the immune
response. This may depend if the induction occurs in a
physiological state or during pathological processes such as
allergy (Pessi et al. 2000; Özdemir 2010), inflammatory
bowel disease (Sartor 2004), or colon cancer (Rafter et al.
2007; Takagi et al. 2001; Ewaschuk et al. 2006; Marotta et
al. 2003), in order to maintain or try to re-establish
intestinal homeostasis. Regarding this point, Maldonado
Galdeano et al. (2007) suggest that, under physiological
conditions, probiotic bacteria can act as mucosal and
systemic adjuvants.
Under pathological states, such as colon cancer or
intestinal chronic inflammations, probiotics may inhibit
disease via modulation of the mucosal and systemic
immune response and by reduction of the inflammatory
response to host microbiota. It has been shown that
intestinal inflammation is linked to the development of
colorectal cancer (de Visser et al. 2006); for this purpose,
Ann Microbiol (2012) 62:15–30
some studies evidence the importance of probiotic or
synbiotic supplementation since an increase in the produc-
tion of IL-10 (an anti-inflammatory cytokine) has been
observed (Pessi et al. 2000; Di Giacinto et al. 2005; Madsen
et al. 2001). Intestinal inflammation may also result from
exposure to toxic factors such as asbestos or smoke, as well
as from ongoing chemical or physical irritation [acid-reflux
disease, exposure to ultraviolet (UV) light]. Mutation and/
or genetic polymorphism in crucial genes that regulate
cytokine function, metabolism and leukocyte survival have
also been implicated as aetiological factors in chronic
inflammation. During acute inflammation, innate immune
cells form the first line of immune defence and regulate
activation of adaptive immune responses. Conversely, during
chronic inflammation, these roles can be reversed—adaptive
immune responses can cause ongoing and excessive activa-
tion of innate immune cells (de Visser et al. 2006). Probiotic
bacteria can modulate this over-response.
Another possible mechanism of carcinogenesis preven-
tion is the activation of natural killer (NK) cells. NK cells are
large granular lymphocytes derived from bone marrow, and
these cells display non-MHC-restricted cytotoxicity against
a variety of tumours, in part by producing mediators with
anti-angiogenic properties (de Visser et al. 2006). It is well
recognized that NK cells act as cytolytic effector cells of the
innate immune system. Oral feeding of Lactobacillus casei
Shirota (LcS) to MC-treated mice rendered their NK cells
tumouricidal in terms of both quality and quantity, resulting
in the suppression of tumour incidence (Takagi et al. 2001).
Yet another possible mechanism of action may involve
dendritic cells. Dendritic cells, as previously highlighted, are
thought to be one of the most important types of cells involved
in the presentation of several antigens and in the production of
cytokines. Recent studies show Lactobacillus strain-specific
activity in prevention of murine tumorigenesis and in
induction of IL-12 release by bone marrow-derived dendritic
cells in vitro (Takagi et al. 2008). Recently, Matsumoto et al.
(2009) described the role of the LAB strain LcS in
prevention of colon cancer by targeting the immune system.
Using a mouse model, they report that L. casei produces a
polysaccharide that inhibits colon carcinogenesis by sup-
pressing synthesis of IL-6 and signal transducer and activator
of transcription 3 (STAT3) (Kumar et al. 2010). Usually, IL-6
induces phosphorylation of STAT3, which mediates the
expression of a variety of genes and plays a key role in
many cellular processes such as cell growth and apoptosis.
