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Probiotics: Production, Evaluation and Uses in Animal Feed, 2009: 47-73

ISBN: 978-81-308-0323-4 Editors: Nelson Pérez Guerra and Lorenzo Pastrana Castro

Effects of separate
supplementation of four
potentially probiotic lactic acid
3 bacteria on both the
production parameters and
faecal coliform counts in
weaned piglets
Paula Fajardo Bernárdez, Clara Fuciños González
Isabel Rodríguez Amado, Lorenzo Pastrana Castro
and Nelson Pérez Guerra
Departamento de Química Analítica y Alimentaria, Facultade de Ciencias
de Ourense, Universidade de Vigo. As Lagoas s/n, 32004 Ourense, Spain

Abstract
In this study, some probiotic characteristics of
four lactic acid bacteria (Pediococcus acidilactici
NRRL B-5627, Lactococcus lactis subsp. lactis
CECT 539, Lactobacillus casei subsp. Casei CECT
Correspondence/Reprint request: Dr. Nelson Pérez Guerra, Departamento de Química Analítica y
Alimentaria, Facultad de Ciencias de Orense, Universidad de Vigo. Las Lagunas s/n, 32004 Orense, Spain
E-mail: nelsonpg@uvigo.es
48 Paula Fajardo Bernárdez et al.

4043 and Enterococcus faecium CECT 410) were assayed for their possible
use as probiotic strains in piglet feeds. The four bacteria were evaluated for
their gastrointestinal transit tolerance, ability to produce high amounts of
biomass and antibacterial substances, stability during freezing storage
(at -20ºC) and during storage with the carrier feed. Subsequently, the effects of
the supplementation of separate potentially probiotic cultures (cells +
antimicrobial substances + skimmed milk) and an antibiotic (colistin sulfate) to
piglet diets on the performance (body weight gain (BWG), feed intake (FI), feed
conversion efficiency (FCE)) and on the faecal coliform counts (FCC) of
weaned piglets were comparatively evaluated during 42 days of diets
administration. The groups receiving the antibiotic exhibited the best results,
meanwhile the probiotic groups showed a significant increase in BWG, FI and
FCE in comparison with the non-treated (controls) groups (P<0.05). The
changes in the FCC in the control groups over time were not significant
(P<0.05), while the FCC values in the groups fed probiotics and antibiotic
significantly dropped at the last sampling time (P<0.05).
To determine the effect of probiotic or antibiotic (avilamycin)
administration during both the administration (0-28 days) and the post-
administration (28-42 days) periods on performance and FCC of weaned
piglets, the production of a viable culture concentrate of Lact. casei CECT
4043 was carried out in MRS broth. The highest average BWG values were
obtained in the groups fed probiotic and avilamycin by day 28. However, the
mean FI and FCE values did not show significant differences among the
groups (P < 0.05). By day 42, the antibiotic group presented the highest
BWG, the group fed Lact. casei exhibited the highest FCE meanwhile the
control group showed the lowest FI value (P < 0.05). Interestingly, at the end
of the experiment (42 days), only the probiotic group presented FCC
significantly lower than those of the first day (P < 0.05).

1. Introduction
Antibiotics have been routinely used in animal feed not only to control
the growth of bacteria that causes diseases in animals but also to enhance the
growth of animals. Although the antibiotics seem to be able to increase the
efficiency of animals’ digestion, the use of these antimicrobial agents in
animal feed has increasingly become the focus of concern. Thus, it has been
reported that the use of antibiotics in animal feed leads to the development of
antibiotic-resistant bacteria which can spread among animal species and be
transmitted to humans by food of animal origin [1,2,3,4].
For these reasons, in recent years, antibiotic growth promoters have been
gradually removed from farm animal feeds. This has led to a renewed interest
Effect of four lactic acid bacteria in weaned piglets 49

in the use of probiotic preparations and organic acids as a replacement.

Probiotics are live microbial cultures that once ingested by humans and
animals in sufficient numbers, can beneficially influence the health of the
host [5,6,7,8,9]. The mode of action of probiotics is strain-dependent and
may include the modulation of the host intestinal microbiota, modifications
of the structure and function of the intestinal epithelium and stimulation of
the immune response by activation and regulation of mucosa-associated and
immune system responses. The mechanisms by which probiotics can interact
with the microbial population in the gut may be based on i) competitive
adhesion with pathogens to epithelial receptors to prevent their adhesion to
the intestine, ii) aggregation with pathogenic bacteria, iii) competition for
nutrients necessary for pathogen survival or iv) production of antimicrobial
substances, which reduce not only the number of viable pathogenic
organisms but may also control their metabolism and toxin production [10].
The probiotics can modulate the intestinal epithelium by stimulating the
production of defensive molecules such as mucins, by reducing the secretory
and inflammatory consequences of bacterial infection and by promoting tight
contact between epithelial cells forming a functional barrier. Mechanisms of
action dealing with the effect of probiotics on the host immune response
include the transport of anti-inflammatory substances to the intestine,
reduction in the inflammatory response and allergy, as well as the reduction
in production of inflammatory substances [11,12].
For an effective application as additives in animal feed, potential probiotic
strains should fulfil many technological and health criteria. Then, these strains
must be: i) non-pathogenic and non-toxic, ii) beneficial to the host in some way
after consumption, iii) resistant to gastric acidity and bile toxicity, iv) able to
attach to the gut epithelial tissue, v) able to colonise and persist within the
gastrointestinal tract, vi) amenable to cultivation on an industrial scale, vii) able
to produce high amounts of antimicrobial substances and viii) stable during
preparation and storage of the carrier feed [5,6,13,14,15].
Some lactic acid bacteria (LAB) strains including species belonging to
the genus Bifidobacterium, Lactobacillus, Pediococcus, Streptococcus and
Enterococcus have proven to be effective in prevention or treatment of some
diseases in humans and animals [9,16,17,18,19]. The effectiveness of LAB to
produce beneficial effects in the host, has been associated not only with their
adhesion capability to gut intestinal epithelial cells, but also with their ability
to produce antibacterial products (mainly organic acids and bacteriocins)
[7,9,20].
The use of probiotic bacteria and their metabolites as additives in animal
feeds has proven to be an effective way to promote body weight and feed
conversion in farm animals [1,16,21]. However, the use and functionality of
50 Paula Fajardo Bernárdez et al.

