Animal Feed Science and Technology

134 (2007) 89–107

Production of four potentially probiotic lactic acid

bacteria and their evaluation as feed additives
for weaned piglets
Nelson Pérez Guerra a , Paula Fajardo Bernárdez a , Jesús Méndez b ,
Pilar Cachaldora b , Lorenzo Pastrana Castro a,∗
a Departamento de Bioquı́mica, Xenética e Inmunoloxı́a, Facultade de Ciencias de Ourense,
Universidade de Vigo, As Lagoas s/n, 32004 Ourense, Spain
b Cooperativas Orensanas Sociedad Cooperativa Ltda (COREN),

Polı́gono San Ciprián de Viñas, 32901 Ourense, Spain

Received 26 January 2006; received in revised form 14 April 2006; accepted 8 May 2006

Abstract

The present study was conducted to study the production and evaluation of potentially probiotic
additives containing both live lactic acid bacteria (Pediococcus acidilactici NRRL B-5627, Lactococ-
cus lactis subsp. lactis CECT 539, Lactobacillus casei subsp. casei CECT 4043 and Enterococcus
faecium CECT 410) and antimicrobial metabolites with could be used as a replacement for antibiotics
in weanling pig diets. The gastrointestinal transit tolerance of the four bacteria was determined by
exposing washed cell suspensions at 30 ◦ C to acidic conditions (pH 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. These studies showed that the four strains are capable of
surviving the passage through the gastrointestinal conditions. Therefore, the production of biomass
and antimicrobial products by these bacteria was performed in whey using a fed-batch fermentation

Abbreviations: BWG, body weight gain; CECT, Spanish Type Culture Collection; CMPW, concentrated
mussel-processing wastes medium; CW, concentrated whey medium; CWYE, concentrated whey medium sup-
plemented with a 2% (w/v) yeast extract; DW, diluted whey medium; DWYE, DW medium supplemented with a
2% (w/v) yeast extract; FCE, feed conversion efficiency; FI, feed intake; LAB, lactic acid bacteria; MRS, de Man,
Rogosa and Sharpe medium; PBS, phosphate-buffered saline
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.

E-mail address: pastrana@uvigo.es (L. Pastrana Castro).

0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2006.05.010
90 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

technique that provided enhanced productions compared to those obtained in batch fermentations.
The obtained fed-batch cultures preserved at −20 ◦ C with skim milk showed a good viability after 3
months of storage. The four cultures exhibited low losses of viability in the piglet feed during their
storage at room temperature for 8 days. These results offered the possibility of using the piglet feed
as a vehicle to administer the four probiotic bacteria. The effects of the supplementation of separate
potentially probiotic cultures and an antibiotic (colistin sulfate) to piglet diets on body weight gain,
feed intake, feed efficiency and on the faecal coliform counts of weaned piglets were also studied.
Although the best results were obtained in the groups receiving the antibiotic, a significant increase in
body weight gain and final body weight was obtained in the groups fed diets supplemented with lactic
acid bacteria as compared with the non-treated (controls) groups (P=0.05). The changes in the total
coliform population in the control groups over time were not significant (P<0.05), while in the groups
fed probiotics and antibiotic, the viable coliform counts significantly dropped at the last sampling
(P<0.05). These results suggest that the lactic acid bacteria used in this study could be used as suitable
strains for widespread use in the pig industry.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Body weight; Feed intake; Gastrointestinal tract; Lactic acid bacteria; Piglets; Probiotic

1. Introduction

The efficiency of animal digestion depends on the microorganisms which live naturally in
its digestive tract. Growth-promoting antibiotics have been used therapeutically in animal
feed to improve the health and well-being of animals and for prophylactic purposes to
improve the production results extensively in poultry and piglet industries. However, the
use of these growth-promoting substances has led to an imbalance of the beneficial intestinal
flora and the development of antibiotic-resistant bacteria which are pathogenic to humans
or animals (Abe et al., 1995; Pascual et al., 1999).
In recent years, there has been a considerable interest in using some probiotic microor-
ganisms and organic acids as an alternative to the use of antibiotics in feeds. Probiotics are
viable microorganisms and supportive substances that once ingested by humans and ani-
mals, produce beneficial physiology effects by assisting in the establishment of an intestinal
population which is beneficial to the host entity and antagonistic to harmful bacteria. The
natural adaptation of many lactic acid bacteria (LAB) to the gut environment and the antimi-
crobial substances produced by them (organic acids and bacteriocins) have provided these
organisms with a competitive advantage over other microorganisms to be used as probiotics
(Salminen et al., 1998; Bogovič Matijašić et al., 2004).
From a practical point of view, the administration of probiotic bacteria and their metabo-
lites has found to be an effective way to promote body weight and feed conversion in farm
animals (Abe et al., 1995; Denli et al., 2003). However, the relatively high prices of both
the commercial probiotic products and organic acids along with the high concentrations
in which they are needed in the feed in order to observe a significant effect, make the use
of these products uneconomic in the pig industry. The joint production of high amounts
of biomass and antimicrobial metabolites in a cheaper culture medium using an adequate
cultivation method (like the fed-batch mode) could be an appropriate alternative to solve
this problem (Guerra and Pastrana, 2003; Guerra et al., 2005).
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 91

