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Archives of Animal Nutrition
ISSN: 1745-039X (Print) 1477-2817 (Online) Journal homepage: http://www.tandfonline.com/loi/gaan20
The effect of eubiotic feed additives on the
performance of growing pigs and the activity of
intestinal microflora
Piotr Nowak, Małgorzata Kasprowicz-Potocka, Anita Zaworska, Włodzimierz
Nowak, Barbara Stefańska, Anna Sip, Włodzimierz Grajek, Wojciech Juzwa,
Marcin Taciak, Marcin Barszcz, Anna Tuśnio, Katarzyna Grajek, Joanna
Foksowicz-Flaczyk & Andrzej Frankiewicz
To cite this article: Piotr Nowak, Małgorzata Kasprowicz-Potocka, Anita Zaworska, Włodzimierz
Nowak, Barbara Stefańska, Anna Sip, Włodzimierz Grajek, Wojciech Juzwa, Marcin Taciak, Marcin
Barszcz, Anna Tuśnio, Katarzyna Grajek, Joanna Foksowicz-Flaczyk & Andrzej Frankiewicz
(2017): The effect of eubiotic feed additives on the performance of growing pigs and the activity of
intestinal microflora, Archives of Animal Nutrition, DOI: 10.1080/1745039X.2017.1390181
To link to this article: http://dx.doi.org/10.1080/1745039X.2017.1390181
Published online: 23 Oct 2017.
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Date: 26 October 2017, At: 04:38
ARCHIVES OF ANIMAL NUTRITION, 2017
https://doi.org/10.1080/1745039X.2017.1390181
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The effect of eubiotic feed additives on the performance of
growing pigs and the activity of intestinal microflora
Piotr Nowaka, Małgorzata Kasprowicz-Potockaa, Anita Zaworska a,
Włodzimierz Nowaka, Barbara Stefańska a, Anna Sipb, Włodzimierz Grajekb,
Wojciech Juzwab, Marcin Taciakc, Marcin Barszczc, Anna Tuśnioc, Katarzyna Grajekd,
Joanna Foksowicz-Flaczykd and Andrzej Frankiewicza
a
Department of Animal Nutrition and Feed Management, Poznan University of Life Sciences, Poznań,
Poland; bDepartment of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań,
Poland; cThe Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences,
Jabłonna, Poland; dDepartment of Innovative Biomaterials and Nanotechnologies, Institute of Natural
Fibres and Medicinal Plants, Poznań, Poland
ABSTRACT
ARTICLE HISTORY
The aim of this study was to compare the effect of probiotic
bacteria, prebiotics, phytobiotics and their combinations on
performance and microbial activity in the digestive tract of
growing pigs. The experiment was conducted over 28 d on
48 male pigs of about 12 kg body weight (BW), which were
allocated to following treatments.: (1) Control Group (Con)
without additive, (2) Group I, addition of a prebiotic (inulin),
(3) Group Ph, a phytobiotic (herbal water extracts), (4) Group P,
a probiotic composed of four strains of lactic acid bacteria, (5)
Group PhP, phytobiotic and probiotic bacteria and (6) Group
PhPI, a phytobiotic, probiotic bacteria and a prebiotic. Animal
performance was recorded and at d 28 six pigs from each
group were euthanised to collect digesta samples. In all
groups except for Group I, diarrhoea incidents were observed.
Groups Ph and P had significantly higher daily gains and final
BW, and Group Ph utilised feed better than other groups. The
pH of ileal digesta was significantly lower in Group PhPI. In the
caecal digesta of Groups I, P and PhP, the pH level was lower
than in the other groups but dry matter contents was significantly higher in Groups Con and I. The short-chain fatty acids
and particular acid content differed significantly only in the
colonic digesta. The yeast and mould numbers in caecal
digesta was highest in Group Con. No treatment effects were
observed for the number of lactic acid bacteria, coli group
bacteria or Clostridium. However, the observed significantly
higher number of total bacteria suggests that a multi-component eubiotic treatment changes the bacterial composition
and distribution more effectively. Our findings indicated that
all used additives changed the intestinal microflora, but the
multi-component eubiotics were not beneficial as feed additives offered separately. Moreover, supplementation of phytobiotics and probiotic bacteria also improved the animal
performance significantly.