many commercial animal probiotic products in practice have been questioned

due to the deficiencies in microbiological quality and labelling found in
some probiotic supplements. These deficiencies include low bacterial counts
(zero in some cases) as well as the presence of strains or species not stated on
label [22,23]. On the other hand, the relatively high prices of these probiotic
supplements make uneconomic their use in the animal industry.
The production of probiotic culture concentrates (containing high
concentrations of viable cells and antimicrobial substances) in cheaper
culture media by using an efficient cultivation method could be an
appropriate alternative to solve this problem [24]. Considering that
substantial amounts of whey and mussel-processing wastes are available free,
or commercialized at very low cost by local dairy and mussel-processing
plants, the use of these wastes as culture media could provide a profitable
substrate for a low cost production of probiotic culture concentrates [24,25].
With regard to the cultivation method, it has been reported the use of a fed-
batch technique based on periodical re-alkalizations of the culture medium
for successfully enhancing biomass production by Lactococcus lactis and
Pediococcus acidilactici strains in different culture media [24,26,27,28,29].
In fact, the concentrations of biomass and antibacterial products obtained in
these cultures were higher than those obtained in the corresponding batch
cultures on the same culture media.
The aim of the present study was to asses the probiotic characteristics of
four LAB (Pediococcus acidilactici NRRL B-5627, Lactococcus lactis subsp.
lactis CECT 539, Lactobacillus casei subsp. casei CECT 4043 and
Enterococcus faecium CECT 410). The strains were firstly characterized by
their tolerance to simulated gastric and small intestine transit conditions.
Secondly, the production of viable culture concentrates of each strain was
carried out in whey-based media using a fed-batch fermentation technique
with successive re-alkalizations of the culture medium. Thirdly, the stability
of the four LAB during their storage with skimmed milk at –20ºC for 3
months and in piglet feed at room temperature was evaluated. Finally, the
effects of 42 days administration of separate probiotic strains or an antibiotic
on the performance (body weight gain, feed intake, feed conversion
efficiency) and faecal coliform counts of weaned piglets were investigated.
The second part of the present study was conducted to determine the
impact of 28 days administration of probiotic and antibiotic on the
performance and faecal coliform counts of weaned piglets during both the
administration and the post-administration (from 28 to 42 days) periods. In
this study, Lact. casei CECT 4043 and avilamycin were respectively used as
the probiotic and the antibiotic.
Effect of four lactic acid bacteria in weaned piglets 51

2. Tolerance to acidic pH and to simulated

gastrointestinal conditions
For a successful treatment with probiotic LAB, viable cells must be able
to survive transit through the stomach and small intestine and colonize in the
animal gut [5,7,13,14,15]. Therefore, the four LAB strains (Ped. acidilactici,
Ent. faecium, L. lactis and Lact. casei) were firstly characterized by
determining their resistance to conditions simulating those existing in the
gastrointestinal tract (GIT). Thus, the in vitro acid and bile stabilities of the
four LAB were assayed by exposing washed cell suspensions at 30ºC to
acidic conditions (pHs of 1.0, 2.0, 3.0, 4.0 and 5.0), to a simulated gastric
juice (pH 2.0) containing pepsin (3 g/L) and sodium chloride (5 g/L), and to a
simulated small intestinal juice (pH 8.0) containing pancreatin (1 g/L) and
sodium chloride (5 g/L), mimicking the gastrointestinal environment.
The results obtained in this assay for each LAB strain are shown
in Figure 1. LAB counts declined significantly (P<0.05) at pH 1.0 as time

Figure 1. Survival of the four LAB after incubation in phosphate buffered saline at
different acidic pH values. Values are means ± standard deviations.
52 Paula Fajardo Bernárdez et al.

Figure 2. Survival of the four LAB after incubation in simulated gastric (GC) and
intestinal (IC) conditions. Controls (Cont.) in each case consisted on samples at the
same pH values without enzymes. Values are means ± standard deviations.

progressed. Although in general, the survival increased with the increase in

the incubation pH, sensitivities of L. lactis and Ent. faecium strains to acidic
pH were slightly higher than those observed for Lact. casei and Ped.
acidilactici.
The effects of simulated gastric and small intestinal transit on the
viabilities of the four LAB are shown in Figure 2. Although Ent. faecium
was the most sensitive strain, all bacteria showed a significant lost (P<0.05)
in viability after being incubated for 180 min with the simulated gastric juice
(upper part of Figure 2). The total declines in the initial bacterial counts
at the end of this assay were: 3.01-log10 (in case of Ped. acidilactici),
3.04-log10 (in case of L. lactis), 3.07-log10 (in case of Lact. casei) and
Effect of four lactic acid bacteria in weaned piglets 53

3.65-log10 (in case of Ent. faecium). Although the LAB counts decreased after
gastric transit, the four strains exhibited acceptable levels of survivability in
these conditions, since at least 2×106 CFU/mL (in case of E. faecium strain)
survived after 180 min of treatment.
Ped. acidilactici, L. lactis and Lact. casei retained viability during
simulated small intestinal juice and were considered intrinsically tolerant to
intestinal transit. Contrarily, Ent. faecium showed a 1.4-log10 cycle reduction
in viability and was considered intrinsically sensitive (lower part of Figure
2). Thus, there will, inevitably, be a loss of viability of probiotics during the
passage through the animal stomach and digestive system, due to pH, bile
acids and other factors. Consequently, successful colonization in the gut will
depend very much on the amounts of probiotic cells able to survive passage
through the animal stomach and digestive system.
These observations suggest that the survivability of the tested probiotic
cells in the porcine GIT could be increase through the administration of
viable culture concentrates incorporated into the feed. Thus, the feed could
act as a safe and protective vehicle for delivering the probiotic bacteria into
the gastrointestinal tract of piglets.

2.1. Fed-batch cultures of the four LAB

Production of highly concentrated cultures of probiotic bacteria allows to
supplement the piglet feed with a high number of viable probiotic cells while
keeping the feed moisture content low. With this procedure the growth of
undesirable mycotoxin-producing moulds in the feed could be controlled.
After performing the re-alkalized fed-batch cultures, a serie of batch
cultures of the four LAB were carried out during 18 h in complex culture
media (MRS or Rothe) and in whey-based media not only to obtain data for
comparisons but also to obtain information about their kinetic behaviour in
these media.
Whey was obtained from a local dairy plant in two forms: as
concentrated whey (CW: the liquid remaining after the first cheese pressing)
and as diluted whey (DW: CW mixed with wash waters). Before being used
as culture media, both wastes were deproteinized as described previously
[25]. The resulting mean composition of the DW medium after
deproteinization was (in g/L): lactose, 20.06; total nitrogen, 0.45; total
phosphorous, 0.25 and soluble proteins, 2.04. The deproteinized CW medium
contained (in g/L): lactose, 48.51; total nitrogen, 1.05; total phosphorous,
0.43 and soluble proteins, 5.02. Mussel processing wastes (MPW) containing
1% (w/v) glycogen were obtained from a local mussel processing plant.
Before being used as culture medium, this waste was deproteinized as
54 Paula Fajardo Bernárdez et al.

