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Interciencia
ISSN: 0378-1844
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Asociación Interciencia
Venezuela
Gusils, Carlos; Bujazha, Marina; González, Silvia
Preliminary studies to design a probiotic for use in swine feed
Interciencia, vol. 27, núm. 8, agosto, 2002, pp. 409-413
Asociación Interciencia
Caracas, Venezuela
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PRELIMINARY STUDIES TO DESIGN A PROBIOTIC FOR
USE IN SWINE FEED
Carlos Gusils, Marina Bujazha and Silvia González
SUMMARY
During inhibitory activity screening of 100 strains of lactic
acid bacteria isolated from the gastrointestinal tract of pigs only
six, four identified as Enterococcus faecium and two as Lactobacillus acidophilus, showed inhibition against enteric indicator
strains: Salmonella enteritidis, S. cholera suis, S. typhimurium
and Yersinia enterocolitica. The inhibitory action was not affected by the addition of catalase and no inhibition was detected
after neutralizing the supernatant culture fluid. Furthermore,
Introduction
Infectious diarrhea of neonatal animals is one of the
most common and economically devastating conditions
encountered in the animal agriculture industry (Muralidhara et al., 1977). Traditionally,
lactic acid was added (1%) to
the drinking water for 10
days after weaning to avoid
pathogen multiplication.
Probiotic foods can be administered to humans or ani-
mals in order to prevent infectious diseases, to strengthen the barrier function of
the gut microflora and/or for
a non-specific enhancement of
the immune system (Sögaard
and Suhr-Jessen, 1990). In the
specific case of swine livestock they should be administered immediately before the
weaning, at the beginning of
the breeding stage, which represents the second unit in the
program of intensive breeding. Probiotic foods can help
these two L. acidophilus showed agglutination with treated yeast.
The agglutination of one strain was inhibited by maltose, this
suggests the presence of a lectin-like structure in their cell walls,
which could be responsible for its adhesion ability. The selected
strains were resistant to pH 3.0 and bile salts. These strains fulfil the conditions of probiotic bacteria and could be selected for
elaborating pig probiotic feed, in order to prevent infectious diseases.
pigs develop local immunity
in the intestine and lactobacilli, generally included in
this type of foods, ferment
carbohydrates with lactic acid
production.
The composition of the intestinal microflora in healthy
animals remains steady, but if
the stability is broken, pathogenic microorganisms, such as
Yersinia or Salmonella can
colonize the intestinal tract,
leading to serious infections
(Garriga et al., 1998).
According to Fuller (1989)
a probiotic is a feed with live
microorganisms, which beneficially affects the host animal by improving its intestinal microbial balance. Most
probiotics contain single or
multiple strains of lactic acid
bacteria (LAB), which are
considered as GRAS (generally regarded as safe) microorganisms.
Probiotic strains can be selected due to their condition
of normal intestinal inhabit-
Silvia González. Researcher, CONICET-CERELA. Associate Professor of Public Health, UNT. Address: Centro de Referencias para
Lactobacilos (CERELA-CONICET), Chacabuco 145, 4000,
S.M. de Tucumán, Argentina.
e-mail: [email protected]
KEYWORDS / Mixed Cultures / Pig / Probiotic /
Received: 02/28/2002. Accepted: 06/12/2002
Carlos Gusils. Researcher, National Research Council of Argentina and Centro de Referencia para Lactobacilos (CONICET-CERELA). Instructor of
Virology and Microbiology,
National University of Tucumán (UNT).
Marina Bujazha. Researcher, Research Council of Tucumán National University (CIUNT), Tucumán, Argentina.
AUG 2002, VOL. 27 Nº 8
0378-1844/02/08/409-05 $ 3.00/0
409
RESUMEN
De 100 cepas de bacterias lácticas aisladas del tracto gastrointestinal de cerdos, solamente seis, cuatro identificadas como
Enterococcus faecium y dos como Lactobacillus acidophilus,
mostraron acción antagonista frente a bacterias patógenas
entéricas: Salmonella enteritidis, S. cholera suis, S. typhimurium
y Yersinia enterocolitica. La capacidad inhibitoria no fue
afectada por el agregado de catalasa o por neutralización. Dos
cepas de L. acidophilus presentaron capacidad de aglutinar con
levaduras tratadas. Solamente, la aglutinación de una de ellas
fue inhibida por el agregado de maltosa, esto sugiere la
presencia de una estructura en su pared celular responsable de
la capacidad de adhesión. Las cepas seleccionadas fueron
resistentes a pH bajos (3,0) y presencia de sales biliares. Estas
cepas podrían ser seleccionadas para el diseño de un alimento
probiótico para cerdos con el objeto de prevenir enfermedades
infecciosas.
