Probiotic Strains Influence on Infant Microbiota in the In Vitro Colonic Fermentation Model GIS1 Veronica Ionela Moroeanu1•Emanuel Vamanu2•Gabriela… [600159]

ORIGINAL ARTICLE
Probiotic Strains Influence on Infant Microbiota in the In Vitro
Colonic Fermentation Model GIS1
Veronica Ionela Moroeanu1•Emanuel Vamanu2•Gabriela Paun1•
Elena Neagu1•Oana Rodica Ungureanu1•Sandra A. V. Eremia1•
Gabriel-Lucian Radu1•Robertina Ionescu3•Diana Roxana Pelinescu3
Received: 11 May 2015 / Accepted: 17 July 2015
/C211Association of Microbiologists of India 2015
Abstract The main goal of our study was to evaluate
the effect of the individual administration of five lyo-
philized lactic acid bacteria strains ( Lactobacillus fer-
mentum 428ST, Lactobacillus rhamnosus E4.2,
Lactobacillus plantarum FCA3, Lactobacillus sp. 34.1,
Weissella parame senteroides FT1a) against the in vitro
simulated microbiota of the human colon using the GIS1
system. The influence on the metabolic activity was also
assessed by quantitative de termination of proteins and
polysaccharides at each segment of human colon. The
obtained results indicated t hat the lactic acid bacteria L.
rhamnosus E4.2 and W. paramesenteroides FTa1 had
better efficiency in synthesisi ng exopolysaccharides and
also a better probiotic pote ntial and therefore could be
recommended for use in probiotics products or food
industry.
Keywords Lactic acid bacteria /C1Probiotics /C1Prebiotics /C1
Fermentation biotechnologyIntroduction
The human gastrointestinal tract (GIT) represents the
densest, complex and diverse community of microorgan-
isms. The complex found inside the human gut is an
ecosystem where the microbiota, the nutrients and the host
cells interact intensively. Many researchers have detailed
the positive interactions between the commensal micro-
biota and the human body, considering the microbiota as a
powerful partner for a good health [ 1].
The very complex microbiota is known as an active
element of the colon physiology having numerous func-
tions, the most important being: trophic functions, meta-
bolic functions and ‘‘barrier effect’’. The gut microbiota is
critical to human health, a microbial imbalance called
dysbiosis leading to the apparition of diseases; however, it
is not always easy to determine whether the changes in the
gut microbiota are a cause or a consequence of a disease.
The relationship between the microbiota and the health
condition is of great importance for the positive modu-
lation of the gut flora by administering live bacteria
(probiotics) or non-digestible compounds (prebiotics) to
prevent and sometimes to cure several diseases associated
to dysbiosis [ 2].
The Food and Agriculture Organization of the United
Nations (FAO) defined probiotics as ‘‘live microorganisms,
that, when administered in adequate amounts, confer a
health benefit on the host’’ [ 3,4]. Probiotics exert several
beneficial effects on human health by stimulating the
immune system, producing antimicrobial substances,
improving the barrier function of the intestinal mucosa and
competing with pathogenic bacteria to receptors on
epithelial cells [ 5].
Probiotics formed from microorganisms belonging to
the genera Lactobacillus (L.) and Bifidobacterium (B.) are a
&Emanuel Vamanu
[anonimizat]
1Centre of Bioanalysis, National Institute of Research and
Development for Biological Sciences, 296 Splaiul
Independentei, 060031 Bucharest, Romania
2Department of Industrial Biotechnology, Faculty of
Biotechnology, University of Agronomical Sciences and
Veterinary Medicine Bucharest, 59 Marasti blvd, 1 district,
011464 Bucharest, Romania
3Faculty of Biology, MICROGEN – Center for Research in
Microbiology, Genetics and Biotechnology, University of
Bucharest, 1-3 Portocalilor Str., 5 district, 060101 Bucharest,
Romania
123Indian J Microbiol
DOI 10.1007/s12088-015-0542-8

group of functional products that are intended to be con-
sumed by larger groups of population. Viability, the main
characteristic of probiotics, is determined by their ability to
survive in a large number after the transit through thestomach (acid pH) and the small intestine (enzymes and
bile acids), respectively. Moreover, the capability to
release substances having antimicrobial effect and to formbiofilms is extremely important as they can increase the
persistence in the colon segments [ 6].