Received 14 July 2017
Accepted 6 October 2017
CONTACT Małgorzata Kasprowicz-Potocka
malgokas@poczta.onet.pl
© 2017 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Digestive tract; microbial
flora; performance; pigs;
prebiotics; probiotics;
synbiotics
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P. NOWAK ET AL.
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1. Introduction
Feed additives such as phytobiotics, probiotics and prebiotics are considered as safe
because they naturally occur in the environment (Wang et al. 2016). These products
belong to the group of so-called “Eubiotics” (Greek “Eubiosis”), which refers to a
healthy balance of microbiota in the gastrointestinal tract. It is well known that the
microbiota play important role due to the production of short-chain fatty acids
(SCFA), which exert multiple beneficial effects on the host. On the other hand,
microbial metabolism of proteins generates a variety of compounds including
ammonia, phenol, p-cresol and indole, many of which have been shown to have a
negative impact on animals (Pieper et al. 2016). The phytobiotics containing the
bioactive substances are commonly used in pharmacology as fragrances and preservatives for foods (Carson et al. 2006; Grashorn 2010; Grela et al. 2013), but they
may also present antibacterial, antiviral and antifungal properties (Wenk 2003;
Vidanarachchi et al. 2005). Probiotics are live or lyophilised strains of different
microorganisms, which may have a beneficial effect on the host when used in the
appropriate amounts (Grashorn 2010, Santini et al. 2010; Vondruskova et al. 2010;
Han et al. 2016). The probiotic bacteria should be harmless, have no antibiotic
resistance, have high survival and resistance to low pH and good adhesion to the
intestinal walls; they also should rapidly reproduce and exhibit antagonistic activity
against pathogenic microorganisms (Shim 2005; De Lange et al. 2010; Vondruskova
et al. 2010; Liu et al. 2014). Moreover, the use of a mixture of several strains of
microorganisms increases its efficacy, especially if the bacteria differ in the fermentation profile and prevent the development of different pathogens (Barszcz et al.
2016). Prebiotics, such as inulin, play a supportive role to probiotics (Grela et al.
2013). Using more than one eubiotic substance (multi-eubiotic) as a feed additive
could be more efficient than using them separately (Namkung et al. 2004). The
efficacy of natural additives, however, is highly dependent on the type, composition
and form of the administered preparation (Botsoglou et al. 2002; Windisch et al.
2008; Wang et al. 2016). Because of that fact, it was hypothesised that a multieubiotic preparation containing a composition of probiotic bacteria strains targeted
against E. coli and C. perfringens, a prebiotic and a phytobiotic, all recognised as
active promoters of metabolic function and microflora, could be an effective feed
additive in pig production. Therefore, the aim of this study was to assess the
response of growing pigs to selected feed additives such as probiotic bacteria,
prebiotics, phytobiotics and their combinations, taking into consideration growth
performance and digestive tract physiology.
2. Materials and methods
2.1. Animals and diets
All the experimental procedures complied with the guidelines of the Local Ethical
Committee for Experiments on Animals in Poznan regarding animal experimentation and
the animal care under study (EU Directive 2010/63/EU for animal experiments). The pigs
received all the necessary veterinary vaccinations and had unlimited access to water and feed.
ARCHIVES OF ANIMAL NUTRITION
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2.2. Growth experiment
The growth experiment was conducted on 48 male pigs (P76 × Naima) of about 12 kg
body weight (BW). The pigs were allocated to six dietary treatments according to their
BW (eight replications each) and were kept in individual pens on straw bedding. All
diets were offered in mash form according to the experimental design (Table 1). The
diets were prepared according to pig requirements (GfE 2006) (Table 2). In the
experimental diets, wheat was replaced by the respective feed additives. Native chicory
inulin (prebiotic), with an average degree of polymerisation of 10, was given at 30 kg/t
(Inulin ORAFTI®GR, BENEO Belgium, Tienen, Belgium). A multispecies probiotic
bacteria preparation (Leuconostoc mesenteroides, two strains of Enterococcus faecium
and Carnobacterium divergens at a ratio of 1:1:1:1:0.01) with maltodextrin as a protector
was dosed in the total amount of 1012 CFU/t feed. The preparation formula was
Table 1. Experimental design.
Experimental diets
Supplement
Inulin [%]
Phytobiotic (watery extract of herbs) [%]
Probiotic bacteria [CFU/kg]
Con
-
I
3
-
Ph
0.02
-
P
109
PhP
0.02
109
PhPI
3
0.02
109
Table 2. Composition of the basal diet.