RESUMO
De 100 cepas de bactérias lácticas isoladas do tracto gastrointestinal dos cerdos, somente seis, quatro identificadas como
Enterococcus faecium e dois como Lactobacillus acidophilus,
mostraram ação antagonista frente a bactérias patógenas
entéricas: Salmonella enteritidis, S. cholera suis, S. typhimurium
e Yersinia enterocolitica. A capacidade inibitória não foi afetada
pelo agregado de catalasa ou por neutralização. Duas cepas de
L. acidophilus apresentaram capacidade de aglutinar com
ants of the host and several
beneficial properties such as
being active antimicrobial
agents against pathogens (hydrogen peroxide, bacteriocins,
and some organic acid such
as lactic, acetic and propionic
acids), the hydrophobic nature
of the bacterial surface, stimulation of the immune system, presence of substances
with adherence capacity to
the epithelium (polysaccharides, lectins), coaggregation
ability, and pH and bile salts
resistance, among others.
Antimicrobial peptides, bacteriocins, produced by LAB
are adsorbed on the cells of
producing strains and other
gram-positive bacteria. Adsorption of bacteriocin molecules by the Pediococcus,
Lactobacillus, Lactococcus
and Leuconostoc producer
strains, as well as by other
sensitive and resistant Grampositive bacteria, has been reported (Klaenhammer, 1988;
Yang et al., 1992).
Some microorganisms are
able to bind to epithelial cells
of the gastrointestinal tract
through lectins present in
their surface structures. Lectins are carbohydrate-binding
proteins or glycoproteins from
non-immune origin which agglutinate cells with receptors
such as yeast and red blood
410
cells (Slifkin and Doyle,
1990).
An important factor controlling in vivo adhesion and
colonization is the animal
species specificity of microorganisms, which indicates
that bacterial strains isolated
from the indigenous microflora of one animal species
will not necessarily colonize
the same site in another animal species.
Morata de Ambrosini et al.,
(1999) determined that Lactobacillus casei from human
origin showed higher adhesion ability to ileal epithelial
cells than L. casei from dairy
origin (50% and 8% respectively). Furthermore, the pH
range with highest adhesion
capacity was between 6 and
7.5, which is within the normal pH range of the intestinal
environment, and adhesion
was only observed at 37ºC.
On the other hand, in previous experiments we found
higher adhesion values after
incubation at 42ºC than at 37
or 30ºC, but in that case the
study was performed on poultry, where the normal body
temperature is 42ºC. This observation can be deemed hostspecific, as lactobacillus
strains and tissue fragments
were obtained from chickens
(Gusils et al., 1999).
levaduras tratadas. Somente, a aglutinação de uma delas foi
inibida pelo agregado de maltosa, isto sugere a presença de uma
estrutura em sua parede celular responsável da capacidade de
adesão. As cepas selecionadas foram resistentes a pH baixos
(3,0) e presença de sais biliares. Estas cepas poderiam ser
selecionadas para o desenho de um alimento probiótico para
cerdos com o objetivo de prevenir doenças infecciosas.
The present study was designed to isolate, characterize
and further select beneficial
lactobacillus strains for the
elaboration of a swine probiotic feed.
