Open Journal of Veterinary Medicine, 2012, 2, 104-112
http://dx.doi.org/10.4236/ojvm.2012.23018 Published Online September 2012 (http://www.SciRP.org/journal/ojvm)
Production and B i o chemestry -Molecular Analysis of
Microbial Community Fermenting Whey as a Potential
Probiotic for Use Animals
Eduardo N. Esteban1, Mirentxu Indart2, Silvia Cerone2, G. de Yaniz2, Ana G. Inza2, Herminia Landi3,
Silvina Mogni2, Marcela Juliarena1, Leticia Igarza2*
1Department of A n i mal Health, Fa c u l ty of Veterinary Science, Universidad Nacional del Centro de la Provincia de Buenos Aires,
2Department of Physiology and Pathology, Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de
Buenos Aires, Tandil, Argentina
3Department of Animal Production, Faculty of Veterinary Science, Universidad Nacional del Centro de la Provincia de Buenos Aires,
Received May 29, 2012; revised June 28, 2012; accepted July 10, 2012
The aims of this work were: To achieve a simple and low cost propagation of potential probiotic agents using plain whey
as a culture medium, study the diversity of the members of the bacterial community (MC) in plain whey and to evaluate
the probiotic capacity of this MC. After a systematic selection of agents according to their growing capacity in whey, the
constituted MC was considered as a unit. Biochemical characterization of the lactic acid bacteria were performed using
the API system. Molecular characterization of the lactic acid bacteria was realized using AFLP™ DNA- fi ngerprinting,
partial 16S rDNA sequence analysis and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The physiological
characterization of yeast was determined with the automated microplate method Allev/Biolomics and using yeast charac-
terization system based on standard taxonomic criteria. The identification molecular was realized by PCR-fingerprinting.
The resistance of MC to pH and bile salts were evaluated. The MC was composed of agents from different separated
Dominium like Bacteria (Lactobacillum) and Eukaria (yeast). They are multispecies and also multistrain assuring high
biodiversity. The MC grew at low pH and different co ncentrations bile salts.
Keywords: Probiotic; Multispecies; Multistrain; Plain Whey
Whey is a cheese industry effluent and a powerful envi-
ronmental contaminant, in most cheese making regions
in Argentina. In recent years, the recovery and marketing
of whey components like proteins, minerals and lactose
has diminished the quantities of th is effluent discarded to
the environment . The fermentation of whey by dif-
ferent procedures as an alternative to produce single cell
protein, beverages, ethanol and methane has been com-
municated by various authors [2-4]. In our case the pro-
duction of a microbial community fermenting whey
could be utilized as potential probio tic in animals. A pro-
biotic is a “live microbial feed supplement which benefi-
cially affects the host animal by improving its intestinal
microbial balance” . Thus, using a probiotic means an
intervention on the intestinal ecosystem. Probiotic bacte-
ria would have antagonistic impact against intestinal in-
fectious bacteria just by competitive exclusion , mo du-
lating the intestinal medium, favoring growth of friendly
bacteria or producing natural antibiotics or bacteriocines
The aim of this work was: To conform a microbial
community of simple and low cost propagation with po-
tential probiotic agen ts using plain whey as a culture me-
dium, to analyze the species that constitute the micro bial
community (MC) by means isolating and identifying
strains through the application biochemistry and molecu-
lar techniques and to evaluate the probiotic capacity of
2. Materials and methods
Development of the microbial community: Fresh
pooled whey from cheese producing companies located
in Tandil, Argentina, was collected and transported to
destination in food grade stainless steel tanks. Average
opyright © 2012 SciRes. OJVM
E. N. ESTEBAN ET AL. 105
protein concentration was 3.5 ± 0.5 mg/ml and pH 5.7 ±
0.3. Immediately after arriving, whey was centrifuged up
to 2800 xg keeping a continuous flow of 4.167 l/min, at
room temperature. Fat free whey was sterilized by micro
filtering successively through 1 µ, 0.5 µ and 0.1 µ pore
size Koch membranes and was used for MC develop-
We examined whey fermentation capacity of some
Caucasian Kefir agents and then added local lactic acid
bacteria obtained from pooled whey in local cheese pro-
ducing plants. Criteria for the addition of new local
strains were as follows: The final pH had to be lower and
the biomass (g/l) had to be equal or larger than obtained
by Kefir agents. It was considered a satisfactory commu-
nity fermenting whey which consistently d rops pH down
to 3.6 ± 0.1 and biomass increases to 6 ± 0.5 g/l after 24
h culture. Cultures were incubated 24 h in stationary
conditions at 37˚C. Biomass was harvested by centrifu-
gation at 10˚C and 2800 xg keeping a continuous flow of
Freeze-drying: Compact biomass obtained by MC
centrifugation was suspended (1:2) in 10% (w/v) recon-
stituted skim milk and 5% (w/v) sodium glutamate (pH
was adjusted to 5 with 1M NaOH). Bacterial suspension
was freeze-drier. After the freeze-drying cycle has been
complete, dried MC was stored at 4˚C unde r va cuum.