Apoptotic deletion
Apoptosis is an important innate regulatory process in the
protection against the development of cancer. Apoptosis
leads to the removal of cells with genomic instability
caused by tumor development or by exposure to carcino-
23
gens (Brown and Attardi 2005). Up-regulation or facilita-
tion of apoptosis during initiation events might increase the
elimination of mutated cells that might otherwise progress
to malignancy. Such an effect might be one of the
mechanisms by which prebiotics and/or probiotics act to
protect against colorectal cancer. Le Leu et al. (2005)
registered a significant apoptotic response to a genotoxic
carcinogen in the distal colon of rats using a synbiotic
combination of resistant starch and Bifidobacterium ani-
malis subsp. lactis. A possible mechanism by which
probiotic bacteria may induce apoptotic deletion could be
through their immunomodulating properties. Cytokines
such as TNF-α are capable of inducing apoptosis. Although
cytokine levels were not measured by Le Leu et al. (2005),
other studies show that the levels of TNF-α, interferon-γ
and interleukin-10 may be increased by probiotic supple-
mentation (Jiang et al. 2009; Lee et al. 2008). Lan et al.
(2008) reported increased induction of apoptosis by
Propionibacterium freudenreichii TL133 in colonic muco-
sal crypts of human microbiota-associated (HMA) rats
treated with DMH (1,2-dimethylhydrazine). HMA rats con-
stitute a well-validated model for experimental studies, aimed
at evaluating the effects of functional foods and probiotics in
GI physiology. The induction of apoptosis occurs only in
animals treated with DMH, suggesting that it specifically
targets damaged cells. The authors suggest that daily
administration of P. freudenreichii may help in the elimination
of damaged cells by apoptosis within the colon epithelium.
This protective role against colon cancer should be further
confirmed by long-term in vivo carcinogenesis assays in
order to evaluate their effect on colorectal tumour incidence.
Altonsy et al. (2010) compared the role of pathogenic
bacteria, probiotic and commensal bacteria on the induction
of apoptosis in Caco-2 cells in vitro. The results show how
the two probiotic strains studied (Lactobacillus rhamnosus
LGG and B. animalis subsp. lactis Bb12) are able to induce
the apoptotic pathway, albeit to a lesser extent compared to
pathogenic bacteria (EPEC, VTEC), via the mitochondrial
route rather than through the Fas receptor pathway. It is
possible that the weak apoptotic effects induced by these
strains may be important in driving turnover of the
intestinal epithelium, which could reduce accumulation of
mutations in long-lived epithelial cells.
Putative molecular mechanisms responsible for colon
cancer reduction by probiotic bacteria
The mechanisms by which probiotics affect human health
beneficially can be divided into different categories, as
delineated in the previous paragraphs, including: strength-
ening of the intestinal barrier, modulation of the immune
response, antagonism of pathogens. The molecular details
behind these mechanisms are now being investigated
24
through genomics-based approaches to identify the relevant
molecular interaction between probiotics and host cells both
in vitro and in vivo.
– Probiotic microorganisms may interact directly with
dendritic cells (DCs). As already illustrated in the
section above on Immune system stimulation, DCs
intervene in early bacterial recognition and consequently
have a role in shaping T-cell responses. Several probiotic
species have been shown to induce the in vitro maturation
and cytokine expression of DCs. It seems that Lactoba-
cillus–DC interactions are mediated by the bacterium
binding to the dendritic lectin (DC-SIGN). Lactobacillus
strains that are able to interact with this lectin led to the
development of populations of regulatory T-cells that
produce interleukin-10. They can also induce hypores-
ponsive CD4+ T-cell populations.
– Probiotic microorganisms could be recognized by
intestinal epithelial cells (IEC) or colon epithelial cells
(CEC) through interaction between Toll-like receptors
(TLRs) and bacterial components. The signal thus
produced arrives in the nucleus through a signal
transduction pathway that can modulate gene expres-
sion of different cytokines.
– Modulation of inflammatory responses may also be
effected through inactivation of the NF-κB signalling
pathway; there are two possible mechanisms for this.
Soluble components produced by probiotic bacteria are
recognized by IEC, leading to inhibition of protea-
somes, and consequently to inhibition of the transcrip-
tion factor NF-κB. The proteasome is in fact a protein
complex whose main function is to degrade unneeded
or damaged proteins by proteolysis. This complex
usually degrades NF- κB inhibitor (I-κB), enabling
NF-κB to access the nucleus. If I-κB is not degraded,
it can bind to NF-κB and inhibit its translocation
across the nuclear membrane. Another way to inhibit
NF-κB is through interaction of bacterial CpG DNA
with TLR9; the result is, again, the prevention of I-κB
degradation.
