INTRODUCTION
Every second of our life we are in contact with the microbes surrounding us. In
fact, we cannot live without microbes as they are responsible for recycling the elements
that are crucial for our life. Our gastrointestinal (GI) tract is inhabited by large numbers of
bacteria that collectively outnumber host cells by a factor of ten (Savage, 1977). The
ecology of the GI tract is currently a hot research topic. The complexity of interactions
between these microbes and our intestinal cells varies tremendously and includes
pathogenic, competitive and symbiotic interactions. Intriguingly, only one thin layer of
epithelial cells separates the GI tract microbes from our other organs. The microbial
community in the GI tract is very complex and consists of different groups of microbes,
such as bacteria, archaea, ciliate and flagellate protozoa, anaerobic phycomicete fungi and
bacteriophage; of these group bacteria have received most attention.
Gut microbiota
The intestine’s normal microbiota is as yet an unexplored organ of host defence.
Although bacteria are distributed throughout the intestine, the major concentration of
microbes and metabolic activity is found in the large intestine (Berg, 1996; Salminen et al.,
1998; Guarner and Malagelada, 2003). The mouth harbours a complex microbiota
consisting of facultative and strict anaerobes including streptococci, Bacteroides,
lactobacilli and yeasts. The upper bowel is sparsely populated, and from the ileum on
bacterial concentrations gradually increase, reaching 1011–1012 colony-forming units
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(CFU)/g in the colon (Figure 1). Up to 500 species of bacteria may be present in the adult
human large intestine; it has been estimated that bacteria account for 35–50% of the
volume of the contents of the human colon.
The bacteria of the human gut include transient and indigenous types. The
indigenous bacteria have sometimes been classified as potentially harmful or healthpromoting; most of them, however, constitute part of the commensal microbiota. The
strains with beneficial properties, potential sources of probiotics, most frequently belong to
the genera Bifidobacterium and Lactobacillus, and some of these exhibit powerful antiinflammatory properties (Isolauri et al., 2002). Moreover, members of these genera have
been attributed with other beneficial aspects such as stimulation of the immune response
and competitive exclusion of pathogens whereby non-specific host resistance to microbial
pathogens is promoted. Establishment of the microbiota provides the host with the most
substantial antigen challenge, with a strong stimulatory effect for the maturation of the gutassociated lymphoid tissue (Cebra, 1999; Grönlund et al., 2000). Realization of this has led
to the introduction of novel modes of probiotic intervention to strengthen the endogenous
host defences, particularly in early childhood, when the risk of developing infectious,
inflammatory and allergic diseases associated with impaired gut barrier function continues
to present a formidable challenge confronting clinicians and scientists.
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Fig. 1. The numerically dominant microbial genera in the adult human gastrointestinal tract.
Functions of the intestinal microbiota
The activity of the intestinal microbiota is comparable to that of the liver, our
metabolically most active organ. The metabolic activity of the intestinal microbiota is
involved in the fermentation of exogenous and endogenous carbon and energy sources.
Fermentation of different types of oligosaccharide is beneficial to the host as it provides
additional energy in the form of short-chain fatty acids. Of these, butyric acid, as a main
energy source for the intestinal epithelium, is important in maintaining mucosal health in
the colon (Brouns et al., 2002). Furthermore, several members of the intestinal microbiota
produce vitamins. The significance of the microbiota in salvaging energy and producing
vitamins is most clearly seen in germ-free animals. Compared to conventional animals,
germ-free animals require 30% more energy in their diet, supplementation of which with
vitamins K and B is mandatory to maintain their body weight (Hooper et al., 2002).
However, the intestinal microbiota can also utilize other substrates such as proteins and
3
amino acids. Fermentation of these may lead to the production of a variety of toxic
substances such as tumour inducers and promoter (Mykkänen et al., 1998). Another
important function of the intestinal microbiota is to provide protection against incoming
microbes. Studies have demonstrated that animals bred in a germ-free environment are
highly susceptible to infections; thus, the intestinal microbiota is considered an important
constituent in the mucosal defence barrier. The phenomenon in question is termed
colonization resistance: bacteria in the gut mucosa compete for the same attachment sites
as pathogenic bacteria, they use the same nutrients as these bacteria, and bacteria present in
the gut produce several compounds inhibiting the growth of pathogens and other transient
incoming bacteria which are not members of the residing intestinal microbiota. Finally, the
intestinal microbiota provides an important stimulus for the maturation of the immune
system. At birth, the immune system is immature and develops upon exposure to microbes;
the number of Peyer’s patches and immunoglobulin (Ig) A-producing cells increases in
their presence, thereby promoting the immunological barrier of the gut mucosa. Recent
insight into the interaction between bacteria and mucosal innate and adaptive immune
systems provides a basis for understanding the role of the gut microbiota in achieving a
disease-free state in the host, in spite of the constant presence in the gut lumen of a myriad
of antigens from food and microorganisms.
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Factors influencing the composition of the gut microbiota
The diet may exert a major effect on the composition and activity of the gut
microbiota. As long as infants are breast-fed and/or formula-fed, the faecal microbiota will
be dominated by bifidobacteria. Breast-feeding tends to contribute to higher levels of
bifidobacteria, although with modern infant formulas the differences are now less
pronounced than in the past (Harmsen et al., 2000). Nevertheless, new molecular methods
indicate that lactic acid-producing bacteria account for less than 1% of the total microbiota
in infants, while bifidobacteria can range from 60 up to 90% of the total faecal microbiota
in breast-fed infants (Favier et al., 2002; Vaughan et al., 2002). The new techniques thus
indicate that the greatest difference in the microbiota of breast-fed and formula-fed infant
lies in the bifidobacterial composition of the intestinal microbiota, while the lactic acid
bacteria composition appears to be fairly similar. With the introduction of solid foods, the
microbiota undergoes a more dramatic change and becomes diverse. This diversity results
in an adult-like microbiota approximately by the age of 2 (Favier et al., 2002). Because the
environment changes along the gastrointestinal tract, different microbes are found at
different sites (Figure 1). The high flow of the contents in the upper part of the
gastrointestinal tract does not allow for the accumulation of a large microbiota, and
secretions from the stomach, liver and pancreas further contribute to this end. In the lower
part of the gastrointestinal tract, the flow of the digesta becomes slower and its
composition is less antimicrobial, supporting the establishment of a larger microbiota.
Because of the anaerobicity of the lower gastrointestinal tract, anaerobic microbes start
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to outnumber the aerobic (Tannock, 1999). Dramatic differences also develop between the
luminal and the mucosal milieu. While the environment in the lumen may be anaerobic, at
the mucosa some oxygen may leak from the tissue, creating a microaerobic environment.
Furthermore, Paneth cells in the mucosa secrete antimicrobial substances such as lysozyme
and defensins (Hornef et al., 2002), whereas enterocytes transport secretory IgA from the
lamina propria to the lumen (Lloyd, 2003). This translates into a different composition of
the luminal and mucosal microbiota (Zoetendal et al., 2002). Consequently, even within
the same microbial species, different strains can be found in the lumen as compared to the
mucosa (Nielsen et al., 1994). The variation in the microbiota composition at different sites
in the gastrointestinal tract clearly has implications for the information obtained from
samples collected from different sites. From Figure 1 it is also obvious that the
composition of the intestinal microbiota is complex. The function of most of the members
of the intestinal microbiota, however, still remains unknown. Thus, although it is tempting
to label certain genera as pathogens and others as beneficial, this is not justified. At low
levels opportunistic pathogens such as Candida sp and Clostridium sp may in fact fulfil a
beneficial role in the gastrointestinal tract by contributing to the maturation of the immune
system; nevertheless, clostridia are often considered less desirable, while bifidobacteria
and lactobacilli are considered beneficial.
During adult life, the intestinal microbiota is relatively stable (Zoetendal et al.,
1998). In old age, however, changes again take place. Levels of Bifidobacterium tend to
decrease (Isolauri et al., 2002) (Figure 2) and the diversity of the Bifidobacterium
microbiota tends to wane (Mitsuoka, 1990; Hopkins and Makfarlane, 2002). Despite the
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differences in composition, however, no significant differences in metabolic activity of the
faecal microbiota between children, adults and the elderly have been observed (Andrieux
et al., 2002). Diseases—gastrointestinal and extraintestinal—can influence the intestinal
microbiota, and vice versa. The most fully documented disease altering the gut microbiota
is acute infectious diarrhoea (Isolauri et al., 2002). In allergic disease, lower levels of
bifidobacteria and higher levels of clostridia have been observed in infancy as compared to
healthy infants (Isolauri et al., 2002). Furthermore, allergic infants appeared to be
colonized mainly with Bifidobacterium adolescentis, healthy infants, again, mainly with B.
bifidum (Ouwehand et al., 2001). These two groups of bifidobacteria induced different
cytokine profiles. Bifidobacteria from allergic infants induced in vitro the secretion of
TNF-a, interleukin(IL)-1b, IL-6 and IL- 12 by macrophages, while bifidobacteria from
healthy infants, and those of dairy origin, stimulated the secretion of IL-10 (He et al.,
2002). Thus the initial composition of the gut microbiota may affect the immunological
development of the host before the immune responder phenotype becomes consolidated.
