Biologia, Bratislava, 61/6: 783—787, 2006Section Cellular and Molecular BiologyDOI: 10.2478/s11756-006-0158-x

The effect of probiotic BioPlus 2B on feed efficiency and metabolicparameters in swine*

Róbert Link** & Gabriel Kováč

II Clinical Department for Internal Diseases, University of Veterinary Medicine, Komenského 73, SK-04181 Košice,Slovakia, e-mail: robolin65@hotmail.com

Abstract: The purpose of our work was to observe the influence of probiotic preparation BioPlus 2B on average dailygains of weaned pigs, feed efficiency and to evaluate some metabolic indices. The weaned pigs, at the age of 42 days, wereincluded into the trial and divided into four groups. Pigs in groups A (n = 5) and B (n = 4) received BioPlus 2B alsobefore weaning. Only group A received BioPlus 2B, at the concentration of 3.2 × 109 CFU per kg of feed, after weaningcontinually. Groups C (n = 5) and D (n = 4) did not receive BioPlus 2B until the start of the trial, but group C was givenBioPlus 2B at the same concentration as group A during the experiment. Blood samples for determination of metabolicindices were collected at the start of experiment, i.e. on 42nd day of pigs life, and then on 56th, 70th, 84th and 91st daysof pigs life. The following biochemical indices were evaluated within the trial: serum levels of total proteins, albumin, urea,total cholesterol and total lipids. Total serum protein level (p < 0.0001) and serum albumin level (p = 0.0024) in groups Aand B were significantly higher in comparison with groups C and D on day 56 of pigs life. Serum urea level in group D wassignificantly (p = 0.049) higher than in group A on 70th day of pigs life. Serum level of total cholesterol in group B on day56 and 84 of pigs life was significantly (p = 0.0004) higher than in groups C and D. Total serum lipid level was significantly(p < 0.05) higher in B group compared to other groups on 56th, 70th, 84th days of pigs life. Average daily gains (ADG) inA group, even if non-significantly, were about 10% better than in groups B, C, D between 57th and 77th days of pigs life.ADG in groups A and B were 14% better in comparison with that in groups C and D between 78th and 91st days of pigslife (p = 0.036). The best feed efficiency in the trial was in group A, approximately 13%, 16% and 21% better than that inthe groups B, C and D, repectively.

Key words: Bacillus licheniformis; Bacillus subtilis; total protein; urea; total cholesterol; average daily gains.

Abbreviations: ADG, average daily gain.

Introduction

It has been proven that the microorganisms of theintestinal tract influence health and productivity of an-imals. Only a few species are able to survive perma-nently in gut, however, after weaning the intestinal mi-croflora is getting stable and typical for each particularanimal (Butine & Leedle, 1989).In the gut of humans, more than 1014 bacteria ex-

ist, which means ten times more than is the number ofall cells of the host. Undamaged gut epithelium and op-timal composition of gut microflora prevent pathogensfrom growing and translocating (Holzapfel et al.,1998). Consequently, scientific results show that se-lected probiotics at the concentration of 109–1011 de-crease the incidence of diarrhoea (Sanders, 1999).Salminen et al. (1999) defined probiotics as micro-

bial cell preparations or components of microbial cell,

which have positive effect on health and performanceof the host. Chesson (1993) asserts that a probioticstrain should come from the intestinal tract (or toler-ate conditions of the intestinal tract), adhere, maintainhigh vitality after freeze-drying, depositing, produce in-hibitors against pathogens and stimulate immunity ofthe host.In the past decade the genus Bacillus along with

Lactobacillus, non-enterotoxic Escherichia coli, Bifi-dobacterium and Streptococcus have been used as pro-biotics.According to taxonomy studies, the genus Bacillus

can be divided into 5 or 6 groups (Claus & Berke-ley, 1986). In the Bacillus subtilis group usually fivephysiologically similar species are described – Bacillusamyloliquefaciens, Bacillus atrophalus, Bacillus licheni-formis, Bacillus pumilus and Bacillus subtilis.Barbosa et al. (2005) described the isolation of

* Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.** Corresponding author

c©2006 Institute of Molecular Biology, Slovak Academy of Sciences

784 R. Link & G. Kováč

237 presumptive gut-associated Bacillus spp. isolatesthat were obtained from organically reared broilers.The Bacillus species identified included B. subtilis, B.pumilus, B. licheniformis, B. clausii, B. megaterium,B. firmus and species of the B. cereus group. Isolatessporulated efficiently in the laboratory, and the result-ing spores were tolerant to simulated gastrointestinaltract conditions. They exhibited also antimicrobial ac-tivity against a broad spectrum of bacteria, includingfood spoilage and pathogenic organisms. Results sug-gest that some of the sporeformers have the potentialto persist in or transiently associate with the complexgut ecosystem.Bacillus species influence also metabolic indices

in the blood of host. Samanya & Yamauchi (2002)revealed that chickens fed dried Bacillus subtilis spp.natto for 28 days had decreased blood ammonia con-centration in the experimental group (P < 0.05). Theseresults suggest that Bacillus subtilis spp. natto was re-sponsible for decreased ammonia concentration.The aim of our study was to evaluate some

metabolic indices in blood of weaned pigs after adminis-tration of probiotic preparation BioPlus 2B, which con-tains equal proportions of Bacillus licheniformis andBacillus subtilis at the concentration of 3.2 × 109 bac-teria per gram. We observed also average daily gainsand feed efficiency.

Material and methods

AnimalsEighteen weaned, cross-bred (Landrace × Slovak White)piglets, at the age of 42 days, were included into the ex-periment. Experiment lasted seven weeks, up to the age of91 days. All groups of piglets have approximately the sameweight at the start of the trial without significant differ-ences. Average weights in groups A and B were ∼13.1 kgand weights in groups C and D were 11.9 kg and 12.1 kg,respectively. Piglets in each group were kept together in onepen throughout the trial; they were fed commercial mixedfeed ad libitum.

Experimental designGroup A (n = 5) and group B (n = 4) consisted of pigs,which were given probiotic preparation BioPlus 2B at theconcentration of 3.2 × 107 Bacillus licheniformis and Bacil-lus subtilis pro toto every day also before weaning. In theexperiment, after weaning at the age of 42 days, only groupA was given BioPlus 2B in dry mixed feed at the concen-tration 3.2 × 109 bacteria per kg of mixed feed continually.

Group C (n = 5) and group D (n = 4) did not re-ceive any probiotics until weaning. In our experiment, afterweaning at the age of 42 days, pigs in group C received thesame concentration of BioPlus 2B as group A, i.e. 3.2 × 109bacteria per kg of feed.

Groups B and D did not receive preparation BioPlus2B in the experiment after weaning.

Pigs were fed an appropriate dry feed mixture depend-ing on the age. At the beginning of the trial they were feda starter feed mixture and at the end of the experimentthey were given feed mixture for fattening. That means,

that at the age of 42 days starter mixture (OŠ-02) was givento weaned pigs and at the age of 56 days starter feed waschanged to pre-fattening feed mixture (A1). Both types ofmixed feed are based on barley and wheat, A1 containedmuch less maize than OŠ-02. As the trial was carried out be-fore Slovakia has joined the European Union, it was allowedto use meat-bone meal in mixed feed (Tajba a.s., Čaňa, Slo-vakia). Starter feed contained crude protein (18.6%), lipids(3.7%), crude fibre (3.5%) and ash (6.7%); the diet pro-vided 13.0 MJ of DE. Fattening feed contained crude pro-tein (17%), lipids (3.1%), crude fibre (4%) and ash (5.8%);the diet provided 12.5 MJ of DE.

Average daily weight gains, feed efficiency and someindices of protein and lipid metabolism were evaluated inthe experiment. In order to monitor daily weight gains inaccordance with growth capacity of pigs, we divided theexperiment into three parts: (i) the first period lasted from42nd to 56th day of pigs life, i. e. up to 17 kg of body weight;(ii) the second period lasted from 57th to 77th day of pigslife, i. e. up to 30 kg of body weight; and (iii) the thirdperiod lasted from the 78th to 91st day of pigs life. Pigs wereweighed every week and the feed efficiency was calculatedat the end of each period of the experiment.