Lactic acid bacteria (LAB) producing exopolysaccha-
rides have gained considerable attention in the dairy
industry due to their thickening properties that contribute tothe improvement of the texture and appearance of the dairy
products obtained by fermentation (yogurt, cheese).
Moreover, these exopolysaccharides have beneficial effectson human health by lowering cholesterol assimilation, by
their immunostimulatory effects and prebiotic effects on
the intestinal microflora [ 7,8]. Ruas-Madiedo and De Los
Reyes-Gavila ´n[9] have correctly noted that the ability to
produce exopolysaccharides is widespread among LAB.
Therefore there is need to better understand their physio-logical role and to search new strains able to produce
exopolysaccharides.
The viability of these strains during the industrial
manufacturing process and also during the shelf life of food
must be carefully examined, at the end the total number of
viable bacteria should be 10
5CFU/g. Functional food has
to meet a series of sensory characteristics when compared
to a lyophilized product. Accordingly, it is expected that a
probiotic product composed by one or more bacterialstrains meet all these requirements, naturally increasing the
degree of consumer acceptability. Therefore viability is
also influenced by the interactions between strains that insome cases may cause additional losses [ 10,11].
The aim of the present work was to study the effect of
administering several lyophilized lactic acid bacteriastrains on the GIS1 system used for in vitro simulation of
the colon. The analysed strains were previously selected for
their probiotic potential by in vitro assays involvingantimicrobial activity, adherence to eukaryotic cell lines,
immunomodulatory activity and resistance to different pH
values and bile salts concentrations (unpublished data). Thecomparative study aimed to determine the strain(s) that
have a significant positive influence on the number of
beneficial strains (especially the number of LAB) corre-lated to a decrease in potentially pathogenic strains. The
influence on the metabolic activity was also monitored at
each segment of the human colon. The studies were carriedout in parallel with the administration of the two lyophi-
lized control strains— Kluyveromyces marxianus and L.
casei 431.Materials and Methods
Microbial Strains
Five lactic acid bacteria strains were used for the tests: L.
fermentum 428ST, L. rhamnosus E4.2, L. plantarum FCA3,
Lactobacillus sp. 34.1, Weissella (W.)paramesenteroides
FT1a and the control yeast K. marxianus from the Faculty
of Biology collection, University of Bucharest, Romania.
For comparison, the strain L. casei 431 was isolated from
diary product—Probiotic Yogurt Drink Vivacto, Tesco-Hungary [ 12]. The bacterial strains were maintained in the
de Man Rogosa Sharpe (MRS) broth (Oxoid, Basingstoke,
Hampshire, England) at 37 /C176C. The yeast strain was
maintained in YPG broth at 37 /C176C. The cells were collected
by centrifugation at 5000 9gfor 10 min and washed three
times. The isolated biomass was freeze-dried in a Christ-Alpha 1-2 LD plus freeze dryer (Martin Christ
Gefriertrocknungsanlagen GmbH). After lyophilization all
the strains were introduced into hard gelatine capsules thatwere gastro-resistant in order to simulate as accurate as
possible the effect when in vivo administered. Each capsule
contained approximately 0.25 g of lyophilized biomass
having a minimum viability of 10
7CFU/g.
In Vitro Human Colon Simulation
The conditions in the colon were replicated in a single-
chamber system, GIS1, inoculated with 10 % ( wt:v) fecal
homogenate from a child (7 years old) in peptonate water
(control). After inoculation, the GIS1 was left for approx-imately 24 h to allow stabilisation. The system was oper-
ated for 20 h. The GIS1 system was described in previous
studies [ 12,13]. Each bacterial strain was individually
tested for better characterization.