In this study, the effect of simulated gastric and small intestine transit on the viability of
four potentially probiotic LAB (Pediococcus acidilactici NRRL B-5627, Lactococcus lactis
subsp. lactis CECT 539, Lactobacillus casei subsp. casei CECT 4043 and Enterococcus
faecium CECT 410) was assessed. Subsequently, the production of high amounts of biomass
and antimicrobial products by the four LAB was carried out using re-alkalized fed-batch
fermentations and cheaper culture media (whey and mussel-processing wastes). Then, the
survival of the four cultures after storage with skim milk at −20 ◦ C during 3 months and
in piglet feed at room temperature were studied. Finally, the potential of the four strains to
be used as probiotic cultures was investigated by determining their impact on body weight
gain, feed intake, feed conversion efficiency and faecal coliform counts in piglets.

2. Materials and methods

2.1. Bacterial strains

Pediococcus acidilactici NRRL B-5627 (a pediocin-producing strain) was obtained from

the Northern Regional Research Laboratory (NRRL, Peoria, IL, USA). Lactococcus lactis
subsp. lactis CECT 539 (a nisin-producing strain), Lactobacillus casei subsp. casei CECT
4043 (a high lactic acid-producing strain), Enterococcus faecium CECT 410 and Carnobac-
terium piscicola CECT 4020 (the target organism in the antibacterial-activity assay) were
obtained from the Spanish Type Culture Collection (CECT). Stock cultures of the four LAB
were maintained at −40 ◦ C in Nutrient broth supplemented with 15 mL of glycerol per litre
of medium. Working cultures maintained at 4 ◦ C on de Man, Rogosa and Sharpe (MRS)
agar (for Ped. acidilactici, L. lactis, Lact. casei and C. piscicola) or Rothe agar (for Ent.
faecium) were prepared monthly from frozen stock cultures.

2.2. Tolerance to acidic pH

The four potentially probiotic strains were grown in MRS (for Ped. acidilactici, L. lactis
and Lact. casei) and Rothe broth (for Ent. faecium) at 30 ◦ C overnight in culture tubes, then
subcultured into the corresponding medium for each strain during 18 h. The cultures were
centrifuged at 5000 × g for 10 min at 4 ◦ C, the pellets washed in sterile phosphate-buffered
saline (PBS: 10 mM sodium phosphate monobasic, 10 mM sodium phosphate dibasic,
130 mM sodium chloride, pH 7.2), and resuspended in sterile PBS. Each strain was diluted
1/100 in sterile PBS at pH 1.0, 2.0, 3.0, 4.0 and 5.0. Incubation times were 1, 2 and 4 h. Serial
decimal dilutions in sterile PBS were prepared, and aliquots (0.1 mL) from the dilutions were
then spotted on MRS or Rothe agar plates for determining the surviving cells number. The
experiment was repeated twice and each reading represents the mean of three observations.

2.3. Preparation of simulated gastric and pancreatic juices

Gastric and pancreatic juices were prepared fresh by dissolving pepsin (Sigma) from
porcine stomach mucosa (3 g/L) and pancreatin (Sigma) from porcine pancreas (1 g/L) in
sterile saline (5 g/L) according to Charteris et al. (1998). Subsequently, the pHs of the gastric
92 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

and pancreatic preparations were, respectively, adjusted to 2.0 and 8.0 with 12 M HCl or
0.1 M NaOH.

2.4. Exposure of the four bacteria to simulated gastrointestinal juices

The four bacterial strains were incubated in their respective broths (MRS or Rothe) at
30 ◦ C for 24 h. A 1-mL aliquot of each culture was centrifuged at 5000 × g for 10 min at
4 ◦ C and washed three times in sterile PBS. The washed cells were resuspended in 300 ␮L
PBS. The total viable counts of the washed cell suspensions were determined as described
above prior to assay of gastrointestinal transit tolerance.
The tolerance of the four LAB to simulated gastrointestinal juices was determined by
mixing 0.2 mL of each washed cell suspension with 1 mL of gastric or intestinal juice.
After brief vortexing, the mixtures were incubated at 37 ◦ C. When assaying gastric transit
tolerance, aliquots of 0.1 mL were removed after 5, 40 and 180 min for determination of
total viable count. When assaying for small intestinal transit tolerance, the sampling times
were 5, 240 and 360 min. The experiment was repeated twice and each reading represents
the mean of three observations.