Probiotic preparationBioPlus 2B consists of equal part of Bacillus licheniformisand Bacillus subtilis at the concentration of 3.2 × 109 CFUper gram of preparation (Chr. Hansen A/S, Hørsholm, Den-mark). Both bacteria are tested for stability and qualitiesand they are deposited in German Collection of Microorgan-isms as DSM 5749 and DSM 5750. According to distributor,the recommended ratio in feed mixture for weaned pigs is400 grams of preparation per ton of feed.

Laboratory testsBlood samples for determination of protein and lipid param-eters were collected in two-week periods. Blood was sampledat the start of the experiment, i.e. at the age of 42 daysof piglets life, and then on 56th, 70th, 84th and 91st day ofpiglets life. The blood was taken from the eye sinus (KOVÁČet al., 1990). All blood samples were collected between 8.00and 9.00 hours, i.e. at the same time after morning feeding.Metabolic parameters were determined from blood serum,which was obtained after coagulation and centrifugation ofblood.

Total protein, urea, albumin and cholesterol levels inserum were determined with multiparametric photocolori-metric analyser ALIZE (Lisabio, France) with the use ofBioMerieux kits (France). The kits use for determinationof total proteins in serum the principle of the Biuret reac-tion and for determination of serum albumin the bromcresolgreen at pH 4.2. Urea was determined using the urease andmodified Berthelot reaction, and cholesterol by enzymaticdetermination.

Total lipids were analysed using colorimetric determi-nation at the wave length 530 nm, with LACHEMA tests(Czech Republic).

Statistical analysisResults were analysed in order to obtain the mean valuesand standard deviation (SD), using GraphPad Prism statis-tical software. One-way ANOVA test was used for statisticalevaluation between the groups; the significance level to dif-ferentiate parameters was set to α = 0.05.

The effect of probiotic BioPlus 2B in swine 785

Table 1. Protein profile in blood of weaned piglets.a

Days of piglets life

42nd 56th 70th 84th 91th

Total protein – A (g/L) 56.3 ± 4.1 60.8 ± 3.9* 52.9 ± 2.9 62.7 ± 3.7 63.0 ± 2.8Total protein – B (g/L) 60.5 ± 3.7 64.5 ± 3.2* 56 ± 4.3 64.1 ± 4.1 63.0 ± 6.1Total protein – C (g/L) 55.5 ± 1.84 51.9 ± 1.55* 52.6 ± 2.1 58.7 ± 2.1 58.3 ± 5.2Total protein – D (g/L) 56.7 ± 1.6 51.7 ± 1.3* 55.4 ± 3.9 63.4 ± 3.2 59.2 ± 1.8Urea – A(mM) 3.22 ± 0.25 4.39 ± 0.7 5.05 ± 0.67* 6.18 ± 1.01 6.16 ± 1.37Urea – B (mM) 4.02 ± 0.41 4.94 ± 0.63 5.6 ± 0.64* 5.31 ± 0.72 5.9 ± 0.63Urea – C (mM) 4.19 ± 0.49 4.7 ± 0.61 5.36 ± 0.54* 6.63 ± 0.79 5.5 ± 0.79Urea – D (mM) 3.95 ± 0.8 5.22 ± 1.11 6.2 ± 0.14* 5.48 ± 0.58 5.34 ± 0.42Albumin – A (g/L) 38.3 ± 3.0 40.6 ± 2.5* 36.3 ± 2.5 39.9 ± 3.5 40.5 ± 1.6Albumin – B (g/L) 40.4 ± 1.6 42.2 ± 1.7* 35.5 ± 1.6 38.4 ± 2.9 37.6 ± 2.1Albumin – C (g/L) 39.3 ± 2.1 35.6 ± 3.0* 35 ± 2.1 38.5 ± 2.7 40.6 ± 3.4Albumin – D (g/L) 39.4 ± 1.8 34.9 ± 1.2* 34.6 ± 2.7 38.9 ± 3.3 40.5 ± 2.6

a A, B, C, D – groups A, B, C, D. * – p < 0.05 among the groups.