described previously [30]. To obtain a concentrated MPW medium (CMPW),

the deproteinized MPW medium was concentrated by ultrafiltration-dialysis
at 100 kD until obtaining a medium with an average glycogen concentration
of 100 g/L and a low content of NaCl, non-protein nitrogen and phosphorous
in comparison to that of the MPW medium. The glycogen contained in the
CMPW media was saccharified to glucose by enzymatic hydrolysis at 40ºC
for 1 h, using a San Super 240L commercial preparation of α-amylases
obtained from Novo Nordisk, Denmark. The enzyme:medium ratio was
1:1000 (v/v). The mean composition of the saccharified CMPW medium (in
g/L) was: glucose, 101.3; total nitrogen, 0.54; total phosphorous, 0.06 and
soluble proteins, 3.47. The media (DW, CW and CMPW) were supplemented
with yeast extract if needed, adjusted at pH 7.0, sterilised at 121ºC for 15 min
and then used as culture media.
The batch cultures were performed in 250 mL Erlenmeyer flasks
containing 50 mL of the corresponding fermentation medium (Tables 1
and 2) at initial pH of 7.0, without pH control, on a rotary shaker (Innova
4330, New Brunswick Scientific Co., Inc., New Jersey) at 30ºC and 200 rpm
for 18 h. In case of P. acidilactici, the fermentation was carried out in DWYE
medium (DW medium supplemented with a 2 % (w/v) yeast extract)
(Table 1), because this strain produced negligible amounts of biomass and
antibacterial substances in the non-supplemented DW medium [25].
The final productions of biomass (X), antibacterial activity (AA) and
lactic acid (LA) obtained in the batch cultures after 18 h of incubation in both
the complex culture media (MRS or Rothe broth) and whey-based media are
shown in Tables 1 and 2. As it can be observed, the complex culture media
yielded the higher concentrations of X, AA and LA for all strains than the DW
media. However, production of probiotic cells in complex culture broths at an
industrial scale could be very expensive due to the high cost of these media.
A possible alternative to solve this problem could be the production of the
probiotic cultures in whey-based media by using a more productive
fermentation method than the batch mode.
The fed-batch fermentation was found to be superior to batch processing
for many productions [31]. For this reason, the productions of highly
concentrated cultures of the four LAB were carried out in whey-based media
by using a repeated-batch fermentation technique with additional supply of
fresh substrates (containing glucose and/or lactose), and periodical re-
alkalizations of the culture medium [24,26,27,28,32].
The re-alkalized fed-batch cultures were carried out at a controlled
temperature of 30ºC in a 6 L bench top fermentor (New Brunswick Scientific,
Edison, New Jersey, U.S.A.) with an agitation speed of 200 rpm and
Effect of four lactic acid bacteria in weaned piglets 55

continuous-record of pH. The fed-batch fermentations were initiated as batch

processes with a working volume of 4 L of fermentation medium. The initial
pH was fixed in 7.0 and the aeration flow rate was maintained in 0.5 L/h. The
fermentation media used in the different re-alkalized fed-batch cultures were
DW medium (in case of L. lactis, Lact. casei and E. faecium) and DWYE
medium (in case of Ped. acidilactici). The feeding media consisted of a
mixture of CW medium and a 400 g/L concentrate lactose (in case of L. lactis
and E. faecium), CMPW medium and a 310 g/L concentrate glucose (in case
of Lact. casei) and CWYE (CW supplemented with a 2% (w/v) yeast extract)
medium and a 400 g/L concentrate glucose (in case of Ped. acidilactici).
The batch fermentations were converted into repeated re-alkalized fed-
batch mode by rapidly withdrawing of 100 mL of the culture from the
fermentor, when it reached the lower steady pH (12 h) as it was observed in
the batch cultures of each strain. After determining the total sugars
concentration in the sample withdrew, the amounts of sugars consumed by
each strain were calculated. The same volume (100 mL) of the feeding
substrates was fed to the residual growing culture in the fermentor to restore
the initial total sugars concentration in the fermentation medium.
Immediately after feeding, the medium was re-alkalized up to set pH of 7.0
with 4M NaOH. The re-alkalized fed-batch cultures were stopped when the
production of biomass slowed down.
The final amounts of biomass, antibacterial activity and other products
(lactic acid, acetic acid, ethanol and butane-2,3-diol) reached in each re-
alkalized fed-batch culture on DW are shown in Tables 1 and 2. In these
cultures, the active period increased from 18 h (in the batch cultures) to 264 h
(in case of L. lactis), to 240 h (in case of P. acidilactici), to 384 h (in case of
Lact. casei) and to 348 h (in case of E. faecium).
The concentrations of biomass (4.9 g/L), viable cells (2.62×1010 CFU/mL),
lactic acid (30.2 g/L) and antibacterial activity (374.0 AU/mL) produced by
Ped. acidilactici in the re-alkalized fed-batch culture were higher than those
produced in the corresponding batch fermentation in DWYE medium
(Table 1). As it was observed in previous re-alkalized fed-batch cultures
[24,27,28,29], a metabolic shift from homolactic to mixed-acid fermentation
was observed from 24 h of incubation, and other end products (acetic acid
and ethanol) other than lactate accumulated in the medium (Table 1). Thus,
the concentrations of ethanol and acetic acid reached values of 3.9 and 7.9
g/L, respectively at the end of the fermentation. The culture was stopped after
240 h of fermentation when the bacterium completely lost its ability to
recover the acidic pH.
56 Paula Fajardo Bernárdez et al.

Table 1. Fermentation and feeding culture media used for the different batch and re-
alkalized fed-batch fermentations of Ped. acidilactici and Lact. casei and average
concentrations of biomass (X), antibacterial activity (AA), lactic acid (LA), acetic
acid (Ac), ethanol (Et) and butane-2,3-diol (B) obtained at the end of the cultures.

DW: diluted whey, DWYE: DW supplemented with 2% (w/v) yeast extract, CW:
concentrated whey; CWYE: CW supplemented with 2% (w/v) yeast extract, CMPW:
concentrated mussel processing waste, CL: concentrated lactose, CG: concentrated glucose,
ND: not detected.

Table 2. Fermentation and feeding culture media used for the different batch and re-
alkalized fed-batch fermentations of L. lactis and Ent. faecium and average
concentrations of biomass (X), antibacterial activity (AA), lactic acid (LA), acetic
acid (Ac), ethanol (Et) and butane-2,3-diol (B) obtained at the end of the cultures.