2.5. Determination of total viable counts

Following exposure to juices, total viable counts of Pediococcus, Lactococcus, Lacto-

bacillus and Enterococcus strains were determined by a pour plate method (in triplicate)
using MRS or Rothe agar after serial 10-fold dilution in PBS. Plates were incubated at 30 ◦ C
for 48 h.

2.6. Culture media, inocula preparation and fermentation conditions

The compositions of the different media used in the different batch and re-alkalized
fed-batch fermentations are shown in Table 1. The preparation of these wastes to be used as
culture media was described previously (Guerra et al., 2001; Guerra and Pastrana, 2002).
Working cultures were propagated twice at 30 ◦ C for 12 h in diluted whey medium (DW;
initial pH 7.0) containing 1 g of yeast extract/100 mL of DW, prior to use as the inoculum.
All fermentations were inoculated with 20 mL of inoculum/L of medium.

Table 1
Mean composition of the culture media obtained from whey and mussel-processing wastes
Medium components Mean composition (g/L)

DW CW DWYE CWYE CMPW

Total sugars 20.54 48.11 24.34 51.65 101.33
Proteins 2.04 5.02 12.42 16.13 3.47
Total nitrogen 0.45 1.05 3.10 3.69 0.54
Total phosphorus 0.25 0.43 0.89 1.15 0.06
DW, diluted whey medium; CW, concentrated whey medium; DWYE, DW medium supplemented with 20 g of
yeast extract/L of medium; CWYE, concentrated whey medium supplemented with 20 g of yeast extract/L of
medium; CMPW, concentrated mussel-processing wastes medium.
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 93

Table 2
Culture media used for the different re-alkalized fed-batch fermentations
Strain Fermentation medium Feeding media
Ped. acidilactici DWYE CWYE medium and a 400 g/L concentrate glucose
L. lactis DW CW medium and a 400 g/L concentrate lactose
Lact. casei DW CMPW medium and a 310 g/L concentrate glucose
E. faecium DW CW medium and a 400 g/L concentrate lactose

Batch cultures for each strain were carried out to obtain data for comparison. These
cultures were performed in 250 mL Erlenmeyer flasks containing 50 mL of the correspond-
ing fermentation medium, on a rotary shaker (Innova 4330, New Brunswick Scientific Co.
Inc., NJ) at 30 ◦ C and 200 rpm for 32 h. L. lactis, Lact. casei and Ent. faecium strains were
grown in DW medium. Ped. acidilactici was grown in DW supplemented with 20 g of yeast
extract/L of medium (DWYE medium) because this bacterium produced negligible amounts
of biomass in DW medium (Guerra et al., 2001).
The re-alkalized fed-batch cultures were carried out at a controlled temperature of 30 ◦ C
in a 6 L bench top fermentor (New Brunswick Scientific, Edison, NJ, USA) with an agitation
speed of 200 rpm and 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. Table 2 shows the
different culture media used as fermentation and feeding substrates.
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 4 M NaOH.
At the end of each re-alkalized fed-batch culture, the medium was adjusted to pH 7.0 to
facilitate the adsorption of the bacteriocin onto the producer strains (Guerra et al., 2001).
After mixing the cultures with skim milk (300 g/L of fermented medium), the mixtures
were stored frozen at −20 ◦ C. These stock cultures were used to prepare the probiotic
supplemented piglet feeds.

2.7. Analytical determinations

The concentrations of biomass, total sugars, phosphorous, nitrogen, protein, lactic acid,
acetic acid, butane-2,3-diol, ethanol and bacteriocins were determined by methods described
in previous works (Guerra et al., 2001; Guerra and Pastrana, 2003).

2.8. Survival of the four LAB during freezing storage

The viability of the frozen stock cultures was checked before and after freezing. For this
purpose, a screw cap glass tube was defrosted weekly at room temperature. Subsequently,
94 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

serial 10-fold dilutions were made in sterile PBS, plated in triplicate on MRS or Rothe agar
depending on the strain, and then incubated at 30 ◦ C for 2 days.