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Fig. 2. The development of the gut microbiota (Mitsuoka et al., 1974)
Probiotics
Probiotics are live microbial food supplements which benefit the health of consumers by
maintaining or improving their intestinal microbial balance (Fuller, 1989; FAO/WHO,
2001). Due to their perceived health benefits probiotic bacteria have been increasingly
included in yoghurts and fermented milks during the past two decades. Most commonly
they have been lactobacilli such as Lactobacillus acidophilus, and bifidobacteria often
referred to as ‘bifidus’ (Daly and Davis, 1998). A major development in functional foods
pertain to foods containing probiotics and prebiotics which enhance health promoting
microbial flora in the intestine. There is growing scientific evidence to support the concept
that the maintenance of healthy gut microflora may provide protection against
gastrointestinal disorders including gastrointestinal infections, inflammatory bowel
diseases, and even cancer (Haenel, 1975; Mitsuoka, 1982). The use of probiotic bacterial
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cultures stimulates the growth of preferred micro-organisms, crowds out potentially
harmful bacteria, and reinforces the body's natural defence mechanisms. Today, plenty of
evidence exists on the positive effects of probiotics on human health. However, this has
usually been demonstrated in diseased human populations only (Salminen et al., 1998a).
Thus there is an urgent need for evidence for probiotic health benefits in average (generally
healthy) populations.
Before a probiotic can benefit human health it must fulfil several criteria: it must
have good technological properties so that it can be manufactured and incorporated into
food products without loosing viability and functionality or creating unpleasant flavours or
textures; it must survive passage through the upper gastrointestinal (GI) tract and arrive
alive at its site of action; and it must be able to function in the gut environment. To study
the probiotic strain in the GI-tract, molecular techniques must be established for
distinguishing the ingested probiotic strain from the potentially thousands of other bacterial
strains that make up the gastrointestinal ecosystem. Additionally, techniques are required
to establish the effect of the probiotic strain on other members of the intestinal microbiota
and importantly on the host. This includes not only positive health benefits, but also
demonstration that probiotic strains do not have any deleterious effects. Armed with this
knowledge, the probiotics can then enter human pilot studies that attempt to assess their
health benefits to consumers (Mattila-Sandholm et al., 1998).
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Selecting probiotic strains: important aspects
The theoretical basis for the selection of probiotic micro-organisms including
safety, functional and technological aspects is illustrated in Figure 3. The significance of
human origin has been debated recently, but most current successful strains are indicated to
be of human origin. It can also be argued that a probiotic strain can function better in a
similar environment (e.g. human GI-tract) to where it was originally isolated from. Safety
aspects include the following specifications:
1. Strains for human use are preferably of human origin.
2. They are isolated from healthy human GI-tract.
3. They have a history of being non-pathogenic.
4. They have no history of association with diseases such as infective endocarditis or GIdisorders.
5. They do not deconjugate bile salts (bile salt deconjucation or dehydroxylation would be
a negative trait in the small bowel (Marteau et al., 1995).
6. They do not carry transmissible antibiotic resistance genes.
The functional requirements of probiotics should be established by using in vitro
methods and the results of these studies should be reflected in controlled human studies.
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While selecting a preferable probiotic strain several aspects of functionality have to
be considered:
1. Acid tolerance and tolerance to human gastric juice.
2. Bile tolerance (an important property for survival in the small bowel).
3. Adherence to epithelial surfaces and persistence in the human GI-tract.
4. Immunostimulation, but no proinflammatory effect.
5. Antagonistic activity against pathogens such as Helicobacter pylori, Salmonella sp.,
Listeria monocytogenes and Clostridium difficile.
6. Antimutagenic and antigarcinogenic properties.
Feeding trials with different probiotic strains have shown that the probiotic strain
usually disappears from the GI-tract within a couple of weeks after the ingestion is
discontinued (Donnet-Hughes et al., 1999; Alander and Mattila-Sandholm, 2000). The role
of the probiotic persistence in the human GI-tract has therefore been questioned. However,
even temporary persistence, which has been noted for several ingested probiotic strains,
may enhance their chances for beneficial functions in the GI-tract, and is therefore
considered a desirable trait.
Even though a probiotic strain fulfils the necessary safety and functional criteria the
aspects related to probiotic production and processing are also of utmost importance.
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Several technological aspects have to be considered in probiotic selection. These
include the following:
1. Good sensory properties.
2. Phage resistance.
3. Viability during processing.
4. Stability in the product and during storage.
Figure 3. The theoretical basis for selection of probiotic micro-organisms includes safety, functional
(survival, adherence, colonisation, antimicrobial production, immune stimulation, antigenotoxic activity and
prevention of pathogens) and technological aspects (growth in milk, sensory properties, stability, phage
resistance, viability in processes).
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AIMS OF THE RESEARCH
The objective of this work were:
1. To investigate human intestinal isolated strains as potential probiotics in in vitro and in
vivo studies. This work is a continuation of an EU project called Crownalife in which
the University of Camerino was partner. The project aimed to improve the quality of
life of the elderly throughout the third age, with emphasis on the preservation of the
period of independence recognized as “crown of life”. The focus was on preventive
nutrition and the application of functional food to derive health benefits for the ever
increasing European elderly population. Based on hypothesis driven human studies, the
projects’ specific objectives were:
a) to assess structural and functional alterations of the intestinal flora with ageing,
across Europe;
b) to validate functional foods based preventive nutrition strategies to restore and
maintain a healthy intestinal flora in the elderly.
Implementations include nutritional recommendations as well as new concepts and
prototype functional food specifically adapted for health benefits to the elderly
population.
From the lactic acid bacteria isolated during the project, it has been screened some
candidate probiotics.
These isolates were identified and investigated for their technological and
functional characteristics as potential novel probiotic strains.
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2. Recently, it has been suggested that the ability of probiotic bacteria to ferment
oligosaccharides may be an especially important characteristic. This is because the
availability of carbohydrates that escape metabolism and adsorption in the small intestine
have a major influence on the microflora that become established in the colon. If certain
carbohydrates, such as oligosaccharides, are fermented only by specific strains of
bifidobacteria and lactobacilli, then diets containing so-called "prebiotic" substrates could
select for those strains of probiotic bacteria. One specific group of oligosaccharides that
has attracted much commercial interest as prebiotics is the fructooligosaccharides (FOS).
These compounds, which are marketed commercially as Raftilose and Nutraflora, can be
obtained from natural sources (e.g., inulin) or synthesized naturally from sucrose. Inulintype fructans are known for their so-called bifidogenic effect, meaning their ability to
selectively increase the number of bifidobacteria in the human colon, as bifidobacteria
are able to use inulin-type fructans as the sole energy source. It has also been
demonstrated that certain Lactobacillus strains are able to grow on these prebiotics.
Some in vivo studies with animal models or clinical trials have demonstrated an increase
of the number of lactobacilli when inulin-type fructans are applied (Kleessen et al., 2001;
Langlands et al., 2004), but in other reports the number of lactobacilli remains stable
after administration of such prebiotics (Gibson et al., 1995; Tuohy et al., 2001). These
results indicate that the ability to ferment inulin-type fructans is strain specific for
lactobacilli, in contrast with bifidobacteria, where this property is more widespread
(Hopkins et al., 1998; Bielecka et al., 2002). The aim of this work was to investigate the
ability of lactobacilli to ferment inulin and to study their kinetics of growth.
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3. Reliable determination of viability of bacteria in probiotic products is important as the
definition of probiotics calls for viable microbes. Plate count method has traditionally
been used for determination of viability of bacteria, but there are obvious disadvantages.
First, plate count requires long incubation times. Plate count method is often hampered
by technical difficulties such as clumping and inhibition by neighbouring cells. The
choice of enumeration medium and incubation conditions for specific species may also
be challenging. For many species a suitable growth medium is not known. The aim of
this work is to develop and evaluate the application of real-time quantitative PCR to the
specific detection and quantification of the Lactobacillus strains in different kind of
products. The results of molecular quantification were compared with those obtained
using the classic plate count method, in order to evaluate the accuracy and robustness of
the molecular approach.
4. To colonize the gastrointestinal tract, probiotic strains need to be ingested as large
populations and on a daily basis. Therefore, food manufacturers are trying to include
probiotic strains in foods and beverage which are part of a normal diet to provide health
defense while enjoying meals and to differentiate such functional products from
concentrated probiotic preparations available as capsules, powders, or liquids. We
applied the probiotic strains in several kind of products and we studied the suitability of
them as biological carriers for a selected strain which survived passage to through the
gastrointestinal tract and maintained colonization.
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MATERIALS AND METHODS
Selecting probiotic strains
Bacterial strains
The Lactobacillus strains used in this study are those isolated from the Crownalife
project using the methods described in Silvi et al. (2003). The potential probiotic strains
used in this study are also listed in Table 1.
Table 1. Strains included in this study
Origin
Strain
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
Human faeces
115
117
202
203
204
216
303
319
401
403
404
901
902
903
904
907
1102
1303
1304
1305
Phenotypical
characterisation
Lactobacillu paracasei
Lactobacillus curvatus
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus spp.
Lactobacillus paracasei
Lactobacillus plantarum
Lactobacillus fermentum
Lactobacillus rhamnosus
Lactobacillus salivarius
Lactobacillus delb.bulgaricus
Lactobacillus brevis
Lactobacillus plantarum
Lactobacillus cellobiosus
Lactobacillus brevis
Lactobacillus rhamnosus
Lactobacillus spp.
Lactobacillus spp.
Lactobacillus spp.
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Low pH and bile salt tolerance
The isolated Lactobacillus strains were tested for their ability to resist to low pH and
bile salt.
The pH value of gastric acid varies in the range of about 1.5-4.5 in a period of 2 hours,
depending on the entering time and the type of gastric contents. In the present study pH 3
was used as a representative gastric pH value. A 24-h-old culture of each Lactobacillus
(108 CFU/ml) was suspended in a citrate buffer pH 3 for 5 hours at 37°C. The suspensions
were then centrifugated at 3000 rpm for 10 min at 4°C twice and washed in sterile saline
solution to eliminate the citrate buffer. Cells were suspended in physiological solution and
a series of 10-fold dilution (10-2-10-10) was prepared. A given amount of each dilution (50
µl) was plated on to de Man Rogosa Sharpe (MRS) agar (Oxoid, Basingstoke, Hampshire,
UK) and incubated anaerobically at 37°C for 24-48 h. The percentage of the viable bacteria
was calculated.