Strains L. lactis Ent. faecium

Fermentation mode Batch Batch Fed-batch Batch Batch Fed-batch
Fermentation medium MRS DW DW Rothe DW DW
Feeding media - - CW and CL - - CW and CL
(400 g/L) (400 g/L)
Fermentation parameters
X (g/L) 1.6 0.4 2.4 1.6 0.2 3.6
9 9 10 9 8
CFU/mL 9.26 × 10 2.21 × 10 1.37 × 10 5.03 × 10 5.80 × 10 1.10 × 1010
AA (AU/mL) 49.6 22.5 84.0 ND ND 4.0
LA (g/L) 1.2 4.5 11.9 4.4 0.5 31.7
AcA (g/L) ND ND 1.3 ND ND 4.5
Et (g/L) ND ND ND ND ND 2.8
B (g/L) ND ND 3.6 ND ND 4.0
Active period (h) 18 18 264 18 18 348

DW: diluted whey, CW: concentrated whey, CL: concentrated lactose, ND: not detected.
Effect of four lactic acid bacteria in weaned piglets 57

For Lact. casei, a first re-alkalized fed-batch fermentation with feeding

with CW medium and a 400 g/L concentrated lactose was carried out in DW
medium (Table 1). However, the results obtained in this culture were not
satisfactory. Although the productions of antibacterial activity (10.0 AU/mL),
lactic acid (10.1 g/L) and acetic acid (0.6 g/L) were improved with this
fermentation technique, the concentrations of biomass (0.8 g/L) and viable
cells (7.9×109 CFU/mL) were only slightly higher than those obtained in the
batch culture in DW. In addition, the strain completely lost its ability to
recover the acidic pH after 156 h of incubation.
For these reasons, a second re-alkalized fed-batch fermentation with
Lact. casei on DW was performed, using a mixture of substrates containing
glucose (CMPW medium and a 310 g/L concentrated glucose) instead of
lactose as feeding media. The results obtained (Table 1) showed that the
addition of glucose improved the productions of biomass (1.2 g/L), viable
cells (1.26×1010 CFU/mL), antibacterial activity (57.0 AU/mL), lactic acid
(19.6 g/L) and acetic acid (3.1 g/L) as compared to the first fed-batch culture.
In addition, the strain retained its capacity for acidifying the culture medium
at the end of the fermentation. These observations suggest at least in part, that
glucose is a better carbon source than lactose for Lact. casei [33].
In the re-alkalized fed-batch culture of L. lactis [24], the shift towards
mixed-acid fermentation was observed from the 84 h of incubation. The
concentrations of biomass (2.4 g/L), viable cells (1.37×1010 CFU/mL),
antibacterial activity (84.0 AU/mL), lactic acid (11.9 g/L), acetic acid (1.3 g/L)
and butane-2,3-diol (3.6 g/L) obtained were also higher than those amounts
produced in the corresponding batch culture (Table 2). In addition, L. lactis was
able to bring about the decrease of pH at the end of the fermentation (264 h).
The final results obtained in the re-alkalized fed-batch culture of Ent.
faecium are shown in Table 2. Again a homolactic fermentation phase
(0-192 h) was followed by a mixed acid fermentation phase (192-348 h) and
increased amounts of biomass (3.6 g/L), viable cells (1.10×1010 CFU/mL),
lactic acid (31.7 g/L), acetic acid (4.5 g/L), ethanol (2.8 g/L), butane-2,3-diol
(4.0 g/L) and antibacterial activity (4.0 AU/mL) accumulated in the medium
after 348 h of fermentation.
In general, the use of this fed-batch culture technique led to an increase
in the metabolically active period of the cells. As a consequence, increased
final concentrations of viable cells and antimicrobial metabolites
(bacteriocins, lactic acid, acetic acid, butane-2,3-diol and ethanol) were
obtained as compared with the batch processes in both the complex culture
media (MRS or Rothe broth) and whey-based media. The shift from
homolactic to heterolactic or mixed-acid fermentation observed in all re-
58 Paula Fajardo Bernárdez et al.

alkalized fed-batch cultures has been observed before under certain

fermentation conditions and it was associated to a modification of pyruvate
metabolism with a decreased activity of lactate dehydrogenase [24,27,34].
Once stopped the re-alkalized fed-batch fermentations, the cultures were
adjusted to pH 7.0 to facilitate the adsorption of the bacteriocin onto the
producer strains [35]. Since the freezing method was found to be a more
effective method for preserving bacterial strains than the freeze-drying
method [4], the cultures of the four LAB were mixed with 30% (w/v)
skimmed milk and then stored frozen at -20ºC. These stock cultures were
used to prepare the probiotic supplemented piglet feeds.

2.2. Survival of the four LAB during freezing storage at -

20ºC
After producing the concentrated viable cultures of the four LAB, it is
essential to assure their storage for a long period of time to maintain their
properties. Therefore, the LAB strains must be adequately preserved in
order to supplement the animal feed with a high number of viable cells. In
addition, the use of probiotic freezing cultures has been proposed as an
alternative to improve their survival in the feed [4].
For these reasons, the four LAB cultures were preserved at -20ºC with
skimmed milk. The cell counts of the frozen cultures were checked before
freezing and then after freezing every two weeks for 12 weeks. To evaluate
the survival of frozen cultures, a screw cap glass tube was allowed to defrost
at room temperature. Subsequently, serial ten-fold dilutions were made in
sterile saline (0.8% of NaCl), plated in triplicate on MRS (in case of L. lactis,
Lact. casei and Ped. acidilactici) or Rothe (in case of Ent. faecium) agar, and
then incubated at 30ºC for 2 days. The results obtained are shown in
Figure 3.
The survivability of the four strains was not significantly affected after
being stored frozen at -20ºC with skimmed milk for 12 weeks (P<0.05).
Thus, the initial bacterial counts of each strain decreased slightly by day 84:
0.22-log10 (in case of Ped. acidilactici), 0.26-log10 (in case of Ent. faecium),
0.70-log10 (in case of L. lactis) and 0.25-log10 (in case of Lact. casei). Then,
skimmed milk proved to be an adequate cryoprotective agent not only for
preserving the four LAB cultures but also for maintaining their stability
during freezing storage for a long period of time. The good cryoprotective
capacity of the skimmed milk have been observed before by other researchers
[4,36], when they assayed skimmed milk and glycerol as cryoprotective
agents to preserve bacterial cultures.
Effect of four lactic acid bacteria in weaned piglets 59

Figure 3. Survival of Ped. acidilactici, Ent. faecium, L. lactis and Lact. casei during
storage at –20ºC with skimmed milk for 84 days. Values are means ± standard
deviations.