Table 2. Serum level of cholesterol and total lipids in piglet blooda.

Days of piglets life

42nd 56th 70th 84th 91th

Cholesterol – A (mM) 2.5 ± 0.2 2.39 ± 0.35* 2.42 ± 0.17 3.14 ± 0.29* 3.1 ± 0.37*Cholesterol – B (mM) 2.6 ± 0.34 2.9 ± 0.28* 2.9 ± 0.43 3.65 ± 0.12* 2.9 ± 0.23*Cholesterol – C (mM) 2.25 ± 0.2 2.3 ± 0.11* 2.29 ± 0.39 2.71 ± 0.14* 2.54 ± 0.35*Cholesterol – D (mM) 2.11 ± 0.34 1.9 ± 0.17* 2.72 ± 0.53 2.5 ± 0.49* 2.39 ± 0.34*Total lipids – A (g/L) 2.48 ± 0.85 1.93 ± 0.74* 2.49 ± 0.68* 5.1 ± 0.42* 2.58 ± 0.45Total lipids – B (g/L) 2.74 ± 0.9 3.43 ± 0.7* 3.71 ± 0.56* 5.1 ± 0.24* 2.3 ± 0.26Total lipids – C (g/L) 3.2 ± 0.73 2.65 ± 0.23* 2.49 ± 0.45* 2.1 ± 0.16* 2.35 ± 0.19Total lipids – D (g/L) 2.94 ± 0.4 1.89 ± 0.67* 3.07 ± 0.39* 2.4 ± 0.53* 2.28 ± 0.17

a A, B, C, D – groups A, B, C, D. * – p < 0.05 among the groups.

Results

Total serum protein level in groups A and B were signif-icantly (p < 0.0001) different compared with the groupsC and D on day 56 of pigs life. In group A the aver-age level of total proteins was 60.8 g/L, in group B 64g/L and in groups C and D only 51 g/L. In general,the highest levels of total proteins were in all groups ondays 84 and 91 of pigs life (Table 1).Serum urea level ranged within 3.2–6.2 mM, with

the lowest levels on days 42 and 56 of pigs life. Duringthe trial we revealed continuous increase in urea levelsin all groups. We found significant differences amongthe groups on day 70 of pigs life (p = 0.049), when urealevel in group D was 6.2 mM whereas in group A 5.05mM (Table 1).Serum albumin levels in groups A and B were the

highest on 56th day of pigs life. Serum albumin levelsin groups A and B were significantly (p = 0.002) higheron day 56 pig life in comparison with groups C and D(Table 1).During the trial, serum levels of total cholesterol

generally ranged from 1.8 to 3.1 mM; in one case itreached 3.6 mM. In the group B total cholesterol levelwas generally higher compared with groups C and D,which performed significant differences on days 56, 84

and 91 of pigs life (p = 0.0004 and p = 0.012, respec-tively).Similar to cholesterol level, also the total lipid level

in group B increased until the day 84 of pigs life andthen also in the group A decreased on 91st day of pigslife. On the contrary, serum lipids in group C had de-creasing tendency during the trial, and serum lipids ingroup D were not stable during the experiment. Werevealed significant differences (p = 0.006, p = 0.02,p < 0.001) among the groups on 56th, 70th, 84th day ofpigs life (Table 2).Average daily gains (ADG) were evaluated in three

periods of the trial. In the last period of the trial, whenADG in groups A and B reached approximately 0.8 kgper day and in groups C and D only 0.66, significantdifferences (p = 0.36) were revealed among groups (Ta-ble 3).Utilisation of feed in the group A was the best of all

groups. In this group we found that conversion of feedwas 2.07 kg of dry mixed feed per kilogram of bodyweight gain. In group B it was 2.35 kg, in group C 2.48kg, and in group D 2.63 kg. After evaluating the resultswe found that conversion of feed was about 13% betterin group A in comparison with the group B, whereas ingroup C the conversion of feed was 6% better than thatin the group D (Table 4).