Tolerance to bile salts was verified inoculating 100 µl of bacterial suspension of each
strains
(108 cells/ml) on to MRS agar containing Bile salt (Oxoid) at different
concentrations (0.1%; 0.3%; 0.5%) and on to MRS agar containing Bile salt N.3 (Oxoid) at
different concentrations (0.05%; 0.1%; 0.2%). Survival of the Lactobacillus strains was
examined by counting the cells after 24 and 48 h of incubation at 37°C.
Only those strains which survived these two resistance tests were unequivocally
identified and further investigated for in vitro probiotic properties.
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Resistance to 0.4% phenol
Some aromatic amino acids derived from dietary or endogenously produced proteins
can be deaminated in the gut by bacteria leading to the formation of phenols. These
compounds can exert a bacteriostatic effect against some Lactobacillus strains. Thus
testing for the resistance to phenol may generate further information on the potential for
survival of lactobacilli in gastrointestinal conditions. Therefore, we tested the ability of
Lactobacillus strains to grow in the presence of phenol by inoculating cultures (1% of an
overnight culture) in MRS broth with and without 0.4% phenol. Serial dilutions were
spread-plated (100 µl aliquots) onto MRS agar at time 0 and after 24 hours of incubation at
37°C to enumerate surviving bacteria.
Genotypic characterization
The 16S rDNA of the selected strains were amplified by PCR using P0 and P6
universal primers
corresponding to position 27f (forward) and 1495r (reverse) of
Escherichia coli 16S rDNA. The DNA extraction was conducted using the Qiagen Dneasy
Tissue kit (Qiagen, Hilden, Germany). One µl of cell lysate was added to 49µl of PCR
mixture containing 45µl of PCR supermix (Invitrogen srl, Milan, Italy) and 1 µl of each
primers (18 pmol/ml). The reaction mixtures, after incubation at 94°C for 1 min and 30
sec, were cycled through the following temperature profile: 5 cycles of 30 s at 95°C, 30s
at 60°C and 4 min at 72°C; 5 cycles of 30s at 95°C, 30s at 55°C and 4 min at 72°C; 25
cycles of 30s at 92°C, 30s at 50°C and 4 min at 72°C; one final cycle of 10 min at 72°C
and 10 min at 60°C. The PCR was conducted in a Tpersonal Thermal Cycler (Biometra,
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Gottingen, Germany). The PCR products were separated by electrophoresis in 2% agarose
gel containing 0.5 μg/ml (w/v) of ethidium bromide (Life Technologies, Italia Srl, San
Giuliano Milanese, Italy). The PCR products were purified using QIAquick PCR
Purification Kit (Qiagen), sequenced by MWG The Genomic Company (M-Medical,
Milan,
Italy)
and
aligned
on
GeneBank
(www.ncbi.nml.nih.gov/Web/Genebank/index.html) using BLAST algorithm.
Functional aspects of probiotics
In vitro adhesion assays
The adhesion of Lactobacillus strains was studied using HT-29 intestinal epithelial cell
line (Adlerberth et al., 1996; Blum and Reniero, 2000). The HT29 cell-lines were grown
routinely in Dulbecco’s modified Eagle’s Medium (DMEM) 4500mg/ml glucose
supplemented with 2mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 10%
fetal bovine serum. For adhesion assays, monolayer of HT-29 cells were prepared on tissue
culture plates. After incubation at 37°C under 5% CO2 atmosphere for 24 h the HT-29 cell
cultures were washed twice with PBS and 10 ml of bacterial suspension at a concentration
of 108 cells/ml was applied to each plate. The plates were incubated at 37°C for 2 h
followed by washing three times with PBS to collect non-adhering bacteria. The adherent
bacteria were released by applying a solution of PBS and EDTA (0.2%) and resuspended
in 10 ml of saline solution. After a centrifugation for 5 min at 3000 rpm, the cells were
suspended in 5 ml of saline solution and a series of 10-fold dilution (10-1-10-5) was
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prepared. A given amount of each dilution (50 µl) was plated on to MRS agar (Oxoid) and
incubated anaerobically at 37°C for 24-48 h.
The adhesion percentage was calculated by comparing the number of adhered cells to
the total cells of the bacterial suspension used. Each adherence assay was conducted in
triplicate.
The adhesion assay was applied, using the same protocol, to a combination (1:1) of the
best strains selected after the screening tests.
Antimicrobial activity assay
Antimicrobial activity of the selected strains was tested against Escherichia coli ATCC
11775, Staphylococcus aureus ATCC 25923, Clostridium perfringens ATCC 13124,
Candida albicans ATCC 10261 and Streptococcus mutans ATCC 20523 using a
modification of the “deferred cross-streak” technique (Fang et al. 1996). Briefly, MRS
agar plates were streaked with the probiotic strain tested (106 CFU/ml) in the centre of the
plate covering a 1cm x 2cm area and then incubated anaerobically at 37°C until grown to
confluence. After incubation the probiotic growth was outlined and then removed. The
plate was incubated again over chloroform for 1 h to inactivate any remaining cells and air
dried for 45 min. The plate was then spread with 100 µl of potential pathogen tested at 107
CFU/ml and incubated at 37°C for 24 h. The inhibition activity of the probiotic strains was
evaluated measuring the zone of inhibition around probiotic growth.
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Antibiotic susceptibility testing
Antibiotic resistance patterns of the selected probiotic strains were studied by disk
diffusion method (Bauer et al., 1966) on MRS agar plates. A total of 12 antibiotic
substances were tested: ampicillin, amoxicillin, colistin sulphate, erythromycin,
gentamicin, kanamycin, neomicin, oxolinic acid, penicillin G, tetracycline, vancomycin,
rifampicin. All the antibiotic substances were from Oxoid. The agar plates were incubated
anaerobically for 24 h at 37°C. The diameters of inhibition zones were measured and the
results (average of 3 readings) were expressed as sensitive (S), resistant (R) and
intermediate (I) according to NCCLS standard (1997).
Plasmid profiles
The isolation of plasmid DNA from the selected bacterial strains and from
Escherichia coli ATCC 13706 as a positive control, was performed with a Qiagen Plasmid
Protocol Kit (Qiagen).
Technological aspects of probiotics
Oxygen tolerance of bacterial strains
We tested the ability of Lactobacillus strains to grow in the presence of oxygen by
inoculating cultures in MRS broth and incubating them at 37°C in aerobic conditions for
24 h. Viable bacteria was analysed by plate count on MRS in anaerobic conditions.
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Determination of the oxidative stress resistance
The oxidative stress resistance of Lactobacillus strains was determined. The
organisms were grown in MRS broth until the culture reached a density of 4 McFarland. A
volume of 2.5 ml of the broth was introduced into 50 ml of 0.4% MRS agar and mixed
well. A volume of 4 ml of inoculated 0.4% MRS agar was poured onto pre-prepared MRS
agar plates and allowed to solidify. A sterile paper disc (6 mm; Wathman International Ltd,
Maidstone, UK) was placed on each Petri dish and a volume of 10 l of 3% H2O2 was
dispensed onto the paper disc. The plates were incubated anaerobically at 37°C for 24 h
and the diameter of the inhibition zones around the paper discs were measured.
Heat resistance
After cultivation in MRS broth for 18 h (stationary phase), cell suspensions (ca. 109
CFU ml-1) of the Lactobacillus strains were heated at 60, 65 and 70°C for 15 min, cooled
and plated on MRS agar. Each experiment was repeated in triplicate, and the average and
standard deviations were calculated.
The following tests were applied only to the best strains selected taking into
account all the analysis performed up to now.
Growth curve of bacterial strains
To evaluate the kinetic of the growth of the selected bacterial strains, glass bottles
(300 ml) containing MRS broth were inoculated with 2% of either strains. The bottles were
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incubated aerobically and anaerobically at 37°C for 9 h. During incubation, samples were
withdrawn at regular time intervals to measure the optical density at 560 nm (OD560).
Growth of Lactobacillus strains in milk
To verify the ability of the selected Lactobacillus strains to grow in milk, which is
the main substrate used to prepare probiotic functional foods, the overnight cultures of the
strains were inoculated separately and together in skim milk (Oxoid) (5% inoculum size,
109 CFU/ml). The products were incubated aerobically at 37°C for 24 h. Survival and/or
growth of Lactobacillus strains and their combination was examined by plating on MRS
agar.
Survival of Lactobacillus strains in milk during cold storage
Ability of the selected Lactobacillus strains to acidify milk was studied by
inoculating three different commercial milk products with 10% bacterial supplement of
either strains. The milk products used were full-cream pasteurized homogenized milk,
partially skim pasteurized homogenized milk and high quality milk. The milk products
were fermented for 20.5 h at 37°C and the process was followed by measurement of pH.
The products were then stored at 4°C for 21 days and the viable bacteria were analysed by
plate count on MRS agar after inoculation, after fermentation process, and after 7, 14 and
21 days of storage.
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Survival of Lactobacillus strains in yoghurt during cold storage
To verify whether the selected Lactobacillus strains could be supplemented in
yoghurt products and could survive during cold storage, two different yoghurt were
produced: (I) yoghurt containing 0.1% fat inoculated with 1% of Lactobacillus strain; (II)
yoghurt height quality inoculated 1% of Lactobacillus strain. The yoghurts were stored at
4°C for 35 days. Viable bacteria were analysed after inoculation and once a week during
the storage period. The samples were plated on MRS agar supplemented with vancomycin
(Sigma-Aldrich Division, Milan, Italy) (30 g/ml) to prevent the growth of yoghurt starter
strains.