2.3. Survival of the four probiotic strains in the piglet feed

stored at room temperature
To exert beneficial effects in the host, probiotic dietary additives must
retain their viabilities and activities in the delivery vehicle before
consumption. However, acidic pH levels (generally pH 2–3) in the stomach
of piglets and exposure to pancreatic secretions such as digestive enzymes
and bile in the small intestine can reduce the number of orally administered
viable probiotics. Although some bacteria are more resistant than others to
this stress, consumption of probiotics with feed, which contains mineral
buffers or alkalinizing agents [37,38,39], can alleviate stomach acid and
increase the survivability of the probiotic cells into the intestine.
In this way, some researchers have observed that the use of food carriers
(e.g. milk, milk proteins and mucin, Cheddar cheese or yogurt) to deliver the
probiotic strains to the porcine GIT, greatly enhance both the gastric and
small intestinal transit tolerance of probiotic cells [40,41,42]. Therefore, with
the use of these protective agents, a high number of probiotic LAB could
survive passage through the animal stomach and digestive system and
colonize in the animal gut. These observations suggest that feedstuff could be
a used as a desirable delivery vehicle for probiotic strains.
For this reason, in the present study, the commercial piglet feed was
mixed with a 2% (v/w) of the corresponding defrosted culture of each LAB
and stored at room temperature. The survival of the LAB strains in these
conditions was then investigated every 2 days. The losses of viability of the
four cultures after 8 days were very similar, with average drops of 0.31-log10
(in case of Ped. acidilactici), 0.32-log10 (in case of L. lactis), 0.29-log10
(in case of Lact. casei) and 0.32-log10 (in case of Ent. faecium), and total
60 Paula Fajardo Bernárdez et al.

Figure 4. Survival of the four LAB in the piglet feed. Values are means ± standard
deviations.

declines of 2.35-log10, 2.57-log10, 2.41-log10 and 2.37-log10, respectively

(Fig. 4).
The results obtained suggest the convenience of supplementing the feed
with the frozen probiotic cultures each 7 days in order to avoid subsequent
losses of viability. With this approach, the piglet feed could be use as a
vehicle for administering the same amounts of LAB cells (about 109 CFU/g
of feed) to the animals at the beginning of each week of treatment.

2.4. Performance of piglets fed with different diets and feed

conversion
On the 21st day of age, a total of 80 piglets were distributed into 4 groups
of 20: the non-treated control group, two probiotic supplemented fed groups
and the antibiotic (colistin sulfate) supplemented fed group. This experiment
was divided in two trials. In the first, the two potentially probiotic strains tested
were Ped. acidilactici and Ent. faecium. In the second trial, which was
developed in the same conditions, the two probiotic strains assayed were
L. lactis and Lact. casei. Each group was housed separately in individual cages.
Throughout the experimental period, all the piglets were fed a basal diet
composed of 30.3% barley, 10% wheat, 16.5% corn, 1% animal fat, 5%
extruded full-fat soybean, 15% soybean meal 47, 3% potato protein, 16% whey
powder, 1% dicalcium phosphate-feed grade, 0.8% calcium carbonate and
1.4% mixture of minerals and vitamins. The diet contained 14.2 MJ
metabolisable energy per kg of feed, 18.5% total protein, 4% crude fat, 5.3%
ash, 0.8% calcium, 0.5% phosphorous, 1.2% lysine and 0.4% methionine. The
probiotic piglet feeds were prepared weekly by inoculation with 2% (v/w) of
the corresponding defrosted culture, in order to administer the same amounts of
probiotic cells to the animals at the beginning of each week of treatment. The
Effect of four lactic acid bacteria in weaned piglets 61

defrosted cultures contained a 30% skimmed milk and consequently, the

probiotic feeds contained 0.6% (v/w) skimmed milk. To be consistent, the
piglets belonging to the control group received the basal diet supplemented
with 0.6% (w/w) skimmed milk. In the group receiving antibiotic, the basal diet
containing 0.6% (w/w) skimmed milk was supplemented with 0.012 g of
colistin sulfate/kg of feed. Each experimental group was fed ad libitum with its
own diet for 42 days. The temperature of the room with continuous lighting
was maintained at 28ºC initially, and reduced 1ºC/week until reached 24ºC, at
which the room temperature was maintained for the end of the experiment.
Body weight (BW), feed intake (FI) and total coliform bacteria counts in faecal
samples were measured before the treatments and at 14, 28 and 42 days
thereafter the animals received the experimental diets. Body weight gain
(BWG) and feed conversion efficiency (FCE, kg of feed consumed per kg of
body weight gain) were then calculated at 14, 28 and 42 days of treatment. The
data concerning growth performance (body weight gain, feed intake, feed
conversion efficiency) were statistically analyzed using the software package
SPSS 12.0 for Windows (Release 12.0.1; Chicago IL: SPSS Inc; 2003). A one-
way analysis of variance (ANOVA) with the step-down multiple-stage F post
hoc test (Ryan-Einot-Gabriel-Welsch multiple F test (P = 0.05)) was used to
distinguish treatment mean differences. Normal distribution for data as well as
the independence and homogeneous variances between treatment groups were
previously verified by looking at the distribution of the data (via histograms)
and the Fisher F-test (which is included in the t-test output), respectively.
All the piglets remained healthy throughout the 42 days of the
experiment and there was no evidence of any detrimental or beneficial effect
on the health of the animals used in these studies due to the different diets
assayed. At the beginning of the experiments, the average weight of the
piglets was 7.74 kg (s.d. 0.01 kg) in the first experiment (left part of Fig. 5)
and 9.55 kg (s.d. 0.04 kg) in the second experiment (right part of Fig. 5).
The results obtained in the first trial (left parts of Figs. 5 and 6) showed
that the groups receiving colistin sulfate and probiotics (Ped. acidilactic and
Ent. faecium) exhibited higher average final BW, BWG, FI and lower FCE
values than the control group (P<0.05) after 42 days of treatment. However,
significantly more favourable results were observed in the group fed antibiotic
(P<0.05). From the comparison between the effects of each probiotic, it can be
observed that the group fed Ped. acidilactici NRRL B-5627 exhibited higher
mean BWG and lower mean FI and FCE values than the group fed Ent.
faecium CECT 410 for the whole experimental period (P<0.05).
In the second trial (right parts of Figs. 5 and 6), the group receiving
antibiotic provided the highest mean final BW, BWG, however the FI and the
62 Paula Fajardo Bernárdez et al.

Figure 5. Initial and final body weight (BW) of the piglets used in the first (left part)
and in the second (right part) trials. Values are means ± standard deviations.