Materials and Methods
Bacterial strains
and culture conditions
Lactic Acid Bacteria (LAB)
were isolated from pig faeces
at the Technological Ecophysiology Laboratory of
CERELA, Tucumán, Argentina. Serial dilutions of faeces
were plated (de Man et al.,
1960) on Man, Rogosa, and
Sharpe (MRS) agar (Merck),
Streptococcus Selective Medium (Merck). Incubation was
at 37ºC in a microaerobic atmosphere. The strains were
identified with API 50 CH
(Biomeriec) and other complementary tests according to
the criteria of Bergey’s Manual of Determinative Bacteriology, 9th Edition. Strains of
Salmonella and Yersinia were
provided by the Enterobacteria Service and the Service of
Special Bacteriology Department of the Instituto Nacional
de Enfermedades Infecciosas
(INEI) Dr. Carlos Malbrán,
Buenos Aires, Argentina. Saccharomyces cerevisae was
provided by the Planta Piloto
de Procesamientos Industriales Microbiológicos (PROIMI), Tucumán, Argentina; and
Mycobacterium sp. was provided by the Instituto de
Microbiología Dr. Luis Verna,
Universidad Nacional de Tucumán, Argentina
All LAB strains were kept
at -20°C in a LAPTg broth
(Raibaud et al., 1961) with
30% glycerol (v/v). Lactobacilli were activated and grown
in a LAPTg medium.
Detection of antibacterial
activity
The antibacterial activity
was tested by the agar spot
test (Klaenhammer, 1988).
The indicator strains used
were Yersinia enterocolítica,
Salmonella typhimurium¸ S.
cholera suis and S. enteritidis
(105-10 6 CFU* ml-1); 75µl of
an overnight culture of indicator microorganisms were
mixed with 12ml of Brain
Heart Infusion agar (BHI-agar,
Merck). The wells (5mm diameter) were filled with a
bacterial suspension of overnight probiotic culture, and
sterile supernatans (50µl) neutralized (pH 5 and 6) with
sterile NaOH 1N or non-neu*
Colony Forming Units.
AUG 2002, VOL. 27 Nº 8
tralized, and treated with
trypsin (1mg·ml-1, SIGMA) or
catalase (0.5mg·ml-1, SIGMA).
The plates were maintained at
room temperature for 3h to
promote the diffusion of this
substance and later incubated
during 24h at 37ºC.
Extraction of adsorbed
bacteriocins from producer cells
was tested by mixing 10µl of
suspension in PBS with 5µl
of the sugar solution (fucose,
galactose, glucose, lactose,
mannose, N-ac. galactosamine, N-ac. glucosamine, sialic acid, sucrose were individually added at 1; 0.5; 0.2;
0.1 and 0.05 M), prior to the
addition of 10µl of the yeast
suspension.
The producer strain was
grown in 1 liter of MRS broth
at 30ºC for 18-20h, without pH
control. The culture was heated
to 70ºC for 30min to inactivate
proteases and to kill cells, and
the pH was adjusted to 6.5
with 4 M NaOH. Cells were
collected by centrifugation
(15,000g, 15 min), the pellet
washed twice in 5mM sodium
phosphate buffer (pH 6.5), resuspended in 10ml of 100mM
NaCl at pH 2.0 (adjusted with
5% phosphoric acid), and
mixed with a magnetic stirrer
(29,000g, 20min at 4ºC), and
the supernatant stored at -20ºC
(Yang et al., 1992).
Hemagglutination assays
Production of lectin-like
substances: Agglutination assay
Cell surface hydrophobicity
was determined by the bacterial adherence to hydrocarbons assay (Rosenberg et al.,
1980). The test bacteria were
grown at 37ºC in LAPTg
broth. Unless otherwise
stated, bacteria were harvested (10,000g, 10min) at
the early logarithmic growth
phase (12-18h), washed twice
and resuspended in physiological solution (PS) to an
optical density (OD600) of 0.50.7. To test tubes containing
3ml of washed cells, 1ml of
test hydrocarbon (Hexadecane, Toluene and Xylene)
were added. The mixtures
were blended on a vortex
mixer for 90s. The tubes were
left to stand for 15min for
separation of the two phases
and the OD of the aqueous
phase was measured. Hydrophobicity was calculated from
three replicates as the percentage decrease in the optical density of the original
bacterial suspension due to
cells partitioning into a hydrocarbon layer. Mycobacterium
sp was used as positive control and Lactobacillus acido-
Bacterial cells were collected by centrifugation
(15,000g for 10min) and suspended to one tenth of the
original volume in phosphate
buffered saline (PBS), pH
7.4. Agglutination was monitored visually on microscopic
slides by mixing 10µl of the
sample with 5µl PBS, and
10µl of a suspension of
treated Saccharomyces cerevisiae (10 8 cells·ml -1 PBS).