Other mechanisms have also been suggested to explain
probiotic strengthening of the intestinal barrier.
– Exposure of IEC (and also CEC) to soluble compo-
nents produced by probiotic microorganisms leads to
the production of specific heat shock proteins (HSPs).
These proteins have various functions and are also
known for their ability to maintain and stabilise
cytoskeletal integrity. Induction of HSPs may be
derived from proteasome (and therefore NF-κB) inhi-
bition or through signal transduction pathway activa-
tion involving mitogen-activated protein kinases
(MAPKs) that leads to HSP expression.
Ann Microbiol (2012) 62:15–30
– Some probiotic strains induce expression of the
antimicrobial peptide β-defensin-2 (hBD-2). β defen-
sins are small antimicrobial peptides of the innate
immune system produced in response to microbial
infection of mucosal tissue and skin; among the
defensin family the β-type is distributed most widely,
being secreted by leukocytes and epithelial cells of
many kinds. Activation of hBD-2 might lead to the
enhancement of the barrier effect of the intestinal
mucosa and could explain, at least in part, the ability
of specific probiotics to inhibit pathogen growth in the
gut (Marco et al. 2006).
– There is also evidence that probiotics can modulate
MUC gene expression. Mucins are a family of large,
heavily glycosylated proteins. Some mucins are
membrane-bound while others are secreted on mucosal
surfaces and play an important role in maintaining
mucosal barriers. MUC genes encode mucin monomers
that are post-translationally modified by exceptionally
abundant glycosylation. The increased expression of
certain specific MUC genes instead of others may
create a highly difficult situation for pathogen binding.
The anti-pathogenic activities (Britton and Versalovic
2008; Thirabunyanon 2011) of probiotics have already been
mentioned; mechanisms of pathogen load reduction are
briefly summarised below:
– competitive exclusion (CE): adhesin-mediated attach-
ment of probiotics excludes pathogens;
– aggregation: probiotics aggregate with pathogens,
leading to expulsion from the gut;
– nutrient competition: probiotics compete with patho-
gens for essential nutrients;
– masking: masking of intestinal receptors for enter-
otoxins binding by probiotic adhesins;
– production of antimicrobial substances: probiotics
produce hydrogen peroxide (H2O2), bacteriocins (e.g.
lactocins, helveticins, lactacins, curvacins, nisin, bifi-
docin) and organic acids, the latter lowering pH, create
a forbidding environment for a wide range of harmful
microorganisms.
It is probable that probiotic strains exert their effects at
different stages of carcinogenesis. As evidenced by Commane
et al. (2005), two different events with their mechanisms
could be distinguished:
(1) anti-initiation events: probiotics may alter carcinogen
metabolism and improve detoxification; they may
enhance DNA repair and scavenge electrophiles.
(2) anti promotion events: probiotics may alter gene
expression, enhance immunity, suppress inflammation,
promote differentiation, suppress proliferation and
increase apoptosis.
Ann Microbiol (2012) 62:15–30
Human Studies
Numerous in vitro studies on human cell lines or animal
studies have investigated the efficacy of probiotic, prebiotic
and synbiotic supplements in reducing the incidence of
colon cancer development. Some studies focus specifically
on enzymatic activities of probiotic bacteria against
carcinogen or genotoxic compounds, or on the immune
stimulation of GALT (as discussed in Mechanisms
underlying cancer prevention).
In vivo human clinical studies, on the contrary, are still
sparse. Several reviews have attempted to analyse and
cluster available case-control studies, or to extrapolate
results from other epidemiological studies not conducted
directly with probiotic and/or prebiotic supplements. The
point at issue here is that it is often not possible to compare
different in vivo experimental results because of the
variability in trial designs or the lack of accurate informa-
tion regarding a particular study.