Figure 6. Effect of dietary probiotics (Ped. acidialctici, Ent. faecium, L. lactis and
Lact. casei) or antibiotic (colistin sulfate) addition on growth performance parameters
of piglets during 42 days after weaning. BWG: body weight gain, FI: feed intake,
FCE: feed conversion efficiency. Values are means ± standard deviations.

FCE were significantly higher than those obtained in the other groups
(P<0.05). From the comparison between the groups receiving probiotics, it
can be observed that the Lact. casei CECT 4043 treated group showed higher
mean final BW, BWG and FI values than the group fed L. lactis CECT 539
Effect of four lactic acid bacteria in weaned piglets 63

(P<0.05). However, no significant differences in feed conversion efficiency

were observed between the groups receiving probiotics and the control group.
Some researchers have observed that the addition of organic acids to
piglet feeds can improve their performance [43,44] and decrease the
intraluminal concentration of coliform bacteria and other microorganisms in
the gastrointestinal tract [45]. These positive effects have been attributed to a
lowering of pH, mainly in the stomach, thus reducing the growth of
pathogenic bacteria [46], which can compete with the host for nutrients in the
stomach and small intestine [47]. Therefore, the direct addition of the
LAB cultures (cells + organic acids + bacteriocins) to the piglet feeds could
contribute to control the growth of pathogenic bacteria not only in the feed
but also in the gastrointestinal tract of the piglets, thus being beneficial for
the animals.
Early weaning represents a stressful condition for piglets, because the
indigenous microflora is still not completely established in young animals.
This commonly contributes to the appearance of infections and diarrhoea,
oedema and other digestive disorders leading to heavy losses for the pig
rearing industry [9,17]. Therefore, the first week after weaning has been
reported to be the most adequate period for establishing a convenient
relationship between the beneficial probiotic bacteria and pathogenic
microorganisms that constitute the intestinal microbiota of the piglets [2,9].
Although the efficacy of probiotic bacteria in promoting growth in post-
weaning piglets and other animals have been studied before [9], there are
discrepancies in the results obtained. Some researchers observed that the
administration of probiotic bacteria in the first days of life produced a
positive effect on growth and on incidence or duration of scouring in piglets
[1,2,3]. However other studies have shown that probiotics have no positive
effects on broilers, neither improving body weight [48,49,50] nor reducing
Salmonella carriage [51,52]. Our results showed that the weight gain of pigs
fed diets supplemented with antibiotic was higher (P<0.05) than that of pigs
fed diets supplemented with probiotic LAB (Ped. acidilactici, Ent. faecium,
L. lactis and Lact. casei) for the whole experiment period (days 1 to 42).
However, LAB supplemented diets resulted in a better performance (P<0.05)
than did the diets without additives (controls). The results obtained in the
present study demonstrated that feeding piglets with 109 UFC of probiotic
bacteria/g of feed/day could be a way for increasing the BWG of these
animals. This probiotic cells concentration in the feed is higher than the
recommended dose of viable probiotic (106 UFC of probiotic/g or mL)
necessary to obtain the beneficial effects [41,53].
64 Paula Fajardo Bernárdez et al.

Since the growth-stimulating effects of probiotic bacteria have been

observed when the piglets were exposed to stresses [1,3,9], a more
pronounced positive effect of the diets supplemented with Ped. acidilactici,
Ent. faecium, L. lactis and Lact. casei on the performance of piglets could be
expected in presence of health problems.

2.5. Total coliform counts of the intestinal content

Coliform counts were determined in the faecal samples before and after
(every two weeks) the piglets received the experimental diets. The faecal
samples were taken directly from the rectum of each animal using a hyssop.
Three replicates of faecal samples of each piglet were taken simultaneously.
Hyssops were weighed before and after taking the faecal samples to
determine the net weights of the samples. To be consistent, the weight of
each faecal sample was multiplied by nine to determine the amount of PBS to
be added to each tube to yield a 101 dilution. Hyssops were shaken vigorously
and then vortexed for 2 min. Ten-fold serial dilutions in sterile PBS were
performed up to 1010 and aliquots (0.1 mL) of 10-fold serial dilutions were
pour plated with eosin methylene blue agar (EMB, Levine Formulation). The
plates were inverted and incubated at 37ºC ± 1°C for 1 to 2 days. Incubated
plates were observed for the optimum number of CFU, between 30 and 300
colonies per plate. The results were expressed as the number of colonies
counted per gram (wet weight) of faeces [2].
Viable counts of coliforms in the faeces were transformed by logarithm
(log10) before statistical analysis of variance. The data were statistically
analyzed by the STAT software (Statsoft, Inc., Tulsa, OK, U.S.A.).
Preliminary analyses for these data were done by the mixed model ANOVA
methodology in the ANOVA/MANOVA procedure. Measurements on the
same piglet at different days were treated as repeated observations, and
unstructured covariance matrix for residuals was assumed. Estimated
covariances between pairs of residuals on different sampling days were not
significantly different from zero. Consequently, independent residuals were
assumed and the mixed model ANOVA methodology was used. The model for
coliform counts data contained the treatment group, the day of sampling and
the interaction between the group and the day of sampling as fixed class effects.
The data on viable plate counts of coliforms in the faeces of individual
animals are shown in Tables 3 and 4. In the first experiment, the
administration of single probiotic strain did not affect the viable counts of
coliform bacteria (Table 3), however the effect of the time of treatment was
evident (P=0.005). Thus, the higher coliform reductions (3.4-log10) were
obtained in the group receiving the antibiotic (P<0.05) followed by the group
Effect of four lactic acid bacteria in weaned piglets 65

Table 3. Effect of administration of concentrated cultures of Ped. acidilactici and Ent.

faecium and colistin sulfate on mean coliform counts in pigs. Values are means ±
standard deviations.

Treatment Day 0 Day 14 Reduction Day 28 Reduction Day 42 Reduction

counta counta (%) after counta (%) after counta (%) after
b c
14 days 28 days 42 daysd
e
Control 6.0 ± 0.2 6.2 ± 0.9 0 6.0 ± 0.5 0 5.5 ± 1.0 68.4
Ped. acidilactici 7.4 ± 0.1 6.6 ± 1.0 84.1 4.3 ± 0.5 99.9 4.1 ± 0.4 99.9
Ent. faecium 6.4 ± 0.2 6.4 ± 1.7 0 5.5 ± 1.3 87.4 4.5 ± 0.2 98.7
Colistin sulfate 7.2 ± 0.6 6.1 ± 1.6 92.1 4.0 ± 0.7 99.9 3.8 ± 0.6 99.9

a
Mean values of results for 20 pigs in log (CFU/g of faeces) ± SD.
b
Calculated as ((No-N)×100)/No, where No is the mean day 0 count and N is the mean day 14 (both
expressed as CFU/g of faeces) .
c
Calculated as ((No-N)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N is the mean
day 28 count (CFU/g of faeces).
d
Calculated as ((N-No)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N is the mean
day 42 count (CFU/g of faeces).
e
Counts increased in this treatment group.