2.9. Survival of the four probiotic strains in the piglet feed stored at room temperature

The survival of the probiotic bacteria in piglet feed was studied using the freezing cultures
of each LAB obtained as described above. The commercial piglet feed was mixed with the
corresponding defrosted culture (20 mL/kg of feed) and stored at room temperature. Daily,
duplicate samples (10 g) of probiotic supplemented piglet feed were mixed 1:10 with sterile
PBS and vortexed for 2 min. Both samples were serially diluted using sterile PBS and each
dilution was plated in triplicate in MRS or Rothe agar. The plates were incubated at 30 ◦ C
for 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 feed.

2.10. Treatment of piglets, preparation of experimental diets and analysis

On the 21st day of age, a total of 80 piglets were distributed into 4 groups of 20: the
non-treated control group, 2 probiotic supplemented fed groups and the antibiotic (colistin
sulfate) supplemented fed group. Each group was housed separately in individual cages.
The composition of the basal diet used is shown in Table 3. 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 28 ◦ C initially, and reduced 1 ◦ C/week until reached 24 ◦ C, at
which the room temperature was maintained for the end of the experiment.
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.

Table 3
Percentage composition of the experimental diets for weaned pigs
Composition % Calculated nutrient content
Barley 30.3 Metabolizable energy (MJ/kg) 14.20
Wheat meal 10.0 Total protein (%) 18.50
Corn meal 16.5 Crude fat (%) 4.00
Animal fat 1.0 Ash (%) 5.30
Integral soybean 5.0 Ca (%) 0.80
Soybean 47 15.0 Available phosphorus (%) 0.46
Potato protein 3.0 Lysine (%) 1.21
Whey powder 16.0 Methionine (%) 0.41
Dicalcium phosphate-feed grade 1.0
Sodium hydrogen carbonate 0.8
Vitamin/mineral premixa 0.21
a Premix contains per kg: Vitamin A 1.5 × 107 IU, Vitamin D 2 × 106 IU, Vitamin E 5 × 104 IU, Vitamin K 1.7 g,

thiamine 2.1 g, riboflavin 6.0 g, pantothenic acid 15.0 g, pyridoxine 3.0 g, cyanocobalamin 25.0 mg, nicotinic acid
36 g, folic acid 0.6 g, biotin 0.2 g, Co 0.15 g, Cu 11 g, Fe 90 g, Mn 55 g, Y 1 g, Se 0.25 g, Zn 110 g.
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 95

In the probiotic supplemented fed groups, the feed was supplemented with 20 mL of the
appropriate probiotic culture/kg of feed. In the group receiving the antibiotic, the basal diet
was supplemented with 0.012 g of colistin sulfate/kg of feed.
Body weight gain (BWG), feed intake (FI), feed conversion efficiency (FCE) 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.

2.11. Coliform counts in faecal samples

Coliform counts were determined in the faecal samples, which were taken directly from
the rectum of each animal with a sterile cotton swab (Collection swab; EUROTUBO,
Madrid, Spain). Three replicates of faecal samples of each piglet were taken simultaneously.
The cotton swabs 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 sterile PBS to be added to each tube to
yield a 101 dilution. The cotton swabs 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 37 ± 1 ◦ C for 1–2 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 (Tortuero et al., 1995).
All media or supplements (Nutrient broth, MRS agar and Rothe agar, methylene blue agar,
glycerol, yeast extract) were obtained from Cultimed Panreac Quı́mica S.A., Barcelona,
Spain.

2.12. Statistical analysis

The data concerning growth performance (body weight gain, feed intake and feed con-
version efficiency) were statistically analyzed using the software package SPSS 12.0 for
Windows (Release 12.0.1; SPSS Inc., Chicago, IL, 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. Nor-
mal distribution for data as well as the independence and homogeneity of 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.
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, USA). 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 covari-
ance matrix for residuals was assumed. Estimated covariances between pairs of residuals
on different sampling days were not significantly different from zero. Consequently, inde-
pendent residuals were assumed and the mixed model ANOVA methodology was used. The
96 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

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.

3. Results

3.1. Effect of physiological saline pH on survival of the four potentially probiotic LAB

The results of acid tolerance (survival at various pH values) showed that relatively high
populations of each strain survived an incubation period of 4 h at pH 2.0, 3.0, 4.0 and 5.0
(Fig. 1). Lact. casei and Ped. acidilactici survived acidic conditions slightly better than L.
lactis and Ent. faecium. For all strains, the increase in the incubation pH led to an increase
in the survival (P<0.05). However, a significant decrease (P<0.05) in the survival was noted
at pH 1.0 when the exposure time progressed for all LAB.