Isolation and biochemical characterization of the lac-
tic acid bacteria were carried out by the Centro de refer-
encia de Lactobacillus, Tucumám, Argentina (CERELA).
Lactic acid bacteria isolation: A twenty grams sam-
ple of the compact biomass obtained by MC centrifuga-
tion were added to 180 ml of saline-2 peptone water (2%
of NaCl, 0.1% of bacteriological peptone and 1% of
Tween 80) and mixed for 3 min into a sterile stomacher
bag (Stomacher Lab-Blender 400, A. J. Seward Lab.,
Serial dilutions were made in 1‰ peptone water and
plated on MRS  agar, MRS pH 5.4, LAPTg agar ,
KF agar and ST medium, all supplemented with cyclo-
heximide (25 µg/ml) to suppress yeast growth. Plates
were incubated at 30˚C and 37˚C anaerobically for 6
days. Isolated colonies that differed microscopically were
randomly selected. The isolates were propagated in MRS
broth and purified. Gram positive rods and negative to
catalase reaction were selected for further studies as pre-
sumptive Lactobacillus species.
Biochemical characterization: Bacteria were first
clustered on the basis of cellular morphology, growth at
15˚C, 30˚C, 37˚C and 45˚C, 0.1% and 0.3% methylene
blue, nitrate reduction, indol production, ammonia pro-
duction from arginina, esculin hydrolysis, CO2 production
from glucose and gluconate, ability to form diacetyl from
citrate. Carbohydrate fermentation tests of selected lacto-
bacilli were performed using the API 50CH system (bio-
Merieux, Marcyl’ Etoile France) according to the manu-
facturer instructions. Tests preparations were incubated at
37˚C and final readings were made after 48 h. Fermenta-
tion profiles were analyzed by APILAB Plus version 4.0
program (bioMerieux, Ma rcy l’ Etoile France).
Molecular characterization of the lactic acid bacte-
ria: AFLP™ DNA-fingerprinting, partial 16S rDNA se-
quence analysis and PCR-denaturing gradient gel elec-
trophoresis (PCR-DGGE) were carried out by the BCCM/
LMG Bacteria Collection (Ghent University. Belgium).
AFLP™ (The AFLP™ technology is subject to patents and
patent applications and AFLP is a registered trademark,
all owned by Keygene N. V., the Netherlands) is a PCR
based technique for whole genome DNA fingerprinting
via the selective amplification of restriction fragments
Five lyophilized individual specie biochemically char-
acterized and 2 lyophilized samples of the whole MC,
obtained 4 months apart, were sent to the BCCM/LMG.
The twelve lyophilized cultures were recovered and
checked for purity on MRS (Oxoid CM361) after an-
aerobic incubation at 37˚C for 72 h. One well isolated
colony was picked for further cultiv ation and subsequent
analyses. Elementary bacteriological tests (cell mor-
phology, gram stain, oxidase and catalase reactions) were
performed for purity check.
DNA was prepared using the method of Gevers et al.
2001 , slightly modified. Purified total DNA was
digested by two restriction enzymes (4 and 6 base cutter).
In this way, only a limited number of fragments with two
different ends and of suitable size for efficient PCR were
obtained. Small ds DNA molecules (15 - 20 bp) contain-
ing one compatible end were ligature to the appropriate
“sticky end” of the restriction fragments. Both adaptors
are restriction half site-specific and have different se-
quences. These adaptors serve as binding sites for PCR
primers. In the current analyses, the following restriction
enzymes and adaptors were used:
Restriction enzyme: EcoR I [hexacutter]
Adaptor: 5 ’-CTCGTAGACTGCGTAC C -3’
Restriction enzyme: Taq I [tetracutter]
Adaptor: 5 ’-GACGAT G AGTCCTGAC - 3’
Selective amplification of some of the restriction
fragments: PCR primers were specifically hybridized
with the adaptor ends of the restriction fragments. Since
the primers contain at their 3’-end one or more so-called
“selective bases” that extend beyond the restriction site
into the fragment, only those restriction fragments that
have the appropriate complementary sequence adjacent
to the restriction site will be amplified. Following primer
combination was used:
Copyright © 2012 SciRes. OJVM
E. N. ESTEBAN ET AL.
CTTATGAGTCCTGACCGAA-3’. PCR products were
separated according to their length using an ABI Prism®
3130XL Genetic Analyzer. Fragments that contain an
adaptor specific for the restriction half site created by the
6-bp cutter are visualized due to the 5’-end labeling of
the corresponding primer with the fluorescent dye FAM.