The yeast cells were prepared by preincubation in
PBS with glutaraldehyde
(1mg·ml -1 ) for 1h at 25ºC,
washed twice with PBS, incubated for 30min at 25ºC
with 10mg/ml glycine and
washed as above. The treated
yeast cells were stored at 4ºC
as a suspension in PBS
(0.1g·ml-1) containing 0.02%
(w/v) sodium azide (Eshdat
et al., 1978).
Inhibition agglutination assay
The ability of different sugars to inhibit agglutination
Bacterial cells were collected
by centrifugation (15,000g,
10min) and suspended to one
tenth of the original volume in
(PBS), pH 7.1. Hemagglutination was carried out at room
temperature with a 96-well
microtiter plate using PBS as a
diluent. 50µl of a 2-fold diluted
sample was mixed with 10µl of
2% pig erythrocyte suspension
(SIGMA) in PBS. The strength
of agglutination was read with
the naked eye after 1h of incubation.
Cell surface hydrophobicity
AUG 2002, VOL. 27 Nº 8
philus CRL 730 as negative
control (Morata de Ambrosini
et al., 1999). The percentage
of hydrophobicity was calculated using the equation
at 560nm. Total counts were
determined on LAPTg agar.
Lactobacilli counts were carried
out on MRS agar (Merck), enterococci on Streptococcus Se-
Resistance to bile salts
lective Medium (Merck) and
Salmonella and Yersinia was
counted on MacConkey agar
(Merck). All plates were incubated at 37ºC for 48h.
At 48h of incubation, bacteria were harvested (15,000g,
10min), supernatants were
used for determination of bacteriocin yield by an agar spot
test, as previously described;
and cellular pellets were
washed and resuspended in
PS for determination of lectin
production by agglutination
assay, as previously described.
In order to examine the resistance to bile salts, lactobacilli were grown in LAPTg
broth supplemented with Oxgall
(Difco) 0.1% or 0.4% (w/v)(1
or 4% bile salts). The cultures
were incubated at 37°C for
24h and growth was monitored by measuring the optical density at 560nm.
Resistance of low pH
Resistance of the isolates to
pH 3.0 was tested as follows:
overnight cultures of the isolated strains were centrifuged
at 5,000g for 10min. After resuspending the pellet in the
same buffer of saline solution,
it was diluted 1/10 in sterile
physiological solution (PS) at
pH 3.0. After 3h at 37ºC, the
appropriate dilutions were
plated in selective agar medium and incubated at 37ºC
for 48h.
Aggregation assays
The aggregation test was
performed according to Reniero et al., (1992). Aggregation was scored positive when
clearly visible sand-like particles, formed by the aggregated cells, gravitated to the
bottom of the tubes, leaving a
clear supernatant fluid within
2h.
Mixed cultures
Mixed cultures (potentially
probiotic strain + pathogen
microorganism) were studied.
10ml of the LAPTg broth
were inoculated with 1 x 107
CFU·ml-1 of individual strains
of lactic acid bacteria and 106
CFU·ml-1 of pathogens. Cultures were incubated for 24h
at 37ºC and followed by
measuring the optical density
Statistical analysis
Experiments were carried
out in triplicate. Significant
differences were tested using
Tukey´s test (Minitab Student
R12) (Rossman and Chance,
1998).
Results and Discusion
The screening of 100 lactic
acid bacteria, isolated from
intestinal content and fecal
swabs of 25 pigs, determined
that only 10 strains presented
antimicrobial activity against
pathogenic indicator bacteria.
Between them 9 strains were
able to inhibit the growth of
Yersinia enterocolitica, S. enteritidis, S. typhimurium and
S. cholerae suis; only one,
the remaining strain was effective only against the two
first pathogens above mentioned (Table I). However,
the supernatants sterilized by
heat, neutralized with NaOH
solution or treated with catalase or proteolytic enzymes,
were unable to inhibit the
growth of pathogens. From
the results it can be assumed
that these lactic acid bacteria
do not have capacity for antimicrobial production, but
some microorganisms adsorb
on their external structures,
411
TABLE I
SELECTION CRITERIA OF POTENTIALLY PROBIOTIC STRAINS ISOLATED FROM FAECES OF PIG
1
:
:
3
:
4
:
2
(-)
(-)
(-)
(-)
growth inhibition negative, (+) growth inhibition positive;
bacterial aggregation negative, (+) aggregation positive after 1h of incubation
agglutination or hemagglutination negative, (+) agglutination or hemagglutination positive
not resistant strain, (+) resistant strain
like-bacteriocin substances.