3.2. Gastrointestinal transit tolerance

The effects of simulated gastric and small intestinal transit on the viabilities of the four
LAB are shown in Fig. 2. Although Ent. faecium showed the lowest level of survival during

Fig. 1. Survival of Ped. acidilactici, L. lactis, Lact. casei and E. faecium after incubation in phosphate buffered
saline at pH 1.0 (), 2.0 (), 3.0 (), 4.0 () and 5.0 ().
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 97

Fig. 2. Effect of simulated gastric () and intestinal () transit on viability of Ped. acidilactici, L. lactis, Lact.
casei and E. faecium. Controls (䊉; ) consisted on samples at the same pH values without enzymes.

simulated gastric transit, all bacteria showed a significant lost (P<0.05) in viability after
their incubation for 180 min with the simulated gastric juice. At the end of this assay, the
total declines in the initial bacterial counts 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 3.65-log10 (in case
of Ent. faecium).
98 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

Table 4
Mean composition of the probiotic preparations obtained from re-alkalized fed-batch cultures of the four LAB
Composition Strains

Ped. acidilactici L. lactis Lact. casei E. faecium

Total sugars (g/L) 14.75 18.29 19.55 20.14
Total nitrogen (mg/L) 430.10 122.20 190.40 69.65
Total phosphorous (mg/L) 210.10 84.30 16.20 60.36
Total protein (g/L) 4.28 0.65 0.30 0.33
Biomass (g/L) 4.88 2.41 1.24 3.61
Colony-forming units (CFU/mL) 2.62 × 1010 1.37 × 1010 1.26 × 1010 1.10 × 1010
pH 7.00 7.00 7.00 7.00
Bacteriocin titres (BU/mL) 374.00 84.00 57.00 –
Lactic acid (g/L) 30.20 11.91 19.62 31.66
Acetic acid (g/L) 3.90 1.28 3.06 4.46
Ethanol (g/L) 7.90 – – 2.81
Butane-2,3-diol (g/L) – 3.56 – 4.03

Ped. acidilactici, L. lactis and Lact. casei retained viability during simulated small intesti-
nal juice and are considered intrinsically tolerant to intestinal transit. Contrarily, Ent. faecium
showed a 1.4-log cycle reduction in viability and is considered intrinsically sensitive.

3.3. Production of probiotic preparations

The mean composition of the probiotic preparations obtained from the re-alkalized fed-
batch fermentations of Ped. acidilactici, L. lactis, Lact. casei and Ent. faecium on whey are
shown in Table 4. The probiotic preparations contained different antimicrobial metabolites
(lactic acid, acetic acid, ethanol, butane-2,3-diol and bacteriocins), which were accumulated
in the culture media as a consequence of the mixed-acid fermentation developed by the four
LAB in these cultures. Although Ped. acidilactici, L. lactis, Lact. casei strains produced
relatively higher amounts of bacteriocins, no bacteriocin activity was detected in the re-
alkalized fed-batch culture of Ent. faecium.

3.4. Viability of LAB cultures after freezing in skim milk at −20 ◦ C

The freeze-drying method has found to be less effective to preserve bacterial strains for
a long period of time than the freezing method (Pascual et al., 1999). For this reason, this
last method was used to preserve the four probiotic LAB used through this work.
The results obtained (Fig. 3) showed that freezing method had not a significant effect
(P<0.05) on the bacterial survival and all strains had a good viability at −20 ◦ C after 3
months with skim milk. Thus, only little total declines in the initial bacterial counts were
observed: 0.22-log10 (in case of Ped. acidilactici), 0.70-log10 (in case of L. lactis), 0.25-
log10 (in case of Lact. casei) and 0.26-log10 (in case of Ent. faecium).

3.5. Survival of LAB during storage of the piglet feed at room temperature

To exert beneficial effects in the host, it is essential that LAB be alive and abundant in
the feed at the time of consumption. Therefore, survival of the LAB strains in the piglet
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 99

Fig. 3. Survival of Ped. acidilactici (), E. faecium (), L. lactis () and Lact. casei () during storage at −20 ◦ C
with skim milk.

feed during storage at room temperature was investigated. The results obtained (Fig. 4)
showed that after 8 days, the losses of viability of the four cultures 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 declines of 2.35-log10 , 2.57-log10 , 2.41-log10 and 2.37-log10 , respectively. So that, the
piglet feeds were prepared weekly by inoculation with the freezing cultures in skim milk in
order to administer the same amounts of LAB (about 109 CFU/g of feed) to the animals at
the beginning of each week of treatment. These results offered the possibility of using the
piglet feed as a way to administer the four probiotic bacteria.