Table 4. Effect of administration of concentrated cultures of L. lactis and Lact. casei

and colistin sulfate on mean coliform counts in pigs. Values are means ± standard
deviations.

Treatment Day 0 Day 14 Reduction Day 28 Reduction Day 42 Reduction

a a a a
count count (%) after count (%) after count (%) after
14 daysb 28 daysc 42 daysd
Control 7.3 ± 0.3 7.3 ± 0.6 0 7.1 ± 1.0 36.9 6.7 ± 0.4 74.9
L. lactis 7.4 ± 0.1 6.4 ± 0.7 90.0 6.0 ± 0.7 96.0 5.6 ± 0.5 98.4
Lact. casei 7.1 ± 0.1 6.3 ± 0.8 84.1 6.0 ± 0.6 92.1 5.7 ± 0.6 96.0
Colistin sulfate 7.3 ± 0.2 5.5 ± 0.5 98.4 5.1 ± 0.6 99.4 5.1 ± 0.2 99.4

a
Mean values of results for 20 pigs in log (CFU/g of faeces) ± SD.
b
Calculated as ((No-N)×100)/No, where No is the mean day 0 count and N is the mean day 14 (both
expressed as CFU/g of faeces) .
c
Calculated as ((No-N)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N is the
mean day 28 count (CFU/g of faeces).
d
Calculated as ((N-No)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N is the
mean day 42 count (CFU/g of faeces).
e
Counts increased in this treatment group.

receiving the Ped. acidilactici strain (3.3-log10), the group receiving the Ent.
faecium strain (1.9-log10) and the control group (0.5-log10). In this last group,
the total coliform counts in the faeces of the animals increased from day 1 to
day 14 and decreased from day 14 to day 42. No significant interaction
between time and treatment was observed in viable plate counts of coliforms
for this group.
66 Paula Fajardo Bernárdez et al.

Then, initial coliform counts (day 0) decreased by day 42 from 2.51 × 107
to 1.26 × 104, from 2.51 × 106 to 3.16 × 104 and from 2.51 × 107 to 1.26 × 104
in pigs fed strains Ped. acidilactici and Ent. faecium, and colistin sulfate,
respectively. Consequently the mean reductions obtained in these groups by
day 42 were respectively 99.9, 98.7 and 99.9% (Table 3). However, mean
coliform counts in the control group only decreased by 68.4% (from
1.00 × 106 to 3.16 × 105) by day 42.
In the second experiment (Table 4), the results showed a significant effect of
time (P=0.002) and the treatment (P=0.006), while the interaction between time
and treatment was not significant (P<0.05). The changes in the total coliform
population in the control group over time were not significant (P<0.05), while in
the groups fed probiotics (L. lactis CECT 539 and Lact. casei CECT 4043) and
antibiotic, the viable coliform counts significantly (P<0.05) dropped on average
for 1.8, 1.4 and 3.2 log units, respectively at the last sampling.
In this assay, initial coliform counts (day 0) decreased by day 42 from
2.51 × 107 to 3.98 × 105, from 1.26 × 107 to 5.01 × 105 and from 2.00 × 107 to
1.26 × 105 in pigs fed L. lactis and Lact. casei, and colistin sulfate,
respectively. Consequently the mean reductions obtained in these groups by
day 42 were respectively 98.4, 96.0 and 99.4% (Table 4). However, mean
coliform counts in the control group only decreased by 74.9% (from
2.00 × 107 to 5.01 × 106) by day 42.
The increase in the coliform population in the post-weaning piglets as it
was observed in the control group has been reported to be as an usual fact
[54]. However, such as increase was not observed in the groups of piglets fed
antibiotic or probiotics supplemented diets. Similarly, Bogovič-Matijašić et
al., [9] have reported an increase in coliform counts in non-treated post-
weaning piglets. Although these researchers did not observed the same
tendency in pigs fed diets supplemented with probiotics (Lactobacillus
gasseri K7 and LF221), the differences were attributed to normal variations
between the animals rather than the probiotic treatment.
The observed capability of the four potentially probiotic strains used
throughout this study to stimulate the growth together with their ability to
reduce coliform counts in the faeces of post-weaning piglets, highlight them
as suitable strains for widespread use in the pig industry.
After determining the impact of the administration of separate probiotic
cultures to piglet diets on performance and faecal coliform counts of weaned
piglets, the following experiment was conducted to investigate the effect of
probiotic administration for a period of time shorter (28 days) than that used
in the previous experiment (42 days). With this approach, the effect of
probiotic inclusion in weaned piglet diets could be evaluated in both the
Effect of four lactic acid bacteria in weaned piglets 67

administration (0-28 days) and the post-administration (28-42 days) periods.

Lact. casei CECT 4043 was selected as the probiotic strain for this study
because the piglets fed this bacterium performed slightly better than piglets
fed diets supplemented with Ped. acidilactici NRRL B-5627, L. lactis CECT
539 and Ent. faecium CECT 410.

2.6. Effects of dietary probiotic Lact. casei CECT 4043 and

avilamycin on performance of piglets during both the
administration and post-administration period
The main objective of this assay was to investigate comparatively the
performance and intestinal coliform counts in weaned piglets fed other
antibiotic (avilamycin) and a more concentrated culture of Lact. casei than
that used in the previous assay. In this case, the feeding trial consisted of two
consecutive periods: administration period (from 1 to 28 days) and post-
administration period (from 29 to 42 days). The highly concentrated culture
of Lact. casei CECT 4043 was obtained in MRS broth by using the re-
alkalized fed-batch fermentation technique described above. The culture was
fed with a mixture of sterile fresh substrates composed of MRS broth and a
400 g/L concentrated glucose. The final composition of this culture was:
biomass, 6.6 g/L; viable cells number, 6.24×010 CFU/mL, antibacterial
activity, 30 AU/mL; lactic acid, 71.7 g/L; proteins, 6.9 g/L; total nitrogen, 1.7
g/L; total phosphorous, 0.2 g/L; total sugars, 12.8 g/L. This culture was
preserved using the same procedure as described above for the four LAB
cultures. Then, the Lact. casei culture in MRS broth was mixed with 30 %
(w/v) skimmed milk and stored frozen at -20ºC until further use. The feed
was weekly supplemented with a 2% (v/w) of the frozen culture. With this
procedure, the viable cell concentration in the feed (1.25×109 CFU/g of feed)
was higher than that of the probiotic feed (2.52×108 CFU/g of feed) prepared
with the culture of Lact. casei in whey (Table 1).
For this assay, a total of 120 piglets were distributed into 3 groups: two
treatment groups and a control group, with 40 piglets (divided in 5 replicates
of 8 piglets) in each. Groups were randomly assigned to following treatment
groups, (1) Basal diet + 0.6% (w/w) skimmed milk (control group), (2) Basal
diet + 0.6% (w/w) skimmed milk supplemented with 20 mL of the Lact. casei
culture/kg of feed and (3) Basal diet + 0.6% (w/w) skimmed milk
supplemented with 0.012 g of avilamycin/kg of feed. The basal diet used in
this experiment had the same mean composition as that of the previous
experiment. Each group was housed separately in individual cages. Each
experimental group was fed ad libitum with its own diet for 28 days. After
68 Paula Fajardo Bernárdez et al.