Metabolic Activity AnalysisExopolysaccharides separation was performed by precipi-
tation with ethanol, according to the method presented byDimitrijevic ´et al. [ 14], while the quantification was per-
formed according to a recent work of authors [ 15]. Protein
concentration in the extracellular extracts of LAB wasdetermined using the Bradford method [ 16]. The cell free
solution was obtained by centrifuging the samples at
5000 rpm for 20 min followed by the filtration of thesupernatant through a 0.2 lm pore size PTFE syringe dri-
ven filter unit (Macherey–Nagel). The chemical reaction
with specific reactive and the absorbance of each samplewere quantified at 595 nm. The standard curve of protein
concentration was obtained using a series of dilutions ofIndian J Microbiol
123

the Bovine Serum Albumin (BSA) protein standard stock
solution.
Microbiological AnalysisThe analyses were made by serial dilution of the culture
sample in physiological saline solution (pH 7.0). The twohighest dilutions were then plated on specific media and
evaluated by an automated colony counter, Colony Quant,
with the corresponding software [ 12,13].
The total number of anaerobes was determined by using
Brain-Heart Infusion (BHI) agar, Mc. Conkey agar forcoliforms; Azide blood agar base for enterococci; Mannitol
salt phenol red agar for staphylococci; Tryptose sulfite
cycloserine agar for clostridia; Bifidus Selective Medium(BSM) agar for bifidobacteria; Rogosa agar for lactobacilli.
The media used were purchased from Sigma-Aldrich,
Germany.
Statistical Analysis
All the microbiological and biochemical experiments were
performed in triplicate, and the results were expressed as
mean ±SD values.
Results and Discussion
The probiotic effect of the newly isolated lactic acid bac-
teria strains depends on their ability to preserve viabilityafter the gastrointestinal transit and also on their influence
on the colonic microbiota. The persistence along the colon
segments depends on the dose and the way of administra-tion that further will directly affect the homeostasis of the
bacteria population at each level [ 17].
The five newly isolated lactic acid bacteria strains were
selected based on: antimicrobial activity against bacterial
strains belonging to Escherichia, Salmonella, Bacillus,
Staphylococcus, Klebsiella and Campylobacter genera;
adherence to eukaryotic cell lines (HCT8 and H29) and
competition with pathogenic microbial strains;
immunomodulatory activity; lack of cytotoxicity; resis-tance to different pH values and resistance to bile salts
concentrations (unpublished data).
Two groups and four bacteria genera were influenced by
the administration of probiotics and/or prebiotics (Table 1).
The main bacterial groups have shown good stability for all
the tested biomass. The yeast K. marxianus was an
exception as its administration resulted in an average
decrease of 0.5 log CFU/mL in the transverse and
descending colon segments.
A major change in the structure of the simulated colon
microbiota was observed when the strains L. fermentum428ST, L. rhamnosus E4.2 and L. plantarum FT1a were
administered. A decrease in the number of potentially
pathogenic strains and Bifidobacterium species was noticed
together with the stabilisation of the total number ofanaerobic microorganisms.
The L. fermentum 428ST strain distinguishes as it sta-
bilizes the bifidobacteria in the descending colon with anincrease of 0.36 log CFU/mL and it decreases the coliform
bacteria colonies in all the colon segments. The L. rham-
nosus E4.2 strain lowers the number of coliforms and
enterococci in the entire colon, while L. plantarum FT1a
strain acts against all pathogenic bacteria. To conclude, astabilisation was noticed between the determined bacteria.
The stability of the microbiota was determined for all
tested strains. The L. fermentum 428ST and L. plantarum
FCA3 strains cause a significant persistence of Bifidobac-
terium strains in the descending segment of the colon when
compared to the reference strain L. casei 431. In the case of
lyophilized strains, the decrease in bifidobacteria number
was explained as a balance of the number of beneficial
anaerobic strains, directly correlated to the number oflactobacilli. As the aim is to increase and stabilise the
number of favourable strains, the effective lyophilic com-
pound (e.g. L. fermentum 428ST) must have an antago-
nistic activity. This effect was also observed by the
decrease in the coliforms number, decrease that was not
achieved for the strains Lactobacillus sp. 34.1 and W.
paramesenteroides FT1a. Moreover, the L. casei 431 strain
has no antagonistic activity on strains from Clostridium
genus. These minor variations are comparable to thosedetermined in untreated microbiota (the first control).