Experimental diets
Con*
Components [%]
Soybean meal
25.35
Corn meal
30.00
Wheat meal
21.20
Barley meal
20.00
Soya oil
0.20
1-Ca phosphate
0.90
Limestone
1.10
L-Lysine (98.5%)
0.40
DL-Methionine (99%)
0.20
NaCl
0.35
0.30
Premix grower♦
Inulin
0.00
Phytobiotics
0.00
Probiotics
0.00
Analysed nutritional value [g/kg]
ME [MJ/kg] (calculated)
13.0
Crude protein
191.7
Crude fibre
34.6
Lysine
11.8
Methionine + cystine
7.7
Ca
7.4
P
6.3
Na
1.6
I#
Ph†
P‡
PhP◊
PhPI§
25.35
30.00
18.20
20.00
0.20
0.90
1.10
0.40
0.20
0.35
0.30
3.00
0.00
0.00
25.35
30.0
21.18
20.00
0.20
0.90
1.10
0.40
0.20
0.35
0.30
0.00
0.02
0.00
25.35
30.00
20.20
20.00
0.20
0.90
1.10
0.40
0.20
0.35
0.30
0.00
0.00
1.00¶
25.35
30.00
20.18
20.00
0.20
0.90
1.10
0.40
0.20
0.35
0.30
0.00
0.02
1.00
25.35
30.00
17.18
20.00
0.20
0.90
1.10
0.40
0.20
0.35
0.30
3.00
0.02
1.00
13.1
188.5
33.6
10.9
7.4
7.2
6.2
1.5
13.0
191.6
34.5
11.8
7.7
7.4
6.3
1.6
13.0
190.7
34.3
11.5
7.6
7.3
6.1
1.6
13.0
190.6
34.3
11.4
7.6
7.3
6.3
1.5
13.1
187.2
33.4
10.7
7.3
7.1
6.2
1.6
*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ♦Provided per kg diet: 1.2 g choline chloride, 450 mg Fe, 120 mg Cu,
1.8 mg Co, 180 mg Mn, 450 mg Zn, 3.6 mg I, 0.9 mg Se, antioxidants (butylated hydroxyanisole, butylated
hydroxytoluene), 45,000 IU vitamin A, 9000 IU vitamin D3; 315 mg vitamin E, 6.6 mg vitamin K3, 6.6 mg vitamin
B1, 18 mg vitamin B2, 13.5 mg vitamin B6, 45 mg pantothenic acid, 90 mg nicotinic acid, 9 mg folic acid, 111 µg
vitamin B12, 450 µg biotin, 7.8 g Ca; ¶content corresponds to 109 CFU/kg feed.
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P. NOWAK ET AL.
prepared at Poznan University of Life Sciences and the individual strains were deposited in the Polish Patent Collection of Microorganisms in Wroclaw under the Accession
Numbers: L. mesenteroides PKM B/00096; E. faecium PKM B/00097; C. divergens PKM
B/00099and E. faecium PKM B/00098. The formulation and dosage of the probiotic
preparations were determined based on the results of in vitro studies. As a phytobiotic,
Oregano vulgaris and Thymus vulgaris watery extracts were used in the total amount of
200 g/t feed. Water extracts of thyme and oregano used in this experiment were
prepared at the Institute of Agricultural and Food Biotechnology, Department of
Food Concentrates and Starch Products (Poznań, Poland). For the extraction of biologically active substances, the method of solid-liquid separation was used. The dry raw
herbal material was subjected to pre-treatment circulation pump maceration. The
extracts obtained were subjected to filtration on plate filters and then concentrated on
a vacuum evaporator and spray dried. The dry extracts were standardised and the assays
were carried out according to pharmacopoeial methods. The extracts of thyme and
oregano contained 0.33 and 0.28% of flavonoids recalculated as hyperoside, 17.8 and
21.5% of polyphenols recalculated as rosemary acid and 5.07 and 5.80% of tannins
recalculated as pyrogallol, respectively. The phytobiotics and inulin used in the experiment were introduced to diets based on literature data (Frankić et al. 2009; Grela et al.
2013, 2014). Bacteria and phytobiotics, encapsulated individually and in multicomponent preparations, were mixed right before use. The experiment lasted 28 d. The
average daily gains (ADG) and feed intake (FI) were recorded at the end of the
experiment and from this data, the average feed conversion ratio (FCR) was calculated.