this period, all the groups were fed with the basal diet until the 42 days of
experiment. The temperature of the room with continuous lighting was
maintained at 28ºC initially, and then reduced 1ºC/week until it reached 24ºC,
at which the room temperature was maintained for the rest of the experiment
as indicated before.
The piglets were weighed on days 1, 14, 28 and 42 of the trial to calculate
body weight gain. Feed intake of each animal was measured on days 14, 28 and
42. Feed conversion efficiency was calculated on days 14, 28 and 42 days as kg
of feed consumed per kg of body weight gain. Experimental results were
statistically analyzed as indicated above in the previous study.
According to the results of the present assay (Fig. 7), the positive effects
produced by the antibiotic on the BWG of the animals remained until the end
of the experiment. The administration of Lact. casei CECT 4043 cells during
28 days was an effective way for promoting BWG of the treated animals as it
was observed before when this strain was administered to the piglets for a
period of 42 days (Fig. 6). However, this positive effect disappeared in the
post-administration period (from 29 to 42 days), when the piglets fed the
experimental diet without probiotics.
Similar results were obtained by other researchers [55], who observed
that feeding the piglets with a probiotic diet during both the growing and at a
part of the finishing stages of growth resulted in a significant improvement in
body weight gain, feed conversion and carcass quality, compared with the
results obtained when the piglets received the probiotic diet only during the
weaning stage. These differences could be probably related with an inability
of the probiotic strains to colonize and persist in the gastrointestinal tract in
the post-administration period. This was explained by the fact that the
probiotic cells are progressively supplanted by the bacteria of the intestinal
microflora once probiotic administration stopped. This leads to a decline in
the numbers of probiotic bacteria in the piglet digestive tract [23,56,57,58].
This decline is a common phenomenon, which has been observed before for
other lactobacilli strains [23,56,57]. In fact, some of these bacteria were
capable of persisting in the porcine gastrointestinal tract for a period of time
between 3 to 10 days post-administration [23,56,57,58].
The results obtained indicated that administration of the potential
probiotic preparation at the dose of 20 ml/kg of feed that is equal to
1.25 × 109 CFU of Lact. casei per g of feed can improve the performance
parameters of the piglets during the administration period. This offers the
possibility of using the piglet feed as a way to administer the probiotic
bacterium at levels higher than the recommended dose of viable probiotic
(106 CFU of probiotic/g or ml) necessary to observe beneficial effects
Effect of four lactic acid bacteria in weaned piglets 69

Figure 7. Effect of dietary probiotic (Lact. casei) or antibiotic (avilamycin) addition

on growth performance parameters of weaned piglets during the administration (1 to
28 days) and the post-administration (28 to 42 days) periods. BWG: body weight gain,
FI: feed intake, FCE: feed conversion efficiency. Values are means ± standard
deviations.

[41,53]. However, the period of administration of the probiotic culture of

Lact. casei should be extended in order to improve its beneficial effect on the
piglets.

2.7. Effects of dietary probiotic Lact. casei CECT 4043 and

avilamycin on intestinal coliform counts of piglets during
both the administration and post-administration period
Some studies showed that the administration of LAB caused a reduction
in intestinal coliform counts in the faeces of the piglets [2,59,60,61,62],
perhaps by a competitive exclusion mechanism [40] or due to the production
of organic acids (lactic and acetic acids) and bacteriocins [29]. But more
often such effects are not significant, except when the animals are challenged
with selected pathogenic strains or in gnotobiotic animals [9]. In contrast,
other studies have shown no effects [40,63].
From the results obtained in this assay (Table 5), it is difficult to draw
clear conclusions on the impacts of probiotic administration on intestinal
coliform counts, not only due to the variations observed in counts between
70 Paula Fajardo Bernárdez et al.

Table 5. Effect of administration of probiotic or antibiotic on mean coliform counts in

pigs.

Treatment Day 0 Day 28 Reduction (%) Day 42 Increment (%)

a a b a
count count after 28 days count after 42 daysc
Control 8.7 ± 1.5 7.3 ± 1.1 96.4 8.0 ± 1.2 80.7
Probiotic 8.8 ± 1.7 7.2 ± 0.6 97.3 7.9 ± 1.3 86.7
Avilamycin 8.5 ± 1.5 6.5 ± 0.9 99.0 7.9 ± 1.5 76.4
a
Mean values of results for 40 pigs in log (CFU/g of faeces) ± SD.
b
Calculated as ((No-N)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N
is the mean day 28 count (CFU/g of faeces).
c
Calculated as ((N-No)×100)/No, where No is the mean day 0 count (CFU/g of faeces) and N
is the mean day 42 (CFU/g of faeces).

individual animals and at different sampling time but also by the fact that the
reduction was also observed in the control group. This apparent contradiction
with the previous results could be related with the individual variations in the
responses of different animals due to the complexity of the intestine [23].
On the other hand, since the growth-stimulating effects of probiotic
bacteria have been observed when the piglets were exposed to stresses
[1,3,9], a more pronounced positive effect of the diets supplemented with
Lact. casei should be expected in presence of health problems.
In conclusion, as probiotics are generally considered to be harmless, the
findings of this study further support the fact that the use of antibiotics for
improving both the health and well-being of animals and the production
results can be reduced. This could be a way for minimizing the risks for
public health, such as the development of antibiotic-resistant bacteria which
are pathogenic to humans or animals as well as the presence of antibiotic
residues in edible animal products.

Acknowledgements
The research presented in this paper was financially supported by the
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
(INIA), Spain (project CAL01-045-C2-2) and The Xunta de Galicia, Spain
(project PGIDT00BIO1E). We thank COREN, S.C.L. for their collaboration
in the elaboration of this work.

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