The presence of the strains belonging to Clostridium
genus showed higher levels (over 5 log CFU/mL) whenlyophilized L. plantarum FCA3 was administered and
partially for L. fermentum 428ST (without the terminal
segment). The obtained results were comparable to thelyophilized yeast strain that was not able to reduce the
microbial species. When L. rhamnosus E4.2 and W.
paramesenteroides
FT1a were administered a significant
reduction (over 0.5 log CFU/mL) in the transverse section
of the colon was observed. For several strains it was
noticed a loss in the antagonist effect on this microbialgroup in the descending segment. A maximum increase of
0.5 log CFU/mL resulted for this section of the colon.
The amount of quantified exopolysaccharides for the
seven tested bacterial strains had variations between the
strains and randomly on the three sections of the colon
where their production was simulated (Fig. 1). In the
ascending colon, the strains L. rhamnosus E4.2 and W.
paramesenteroides FTa1 had significantly synthesised
higher amount of exopolysaccharides compared to control(without the inoculated strain) and reference strains, L
casei 431 andK. marxianus , respectively. The same trendIndian J Microbiol
123

Table 1 The effect of administration of lyophilised probiotic strains on the simulated colon microbiota
Sample Number of microorganisms (log CFU/mL)
Total anaerobes Total aerobes Enterococci Coliforms Clostridia Bifidobacteria
Control (without any strain administration)
Ascending colon 5.26 ±0.01 4.74 ±0.65 4.27 ±0.40 5.07 ±0.10 5.60 ±0.70 4.96 ±0.40
Transverse colon 5.64 ±0.35 4.93 ±0.70 4.57 ±0.01 5.87 ±0.60 5.83 ±0.80 5.29 ±0.60
Descending colon 5.74 ±1.05 4.68 ±0.95 4.73 ±2.30 5.97 ±1.20 5.81 ±1.35 5.05 ±1.25
Control— K. marxianus
Ascending colon 5.20 ±1.00 4.98 ±1.60 4.50 ±2.40 5.03 ±0.01 5.27 ±1.60 4.71 ±0.30
Transverse colon 4.99 ±2.60 4.76 ±1.00 4.76 ±1.20 4.88 ±0.10 5.01 ±0.55 4.79 ±1.00
Descending colon 4.56 ±0.30 4.72 ±0.50 4.72 ±2.20 5.82 ±1.40 5.69 ±0.70 4.56 ±1.00
Control— L. casei 431
Ascending colon 5.62 ±0.70 5.32 ±1.00 5.05 ±0.70 5.09 ±0.20 5.60 ±0.05 5.26 ±0.15
Transverse colon 5.05 ±0.50 4.96 ±0.01 4.74 ±0.01 4.94 ±0.00 4.89 ±0.10 5.02 ±0.30
Descending colon 4.92 ±0.75 4.81 ±0.20 4.60 ±1.00 4.70 ±0.04 4.75 ±0.01 4.86 ±0.55
L. plantarum FCA3
Ascending colon 5.01 ±0.20 5.34 ±0.10 4.67 ±0.01 5.87 ±0.30 5.37 ±0.40 4.81 ±0.02
Transverse colon 5.52 ±0.00 5.65 ±0.55 4.77 ±0.50 5.85 ±0.60 5.88 ±0.02 5.16 ±0.40
Descending colon 5.90 ±1.10 5.19 ±0.80 4.59 ±0.00 5.90 ±0.00 5.35 ±1.00 5.20 ±1.10
Lactobacillus sp. 34.1
Ascending colon 5.23 ±0.65 4.90 ±0.70 5.44 ±0.05 5.50 ±1.00 5.29 ±0.44 4.98 ±0.35
Transverse colon 5.63 ±0.44 4.93 ±0.06 5.54 ±0.40 5.69 ±1.25 4.90 ±0.25 4.88 ±0.50
Descending colon 5.90 ±0.01 5.57 ±0.68 5.23 ±0.35 5.58 ±1.05 5.47 ±0.45 5.23 ±0.00
L. fermentum 428ST
Ascending colon 4.95 ±1.02 5.00 ±1.40 4.63 ±0.70 4.68 ±0.48 5.55 ±0.35 4.71 ±0.01
Transverse colon 5.81 ±1.10 4.94 ±0.60 4.78 ±0.40 4.38 ±0.35 5.54 ±0.28 5.24 ±0.44
Descending colon 5.84 ±1.40 4.98 ±0.45 4.98 ±0.60 4.67 ±0.00 4.92 ±0.25 5.41 ±0.36
L. rhamnosus E4.2