Faecal scores were based on a standard scoring system (0: dry, hard, well-formed faeces;
1: soft but formed faeces; 2: pasty faeces, green or brown in colour; 3: viscous faeces in
light colour, episodic; 4: fluid faeces in light colour; 5: watery faeces, continuous), and
scores 3–5 were identified as diarrhoea events. The next day after the experiment, six
pigs from each group were euthanised. Immediately after euthanasia (about 10 min),
their livers were excised and weighed, and the ileal and caecal digesta were collected for
pH measurements and chemical and microbial analysis. The ileal and caecal digesta
were sampled and frozen (at −20°C) to determine the amount of dry matter (DM),
ammonia and SCFA. The colonic digesta were sampled from its proximal, middle and
distal part (segments C25, C50 and C75, respectively), frozen and analysed for DM,
enzymes and phenolic compounds content.
2.3. Chemical analysis
The pH of the digesta was measured using a microelectrode and a pH metre (model
301, Hanna Instruments, Vila do Conde, Portugal). Ammonia was extracted and
analysed by the spectrometric method using a Nessler reagent (POCh, Gliwice,
Poland).
The SCFA analysis was performed according to the procedure described by Barszcz
et al. (2011) on an HP 5890 Series II gas chromatograph (Hewlett Packard, Waldbronn,
Germany) with a flame ionisation detector and Supelco Nukol fused silica capillary
column (Supelco, Bellafonte, USA; 30 m × 0.25 mm i.d.; 0.25 mm). Helium was used as
the carrier gas. The concentrations of individual SCFA were estimated in relation to an
internal standard (isocaproic acid) using a mixture of SCFA standard solutions.
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ARCHIVES OF ANIMAL NUTRITION
5
Phenol, p-cresol and indole concentrations were analysed based on the method
described by Taciak et al. (2015), using the Shimadzu GC-2010 gas chromatograph
(Shimadzu, Kyoto, Japan) with a flame ionisation detector, Supelco Nukol fused silica
capillary column (60 m × 0.32 mm i.d.; 0.25 µm) and helium as a carrier gas. Phenol,
p-cresol and indole concentrations were calculated using the standard curves and
proportion to 5-methylindole as the internal standard.
Samples of fresh digesta for microbial analysis were prepared by adding 27 ml of
buffered peptone water (Oxoid, Hampshire, UK) to 3 g of samples and homogenising
for 30 s in a laboratory stomacher. Microbial counts were determined using a decimal
dilution series of homogenised samples. The total bacteria count was determined by the
standard plate method using a Columbia LAB-AGAR + 5% KB Agar (Biocorp, Warsaw,
Poland) after a 24-h incubation period at 37°C, and a lactic acid bacteria count using
MRS LAB-AGAR (Biocorp, Warsaw, Poland) after a 72-h incubation period at 30°C.
The yeast content was calculated using YGC Agar (Oxoid, Hampshire, UK) after
incubation at 25°C for 3–5 d. Coliform bacteria were determined using McConkey
agar (Biocorp, Warsaw, Poland) after a 24 h incubation period at 37°C. The number of
bacteria from the Clostridium group in the contents of the caecum was evaluated by the
flow cytometry from refrigerated samples preserved in glycerol and a 1% PBS solution.
FITC-conjugated anti-Clostridium polyclonal antibody purchased from Antibodies
Online (Atlanta, GA, USA) was employed for specific detection of Clostridium sp.
cells. Prior to being stained with antibodies, the samples were filtered using a nylon
net 10 µm syringe filter (assembled with a Swinnex filter holder 25 mm – both from
Merck Millipore, Germany) and washed with a 1% PBS solution containing 1% Tween
20. The cells were then blocked with 5% BSA in a 1% PBS solution to avoid non-specific
binding of anti-Clostridium antibodies. Stained samples were washed three times with a
1% PBS solution containing 1% Tween 20 and analysed with a BD FACS Aria™III
(Becton Dickinson, CA, USA) flow cytometry (cell sorter). The configuration of the
flow cytometry was as follows: a 70 µm nozzle and 70 psi (0.483 MPa) sheath fluid
pressure. The fluorescent signals from FITC conjugated anti-Clostridium antibody were
collected using a 530/30 band pass filter (FITC detector). Flow cytometry analyses were
performed using the logarithmic gains and specific detector settings (10,000 events were
recorded per analysis). The threshold was set on the forward scatter (FSC) and FITC
signals in order to separate microbial cells from the background and put all the cells on
the scale within bivariate FITC vs. side scatter (SSC) dot plots. The data was acquired in
a four-decade logarithmic scale as area signals (FSC-A, SSC-A and FITC-A) and
analysed with the FACS DIVA software (Becton Dickinson). The Q2 regions syndicating the positive sub-populations (stained with anti-Clostridium antibody) were defined
by gating in the dot plots of green fluorescence (FITC-A) versus side scatter signals
(SSC-A). Each sample was analysed in triplicate.