Survival of bacterial strains to lyophilization process
The microorganisms were grown in Brain Hearth Infusion (BHI) broth (Oxoid).
Flasks of BHI medium supplemented with Hemin, Vitamin K and blood were inoculated
with Lactobacillus strains in log phase and incubated at 37°C for 24 h. A little quantity of
sterilized partially skimmed milk was used to harvest the grown bacteria cells which were
subjected to the lyophilization process. Cells are first frozen at -196°C an then dried by
sublimation under high vacuum in a Edwards FreezeDryer Modulyo (England). Viability
of the probiotic strains was determined after the lyophilization and compared with the
concentration of probiotic recovered before the freeze drying to calculate the survival
percentage.
24
RAPD-PCR strain typing
To assess the genetic stability of selected Lactobacillus strains after their
incorporation in functional foods, strains typing was done using as a primer the M13
minisatellite core sequence (5’-GAG GGT GGC GGT TCT-3’). Reactions were carried out
in 25 l amplification mixtures with 12.5 l of 2X Master Mix (Fermentas), 0.5 l of
primer, 1 l of total DNA and 11 l of water. The reaction mixtures, after incubation at 94
°C for 2 min, were cycled through the following temperature profile: 94 °C for 60 s, 42°C
for 20 s and 72°C for 2 min. Final extension was carried out at 72°C for 10 min. The PCR
was conducted in a Tpersonal Thermal Cycler (Biometra, Göttingen, Germany).
Amplification products were separated on a 2% agarose gel, containing 0.5 g/ml (w7v) of
ethidium bromide (Life Technologies).
Prebiotic experiments
Bacterial strains, media, and substrates
Customized MRS medium (de Man et al., 1960), without glucose and
supplemented with 0.5 g liter–1 L-cysteine hydrochloride (VWR International, Darmstadt,
Germany), hereafter referred to as mMRS medium, was used as the fermentation medium
throughout this study. The pH of the medium was adjusted to 6.5 before sterilization (at
121°C and 2.1 x 105 Pa for 20 min). Inulin (Synergy 1) was used as the sole energy sources
(in concentrations of 10 and 20 g liter–1). Inulin was sterilized through membrane filtration
using Sartolab-P20 filters (pore size, 0.20 µm; Sartorius AG, Goettingen, Germany) and
added aseptically to the sterile mMRS medium. Solid medium was prepared by adding
25
1.5% (wt/vol) agar (Oxoid) to the broth. BENEO Synergy 1 was kindly provided by Orafti
N.V. (Tienen, Belgium).
Growth of lactobacilli on prebiotics. (i) Agar plate assays
A first screening of the growth of the studied Lactobacillus strains on inulin as
energy sources, was performed with an agar plate assay. mMRS agar medium containing
the appropriate energy source (1%, wt/vol) and 300 mg liter–1 bromocresol purple (SigmaAldrich Chemie Gmbh, Steinheim, Germany) as a color indicator was used. The
Lactobacillus strains were propagated twice in MRS broth (Oxoid), and cultures obtained
after 12 h of growth at 37°C were centrifuged (at 5,500 x g for 10 min). The pellet was
washed once with phosphate-buffered saline (0.8% NaCl, 0.02% KH2PO4, 0.115%
Na2HPO4 [pH 7.4]) and resuspended in phosphate-buffered saline, followed by spotting of
10 µl of this suspension on mMRS agar plates. The plates were incubated at 37°C for 48 h.
All incubations took place anaerobically in an anaerobic cabinet (Concept 400, Ruskinn
Technology Limited, Leeds, West Yorkshire, UK). Plates were checked for color changes
around the developing colonies. These experiments were performed in triplicate.
ii) Fermentation experiments
To confirm the growth of Lactobacillus strains that were positive for growth on
inulin-type fructans, small-scale fermentations in glass bottles (100 ml) containing mMRS
medium with inulin as the sole energy source (1%, wt/vol), were carried out in duplicate.
Therefore, lactobacilli were propagated twice in mMRS medium with glucose as the sole
26
energy source for 12 h. These precultures were inoculated (2%, vol/vol) in mMRS medium
containing the energy source to be studied. All incubations took place anaerobically at
37°C. During fermentation, samples were withdrawn at regular time intervals to measure
the optical density at 600 nm (OD600) and pH.
Probiotic foods
Probiotic products manufacture
Six different products were used as carriers for delivering probiotic bacterial strains:
1. yoghurt, provided by Centrale del Latte dell’Aquila;
2. ricotta cheese, provided by a local cheese factory (Caseificio Picenum);
3. mozzarella cheese, provided by Sabelli;
4. chocolate, provided by a local bakery;
5. chocolate mousse, provided by a local pastry making;
6. ice-cream, provided by a local ice-cream shop.
All the products were inoculated with the bacterial strains directly on production site
and after a careful analysis of the best method of inoculum for each specific product. The
selected bacterial strains were mixed in the same concentration and inoculated into the
food products to rich a final concentration of approximately 109 CFU/g. Each product was
tested with different concentrations of bacterial strains, separately and together, to reach
the best inoculum concentration that allowed to have a concentration of approximately 109
CFU/g in the final food product and during its shelf life (Fig. 4).
27
FOOD
PRODUCT
STUDY OF
INDUSTRIAL PROCESS
INOCULUM
TRIALS
APPLYING OF DIFFERENT
CONCENTRATIONS OF
STRAINS
BEST INOCULUM
METHOD CHOISE
BEST INOCULUM
CONCENTRATION CHOISE
VIABILITY TEST
AND SENSORY
ANALYSIS
PROBIOTIC FOOD
PRODUCT REALIZATION
Fig. 4. Steps to realize a probiotic food product.
Probiotic traceability
Design of PCR primers
We designed specific primers for the detection of the selected bacterial strains using
an alignment of LAB 16S rRNA gene sequences extracted from the GenBank database
(http://www.ncbi.nlm.nih.gov). The primers were synthesized by MWG-Biotech
(Ebersberg, Germany).
28
Quantitative real-time PCR
The quantitative real-time PCR procedure was used for the study of quantification
of bacteria in different kind of product. For the quantitative real-time PCR analyses 200
mg of the products were used for the DNA extraction by using the NucleoSpin food
(Macherey-Nagel, Germany). Samples (0.4 l) were analysed in 20 l amplification
reactions consisting of 9 l of Brilliant SYBER Green QPCR Master Mix (Stratagene), 0.4
l of each primer and 9.8 l of water. Thermal cycling for the quantification of
Lactobacillus species consisted of an initial cycle of 95°C for 10 min, 35 cycles of 94°C 30
s, 55 °C 30 s and 72°C 2 min. To determine the specificity of amplification, analysis of
product melting curve was performed after the last cycle of each amplification. Real-time
PCR was performed with the Mx3000p (Stratagene). Results of real-time PCR were
compared with those obtained by plate count on MRS agar.
In vivo studies
Recovery of Lactobacillus strains from human faeces after probiotic products intake
Ten healthy volunteers participated in this study. Eligible participants were of both
sexes and aged 24-65 years. Each subject signed an informed consent after he/she had been
made fully aware of the purpose of the study. For the present study we used different kind
of probiotic products and the subjects ingested daily one or more of this products
indifferently for a period of three months (intervention period). Faecal samples were
collected at the start and the end of intervention and at the end of follow-up (a period of 14
days). At each sampling, microbial analysis and reisolation of the strain were done as
29
follows. Faecal samples were suspended (1:10 wt/vol) in physiological solution and 10fold serially diluted and 100 l of appropriate dilutions was plated on Rogosa agar (Oxoid)
with or without 12 g ml-1 of vancomycin (Sigma-Aldrich). Vancomycin-resistant
lactobacilli were enumerated on Rogosa-vancomycin agar. Plates were incubated
anaerobically for 3 days at 37°C. Ten colonies randomly selected from countable agar
plates were isolated and checked for purity. DNA was extracted using the Qiagen kit and
analyzed using the RAPD technique.
30
RESULTS
Low pH and bile tolerance
The viable count of most bacterial strains decrease after 5 h in citrate buffer, pH 3.0
at 37°C (Table 2). Strains 1102, 216, 202, 203, 901, 401, 903, 904 and 1304 were inhibited
from the low pH. Inhibition of the test strains ranged between 36.9% and 99.9%. Strains
1303, 403, 404 and 907 remained almost unaffected by the low pH after 5 h. All the strains
that showed resistance to low pH grew well also in the presence of bile. No high variations
existed among the cultures with regard to their growth in the presence of bile salts. Results
showed that all the strains were, in general, resistant to all the tested concentrations of bile
salts.
Only the strains that showed a percentage of inhibition ranged from no inhibition to
99.9 were considered for the following tests.
31
Table 2. Survival of Lactobacillus strains in citrate buffer pH 3.0 at 37°C, as determined by viable
count.
Strains
115
117
202
203
204
216
303
319
401
403
404
901
902
903
904
907
1102
1303
1304
1305
Viable counta (log10 cfu ml-1)
0h
5h
9.71±0.23
8.64±0.32
9.42±0.04
6.37±0.17
7.98±0.45
0
7.98±0.65
0
8.05±0.57
0
8.91±0.40
0
9.25±0.08
7.96±0.04
9.71±0.12
9.50±0.47
8.42±0.21
0
9.25±0.39
9.23±0.07
9.71±0.08
9.96±0.36
8.86±0.28
0
7.65±0.51
6.07±0.03
9.56±0.03
3.23±1.12
8.08±0.17
0
10.67±0.21
12.50±0.54
9.15±0.38
2.75±0.51
9.08±0.12
9.11±0.35
8.67±0.08
0
9.76±0.23
7.79±0.81
% inhibitionb at 5 h
91.5
99.9
100
100
100
100
94.9
36.9
100
-c
100
97.4
>99.99
100
>99.99
100
98.9
a
Log mean counts of two trials (average ± s.d.)