Ascending colon 4.89 ±0.00 4.85 ±0.30 4.82 ±0.45 4.54 ±0.68 4.93 ±0.60 4.95 ±0.04
Transverse colon 4.69 ±0.05 4.68 ±0.00 4.47 ±0.03 4.36 ±0.48 4.47 ±0.00 4.62 ±0.05
Descending colon 4.64 ±0.01 4.77 ±0.36 4.83 ±0.00 4.36 ±0.56 4.88 ±0.00 4.30 ±0.00
W. paramesenteroides FT1a
Ascending colon 4.88 ±0.40 4.66 ±0.10 4.25 ±0.70 4.27 ±1.60 4.60 ±0.30 4.49 ±0.86
Transverse colon 4.92 ±0.08 4.79 ±0.37 4.20 ±1.30 4.55 ±0.76 4.34 ±0.00 4.70 ±1.25
Descending colon 4.89 ±0.00 4.65 ±0.45 4.44 ±1.46 4.20 ±1.40 4.81 ±0.70 4.59 ±0.35
Fig. 1 The amount of
polysaccharides from simulatedmedia (mg/L). DCdescending
colon, TCtransverse colon, AC
ascending colonIndian J Microbiol
123

was observed even for the transverse and descending colon.
Consequently, it could be concluded that L. rhamnosus
E4.2 and W. paramesenteroides FTa1 have better capacity
in the synthesis of exopolysaccharides together with a goodprobiotic effect such reduction of Clostridium cells num-
bers. Lactic acid bacteria exopolysaccharides are important
for probiotic effect due to their involvement in the adher-ence to human or animal eukaryotic cells and therefore
exclusion of pathogenic microbial cells, immunomodula-
tory and anticarcinogenic activity. During the last decades
exopolysaccharides were used in order to obtain prebiotic
and symbiotic products [ 18,19]. The capacity of the two
lactic acid strains to produce exopolysaccharides could be
important from medical point of view if the strains are used
as probiotics or for food industry.
The experimental results indicated higher quantities of
released extracellular proteins for the bacterial strains
Lactobacillus sp. 34.1, L. rhamnosus E4.2 and W.
paramesenteroides FT1a than for control for all the three
colon segments, but lower than the reference strain L. casei
431 (Fig. 2). The differences that appeared in the
exopolysaccharides amount obtained from Lactobacillus
sp. 34.1, L. rhamnosus E4.2 and W. paramesenteroides
FT1a compared to the amount obtained from L. casei 431
(strain isolated from Probiotic Yogurt Drink Vivacto) could
be the result of the higher capacity of the three strains to
biosynthesise exopolysaccharides under stress conditions(unpublished data). A possible explanation of the high
amount of extracellular proteins obtained for L. casei 431
could be explained by the higher number of the total viablemicrobial cells as shown in Table 1.
The extracellular proteins secreted by the lactic acid
bacteria (in vitro in the culture medium corresponding toin vivo release to the intestinal lumen) can diffuse through
the mucous layer covering the surface of the colon andinteract with the epithelial and immune cells of the host
organism. The role of LAB secreted proteins is not fully
understood-can increase mucus barrier, have immunomod-
ulating action, increase the absorption of electrolytesthrough the gastric mucosa. If the role of LAB secreted
proteins will be understood than the researchers will better
understand how probiotics act on the health of the hostorganism and then treat various inflammatory bowel dis-
eases, allergies and autoimmune diseases [ 5].