3.6. Performance of piglets fed with different diets

The effect of probiotic addition on growth performance parameters of the piglets was
also assessed. 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

Fig. 4. Survival of Ped. acidilactici (), E. faecium (), L. lactis () and Lact. casei () in the commercial feed
during storage at room temperature.
100 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

Table 5
Effect of dietary probiotics (B-5627, CECT 410) or antibiotic (colistin sulfate) addition on growth performance
parameters of piglets during 42 days after weaning
Treatmenta Initial BW (day 1) (g) Final BW (day 42) (g)
Piglet performance
Control 7750 ± 51a 28884 ± 154a
B-5627 7730 ± 19a 29506 ± 195ab
CECT 410 7750 ± 31a 29345 ± 146ab
CS 7750 ± 20a 30750 ± 128b
F 0.92 255.51
d.f. 3, 40 3, 40

Treatmenta BWG (g per piglet) FI (g per piglet) FCE (g of FI/g of BWG)

Piglet performance (days 1–14)
Control 4500 ± 37a 6530 ± 86a 1.451 ± 0.016a
B-5627 4620 ± 39ab 6230 ± 41ab 1.349 ± 0.031ab
CECT 410 4530 ± 34a 6130 ± 83b 1.353 ± 0.020ab
CS 4798 ± 83b 6050 ± 25ba 1.261 ± 0.022b
F 66.06 105.84 115.22
d.f. 3, 40 3, 40 3, 40
Piglet performance (days 1–28)
Control 11902 ± 39a 18480 ± 84a 1.553 ± 0.007a
B-5627 12000 ± 44ab 18130 ± 103ab 1.511 ± 0.008ab
CECT 410 12293 ± 75b 18230 ± 94b 1.483 ± 0.013b
CS 13800 ± 148ba 17790 ± 89ba 1.289 ± 0.012ba
F 1005.31 94.68 1248.41
d.f. 3, 40 3, 40 3, 40
Piglet performance (days 1–42)
Control 21134 ± 154a 33030 ± 86a 1.563 ± 0.014a
B-5627 21776 ± 195ab 31890 ± 130ab 1.464 ± 0.011ab
CECT 410 21595 ± 146ab 32330 ± 98b 1.497 ± 0.012b
CS 23000 ± 128b 32500 ± 320b 1.413 ± 0.020ba
F 254.93 65.26 180.44
d.f. 3, 40 3, 40 3, 40
Means within columns followed by the same letter are not significantly different by Ryan–Gabriel–Welsch Multiple
F-test (P=0.05) after a significant ANOVA (P<0.01).
a The piglets from the control group were not given probiotics or antibiotic. The piglets in the B-5627, CECT

410 and CS groups were given Pediococcus acidilactici NRRL B-5627, Enterococcus faecium CECT 410 and
colistin sulfate, respectively. BWG, body weight gain; FI, feed intake; FCE, feed conversion efficiency.

due to the different diets used. At the beginning of the experiments, the average weight
of the piglets was 7745 g (S.D. 33 g) in the first experiment and 9560 g (S.D. 82 g) in the
second experiment.
The results obtained in the first trial (Table 5) showed that the groups receiving colistin
sulfate and probiotics (Ped. acidilactic and Ent. faecium) exhibited higher average final BW,
BWG and lower FI and FCE values than the control group (P=0.05) after 42 days of treat-
ment. However, significantly more favourable results were observed in the antibiotic group
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 101

(P=0.05). The mean final BW and BWG values were not significantly different between the
groups receiving the probiotics B-5627 and CECT 410 for the whole experimental period
(P=0.05). However, the probiotic NRRL B-5627 group exhibited lower mean FI and FCE
values than the probiotic CECT 410 group.

Table 6
Effect of dietary probiotic (CECT 539, CECT 4043) or antibiotic (colistin sulfate) addition on growth performance
parameters of piglets during 42 days after weaning
Treatment* Initial BW (day 1) (g) Final BW (day 42) (g)
Piglet performance
Control 9510 ± 29a 30400 ± 275a
CECT 539 9570 ± 64a 31933 ± 103ab
CECT 4043 9550 ± 61a 32250 ± 287b
CS 9600 ± 112a 33000 ± 156b
F 31.18 247.51
d.f. 3, 40 3, 40

Treatment* BWG (g per piglet) FI (g per piglet) FCE (g of FI/g of BWG)