786 R. Link & G. Kováč

Table 3. Average daily weight gains.a

Days of piglets life

42–56 57–77 78–91

A group (kg/day) 0.38 ± 0.07 0.66 ± 0.09 0.78 ± 0.07*B group (kg/day) 0.34 ± 0.06 0.60 ± 0.12 0.78 ± 0.1*C group (kg/day) 0.35 ± 0.025 0.60 ± 0.07 0.66 ± 0.04*D group (kg/day) 0.37 ± 0.09 0.58 ± 0.07 0.67 ± 0.11*

a * – p < 0.05 among the groups.

Table 4. Feed efficiency of groups (kg mixed feed/ kg body weightgain).

Days of piglets life

42–56 57–77 78–91 Mean – whole trial

A group 2.21 1.97 2.05 2.07B group 2.47 2.30 2.18 2.35C group 2.40 2.31 2.42 2.48D group 2.27 2.41 2.60 2.63

Discussion

Weight of pigs increased during the experiment frominitial 12–13 kg of body weight to 38–42 at the age91 days. ADG between the 42nd and 56th day of pigslife were 0.34–0.38 kg/day, later between the 57th and77th day of pigs life a continuous increase to 0.58–0.66kg/day was observed, and finally between the 78th and91st day of pigs life it was 0.66–0.78 kg/day.According to Lazar et al. (1984) a norm for weight

gains in category 8–17 kg is 430 g/day, in category 17–30 kg it is 530 g/day and in category 30–40 kg it is 590g/day.According to Straw et al. (2003) we achieved ideal

ADG in our trial, moreover at the age of 60 and 80 dayspigs in our trial had even better growth rate.After evaluating the growth rate among groups we

can say that application of preparation BioPlus 2B hada positive effect on ADG. Group A, which was givenpreparation from time of birth up to the end of trial,achieved the best results in the trial. The results suggestpositive effect of long-term BioPlus 2B administration.Administration of BioPlus 2B to groups B and C

had not such a high-positive effect as in the group A,which can be explained by shorter period of adminis-tration. In group B BioPlus 2B was administrated onlyin the suckling period and so ADG were not as high asin the group A.Alexopoulos et al. (2004b) tested the efficacy

of BioPlus 2B on the health status and productivityof pigs, during weaning, growing and finishing stagesof growth. The results have shown that BioPlus 2B-treated pigs had a lower morbidity and mortality dur-ing the whole trial period – compared with the doublecontrols (9.26–14.81% versus 25.93% and 0.00–3.70%

versus 11.1%, respectively), as a result of the lower inci-dence of post-weaning E. coli-caused diarrhoea. Weightgain, feed conversion and carcass quality of the BioPlus2B-treated pigs were improved compared with the dou-ble controls, whilst the beneficial effects of the probi-otic were more pronounced when the medium and highdoses were used.Stability of total proteins and albumin in blood of

long-term treated piglets was revealed in our experi-ment. It could be caused by better utilisation of feedproteins by microbial enzymes. Ahrens et al. (1992)conducted an experiment on weaned pigs and observedthat supplementation of their rations with BioPlus 2B,at a dose of 1.2 × 106/g feed, resulted in higher weightgains (by 10%) and better conversion of feed in the firstthree weeks of the experiment. Also the ADG were sig-nificantly higher in the experimental group (679 g/day)during the subsequent two weeks in comparison withthe control (570 g/day). Digestion of proteins in thesmall intestine of experimental pigs was significantlybetter (76%) in comparison with that in the control(68%). This effect is the most important during chang-ing of mixed feed.These results are in agreement with assertion that

Bacillus licheniformis and Bacillus subtilis produce anumber of enzymes, among which protease, lipase, amy-lase are the most important for helping the digestion ofproteins from feed (Michalík et al., 1994).Bacillus species are able to germinate in gut after

administration, and then help the host to digest feed.Hoa et al. (2001) revealed that the number of elim-inated Bacillus subtilis bacteria was higher than thatin the inoculum, i.e. one may assume that germinationand multiplication of probiotic spores occurred. Ger-mination of spores of Bacillus cereus var. toyoi in theintestinal samples from both broiler chicks and sucklingpiglets was also confirmed by Jadamus et al. (2001).Nakano & Zuber (1998) revealed that after givingglucose and nitrate as a terminal electron acceptor,Bacillus subtilis has been shown to grow anaerobically.It seems that germination of spores in the digestivetract is a precondition of the potential probiotic effectresulting in the improvement of health and increasedweight gains in farm animals.Decrease in serum cholesterol after administration

of BioPlus 2B was not proven, because cholesterol levelsin the C and D groups were almost the same.