The activity of bacterial enzymes was analysed spectrophotometrically using a
Unicam UV 300 spectrophotometer (Thermo-Spectronic, Cambridge, UK). βGlucuronidase activity was quantified according to the method developed by Barszcz
et al. (2011), based on the analysis of phenolphthalein released from phenolphthalein βD-glucuronide. The absorbance was measured at 540 nm, and the amount of phenolphthalein released was estimated using a standard curve. The activity of β-glucosidase was calculated according to the modified method of Juskiewicz et al. (2002), based
6
P. NOWAK ET AL.
on the analysis of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. The
absorbance was measured at 400 nm. Mucinase activity was analysed according to the
method modified by Barszcz et al. (2016), based on the quantification of reducing
sugars released from porcine gastric mucin during the incubation period. The absorbance was measured at 540 nm, and the activity was calculated from a standard curve
for glucose.
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2.4. Statistical analysis
The data were analysed by a one-way analysis of variance and Tukey’s multiple range
post hoc test using SAS ver. 5.0 (Iowa, USA) and as level of significance p < 0.05 was
used.
3. Results
3.1. Health and growth performance
Although some individuals suffered from diarrhoea, no animal died during the experiment. There were no pigs with diarrhoea in Group I, whereas in the other groups for
1–5 pigs diarrhoea was observed (Table 3).
In comparison with the other groups, animals from Groups Ph and P were characterised by a higher final BW and ADG (p = 0.001), whereas the lowest results were
observed in Groups I and PhP (Table 3). The difference between the final BW and ADG
of Groups Ph and I amounted to about 8 kg and 260 g/d, respectively. The FI in all
experimental groups did not differ significantly in comparison with Group Con, but in
Group P, it was about 10–11 % higher (p = 0.001) than in Groups I and PhP. FCR was
the lowest in Group Ph and differed substantially from the other groups. Also, FCR in
Group P was lower (p = 0.001) than in Groups I and PhP.
3.2. Fermentation indices in ileal digesta
The pH of ileal digesta of Group PhPI was the lowest (5.85) and significantly lower than
in Groups Con, I, P and PhP (Table 4). The highest pH value was found in Groups Con
and I, which was significantly higher than in Groups Ph, PhP and PhPI.
Table 3. Body weight (BW), average daily gain (ADG), feed intake and feed conversion ratio (FCR) of
pigs (n = 8).
Experimental diets
Number of pigs with diarrhoea
Initial BW [kg]
Final BW[kg]
ADG [g]
Feed intake [kg]
FCR [kg/kg]
Con*
1
12.9
34.1c
710c
40.0ab
1.89ab
I#
Ph†
P‡
0
12.9
31.7d
630d
38.2bc
2.04a
2
12.9
39.6a
890a
40.8ab
1.53c
3
12.7
36.7b
790b
42.4a
1.81b
PhP◊
5
12.9
32.1d
640d
38.3bc
2.01a
PhPI§
4
12.9
33.6cd
670 cd
39.1ab
1.95ab
SEM^
0.13
0.48
2.00
0.53
0.04
p-Value
0.997
<0.001
<0.001
0.008
<0.001
*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ^SEM, standard error of mean. a–dMeans with different superscripts within
a row are significantly different (p < 0.05).
ARCHIVES OF ANIMAL NUTRITION
7
Table 4. Liver mass, pH, dry matter (DM) and ammonia content in fresh ileum digesta (n = 6).