% inhibition = [(cfu ml-1initial – cfu ml-1 final)/cfu ml-1 initial] x 100
c
no inhibition
b
Resistance to 0.4% phenol
Phenols may be formed in the gut by bacterial deamination of some aromatic amino
acids derived from dietary or endogenously produced proteins. Our results suggest a
different resistance for Lactobacillus strains tested. In general there are a good tolerance of
all the strains tested towards phenol even if the growth in the presence of phenol was lower
than in MRS broth without phenol. For strain 404 a decrease in viable count was observed
in the presence of phenol after 24 h (Table 3).
32
Table 3. Ability of Lactobacillus strains to grow in the presence of phenol 0.4% at 37°C
Strains
115
117
303
319
403
404
902
907
1303
1305
a
0h
7.95±0.06
8.17±0.16
8.09±0.04
7.87±0.10
7.07±0.03
7.36±0.12
5.16±0.09
7.06±0.04
7.55±0.06
8.00±0.07
Viable countsa (log10 cfu ml-1)
MRS broth
MRS broth + phenol 0.4%
24 h
Increaseb
0h
24 h
Increaseb
10.09±0.02
2.14
8.05±0.14 8.75±0.34
0.70
10.65±0.07
2.48
8.12±0.13 8.52±0.40
0.40
9.88±0.11
1.79
7.98±0.43 8.64±0.51
0.66
8.87±0.24
1.00
8.05±0.08 8.63±0.09
0.58
7.29±0.12
0.22
7.40±0.11 7.42±0.02
0.02
9.12±0.03
1.76
7.24±0.21 5.65±0.28
-1.59
7.83±0.15
2.67
5.71±0.21 7.13±0.40
1.42
8.92±0.27
1.86
7.05±0.32 8.08±0.38
1.03
9.27±0.17
1.72
7.48±0.23 8.00±0.05
0.52
10.03±0.19
2.03
8.05±0.10 8.96±0.28
0.91
Log mean counts of two trials (average ± s.d.)
Increase = log10(final population)-log10(initial population)
b
Genotypic characterization
The 16S rDNA of the bacterial strains that survived from the tests above described
were sequenced and they were identified by alignment (Table 4). The results showed that
there is a percentage of correlation between phenotypical and genotypic characterization of
only 40%.
Table 4. Comparison between phenotypical and genotypic characterization of Lactobacillus strains.
Bacterial
strains
115
117
303
319
403
404
902
907
1303
1305
Phenotypical characterization
Genotypic characterization
Lactobacillus paracasei
Lactobacillus curvatus
Lactobacillus paracasei
Lactobacillus plantarum
Lactobacillus rhamnosus
Lactobacillus salivarius
Lactobacillus brevis
Lactobacillus brevis
Lactobacillus paracasei
Lactobacillus spp.
Lactobacillus casei
Lactobacillus casei
Lactobacillus casei
Lactobacillus plantarum
Lactobacillus rhamnosus
Lactobacillus fermentum
Lactobacillus reuteri
Lactobacillus brevis
Lactobacillus paracasei
Lactobacillus casei
33
In vitro adhesion assays
The Lactobacillus strains were examined for their ability to adhere to human
intestinal cell line HT29. Results for adhesion tests were summarized in Figure 5. Strains
303, 403, 902 and 1303 expressed good in vitro adherence to human HT29 cell line. In
particular, L. rhmnosus 403 and L. paracasei 1303 exhibited an adhesion rate of 14.9% 
3.2 and of 4.7%  1.5 respectively which are higher than the adhesion rate of commercial
Lactobacillus strains belonging to the same species (Fig. 6).
Moreover, the adhesion assay applied to a combination (1:1) of the L. rhamnosus 403
and L. paracasei 1303, showed an increased adhesion on HT29 cells (Fig. 7).
20
18
16
Adhesion (%)
14
12
10
8
6
4
2
0
L. casei 115 L. casei 117 L. casei 303 L. plantarum
319
L.
L. fermentum L. reuteri 902 L. brevis 907 L. paracasei L. casei 1305
rhamnosus
404
1303
403
Bacterial strains
Figure 5. The adhesion percentages of Lactobacillus strains to human intestinal cell lines.
34
L. rhamnosus
20
18
16
14
L. paracasei
Adhesion (%)
12
10
8
6
4
2
0
L. rhamnosus
LC-705
Fyos®,
Nutricia
Lactobacillus
GG ATCC
7469
Lactophilus®,
Laboratoires
Lyocentre
L. rhamnosus BIO®, Danone
403
Actimel®,
Danone
Yacult®,Yacult
L. paracasei
1303
Figure 6. Comparison among the adhesion percentages of L. rhamnosus 403 and L. paracasei 1303 and
some commercial Lactobacillus strains to human intestinal cell lines.
The adhesion values of commercial strains are from Tuomola et al. (1998)
L. rhamnosus 403 - L.
paracasei 1303 mixture
*
L. rhamnosus 403
L. paracasei 1303
0
5
10
15
20
25
30
Adhesion, %
Fig. 7. Adhesion percentages of Lactobacillus paracasei 1303, Lactobacillus rhamnosus 403 and two-strains
mixture (1:1). Each value represents the mean  SD of three measurements.
* Significantly different from the adhesion of the single strain, P<0.05 (t-test)
35
Antimicrobial activity assay
The inhibitory activity of the Lactobacillus strains was ranked according to the size
of zones of inhibition against common human pathogens (Table 5). Weak antibacterial
activity was exhibited only by 907, 902 and 904 strains. No antibacterial activity was
shown by 1305 strain. Strains 403 and 1303 exhibited antibacterial activity against all the
tested pathogens. Strains 403 and 1303 exhibited a particularly enhanced antipathogenic
activity against Candida albicans (inhibition zone > 2.5 x 3 cm) (Figure 8).
Table 5. Degree of inhibition of tested potential human pathogens from Lactobacillus strains.
Bacterial
strains
115
117
303
319
403
404
902
907
1303
1305
E. coli
(ATCC 11775)
+++
+++
+++
NI
+++
NI
NI
+++
+
NI
Inhibition of growtha by
S. aureus
C. albicans
Cl. perfringens
Str. mutans
(ATCC 25923) (ATCC 10261) (ATCC 13124) (ATCC 20523 )
NI
NI
+++
NI
NI
NI
+++
+
+++
+++
+++
NI
++
++
+++
NI
++
++++
+++
+
NI
+++
NI
NI
NI
+++
NI
+
NI
NI
NI
NI
+++
++++
+++
+
NI
NI
NI
NI
a
+ zone of inhibition < 2x1.5 cm, ++ zone of inhibition < 2x2.5 cm, +++ zone of inhibition < 2.4x3 cm,
++++ zone of inhibition >2.5x3 cm, NI no inhibition
36
Figure 8. In vitro inhibition of Candida albicans with L. paracasei 1303 (left, zone of inhibition >2.5x3 cm)
and L. rhamnosus 403 (right, zone of inhibition >2.5x3 cm).
Antibiotic susceptibility testing
The Lactobacillus strains examined in this study and evaluated according to the
NCCLS standard (1997), were susceptible to most often prescribed antibiotics (Table 6).
All strains were found resistant to vancomycin, colistin sulfate, gentamicin (except strain
902), oxolinic acid, neomycin (except strains 404, 907 and 1303) and kanamycin and
susceptible to the other antibiotics tested.
37
Table 6. Antibiotic susceptibility test of Lactobacillus isolates from human faeces
Antibiotic
Ampicillin
Amoxicillin
Colistin sulphate
Erythromycin
Gentamicin
Kanamycin
Neomicin
Oxolinic acid
Penicillin G
Rifampicin
Tetracycline
Vancomycin
a
115
Sa
S
Ra
R
R
R
R
R
S
S
S
R
117
S
S
R
R
R
R
R
R
S
S
S
R
303
S
S
R
R
R
R
R
R
S
S
S
R
319
S
S
R
R
R
R
R
R
S
S
S
R
Bacterial strains
403
404
902
S
S
S
S
S
S
R
R
R
R
S
S
R
R
S
R
R
R
R
Ia
R
R
R
R
S
S
S
S
S
S
S
S
S
R
R
R
907
S
S
R
S
R
R
I
R
S
S
S
R
1303
S
S
R
S
R
R
I
R
S
S
S
R
1305
S
S
S
S
R
R
R
R
S
S
S
R
S: sensitive strain; I: strain of intermediate resistance; R: resistant strain (NCCLS standard, 1997)
Plasmid profiles
Analysis of plasmid profiles revealed that only strains 902 and 1305 contain
plasmids whereas a plasmid was isolated from E. coli (positive control).
Oxygen tolerance of bacterial strains
All tested bacteria strains showed a good resistance to oxygen and they grow well
in aerobic conditions even if more slowly than in anaerobic conditions.
38
Determination of the oxidative stress resistance
All the tested strains showed a good resistance to the oxidative stress (Fig. 9). In
particular, strains 319 and 1305 were the most susceptible to oxidative stress while strains
117, 403 and 1303 showed smaller inhibition zones in the H2O2 disc diffusion assay.
25
Lactobacillus strains
Diameter of the inhibition zone (mm)
20
15
10
5
0
115
117
303
319
403
404
902
907
1303
1305
Fig. 9. Comparison of oxidative stress resistance of Lactobacillus strains. Error bars show the SD of the
mean of six determination in two experiments.