The effect of probiotic BioPlus 2B in swine 787

Xiao et al. (2003) described lowering of theserum concentration of total cholesterol and low-densitylipoprotein cholesterol after administration of Bifi-dobacterium longum in rats in comparison with the con-trol. Streptococcus thermophilus and Lactobacillus del-brueckii ssp. bulgaricus did not have, however, the sameeffect.According to Kiessling et al. (2002) long-term

consumption of Lactobacillus acidophilus 145 and Bi-fidobacterium longum 913 in women did not influ-ence the serum concentration of total cholesterol andthe low-density lipoprotein cholesterol, but the ratioof LDL/HDL cholesterol decrease from 3.24 to 2.48(p = 0.002). Selection of bacterial species seems to playthe most important role in lowering the total serumcholesterol concentration.On the other hand, Alexopoulos et al. (2004a)

described higher serum cholesterol, total lipid concen-trations, milk fat and protein contents at mid-sucklingperiod after BioPlus 2B administration to sows. As aconsequence, a positive effect was also noticed with re-gard to litter health and performance characteristics interms of: (i) decrease in piglet diarrhoea score; (ii) de-crease in pre-weaning mortality thus leading to increasein the number of weaned piglets per litter; and (iii) in-crease in piglet’s body weight at weaning.One of the mechanisms of decreasing the serum

cholesterol level is its conversion to coprostanol in theintestinal tract, which is not absorbed. However, Car-dona et al. (2003) did not find conversion of choles-terol to coprostanol after application ofBacillus licheni-formis NCTC 13123 to germ-free mice. Consequently,they judged that Bacillus licheniformis alone did notinfluence cholesterol metabolism. We suppose that thisnotion was also proven in our trial.Total serum cholesterol in blood of weaned piglets

was not decreased by BioPlus 2B administration. Thecontinuous probiotic administration in group A main-tained the serum levels of total proteins, urea and albu-min of weaned pigs more constant than in the groupsC and D. BioPlus 2B prevented serum urea level fromincreasing in period of changing mixed feed. Long-termadministration of BioPlus 2B appears to be more effec-tive because high daily weight gain could be achieved.

References

AHRENS, F., SCHMITZ, M. & WARLIES, B. 1992. MikrobiellerZusatzstoff in der Ferkelfutterung. Kraftfutter 75: 418–420.

ALEXOPOULOS, C., GEORGOULAKIS, I.E., TZIVARA, A., KRI-TAS, S.K., SIOCHU, A. & KYRIAKIS, S.C. 2004a. Field evalu-ation of the efficacy of a probiotic containing Bacillus licheni-formis and Bacillus subtilis spores, on the health status andperformance of sows and their litters. J. Anim. Physiol. Anim.Nutr. 88: 381–392.

ALEXOPOULOS, C., GEORGOULAKIS, I.E., TZIVARA, A., KYRI-AKIS, C.S., GOVARIS, A. & KYRIAKIS, S.C. 2004b. Field eval-uation of the effect of a probiotic-containing Bacillus licheni-formis and Bacillus subtilis spores on the health status, per-formance, and carcass quality of grower and finisher pigs. J.Vet. Med. A Physiol. Pathol. Clin. Med. 51: 306–312.

BARBOSA, T.M., SERRA, C.R., LA RAGIONE, R.M., WOOD-WARD, M.J. & HENRIQUES, A.O. 2005. Screening for Bacillusisolates in the broiler gastrointestinal tract. Appl. Environ.Microbiol. 71: 968–978.