Experimental diets
pH
DM [g/100g]
Ammonia [mmol/l]
Liver mass [kg/kg BW]
Con*
6.60a
11.84bc
9.40
0.025
#
I
6.46a
14.05a
11.44
0.025
Ph†
6.07cd
9.50d
9.49
0.020
P‡
6.42ab
10.13cd
9.00
0.020
PhP◊
6.18bc
12.82ab
11.20
0.026
PhPI§
5.85d
11.36bcd
8.53
0.024
SEM^
0.07
0.32
0.51
0.013
p-Value
0.001
0.001
0.360
0.256
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*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ^SEM, standard error of mean. a-dMeans with different superscripts within
a row are significantly different (p < 0.05).
With exception of Group Ph, the DM of ileal digesta did not differ significantly in
comparison with Group Con. The highest DM content in ileum digesta (14.05 g/100 g)
was observed in Group I, which was significantly higher than in Groups Ph, P and
PhPI. The ammonia content in ileal digesta was similar in all groups and ranged
between 8.5 and 11.4 mmol/l for Group PhPI and I, respectively. The liver mass did
not differ among the groups.
3.3. Microbial activity indices in caecal digesta
In the caecal digesta of animals from Groups I, P and PhP, the pH level was lower
(p = 0.001) than in Group Con and in other groups (Table 5). DM of caecal digesta was
significantly higher in Groups Con and I than in Groups P and PhP. The ammonia
concentration did not differ significantly between Group Con and the other groups, but
the value in Group Ph was significantly higher than in Groups PhP and PhPI, whereas
in Group P, it was higher than in Group PhPI (p < 0.05). The total SCFA content and
the content of individual acids did not differ among the groups. There were also no
Table 5. Microbial activity indices in a fresh caecal digesta of the pigs (n = 6).
Experimental diets
pH
DM&[g/100g]
Ammonia [mmol/l]
Acetate [μmol/g]
Propionate [μmol/g]
Isobutyrate [μmol/g]
Butyrate [μmol/g]
Isovalerate [μmol/g]
Valerate [μmol/g]
Total SCFA¶ [μmol/g]
β-glucosidase [IU/g]
β-glucuronidase [IU/g]
Mucinase [IU/g]
Phenol [μmol/g]
p-Cresole [μmol/g]
Indole [μmol/g]
Con*
5.42ab
14.06a
15.3abc
42.5
19.7
0.00
8.50
0.00
0.44
71.1
282
21.5
31.2
0.021
0.250
0.087
I#
5.24c
13.50 a
14.5abc
41.0
21.0
0.00
11.53
0.00
2.70
76.2
212
15.5
36.9
0.017
0.241
0.083
Ph†
5.53a
11.46b
18.3a
40.4
17.4
0.00
9.12
0.00
1.46
68.4
216
17.6
33.5
0.019
0.254
0.082
P‡
5.20c
9.98b
15.8ab
36.9
18.5
0.00
7.17
0.00
0.68
63.3
204
10.9
34.7
0.021
0.254
0.089
PhP◊
5.30c
11.75b
13.2bc
35.9
20.3
0.00
11.52
0.00
3.01
70.7
184
10.6
29.0
0.022
0.212
0.086
PhPI§
5.47ab
11.69ab
11.1c
33.6
22.3
0.00
8.36
0.00
2.06
66.3
202
9.5
27.9
0.020
0.231
0.082
SEM^
0.03
0.29
0.67
1.11
0.61
0.00
0.65
0.00
0.31
1.74
12.4
1.60
1.50
0.001
0.012
0.001
p-Value
0.001
0.001
0.030
0.141
0.230
1.000
0.278
1.000
0.064
0.379
0.300
0.175
0.517
0.599
0.928
0.348
*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ^SEM, standard error of mean; &DM, dry matter; ¶SCFA, short chain fatty
acids. a–cMeans with different superscripts within a row are significantly different (p < 0.05).
8
P. NOWAK ET AL.
differences in bacterial enzymes activity and the content of phenolic compounds in the
caecal digesta among the groups (Table 5).
In comparison with Group Con, levels of yeast and moulds in the caecal digesta of all
other groups were reduced (p < 0.05) (Table 6). A significantly higher total bacteria
count than in Groups Con and P was found in digesta from animals of Groups PhP and
PhPI. There were no differences in the lactic acid and coli group bacteria count or the
Clostridium sp. count.
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3.4. Microbial activity indices in colonic digesta
The pH of digesta varied between parts of the colon (Table 7). Generally, the pH of
digesta in Group I was statistically higher (p < 0.05) in comparison with the other
groups, whereas the lowest pH was found in Groups Con and PhP. The total SCFA
content in the colonic digesta differed significantly (p < 0.05) among the groups and
segments, but generally in Group PhP, the SCFA content was the highest and in
Group I the lowest. Also, the acetate, propionate, isobutyrate and butyrate contents
in colonic digesta were diversified. In the digesta of Group I, the acetate concentration was significantly reduced in comparison with Group Con and the other groups.