Pool-Zobel (2005) and Pool-Zobel and Sauer (2007)
review experimental and human data about the effect of
prebiotics, focusing on inulin-type fructans and their
incidence on colon cancer risk. They conclude that animal
studies largely support the assumption that inulin-type
fructans may reduce colorectal cancer (CRC) incidence
when supplemented during the initial stages of cancer
development. Discrepancies can also be found in human
epidemiological studies carried out to correlate high-fibre
diet with the risk of CRC (Liong 2008; Fuchs et al. 1999).
Several such studies deal with the effects of fibre.
Dietary fibres are carbohydrate polymers that are not
hydrolysed by the endogenous enzymes of the human
intestine. They are composed of a soluble fraction
(soluble fibre, which also includes prebiotic compounds)
and an insoluble fraction (insoluble fibre, e.g. lignin) that
can be present in different ratios. Much evidence
supports the view that fibre interacts with the intestinal
microbiota. The magnitude of interaction depends on the
composition. Some of the effects of fibre, e.g. increased
faecal bulk and decreased colonic transit, are linked to
fibre itself, while other beneficial effects are linked to
fibre fermentation by host microbiota (Queiroz-Monici et
al. 2005; Rose et al. 2007), leading to a modulation of
microbiota composition. Works reviewed by Liong (2008)
and the prospective study of Fuchs et al. (1999) stress that
no significant association was found between fibre intake
and the risk of colorectal adenoma. However, these studies
do not evaluate a particular prebiotic compound but rather
the incidence of an high-fibre diet, with no detailed
description of either fibre composition or fibre source.
From the epidemiologic point of view, as stressed by
Friedenreich et al. (1994), methodological factors can
influence the results from pooled analysis of nutritional
25
epidemiological data. Because of the multitude of dietary
assessment methods and food composition tables used in
nutritional epidemiological studies, heterogeneous results
are likely to occur. Standardisation of study methods
would facilitate pooled analysis, thereby permitting more
reliable conclusions regarding diet–disease relationships
(Friedenreich et al. 1994).
An interesting overview of human trials on the effect of
fibre incidence on colon cancer development is reviewed by
Young et al. (2005), where authors deal with the different
types of human epidemiological studies separately (i.e.
correlative studies, case control studies, and prospective
studies). The findings of this work show the importance of
an intake of at least 35 g fibre/day in order to even consider
taking the epidemiological study into consideration; more-
over, it is stressed that dietary fibre might be most
protective when consumed in natural food sources rather
than as a synthetic supplement (Young et al. 2005).
On the other hand, a review by Capurso et al. (2006)
gives an overview of available human studies with pro-
biotic bacteria. This article has a meaningful title: “Pro-
biotics and the incidence of colorectal cancer: when
evidence is not evident”. The authors analysed existing
data from basic science (animal and in vitro models) and
human (epidemiological and interventional) studies to
highlight areas for which more evidence is necessary. They
interrogated Medline for studies analysing the risk of CRC
and the use of probiotics, as well as screening the
references of identified papers. Once again, this review
stresses the paucity of human epidemiological studies
designed specifically to analyse the effect of probiotics on
CRC incidence; moreover, authors underline the presence
of important confounding factors, such as the role of fibre,
dairy products and vitamin D, which are often present.
Conflicting epidemiological data regarding the impact of
fermented dairy product consumption in humans have been
gathered by authors. Also in this case, human epidemio-
logical and interventional studies still do not seem to
support the promising results observed under experimental
conditions. These discrepancies may be explained partially
by the use of bacterial strains lacking the appropriate
characteristics, again stressing the concept that different
probiotics might have specific and different clinical
applications. In fact, epidemiological studies examine the
action of “fermented dairy products”, which do contain
naturally occurring probiotics but of unknown strains and in
undefined amounts. Therefore, even though a large body of
evidence supports the potential anticarcinogenic action of
probiotics on the basis of the results obtained in both in
vitro and in vivo models, further evidence is still needed
(Capurso et al. 2006).