The resulting electrophoreses patterns were normal-
ized and subjected to a band pattern recognition proce-
dure using the GeneMapper 4.0 software (Applera, USA).
A normalized table of peaks, containing fragments of 20
to 600 base pairs, was transferred into the BioNumer-
ics™ 4.61 software. For numerical analysis, a data inter-
val was delineated between the 40 and 580 bp bands of
the internal size standard. The profiles were compared
with the reference profiles of the lactic acid bacteria (in-
cluding bifidobacteria) as currently available in BCCM
database. Clustering of the patterns was done using the
Dice coefficient and the Upgma algorithm.
Partial 16S rDNA sequence analysis and phyloge-
netic study: DNA prepared for the PCR-DGGE analyses
was used. 16S rRNA genes were amplified by PCR using
the forward primer 16F27 (pA) 5’AGA GTT TGA TCC
TGG CTC AG 3’ and 6R1522 (pH) 5’AAG GAG GTG
ATC CAG CCG CA 3’ (hybridizing po sition referring to
E. coli 16S rRNA gene sequence numbering 8-27 and
1541-1522 respectively). PCR amplified 16S rDNAs
were purified using the NucleoFast® 96 PCR Clean-up
Kit (Macherey-Nagel, Düren, Germany). Sequencing
reactions were performed using the BigDye® Terminator
Cycle Sequencing Kit and purified using the BigDye®
XTerminatorT Purification Kit Sequencing was per-
formed using an ABI Prism® 3130XL Genetic Analyzer
(Applied Biosystems, Foster City, CA, USA).
The following forward and reverse primers were used
to get a partial overlap of sequences, ensuring highly
reliable assembled data: 16F358 (Gamma) 5’CTC CTA
CGG GAG GCA GCA GT 3’, 16R339 (Gamma) 5’ACT
GCT GCC TCC CGT AGG AG 3’, 16R519 (BKL1) 5’
GTA TTA CCG CGG CTG CTG GCA 3’ (hybridizing
position referring to E. coli 16S rRNA gene sequence
numbering 339-358, 358-339 and 536-516). Sequence
assembly was performed by using the program Autoas-
sembler™ (Applied Biosystems, Foster City, CA, USA).
A similarity matrix was created using the software
package Bio Numerics (Applied Maths, Belgium) by
homology calculation with a gap penalty of 0% and after
discarding unknown bases and is based on pairwise ali-
gnment using an open gap penalty of 100% and a unit
gap penalty of 0%.
Phylogenetic analysis was performed using the soft-
ware package Bio Numerics after including the consen-
sus sequence in an alignment of small ribosomal subunit
sequences collected from the international nucleotide
sequence library EMBL. A resulting tree was constructed
using the neighbor-joining method.
PCR-denaturing gradient gel electrophoresis (PCR-
DGGE): PCR-DGGE analysis was carried out as de-
scribed in Van Hoorde et al., 2008 .
Physiological and molecular characterization of
Each sample of freezer-died power of MC after addi-
tion of an equal amount of peptone water were cultured
on dextrose yeast extract peptone agar (DYPA) with
0.02% of chloramphenicol. The physiological and mor-
phological profiles of the yeast isolates were determined
with the automated microplate method Allev/Biolomics
(BioAware SA, Hannut, Belgium) , and using yeast
characterization system based on standard taxonomic
criteria [14,15]. The identification molecular was real-
ized according to Kutzman and Robnett 1998  and
Barnett et al., 2000 .
For DNA sequence analyses, the primer pair LR0R-
LR6 was used to amplify the D1/D2 region of the large
subunit (LSU) of the ribosomal gene complex . Suc-
cessful PCR amplifications resulted in a single band ob-
served on a 0.8% (w/v) agarose gel, corresponding to
approximately 600 bp. PCR produ cts were cleaned using
the QIAquick PCR purification kit, following the manu-
facturer’s protocol. Sequencing reactions were performed
using a CEQ DTCS Quick Start Kit (Beckman Coulter,
Fullerton, California, USA), according to the manufac-
turer’s recommendations, with the primers LR0R and
LR3. Nucleotide sequences were determined using a
CEQ 2000 XL capillary automated sequencer. The se-
quences were assembled and edited in Sequencher 4.8
(Gene Codes Corp., Ann Arbor, Michigan, USA). After
initial BLAST searches for the most similar sequences,
alignments were performed to compare type strain se-
quences with the query sequences. The number of sub-
stitutions and potential insertions or deletions (indels)