BUTINE, T.J. & LEEDLE, J. 1989. Enumeration of selectedanaerobic bacterial groups in ceacal and colonic contents ofgrowing-finishing pigs. Appl. Environ. Microbiol. 55: 1112–1116.

CARDONA, M.E., NORIN, E. & MIDTVEDT, T. 2003. Biochem-ical functions of Bacillus licheniformis in gnotobiotic mice.Microb. Ecol. Health Dis. 15: 40–42.

CHESSON, A. 1993. Probiotics and other intestinal mediators, pp.197–214. In: COLE, D.J.A., WISIMAN, J. & VARLEY, M.S.(eds) Principles of Pig Science, Nottingham University Press,Loughbourough.

CLAUS, D. & BERKELEY, C.W. 1986. Genus Bacillus, pp. 1105–1139. In: SNEATH, P.H.A. (ed.) Bergey’s Manual of System-atic Bacteriology, Waverly Press, Inc., Baltimore, MD.

HOA, T.T., DUC, L.H., ISTICATO, R., BACCIGALUPI, L., RICCA,E., VAN, P.H. & CUTTING, S.M. 2001. Fate and disseminationof Bacillus subtilis spores in a murine model. Appl. Environ.Microbiol. 67: 3819–3823.

HOLZAPFEL, W.H., HABERER, P., SNEL, J. & SCHILLINGER, U.1998. Overview of gut flora and probiotics. Int. J. Food Mi-crobiol. 41: 85–101.

JADAMUS, A., VAHJEN, W. & SIMON, O. 2001. Growth behaviourof a spore-forming probiotic in the gastrontestinal tract ofbroiler chicken and piglets. Arch Tierernahr. 54: 1–17.

KIESSLING, G., SCHNEIDER, J. & JAHREIS, G. 2002. Long-termconsumption of fermented dairy products over 6 months in-creased HDL cholesterol. Eur. J. Clin. Nutr. 56: 843–849.

KOVÁČ, G., MARTINČEK, M., MUDROŇ, P., CILÍK, D., DE-MOVIČ, T., PAŠTEK, J., STANISLAVSKÝ, V. & RUŽIČKA, S.1990. Odber vzoriek krvi od prasiat. Porovnanie používanýchmetód a popis novej metódy. Veterinářství 40: 417–420.

LAZAR, J., MAGIC, D. & KOVALČÍK, T. 1984. Výživa a dietetikaošípaných a hydiny. Príroda, Bratislava, 173 pp.

MICHALÍK, I., SZABOVÁ, E., JAŠKA, P., URMINSKÁ, D. &SCHWARZ, M. 1994. Biochemická characteristika bakteriál-nej alkalickej proteinázy. Poľnohospodárstvo/Agriculture 40:95–112.

NAKANO, M.M. & ZUBER, P. 1998. Anaerobic growth of a “strictaerobe”(Bacillus subtilis). Annu. Rev. Microbiol. 52:165–190.

SALMINEN, S., OUWEHAND, A., BENNO, Y. & LEE, Y. 1999.Probiotics: how should they be defined? Trends Food Sci.Technol. 10: 107–110.

SAMANYA, M. & YAMAUCHI, K.E. 2002. Histological alterationsof intestinal villi in chickens fed dried Bacillus subtilis var.natto. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 133:95–104.

SANDERS, M. 1999. Probiotics. Food Technol. 53: 67–77.STRAW, B.E., MEUTEN, D.J. & THACKER, B.J. 2003. Clinicalexamination and diagnostics, pp. 3–61. In: STRAW, B.E.,D’ALLAIRE, S., MENGELING, W. & TAYLOR, D. (eds) Dis-eases of Pigs, Hajko & Hajková, Bratislava.

XIAO, J.Z., KONDO, S., TAKAHASHI, N., MIYAJI, K., OSHIDA, K.,HIRAMATSU, A., IWATSUKI, K., KOKUBO, S. & HOSONO, A.2003. Effects of milk products fermented by Bifidobacteriumlongum on blood lipids in rats and healthy adults male vol-unteers. J. Dairy Sci. 86: 2452–2461.

Received September 14, 2005Accepted April 27, 2006