The propionate content in the colon digesta of Group I was statistically lower in all
parts of the colon than in Group Con. The propionate content in segments C25 and
C50 was also lower in Groups Ph and P than in Group PhP (p < 0.05). The
isobutyrate content, was statistically lower in digesta of Group PhP, but the butyrate
content in segment C50 was significantly lower in Group I (p < 0.05) than in Groups
Con, PhP and PhPI. Only the activity of β-glucuronidase in segment C75 was
reduced in Group PhP in comparison with Groups Con, I, Ph and P. The βglucuronidase activity was also lower in digesta of Groups PhP and PhPI than in
Group P. Phenol, p-cresol and indole contents in the colonic digesta were similar in
all groups but in segment C75 a tendency of a reduced p-cresol and indole contents
was found in Group PhP.
4. Discussion
Young pigs are subjected to several stressors such as nutritional, environmental, social
and microbial imbalances. As a result, a high incidence of diarrhoea, growth depression,
low FI, impaired intestinal morphology and function are observed. Several feed
Table 6. Microbial counts in the caecal digesta [log CFU/g] (n = 6).
Experimental diets
Yeast and moulds
Lactic acid bacteria
Coli group bacteria
Total bacteria number
Clostridium&
Con*
5.63a
7.96
7.15
8.63b
3.15
I#
5.15b
9.04
7.11
9.08ab
3.01
Ph†
4.53b
8.61
8.20
8.87ab
3.16
P‡
4.71b
8.49
6.92
8.66b
3.04
PhP◊
5.11b
8.77
7.46
9.11a
3.04
PhPI§
4.32b
8.79
7.40
9.18a
3.00
SEM^
0.21
0.52
0.45
0.59
0.20
p-Value
0.007
0.158
0.502
0.020
0.256
*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ^SEM, standard error of mean; &Logarithm values from the means of flow
cytometric counts of Clostridium sp. cells stained with specific antibodies. abMeans with different superscripts within a
row are significantly different (p < 0.05).
ARCHIVES OF ANIMAL NUTRITION
9
Table 7. Microbial activity indices in a fresh colonic digesta of pigs (n = 6).
pH
Acetate
[μmol/g]
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Propionate
[μmol/g]
Isobutyrate
[μmol/g]
Butyrate [μmol/g]
Isovalerate
[μmol/g]
Valerate [μmol/g]
Total SCFA¶
[μmol/g]
β-Glucosidase
[IU/g]
β-Glucuronidase
[IU/g]
Mucinase [IU/g]
Phenole
[μmol/g]
p-Cresole
[μmol/g]
Indole
[μmol/g]
Colon segment
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
C25
C50
C75
Con*
6.08b
6.33bc
6.49b
37.8ab
34.1a
31.3a
19.0ab
17.1ab
15.2ab
0.37ab
0.52a
0.65
10.37
9.67a
9.78
0.18
0.52
0.81
1.53
1.47
1.59
69.2ab
63.4a
59.3a
312
326
366
35.9
34.3
42.1ab
36.0
36.2
39.9
0.019
0.017
0.020
0.330
0.358
0.369
0.105
0.103
0.102
I#
6.77a
7.14a
7.09a
28.4c
23.8b
23.5b
13.3c
10.5c
9.7c
0.50ab
0.69a
0.71
7.44
6.03b
5.72
0.48
0.82
0.76
1.68
1.40
1.24
51.8c
43.2b
41.6b
266
237
220
32.9
37.2
44.1ab
35.2
36.4
39.3
0.018
0.016
0.020
0.342
0.381
0.410
0.090
0.093
0.092
Ph†
6.27b
6.45bc
6.63ab
37.7ab
34.2a
32.0a
17.6bc
15.2b
13.2b
0.62a
0.69a
0.91
10.47
8.16ab
7.68
0.52
0.86
1.13
2.21
1.48
1.34
69.1ab
60.6a
56.3a
256
235
227
29.8
34.3
42.1ab
33.8
33.9
33.7
0.016
0.019
0.017
0.401
0.508
0.535
0.091
0.110
0.102
P‡
6.31b
6.63b
6.68ab
36.7ab
33.2a
31.9a
16.8bc
14.6b
13.5b
0.49ab
0.74a
0.88
9.33
8.60ab
8.37
0.41
1.57
1.05
1.32
1.14
1.21
65.1b
59.9a
57.0a
369
333
352
37.9
41.6
49.6a
36.6
37.4
35.0
0.020
0.020
0.021
0.313
0.361