For this reason bacteriocin
presence on cell walls of pig
lactic acid bacteria was studied, but the results determined that the assayed
strains did not present this
kind of substance in its external layers.
The adhesiveness of two
strains isolated from pig was
recorded as positive concerning the aggregation test of
Reiniero et al. (1992), agglutination of treated yeasts and
hemagglutination assays
(Table I). The addition of sucrose (0.05M) inhibited agglutination and hemagglutination from the strain named
4c; these results could be
correlated to the presence of
an external lectin-like structure with sucrose affinity.
a
In previous works we found
lectin-like structures in two
lactobacilli isolated from
chickens; the adhesion characterization of Lactobacillus
animalis indicated that a lectin-like structure has glucose
and mannose as specific binding sugars. The results about
adhesion related to Lactobacillus fermentum showed that
sialic acid and mannose are
involved in the binding (Gusils et al., 1999).
The strains with antimicrobial and/or adhesive activity
were resistant to bile salts (1
and 4%) and pH 3.0 (Table I).
High acidity in the stomach
and high concentration of bile
components in the small intestine are the first host attributes that affect strain selection. Gilliland et al., (1984)
observed a great variability
among Lactobacillus acidophilus strains isolated from calf
intestinal contents in their ability to grow in vitro in the
presence of bile salts. When a
strain exhibiting low tolerance
to bile and another strain exhibiting high tolerance to bile
were administered orally to
calves, the more resistant
strain caused greater increase
in the number of facultative
lactobacilli than the one possessing low tolerance (Nousiainen and Setälä, 1998).
The adhesion ability of the
other lactobacillus strain (13c)
apparently was not related to
a lectin-like structure because
no sugar solutions assayed in
this work inhibited agglutination properties. In this case
hydrophobic strength could be
involved in the bacteria-epithelial cells interactions. However, other lactobacillus
strains (6c and 11c) showed
similar hidrophobicity values
without adhesive properties.
Group A streptococci are hydrophobic and adhere to hydrophobic surfaces but are
unable to colonize all these
surfaces (Courtney et al.,
1990). The adhesion process
is probably a composite of
factors including presentation,
orientation, and substrata. In
addition, the mechanism of
adhesion may require the participation of a number of distinct surface constituents that
interact in a sequential manner to overcome repulsive
forces.
Competition assays were
carried out using different
b
Figure 1. Mixed culture with selected lactic acid bacteria and enteropathogens ( Yersinia enterocilitica,
Salmonella cholera suis,
S.
typhimurium and
S. enteritidis). Control: pure cultures of several Salmonella sertype. a) pig strains with inhibitory capacity, and b) strains
with adherence properties.
412
AUG 2002, VOL. 27 Nº 8
TABLE II
IDENTIFICATION OF POTENTIAL PROBIOTIC STRAINS
Strain
4c
7c
10c
13c
14c
19c
Biochemical Identification
Lactobacillus
Enterococcus
Enterococcus
Lactobacillus
Enterococcus
Enterococcus
double mixed cultures between potentially probiotic
strains and pathogenic microorganisms in order to study
benefical properties. After incubation during 24h at 37°C
the different mixed cultures,
lactobacillus counts and lectin-like substance production
did not present significant differences (P>0.05) with respect to control cultures (data
not shown). On the contrary,
in the same mixed cultures,
partial inhibition of pathogens
was observed when mixed
and control cultures were
compared (Figure 1). The
antipathogenic effect observed
in mixed cultures could be
explained as a nutritional
competition.
After physicochemical assays, four strains were identified as Enterococcus faecium
and two as Lactobacillus
acidophillus by the fermentation pattern in the API test
acidophillus
faecium
faecium
acidophillus
faecium
faecium
and another biochemical determinations (Table II).
Among 100 strain isolates,
only six were selected because
of their potentially probiotic
(adhesion capacity, antipathogenic activity against enteric
bacteria, resistance to bile salts
and pH 3.0, high hydrofobicity
values of cell walls, etc. Studies related to the effect of oral
administration to pigs of these
potentially probiotic strains are
in progress.
ACKNOWLEDGEMENTS
This research was supported by CIUNT under program D26/126 and by
CONICET.
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