was determined from these pairwise comparisons using
BioEdit 22.214.171.124 . The sequences were deposited in
the EMBL databank (http://www.ebi.ac.uk/EMBL; Hinx-
ton, UK) with accession numbers DIV/08 182 A, 182 B,
182 C, 182 D.
High-molecular-mass DNA for PCR-fingerprint ana-
lyses was extracted using the Invisorb® Spin Plant Mini
Kit (Invitek GmbH, Berlin, Germany) as described by
Groenewald et al., 2008 . PCR-based fingerprinting
with the single primer M13, a ubiquitous microsatellite
sequence , was applied as described before by Gro-
enewald et al., 2008 . The resulting M13-based PCR-
fingerprint profiles were compared visually with refer-
“In vitro” evaluation of the MC probiotic capacity.
An aliquot of MC, was inoculated into acidified MRS
broth to pH 2.5, 3 and 4 with HCl (1N) and non-acidified
(pH 6.8) and i n cubat e d f or 3 h at 37˚C.
Copyright © 2012 SciRes. OJVM
E. N. ESTEBAN ET AL. 107
After incubation, serial dilutions in peptone water
were spread onto MRS agar plates. Plates were incubated
anaerobically for 48 h at 37˚C.
Percentage of resistance to each analyzed pH was cal-
culated by the equation: %RpH = [(UFC/ml)MRS pH... ×
100]/(UFC/ml) MRS pH 6.5 .
An aliquot of MC was inoculated into acidified MRS
broth with 0.03, 0.05 and 0.1 concentrations of bile salt
and within bile salts and incubated for 3 h at 37˚C.
After incubation, serial dilutions in peptone water were
spread onto MRS agar plat es at 1h and 3 h p ost i ncubati on.
Plates were incubated anaerobicall y for 48 h at 37 ˚C.
Percentage of resistance to each analyzed bile salt
concentration was calculated by the equation: %RpH =
[(UFC/ml)MRS pH... × 100]/(UFC/ml) MRS pH6.5 .
3.1. MC Development
In optimum anaerobic culture conditions, the biomass
production in creased to 6 ± 0.5 g/l and pH fell to 3.6 ±
0.1 after 24 h culture. Periodic microscopic examination
of the MC showed a homogeneous proportion in the
quantity of yeast and lactic acid bacteria.
The viability after MC rehydration was 3.2 × 109 lac-
tobacilli plus 1.5 × 106 yeast CFU per gram lyophilized
3.2. Isolation and Biochemical Characterization
of the Lactic Acid Bacteria
Most of the random isolates from MC (95%) expressed
general characteristics corresponding to lactic acid bacte-
ria (Gram-positive, catalase-negative, non-motile rods).
From each resulting cluster, one to four strains were
selected for further analysis so twelve lactobacilli were
characterized by fermentation profiles.
Phenotypic characterization of the microbial commu-
nity indicated the following species and strains: Lactoba-
cillus helveticus (ID11609, ID11610, ID11618), Lacto-
bacillus delbrueckii (ID11611), Lactobacillus paracasei
(11614), Lactobacillus fermentum (ID11613, ID11616,
ID11619), Lactobacillus sp (ID11612, ID11615, ID11617),
and Lactobacillus buc h neri (ID11620).
3.3. Molecular Characterization of Lactic
The results of the iden tification using AFLP and 16S and
PCR-DGGE are given.
Two colony types were i s ol ated from ID11615: Smooth
(t1) and rough (t2) col o ny. The Lactobacillus identificated
were: L. helveticus (ID11609, ID11610, ID11611 and
ID11612), L. fermentum (ID11613, ID11615 t1 , ID11616),
L. paracasei ID11614, ID11618), L. casei (ID11617), L.
parabuchneri (ID11620) as are shown in the dendo-
grams of the cluster analyses of AFLPTM profiles (Fig-
For the rough colony and for ID11619, no profile
could be obtained. Parcial 16S rDNA sequence analysis
was carried out. The percentage 16S rDNA sequence
similarity was Lactobacillus gasseri DSM 20243T (100)
and Lactobacill us pa ni s DSM 6035T (99.8) respectively.
Similar DGGE results were obtained for both gradients
(35% - 60% and 35% - 70%). The results are shown in
3.4. Physiological and Molecular
Characterization of Yeast
The results obtained are shown in Table 1.
3.5. “In Vitro” Evaluation of the MC
Obtained percentage of resistance pH and bile salts are
shown in Table 2.