Piglet performance (days 1–14)
Control 4750 ± 289a 6800 ± 110a 1.436 ± 0.079a
CECT 539 5220 ± 87ab 7620 ± 95ab 1.460 ± 0.021a
CECT 4043 5720 ± 281b 7850 ± 147b 1.375 ± 0.067ab
CS 5250 ± 95ab 7970 ± 107b 1.518 ± 0.027b
F 35.01 204.01 11.76
d.f. 3, 40 3, 40 3, 40
Piglet performance (days 1–28)
Control 11070 ± 222a 16650 ± 137a 1.504 ± 0.0027a, b**
CECT 539 12090 ± 307ab 17810 ± 164ab 1.474 ± 0.044a
CECT 4043 12260 ± 38ab, b** 18240 ± 176b 1.488 ± 0.013a
CS 12450 ± 92b 18970 ± 75ba 1.524 ± 0.009b
F 98.89 457.54 6.20
d.f. 3, 40 3, 40 3, 40
Piglet performance (days 1–42)
Control 20900 ± 275a 39770 ± 247a 1.903 ± 0.022a, b**
CECT 539 22363 ± 103ab 42440 ± 159ab 1.898 ± 0.008a
CECT 4043 22700 ± 287b 43040 ± 58b 1.896 ± 0.023a
CS 23400 ± 156ba 44950 ± 66ba 1.921 ± 0.013b
F 229.81 1949.48 4.26
d.f. 3, 40 3, 40 3, 40
Means within columns followed by the same letter are not significantly different by Ryan–Gabriel–Welsch Multiple
F-test (P=0.05) after a significant ANOVA (P<0.01).
* The piglets from the control group were not given probiotics or antibiotic. The piglets in the CECT 539, CECT

4043 and CS groups were given Lactococcus lactis subsp. lactis CECT 539, Lactobacillus casei subsp. casei
CECT 4043 and colistin sulfate, respectively. BWG: Body weight gain, FI, feed intake, FCE, feed conversion
efficiency.
** Means within columns followed by two letters separated by a coma are not significant different to the means

followed by the same letters.

102 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

In the second trial (Table 6), the group receiving the antibiotic showed the highest mean
final BW, BWG and FI, but the FCE value for this group was not significantly different
than the FCE value obtained in the control group (P=0.05). In addition, the mean final BW,
BWG and FI values in the CECT 4043 group were higher than those values obtained for the
CECT 539 group. However, no significant differences in feed conversion efficiency were
observed between the two groups receiving probiotics and the control group (P=0.05).

Fig. 5. Viable plate counts of coliforms in the faeces of piglets in the non-treated control groups, the probiotic
(B-5627, CECT 410, CECT 539 and CECT 4043) treated groups, and antibiotic (CS: colicin sulfate) treated
groups. B-5627: Pediococcus acidilactici NRRL B-5627, CECT 410: Enterococcus faecium CECT 410, CECT
539: Lactococcus lactis susp. lactis CECT 539 and CECT 4043: Lactobacillus casei susp. casei CECT 4043.
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 103

3.7. Total coliform counts of the intestinal content

The data on viable plate counts of coliforms in the faeces of individual animals are shown
in Fig. 5. In the first experiment, the administration of single probiotic strain did not affect
the viable counts of coliform bacteria (upper part of Fig. 5), 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 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 days 1 to 14 and decreased from days 14 to 42. No significant
interaction between time and treatment was observed in viable plate count of coliforms for
this assay.
In the second experiment, 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 dropped
on average for 1.8, 1.4 and 3.2 log units, respectively, at the last sampling (P<0.05).

4. Discussion

The evaluation of LAB for their potential use as probiotics in farm animals is still
increasing (Denli et al., 2003; Bogovič Matijašić et al., 2004). In this work, some important
characteristics of four LAB (Ped. acidilactici, Ent. faecium, L. lactis and Lact. casei) were
evaluated before using them for probiotic use. Since these LAB are non-pathogenic and
non-toxic bacteria, the first trial was focused on the determination of their resistances to
simulated gastrointestinal transit conditions.
Although a reduction in the viable cell number for all LAB after gastric transit was
observed, 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. However, if the gastric and small intestinal transit tolerance of these strains is
increased, a higher number of cells could reach the small intestine and the colon. Two
methods for this purpose are the use of protective agents (Charteris et al., 1998; Gardiner
et al., 1999; Kos et al., 2000) and the use of probiotic preparations containing high con-
centrations of viable cells and antimicrobial metabolites. Therefore, in the present work,
the feed was used not only as a way to administer the probiotics but also as a protective
agent. The probiotic cultures were obtained by using a high efficient fed-batch fermentation
technique (Guerra and Pastrana, 2003; Guerra et al., 2005) and cheaper culture media (whey
and mussel-processing wastes).
The four potentially LAB showed good viabilities at −20 ◦ C for 3 months with skim
milk, thus remaining the bacterial cultures at levels sufficiently higher to offer the above
suggested therapeutic effects. These results agree and confirm the reports of Coppola et
al. (1996) and Pascual et al. (1999) that skim milk was a good cryoprotective agent for
preserving LAB cultures that underwent freezing for a long period of time (18 months).
104 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107