Using the in vitro system GIS1, the interactions between
human microbiota and epithelial cells or components of the
immune system could not been simulated [ 20]. These
technological limitations make necessary, at the end, an
in vivo study to validate the effects of administering a
dietary supplement that will contain lyophilized biomass.The effect of the individual administration of a LAB strain
on the composition of the microbiota that is in formation had
emphasised that the formulation of a competitive probioticproduct cannot be made unless associating microbiological
and biochemical effects of several LAB strains.
Due to the fact that the amount of lactobacilli in the
microbiota is considered to be approximately 1 % [ 21], the
increase in anaerobic microorganism number can be con-
sidered as an exceeding of this percentage coming fromlyophilized compounds administration (Table 1). In terms
of persistence in the colon sections, the exopolysaccharides
synthesis adds to other physiological phenomena that willfavour certain selected strains. These can cause an increase
in the intake of nutrients, but also provides a decrease in
cellular density (Fig. 1). The strains W. paramesenteroides
FT1a and L. rhamnosus E4.2, which are in this situation,
have caused the isolation of significant amounts of
exopolysaccharides in the colon segments, this beingexplained as an improved persistence at the level of
descending colon. This metabolic behaviour was noticed in
the case of gastrointestinal transit for the same strains(unpublished data) and is in opposition to previous studies
that have reported a higher number of favourable
microorganisms in the ascending colon [ 22]. Moreover, the
increase in the level of lactobacilli is correlated to the
decrease in the level of bifidobacteria, generating equilib-
rium between the strains that formed the tested microbiota.
In this scientific research, the reduction of Bifidobac-
terium number, after the administration of W. paramesen-
teroides FT1a and L. rhamnosus E4.2 was inversed with
the amount of exopolysaccharides. Other study demon-
strated the similar reduction of the viable cells related to
the inflammatory effect and proliferation of pathogenicstrains [ 23]. An increase in the metabolic activity could
determine a secondary synthesis of other compounds with
antimicrobial effects.
Stability of Bifidobacterium species is very important in
the microbiota composition as they could stabilize the
Fig. 2 The amount of proteins from simulated media (mg/L). DC
descending colon, TCtransverse colon, ACascending colonIndian J Microbiol
123

microbiota of the host [ 24] by several mechanisms (pro-
duction of inhibitory substances, competition with patho-
genic strains, metabolic activity, toxins inhibitory etc.).
These mechanisms could interact with the positive strainsfrom the normal microbiota and it is possible to determine
the disruption of equilibrium from the different strains.
Interaction can appear between probiotic strains andtherefore it is important to determine the incompatibility
after the ingestion of the end product.
The human colon has a direct influence on health due to
the significant fermentative activities that occur inside.
Therefore, it can be assumed that a negative change in thefermentative balance can cause nutritional dysfunction that
further can transform into obesity and/or diabetes [ 25]. The
viability and colonisation ability at colon level are influ-enced by a series of physico-chemical factors, the micro-
biota fermentative capacity playing a significant role. The
action mechanism is not completely understood, but itprimarily depends on the number of viable strains after the
passage through the stomach and small intestine. It is
possible that the low pH, the enzymes and the action ofbiliary acids to influence the probiotic activity [ 26].
An easy way to improve the health status at gastroin-
testinal tract level is to administer different lyophilizedcomplexes (consisting of compounds having prebiotic effect
and bacterial strains that have modulatory capacity when
applied to microbiota) to target groups of people [ 27].
Conclusions
The high level of beneficial microorganisms and the syn-
thesis of bioactive compounds have transformed the testedstrains into an important element in developing consortia
having probiotic properties for normalising the unbalanced
colon microbiota.
In vitro studies have addressed the role that every lyo-
philized strain has on the human microbiota. Moreover,
these preliminary studies have to be continued with in vivostudies that involve biochemical and molecular biology
analysis.
Acknowledgments This research was supported by the National
Centre for Program Management PN-II-PT-PCCA-2011-3.1-0969/
2012 coordinated by Romanian Agency for Scientific Research.
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