4. Discussion and Conclusions
The Kefir is a complex probiotic. Its lactobacilli and
yeast components were selected over thousands of years
towards milk fermentation . In a similar manner, we
took from Kefir the most adaptable members to grow in
whey. Other lactobacilli and yeast found in our environ-
ment, and which improved whey fermentation, were also
selected and incorporated to the developing consortium.
Based on a consistent fermentation in successive whey
baths, determined by pH modification and sensorial ex-
aminations like smell and taste, we considered the con-
stituted community as a unit. This consistent fermenta-
tion suggested a symbiotic relationship and adaptation
capacity of the MC.
This MC is composed by at least seven lactic acid
bacteria and two yeast species, comprising agents from
separated dominium like Eukaria and Bacteria. The ap-
plication of molecular biology tools to better understand
the integrated process of this complex consortium would
allow us, in the near future, to use specific community
members, or specifically tailored consortia of members,
to target desired interactions, resulting in health benefits
and food production impr ovement [24,25].
Selected lactic acid bacteria and yeast are multispecies
and also multistrain assuring high biodiversity. The ad-
vantage of multistra in and multispecies probiotics is that a
number of favorable characteristics of individual strains
are combined in a single preparation . It has been
clearly shown that multispecies preparations have advan-
tages when compared to monostrain probiotics or, to a
lesser extent, multistrain probiotics . Well-designed
Copyright © 2012 SciRes. OJVM
E. N. ESTEBAN ET AL.
Copyright © 2012 SciRes. OJVM
multispecies probiotics can benefit from a certain amount
of synergism when the effects of different probiotic spe-
cies are combined [28,29]. The activity can also be sti-
mulated through simbiosis among strains in the prepa-
ration [30-32]. How probiotics, whether monospecies or
multistrain/mulitispecies formulations, actually helping
the host to attain such greater protection levels is still
poorly underst ood [32,33].
Figure 1. Dendrogram of the cluster analyses of AFLPTM profiles (ID11609-D11612).
Figure 2. Dendrogram of the cluster analyses of AFLPTM profiles (ID11613, ID11615 t1 and ID11616).
E. N. ESTEBAN ET AL. 109
Figure 3. Dendrogram of the cluster analyses of AFLPTM profiles (ID11614, ID11617 and ID11618).
Figure 4. Dendrogram of the cluster analyses of AFLPTM profiles (ID11620).
Copyright © 2012 SciRes. OJVM
E. N. ESTEBAN ET AL.
Copyright © 2012 SciRes. OJVM
Figure 5. PCR denaturing gradient gel electrophoresis (35% - 70%). Computer out prints of the profiles obtained for the 13
Table 1. Yeast isolates, colony types and the results morphological and physiological characterization and molecular identifi-
Collection numberColony type Morphology and
physiological characterizationMolecular identification by D1/D2
large subunit rDNA sequence
DIV/08-182A MUCL 51663 Surface pulverulent, relief umbonatePichia occidentalis Pichia kudravzevii
DIV/08-182B MUCL 51664 Surface cerebriform Saccharomyces cerevisiae Saccharomyces cerevisiae
DIV/08-182C MUCL 51665 Surface smooth, relief umbonate Saccharomyces cerevisiae Saccharomyces cerevisiae
DIV/08-182D MUCL 51666 Small colonies, surface smooth Saccharomyces cerevisiae Saccharomyces cerevisiae
Table 2. Percentage of resistance of CM to 2.5, 3 and 4 pH and to 0.03, 0.05 and 0.1 bile salts concentrations at time different.
Bile salts concentrations (%) pH
Time of incubation (h) 0.03 0.05 0.1 2.5 3 4
1 99.4 15.2 5.9 - - -
3 70.3 24.7 2.0 23.3 77.2 85.4
The food transit time through th e monogastric animals
is about 90 min . The tolerance at different pH and
bile salt concentration obtained in this work indicate the
possibility to exp lore the usefulness of this MC as probi-
otic in animal model. The MC should be tested to detect
the possible production of adverse effect and be evalu-
ated if this product could increase the health status of
calves through of the stimulation of the immature new-
born immune system preventing the diarrhea responsible
for high mortality and morbidity in neonates [35-40].
Expert technical assistance was provided by Bioq. Glad ys
Martos (CERELA-Argentine), Dr. S. Van Trappen
(BCCM/LMG, Belgium) and Dr. Heidi-Marie Daniel
BCCM/MUCL, Belgium). Economic assistance was pro-
vided by Comisión de Investigaciones Científicas de la
Provincia de Buenos Aires, Agencia Nacional de Pro-
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