Heat resistance
The results of heat resistance of Lactobacillus strains (Table 7) showed that strains
117, 303 and 403 are the only strains able to survive at 65°C after 15 min of incubation
39
even if the reduction in the number of strains 303 cells was considerably greater than the
reduction in the number of strains 117 and 403 at the same temperature and time
conditions. The Lactobacillus strains did not differ greatly in the ability to survive at 60°C
except strains 902 and 1303 which are not able to survive at this temperature. All strains
are killed after incubation at 70°C for 15 min.
Tab. 7. Survival of Lactobacillus strains after heating at 60°C, 65°C and 70°C
Bacterial
strains
115
117
303
319
403
404
902
907
1303
1305
Viable counta (log cfu/ml)
60°C
65°C
70°C
0
15’
0
15’
0
15’
7.65±0.05 7.84±0.13 7.56±0.05 0.00±0.00 7.53±0.08 0.00±0.00
8.67±0.08 8.53±0.02 7.98±0.10 7.3±0.08 7.87±.005 0.00±0.00
8.12±0.06 8.30±0.06 7.87±0.01 1.08±0.14 7.97±0.14 0.00±0.00
7.56±0.08 6.87±0.04 7.32±0.07 0.00±0.00 7.43±0.03 0.00±0.00
7.95±0.12 7.82±0.09 7.63±0.08 6.39±0.09 7.03±0.09 0.00±0.00
7.67±0.10 6.30±0.01 7.63±0.13 0.00±0.00 7.43±0.06 0.00±0.00
7.83±0.07 0.00±0.00 7.56±0.06 0.00±0.00 7.58±0.04 0.00±0.00
7.87±0.12 5.20±0.12 7.73±0.04 0.00±0.00 7.76±0.10 0.00±0.00
7.74±0.13 6.54±0.02 7.91±0.06 0.00±0.00 7.57±0.08 0.00±0.00
7.45±0.05 0.00±0.00 7.65±0.12 0.00±0.00 7.87±0.12 0.00±0.00
a
Values are means of triplicate ± standard deviation
Result of the screening:
On the basis of the tests carried up to now we selected two strains with the best
potential probiotic characteristics to be further investigate for others in vitro and in vivo
probiotic properties. The two selected strains were 403 and 1303 since they exhibited good
resistance to ph and bile salts, good phenol resistance, very good adhesion to human
intestinal cell line, good antimicrobial activity and antibiotic resistance, no plasmid
presence, good oxygen tolerance and resistance of oxidative stress. Furthermore the two
40
bacterial strains showed a different heat resistance which could allow to use them in
different industrial processes. The two bacterial strains were deposited in the culture
collection Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSMZ), the strains
403 DSM 16104 corresponding to Lactobacillus rhamnosus IMC501 and the strains 1303
DSM 16105 corresponding to Lactobacillus paracasei IMC502.
Growth curve of bacterial strains
By using the spectrophotometric method, the bacterial growth curve of strains
Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502 in an MRS liquid
medium was determined, keeping the optimal conditions of pH and temperature and
monitoring the growth under conditions of anaerobiosis as well as of aerobiosis for a time
of nine hours. Figure 10 reports the growth curves of the two bacterial strains.
Lactobacillus rhamnosus IMC 501 exhibits, in the exponential growth stage under
conditions of anaerobiosis, higher O.D. values until the seventh hour; thereafter, the
growth in aerobiosis has higher values. Lactobacillus paracasei IMC 502 exhibits, in the
exponential growth stage under conditions of anaerobiosis, higher O.D. values until the
eighth hour; thereafter, the growth in aerobiosis is found to be better.
41
Lactobacillus paracasei
IMC 502
2,2
2
Anaerobic conditions
1,8
Aerobic conditions
1,6
O.D. (560 nm)
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0
1
2
3
4
5
6
7
8
9
Time (h)
Fig. 10. Growth curve of L. rhamnosus and L. paracasei under aerobic and anaerobic conditions.
Growth of Lactobacillus rhamnosus and Lactobacillus paracasei in milk
Ability of L. rhamnosus IMC 501 and L. paracasei IMC 502 to grow in milk was
studied. L. rhamnosus IMC 501 showed a better growth rate then L. paracasei IMC 502 in
milk, both in aerobic and anaerobic conditions (Table 8). However the growth rate was
42
higher when the two bacterial strains were used in combination which means that the coinoculum of the two Lactobacillus strains is more effective for the probiotic use.
Table 8. Growth of L. rhamnosus IMC 501 and L. paracasei IMC 502 in milk, separately and together, under
aerobic and anaerobic conditions
Bacterial strains
Lactobacillus rhamnosus
Lactobacillus paracasei
L. rhamnosus + L. paracasei
Viable counts (log CFU/ml)
in aerobic conditions
Viable counts (log CFU/ml)
in anaerobic conditions
11.41 ± 0.32
8.87 ± 0.79
13.48 ± 0.59
13.27 ± 0.11
8.74 ± 0.83
13.50 ± 0.34
a
The values are means of triplicates ± standard deviations
Survival of Lactobacillus rhamnosus and Lactobacillus paracasei in milk during cold
storage
Changes in the pH of milk and the number of viable L. rhamnosus IMC 501 and L.
paracasei IMC 502 were followed during fermentation process and cold storage for 21
days at 4°C (Fig. 11). The strains were not able to acidify milk during the 20.5 h
fermentation and therefore they are not suitable to be used alone for production of
fermented milk products. However, both strains remained viable during the cold storage
period and the pH values of the milk products remained fairly constant.
43
High quality
milk
L.rhamnosus IMC 501
12
Full cream
milk
6,8
Partially skim
milk
6,7
6,6
10
High quality
milk pH
6,5
6,3
6,2
6
6,1
pH
log cfu/ml
Full cream
milk pH
6,4
8
Partially skim
milk pH
6
4
5,9
5,8
2
5,7
5,6
0
5,5
0
20,5
168
312
504
Time (h)
High quality
milk
L. paracasei IMC 502
12
Full cream
milk
6,8
Partially
skim milk
6,7
6,6
10
High quality
milk pH
6,5
6,4
Full cream
milk pH
6,3
6,2
6
6,1
pH
log cfu/ml
8
Partially
skim milk pH
6
4
5,9
5,8
2
5,7
5,6
0
5,5
0
20,5
168
312
504
Time (h)
Fig. 11. Viable L. rhamnosus and L. paracasei bacteria and pH changes during fermentation (20.5 h at 37°C)
and cold storage (21days at 4°C).
44
Survival of Lactobacillus rhamnosus and Lactobacillus paracasei in yoghurt during
cold storage
Viability of the strains in yoghurt made with commercial starter strains (L.
delbrueckii sp. bulgaricus and Streptococcus thermophilus) was tested with two different
products. L. rhamnosus IMC 501 numbers remained fairly constant during cold storage
period, ranging between 1x105 and 6.7x105 cfu/g in 0.1% fat yoghurt and between 6.1x105
and 7.8x105 cfu/g in high quality yoghurt. L. paracasei IMC 502 numbers increased with 1
log during the cold storage being at level of 8.85x106 cfu/g in 0.1% fat yoghurt and
1.16x107 cfu/g in high quality yoghurt after 35 days of cold storage (Fig. 12). The growth
and survival of the two Lactobacillus strains was not affected by the different fat contents
of the two yoghurt products tested.
45
7
0.1 fat yoghurt
high quality yoghurt
6
log cfu/g
5
4
3
a
2
1
0
0
7
14
21
35
Time (days)
0.1 fat yoghurt
8
high quality yoghurt
7
6
b
log cfu/g
5
4
3
2
1
0
0
7
14
21
35
Time (days)
Fig. 12. Viable L. rhamnosus (a) and L. paracasei (b) in 0.1 fat and in high quality yoghurt during cold
storage (35 days at 4°C).
Survival of bacterial strains to lyophilization process
The results of the lyophilization test showed that the two Lactobacillus strains did
not varied considerably in their ability to survive during lyophilization process. Both
46
strains showed a reduction that is more marked for L. rhamnosus IMC 501 (4 Log
reduction) than in L. paracasei IMC 502 (2 Log reduction).
Tab. 9. Viability of probiotic strains after lyophilization process
Viable counta (log cfu/g)
before lyophilization
14.49 ± 0.16
12.83 ± 0.31
Bacterial strains
L. rhamnosus IMC 501
L. paracasei IMC 502
Viable counta (log cfu/g)
after lyophilization
10.82 ± 0.23
10.85 ±0.27
a
The values are means of triplicates ± standard deviations
RAPD-PCR strain typing
Using RAPD-PCR the banding pattern of L. rhamnosus IMC 501 could be
distinguished from L. paracasei IMC 502. The RAPD profiles of the two Lactobacillus
strains are shown in Fig. 13.
M
IMC 502
IMC 501 IMC 501 IMC 502
Fig. 13. Agarose gel of the RAPD products from the L. rhamnosus IMC501 and L. paracasei IMC502 using
the primer M13. M, 100-bp DNA ladder.
47
Growth of lactobacilli on prebiotics. (i) Agar plate assays
Using the agar plate assay, Lactobacillus strains that fermented the prebiotic inulin
as the sole energy source, gave a yellow zone against a purple background, due to the
production of significant amounts of organic acids, while the nonfermenting strains did not
cause any colour change of the agar medium. Both Lactobacillus strains fermented inulin
but for L. rhamnosus IMC 501 only slight colour change was observed.
ii) Fermentation experiments
During small-scale fermentations (100 ml) both L. rhamnosus IMC 501 and L.
paracasei IMC 502 grew on inulin even if very slowly in respect to the growth in MRS
with glucose. However L. paracasei IMC 502 showed a better growth rate after 24 h of
fermentation and with a concentration of inulin of 10 mg/l than L. rhamnosus IMC 501
(Fig. 14). The acidification profile of both strains was different to that obtained during
fermentation of glucose in particular the ph decrease slowly.