Concluding, a noteworthy human intervention study that
was performed within the EU project SYNCAN (Van Loo
26
et al. 2005) has yielded some interesting results. This
project involves the integration of an in vitro study to select
the most suitable synbiotic preparation, the application of
this synbiotic in an in vivo rat model of chemically induced
colon cancer, and, as the core of the project, an investiga-
tion of synbiotic effects in a human intervention study. The
assessment of the survival during gastric transit of the
administered probiotics (Bifidobacterium animalis subsp.
lactis Bb12 and Lactobacillus rhamnosus GG) was per-
formed in three healthy volunteers in a separate study.
Subsequently, a 12-week randomised, double-blind,
placebo-controlled trial of a synbiotic food supplement for
reduction in cancer risk bio-markers is carried out. The
synbiotic used in this study was composed of the two
probiotics listed above and the oligofructose-enriched
inulin Synergy1®. Polypectomised and colon cancer sub-
jects having previously undergone ‘curative resection’ for
colon cancer were selected and assigned randomly to
placebo or synbiotic group. The ability of the synbiotic
combination to modulate the gut microbiota was demon-
strated in both the cancer and the polypectomised subjects.
The selectivity of fermentation was shown by the bifido-
genic effect, which was accompanied by a decrease of
coliforms in both polypectomised and cancer patient
groups, and a decrease of Clostridium perfringens in the
polypectomised group. The authors stress that, since the
synbiotic supplement modulates the microbiota, it can be
hypothesised that the changes in biomarkers monitored in
the human anticancer study in test groups vs control group
are likely to be the consequence of the administration of an
active synbiotic preparation. The analyses of various
markers in this human study show reduced genotoxicity,
increased caecal SCFA, particularly butyrate, priming of the
immune system to be more efficient to fight cancer cells
and counteract inflammation, and a reduction in DNA
damage (Van Loo et al. 2005).
Conclusions
The use of probiotics, prebiotics and synbiotics to prevent
colon cancer has gained much attention in the last decade
due to the fact that in vitro systems and studies in a wide
range of animal models provide considerable evidence that
they exert anti-neoplastic effects. This review reports an
overview of the possible mechanisms that could underlie
the preventative action of probiotics and prebiotics against
CRC development. The evolution of CRC is a complicated
multistep process influenced by genetic as well as by
environmental factors. Similar considerations can be
reported for probiotics and prebiotics. Their preventative
effect on CRC development is possibly the sum of a series
of beneficial effects that these supplements exert in the GI
Ann Microbiol (2012) 62:15–30
tract. However, their effect is also influenced by strain
specificity, host (i.e. genetic factors, indigenous microbiota)
and by environmental variants (e.g. diet, lifestyle, stress
conditions etc.). It is of the utmost importance that the
peculiar characteristics of a given probiotic strain are
clearly defined in order to target and associate a particular
strain to a defined disorder. Moreover, an appropriate
prebiotic compound should be selected and associated to a
specific probiotic microorganism to favour a synergistic
effect. Unfortunately, despite the huge quantity of data
available in vitro and on animal models, these outcomes are
not often supported by human data. The complexity of
creating a series of long-term human intervention studies
should be overcome and new human studies on probiotics
and prebiotics should be financed in order to generate
uncontroversial experimental evidence. Further research is
required to identify probiotic, prebiotic or synbiotic
combinations that will be more effective for humans in
different conditions. It is very likely that there will be no
treatment that is ideal for all cases. Probably, the great
efforts of the scientific community to identify the genetic
determinants involved in the health-promoting effects of
probiotic bacteria (i.e. probiogenomics) (Ventura et al.
2009), together with new well-designed human trials, will
yield significant insights into pre- and pro-biotic mecha-
nisms. However, it is feasible to conclude that pro-, pre-
and synbiotics hold great potential as a strategy for the
prevention of colorectal cancer.
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