Taking into account that the initial bacterial counts in the feed decreased with the storage
time, the high viability observed in the freezing cultures offers the possibility of using them
for preparing the piglet feed weekly.
The addition of organic acids to piglet feeds can improve their performance (Paulicks et
al., 1996; Kirchgessner et al., 1997) and decrease the intraluminal concentration of coliform
bacteria and other microorganisms in the gastrointestinal tract (Roth and Kirchgessner,
1997), which can compete with the host for nutrients (Canibe et al., 2001). 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 in the feed as well as in the
gastrointestinal tract of the piglets, thus being beneficial for the animals.
Although the efficacy of probiotic bacteria in promoting growth in post-weaning piglets
and other animals have been studied before (Bogovič Matijašić et al., 2004), 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 (Abe et al., 1995; Tortuero et al., 1995; Shu et
al., 2001). However, other studies have shown that probiotics have no positive effects on
broilers, neither improving body weight (Watkins and Kratzer, 1983, 1984; Maiolino et al.,
1992) nor reducing Salmonella carriage (Adler and DaMassa, 1980; Stavric et al., 1992).
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
1–42). However, LAB supplemented diets resulted in a better performance (body weight
gain, feed intake and feed conversion efficiency) (P=0.05) than did the experimental 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 increased the BWG of
the animals. This value is higher than the recommended dose of viable probiotic (106 UFC
of probiotic/g or mL) necessary to obtain beneficial effects (Charteris et al., 1998; Lee et
al., 2000). Since the growth-stimulating effects of probiotic bacteria have been observed
when the piglets were exposed to stresses (Abe et al., 1995; Shu et al., 2001; Bogovič
Matijašić et al., 2004), 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.
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 (Mathew et al., 1996). However,
such as increase was not observed in the groups of piglets fed antibiotic supplemented diet
or probiotics supplemented diets. Similarly, Bogovič Matijašić et al. (2004) have reported an
increase in coliform counts in non-treated post-weaning piglets. Although these researchers
did not observe the same tendency in pigs fed diets supplemented with probiotics (Lacto-
bacillus gasseri K7 and LF221), the differences were attributed to normal variations between
the animals rather than the probiotic treatment. Other studies have shown that the adminis-
tration of probiotics caused a reduction in coliform counts in the faeces of piglets (Tortuero
et al., 1995; Chang et al., 2001; Shu et al., 2001), perhaps by a competitive exclusion
mechanism (Gardiner et al., 1999). But more often such effects are not significant, except
when the animals are challenged with selected pathogenic strains or in gnotobiotic animals
(Bogovič Matijašić et al., 2004). In contrast with the above results, Gardiner et al. (1999)
 N.P. Guerra et al. / Animal Feed Science and Technology 134 (2007) 89–107 105

did not observe a significant reduction of the coliform population in the pigs fed probiotic
(Enterococcus faecium strain Fargo 688® ) cheese.
The result of the feeding trial showed that the non-bactericinogenic strain of E faecium
have similar impact on the result as the other strains. So that, the observed probiotic effect
of this strain in piglets could be related to: (i) the reduction or inhibition of the proliferation
of coliform and pathogenic bacteria, due to the production of antimicrobial substances like
lactic acid, acetic acid, ethanol and butane-2,3-diol (Saavedra et al., 2003), (ii) an inhibition
of the adhesion of coliform and pathogenic bacteria to host cells (Jin et al., 2000), and/or
(iii) a more rapid clearance of enteropathogens by and elevated immune response, either
specific or innate, resulting from probiotic treatment (Perdigon et al., 1995, 2003).
In general, probiotics may support a more rapid colonization of the intestine with a
healthy, commensal bacterial flora in piglets, most likely “inherited” from the sows. These
commensal bacteria have found to enhance development of both the intestinal epithelia and
the gastrointestinal lymphoid system (Falk et al., 1998; Umesaki and Setoyama, 2000). So
that, a balanced microbial population would support the inherent defense mechanisms of a
healthy intestinal tract, resulting in better control of intestinal pathogens (Pollmann et al.,
2005).

5. Conclusion

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.

Acknowledgements

The research presented in this paper was financially supported by the Instituto Nacional
de Investigación y Tecnologı́a 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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