48
L. rhamnosus IMC 501
MRS
MRS + inulin (10mg/l)
2,5
MRS + inulin (20mg/l)
O.D. (600 nm)
2
1,5
1
0,5
0
0
10
24
70
75
Time (h)
L. paracasei IMC 502
MRS
MRS + inulin (10mg/l)
MRS + inulin (20mg/l)
O.D. (600 nm)
2,5
2
1,5
1
0,5
0
0
10
24
70
75
Time (h)
Fig. 14. Fermentation of L. rhamnosus IMC 501 and L. paracasei IMC 502 in mMRS medium with 10 and
20 mg/l of inulin. The graphs are representative of the results of two experiments.
Design of PCR primers
The sequence of the two Lactobacillus strains is different within the V1 region of
the 16S rRNA gene (Fig.15). Polymerase chain reaction primers were designed (Table 10)
to this variable region which, when used in conjunction with primer Y2, enabled
amplification of a specific product of approximately 290 bp from each of the strains.
49
50
100
L. rhamnosus
GAGTTCTGAT TATTGAAAGG TGCTTGCATC TTGATTTAAT TTTGAACGAG TGGCGGACGG
L. paracasei
GAGTTCTCGT TGATGATCGG TGCTTGCACC GAGATTCAAC ATGGAACGAG TGGCGGACGG
Fig. 15. Alignment of the V1 region of the 16S rRNA genes of L. paracasei IMC 502 and L. rhamnosus IMC
501. The bases comprising the species-specific polymerase chain reaction primers (Table 10) are underlined.
Table 10. Polymerase chain reaction primers used in this study.
Name
Y2
Sequence
5’-CCCACTGCTGCCTCCCGTAGGAGT-3’
Lp
Lr
5’-CACCGAGATTCAACATGG-3’
5’-TGCATCTTGATTTAATTTTG-3’
Comment
Conserved 16S rRNA
(Young et al., 1991)
L. paracasei 16S
L, rhamnosus 16S
Quantitative real-time PCR
We realized different probiotic products using different technological methods to
obtain the best concentration of probiotic strains in the final product. We tested the
products to verify the concentration of probiotic strains after the inoculum and during the
storage period. We inoculated both bacterial strains at the same concentration (1:1) and we
monitored the total count of the two strains both with plate count and Real time PCR.
Results were confirmed by RAPD. The results of real-time PCR compared with the results
of plate counts showed that there was a statistically significant difference between the two
methods of quantification in all types of samples except for ricotta cheese, yoghurt and
milk chocolate at 0 days.
50
Tab. 11. Real-time PCR-based quantification method of L. rhamnosus and L. paracasei compared to
traditional plate count.
Type of sample
Ricotta cheese
- 0 daysb
- 6 daysc
Yoghurt
- 0 days
- 1 month
Ice cream
- 0 days
- 1 month
Mousse
- 0 days
- 1 month
Milk chocolate
- 0 days
- 8 months
Black chocolate
- 0 days
- 8 months
Mozzarella cheese
- 0 days
- 15 days
MRS count
(log CFU/g)a
Real time PCR
quantification
(log CFU/g)a
7.51±0.04
7.52±0.03
8.38±0.05
8.41±0.14*
8.16±0.11
8.10±0.07
8.42±0.22
8.50±0.12*
8.46±0.02
8.60±0.05
10.69±0.03*
9.65±0.09*
9.52±0.01
9.30±0.08
9.87±0.15*
9.74±0.08*
8.80±0.02
8.82±0.06
9.37±0.06
9.41±0.11*
8.74±0.04
8.73±0.06
9.13±0.01*
9.11±0.05*
7.89±0.11
7.75±0.08
8.76±0.04*
8.57±0.07*
a
The values are means of triplicates ± standard deviation
day of production and inoculum of product
c
expire date of specific product
*Significantly different from MRS count (p<0.05, Student t test)
b
Recovery of L. paracasei and L. rhamnosus from human faeces after probiotic
products intake
This test was carried out to validate the food products as carriers for transporting
bacterial cells into the human gastrointestinal tract. This was demonstrated by the recovery
of L. rhamnosus IMC 501 and L. paracasei IMC 502 from 10 out of 10 faecal samples
(collected at the end of the consumption period) (Table 12) while the strains were not
detected in any of the faeces samples obtained before the consumption of probiotic food
products. The strains were also still identified in 9 volunteers at the end of follow-up (day
51
104) (Table 12). The RAPD profiles obtained from colonies isolated from the faecal
samples of volunteer C at day 104 are shown in Fig. 16: the profile reported in lanes
5,6,8,9,12,13 were identical to the pattern obtained from L. paracasei IMC502, used as
positive control (lane 2).
52
Table 12. Total vancomycin-resistant Lactobacillus count in the faeces of ten healthy volunteers fed L.
rhamnosus IMC 501 and L. paracasei IMC 502-containing food products and recovery of the strains
identified by PCR.
Day
of
samplinga
Subject
Lactobacillus spp.
(CFU/g)
0
A
B
C
D
E
F
G
H
I
L
90
104
a
3.2 x 105
1.4 x 103
2.5 x 104
1.7 x 105
3.9 x 104
1.2 x 103
6.7 x 103
1.5 x 104
2.1 x 105
1.9 x 105
No. of L. rhamnosus
IMC501
colonies/no.
analyzed
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
No. of L. paracasei
IMC502
colonies/no.
analyzed
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
A
B
C
D
E
F
G
H
I
L
1.7 x 107
1.2 x 105
3.1 x 106
5.7 x 105
2.0 x 105
7.0 x 104
1.1 x 105
2.3 x 106
4.6 x 105
1.3 x 105
10/10
0/10
0/10
2/10
4/10
3/10
6/10
7/10
0/10
2/10
0/10
8/10
7/10
0/10
4/10
5/10
0/10
1/10
8/10
5/10
A
B
C
D
E
F
G
H
I
L
1.04 x 106
1.30 x 103
2.81 x 105
1.30 x 104
1.29 x 104
2.20 x 103
6.90 x 104
1.73 x 105
5.45 x 105
3.31 x 104
9/10
0/10
0/10
0/10
3/10
1/10
5/10
7/10
0/10
3/10
0/10
6/10
6/10
0/10
6/10
3/10
0/10
0/10
6/10
5/10
Day 0, before consumption; day 90, end of consumption; day 104, 2 weeks after end of consumption.
53
Isolates from faeces
IMC IMC
502 501
1
2
3
4
5
6
7
8
9
10
11
12 13
Fig. 16. Detection of Lactobacillus paracasei IMC502 by RAPD. Lane 1, 100-bp DNA ladder; lane 2,
reference strain L. paracsei IMC502; lane 3, reference strain L. rhamnosus IMC501; lanes 4 to 13, strains
from faecal samples of subject C at day 104: lane 5-6-8-9-12-13, strain IMC502; and lanes 4-7-10-11, other
strains.
54
DISCUSSION
Despite the difficulties encountered in reliably characterizing probiotic strains
using in vitro methods, the initial screening of strains in this manner remains a useful
preliminary step in the detection of probiotic candidates.
Among the screened bacterial strains isolated from human faeces, L. rhamnosus
IMC 501 and L. paracasei IMC 502 showed good probiotic characteristics.
They survived under low pH conditions for five hours and they tolerated well bile
acids under in vitro conditions even at concentrations higher than those previously used by
other authors (Jacobsen et al., 1999; Fernández et al., 2003). Acid tolerance of bacteria is
an important factor as well as to assure their resistance of gastric stresses also for their use
as dietary adjuncts in acid foods such as yoghurt. Bile resistance is also important for an
organism that is expected to grow in the intestinal tract. In addition, bile resistance is an
important characteristic to consider in selection of a culture as a dietary adjunct. The
necessary degree of bile tolerance is not known, but it seems reasonable that the most bileresistant cultures that also possess other desiderable characteristics should be selected.
Adhesion and colonisation of probiotic bacteria in the gastrointestinal tract of the host is
believed to be one of the essential features required for the delivery of their health benefits
(Bernet-Carnard et al., 1997). It is known that good adhesion of probiotic microorganism
to the intestinal cells is related to many beneficial effects. In fact the adhesion is a
prerequisite for colonisation (Alander et al., 1999), stimulation of the immune system
(Schiffrin et al., 1995) and for antagonistic activity against enteropathogens (Coconnier et
al., 1993). L. rhamnosus IMC 501 and L. paracasei IMC 502 expressed higher values of in
55
vitro adhesion to HT29 cell line than other commercial strains belonging to the same
species (Tuomola et al., 1998). If this result is directly comparable with the in vivo
situation, a smaller quantity of the two Lactobacillus strains need to be consumed to have
the same number of organisms adhere to the intestinal epithelium as obtained with
Lactobacillus strains isolated from commercial products. The property of the probiotic
microrganisms to adhere to intestinal mucosa is of a great relevance because it is related to
many of the health effects. Combination of the two probiotic strains may have synergistic
adhesion effects. The property of co-adhesion, having an improved adhesion effect, is a
peculiar characteristic of the strain, Lactobacillus paracasei IMC502 and Lactobacillus
rhamnosus IMC501 in our case. In fact, as reported in literature (Ouwehand et al., 2000;
Azuma and Sato, 2001; Collado et al., 2007), not all probiotic bacteria, with good
individual property of adhesion, are able to improve this characteristic in combination with
other probiotic microrganisms.
An important aspect of the function of probiotic bacteria is the protection of the
host gastrointestinal micro-environment from invading pathogens. It is generally believed
that the resident gastrointestinal microflora in vivo provides protection for the host against
possible colonisation by pathogenic bacteria (Reid et al., 1990). Several reports have been
documented on the ability of probiotic lactobacilli and bifidobacteria to inhibit the cell
association and invasion by pathogenic bacteria (Chan et al., 1985; Hudault et al., 1997).
Lactic acid bacteria have been shown to inhibit the in vitro growth of many enteric
pathogens and have been used in both human and animals to treat gastrointestinal disorders
(Rolfe, 2000). L. rhamnosus IMC 501 and L. paracasei IMC 502 have been shown in vitro
56
to strongly inhibit some of the usual potentially harmful microrganisms and also
particularly evident was the effect against Candida albicans. For this reason the two
bacterial strains could be considered promising also in improving the response to Candida
infections. Further research will investigate the nature of the antimicrobial agents produced
by the bacteria.
The two bacterial strains were tested for antibiotic susceptibilities and for the
presence of plasmids to exclude the possibility that they may carry potentially
transmissible plasmid-encoded antibiotic resistance genes, as shown for example in some
Lactobacillus strains (Ahn et al., 1992; Tannock et al., 1994; Fons et al., 1997). It is
important to underline that the antibiotic resistance of some probiotic strains could be
beneficial for people with an unbalanced intestinal microflora due to the administration of
various antimicrobial agents (Salminen et al., 1998). At the same time, the presence of
antibiotic resistance plasmids is considered a factor excluding the use of the strain as
probiotics (Salminen et al., 1998). Among antibiotic resistances, vancomycin resistance is
of major concern because vancomycin is sometimes the only available antibiotic left, that
is effective against strains of microorganisms that have multiple resistance to antibiotics
(Woodford et al., 1995). The antibiotic susceptibility tests indicated that both strains were
resistant to vancomycin. The results were as expected as lactobacilli are known to be
naturally resistant toward vancomycin (Klein et al., 2000) and such resistance is usually
intrinsic, chromosomally encoded and no transmissible (Klein et al., 1998).
Moreover, our results indicates the absence of plasmids in both tested strains. The
presence of plasmid DNA is also assessed during this preliminary stage in order to obtain
57
information on the genomic stability of the strain. As a general rule, the presence of
plasmids is not a reason to discard the strain as a potential probiotic, but the role of this
extrachromosomal DNA in estabilishing phenotypes relevant for technological and
probiotic properties must be assessed.
Lactobacilli are anaerobic and under aerobic conditions highly toxic reactive
oxygen intermediates such as superoxides, hydroxy radicals and peroxides are formed
within the cells (Talwalkar and Kailasapathy, 2004). Exposure to oxygen may occur during
processing and after opening pots before consumption. Oxidative stress resistance is a
mesure of the ability of bacteria to survive such conditions. In this study, L. rhamnosus
IMC 501 and L. paracasei IMC 502 showed generally higher oxidative stress resistance
that the others Lactobacillus strains demonstrating its technological superiority.
The present study indicates that L. rhamnosus IMC 501 and L. paracasei IMC 502
possess a number of interesting properties that constitute the basis for their use as healthpromoting bacteria in functional foods.
The two strains have been deposited in the culture collection Deutsche Sammlung
von Mikroorganismen und Zelkulturen (DSMZ), the numbers DSM 16104 and DSM
16105 and they are Italian patent n. RM2004A000166.
The test to screening the ability of the two Lactobacillus strains to ferment inulin
showed that both strains were able to ferment inulin even if L. paracasei IMC 502 showed
a better growth rate. In the Western diet, the daily per capita intake of oligofructose and
inulin has been estimated to range from 1 to 10 g (Van Loo et al., 1995). It has been
demonstrated that the average recovery of such prebiotics in the colon varies between 86
58
and 89% of the material fed (Cummings et al., 2001). Thus, the degradation ability of L.
paracasei IMC 502 could be an important advantage for this strain to survive in the
competitive environment of the colon.
Probiotic bacteria are often selected on the basis of human intestinal origin and
their potential health associated properties. However the effects of these strains on the
overall nature of the final products in which they are supposed to be consumed are
important. Not surprisingly, strains of human intestinal origin often have poor survival and
negative effects on the characteristics of final products. To successfully commercialize a
probiotic product, good sensory properties is as important as the health properties. For the
application of probiotic cultures, two main objectives need to be fulfilled, i.e. a good
viability of probiotic and the organoleptic quality of the end product. For food product an
acceptable level of probiotic bacteria is >106 cfu/ml (Cummings et al., 2001). These limits
are easily achieved in products with L. rhamnosus IMC501 and L. paracasei IMC502 since
their survival during processing, storage and shelf life of the products are high. Moreover,
in some of the products tested, like mozzarella cheese and chocolate mousse, the probiotic
extend the shelf life increasing the product preservation. In particular, in chocolate mousse
(as control) we detected yeasts after 7 days of storage while in probiotic chocolate mousse
we didn’t detect yeasts during the same storage period and until the end of storage (data
not shown). This finding is very important since it indicates that the presence of probiotic
in chocolate mousse avoid the use of preservative or biopreservative in the product.
Many probiotic bacteria have a negative effect on the taste of various food
products. This make it necessary to use special aromas, flavours, and/or fruit preparations
59
trying to mask the off-flavors. A big advantage of L. rhamnosus IMC501 and L. paracasei
IMC502 is their ability to don’t modify the organoleptic parameters of the tested products
making it possible to be used both with or without any added flavours or fruit preparation.
Our results demonstrate that the tested products are suitable substrates for ensuring a
regular consumption of probiotics in foods that are part of a daily diet.
Although still a matter of debate, it is assumed that a minimal concentration of 106
CFU per ml or g of product is needed for probiotic bacteria to exert a health-promoting
effect. Consequently, the correct enumeration of probiotic bacteria in commercial products,
on a routine basis is indispensable in the process of delivering a functional product. We
used two different method for the quantification of probiotic lactobacilli in foods.
Traditional plate count was compared with real-time PCR. Quantitative real-time PCR is
based on quantification of bacterial DNA. It is generally accepted that the DNA levels are
not associated with viability, as dead cells may also retain significant amounts of DNA.
Our results showed that Real-time PCR quantification gave usually higher values in terms
of 16S rDNA than CFU. This discrepancies could be related to the multiplicity of 16S
rRNA gene copies. Although frequently used as target molecule for many bacteria, it is
well known that in many bacteria, the 16S rRNA gene can be presented in multiple copies,
possibly resulting in an overestimation of the number of bacteria in a product sample
(Masco et al., 2007). Another reason for real-time PCR overestimation could be related to
the amplification of DNA from dead cells. However, as indicated by Lahtinen et al. (2006),
although the bacteria are not readily culturable, they are not necessarily dead. Since the
clinical impact of dormant probiotic cells has not been determined, such studies are
60
urgently needed for assessing the efficacy of probiotics in food products during storage.
However, the results obtained indicate that both methods are suitable for the quantification
of total cells at the beginning and at the end of the storage of the products.
In order to confirm intestinal transit survival in humans, analysis of faecal samples
was conducted prior and following probiotic ingestion. The results on recovery of L.
rhamnosus IMC 501 and L. paracasei IMC 502 from human faeces after different food
products intake showed that both strains were recovered from the faecal samples of the
volunteers even if in different proportion for each volunteer. This could indicate that the
probiotic strains colonization is host specific and confirm the high adhesion ability of the
two bacterial strains and their persistence also for two weeks after the treatment. Included
in human trial were observation monitoring of potential side-effects of probiotic
consumption. These included intestinal discomfort, increased flatulence, and changes in
stool consistency and frequency. No adverse effects of probiotic administration were
observed in the pilot study. The probiotics were well tolerated by all individuals and the
fact that no side-effects were detected in subjects ranging in age from 24 to 65 suggests
that L. rhamnosus IMC 501 and L. paracasei IMC 502 are safe microbial food
supplements. Adding weight to this argument are the strain’s origin from the intestinal tract
of a healthy human.
L. rhamnosus IMC 501 and L. paracasei IMC 502 are indigenous to the intestinal
tract of a portion of the population in Italy (Silvi et al., 2003). This, combined with the
absence of deleterious effects during the human feeding trials, suggests that the strains are
safe for use as a human probiotic. The way forward is open for further clinical testing of
61
these strains to assess their efficacy in contributing to improved human health. As for all
probiotics, identifying health benefits stemming from ingestion of L. rhamnosus IMC 501
and L. paracasei IMC 502 against specific intestinal disorders and determining their
mechanisms of action are the pending research challenges.
62
CONCLUSIONS
This study demonstrate that Lact. rhamnosus IMC501 and Lact. paracasei IMC
502 may be used for production of several types of functional probiotic food products with
desirable technological properties. These cultures may contribute to increased protection
against infection and safety of product that occasionally contain potentially pathogenic
bacteria. Actually the two Lactobacillus strains showed adequate properties for probiotic
applications. This contention is based on (i) in vitro evaluation of technological and
organoleptic properties, adhesion competence, resistance to abiotic stress; (ii) in vivo
physiological tests, including intestinal survival of L. rhamnosus IMC 501 and L.
paracasei IMC 502. We have shown that the two Lactobacillus strains survive passage
through the gastrointestinal tract and that a regular consumption of probiotic food products
containing our bacterial strains is able to maintain the probiotic strains at a physiologically
significant level. Indeed, in 9 of 10 volunteers L. rhamnosus IMC 501 and L. paracasei
IMC 502 survived while passing through the gut at a high population level for at least 14
days after cessation of probiotic foods consumption.
63
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