0.414
0.096
0.102
0.103
PhP◊
5.95b
6.07c
6.42b
40.5a
36.6a
34.4a
22.2a
20.6a
17.7a
0.12b
0.00b
0.49
12.51
10.33a
8.96
0.00
0.00
0.21
3.15
2.47
1.90
78.5a
70.4a
63.7a
295
322
324
24.1
28.9
23.1c
34.8
34.5
37.0
0.025
0.022
0.017
0.269
0.251
0.293
0.089
0.083
0.085
PhPI§
6.34b
6.35bc
6.70ab
34.3b
31.6a
29.1a
18.7ab
17.6ab
14.5ab
0.25b
0.53a
0.63
8.93
9.17a
7.84
0.00
0.46
0.67
1.58
2.29
1.49
63.8b
61.6a
54.3a
359
339
284
25.6
24.5
28.2bc
34.9
33.8
33.9
0.020
0.021
0.015
0.375
0.313
0.366
0.095
0.095
0.106
SEM^
0.07
0.09
0.06
0.97
1.10
0.95
0.74
0.77
0.63
0.05
0.07
0.06
0.52
0.45
0.43
0.07
0.17
0.11
0.21
0.18
0.14
1.98
2.05
1.80
16.1
18.5
19.1
1.90
2.20
2.70
0.70
0.60
0.80
0.001
0.001
0.008
0.021
0.027
0.024
0.002
0.003
0.003
p-Value
0.004
0.002
0.020
0.001
0.007
0.009
0.006
0.001
0.003
0.085
0.003
0.340
0.080
0.040
0.115
0.070
0.160
0.235
0.100
0.160
0.733
0.001
0.001
0.004
0.217
0.340
0.092
0.267
0.309
0.027
0.926
0.517
0.122
0.209
0.548
0.339
0.561
0.112
0.088
0.296
0.070
0.453
*Con, Control; #I, inulin; †Ph, phytobiotics; ‡P, probiotic bacteria; ◊PhP, phytobiotics and probiotic bacteria; §PhPI,
phytobiotics, probiotic bacteria and inulin; ^SEM, standard error of mean; ¶SCFA, short-chain fatty acids. a–cMeans
with different superscripts within a row are significantly different (p < 0.05).
additives are recognised as effective substances in pig production, preventing diarrhoea
and improving the results of growing pigs.
4.1. Effect of the phytobiotic additive
Spices and herbs are commonly used in animal nutrition as flavouring additives and
also for their antiseptic or medicinal properties. These properties are generally the effect
of bioactive substances such as essential oils, organic acids, flavonoids, saponins, etc.
The water extracts of thyme and oregano used in this experiment were chosen for their
activity against several pathogenic bacteria and lack of activity against selected probiotic
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10
P. NOWAK ET AL.
bacteria (data not published). Generally, herbs stimulate feed consumption (Frankić
et al. 2009; Hanczakowska and Swiatkiewicz 2012), but some authors found that thyme
or oregano, or their extracts used as a flavouring additive, did not improve or even
reduced feed consumption (Namkung et al. 2004; Jugl-Chizzola et al. 2006). It is in
agreement with our own observations, where the phytobiotics did not improve FI,
compared to the Control diet. Despite this, the pigs’ performance was satisfactory, due
to the fact that daily gains in pigs offered phytobiotics were higher, which was a result
of better feed utilisation. Finally, the pigs from this group achieved about 16% higher
BW in comparison with Control group. It is in accordance with the scientific literature,
since phytobiotics, thyme and oregano, in particular, are recognised as digestion
stimulants (Vidanarachchi et al. 2005; Frankić et al. 2009; Han et al. 2016). Active
substances present in herbs enhance the synthesis of bile acids in the liver and their
excretion in bile, which has a beneficial effect on the digestion and absorption of lipids.
Moreover, plant spices stimulate the functioning of pancreatic enzymes and increase the
activity of the digestive enzymes of gastric mucosa (Costa et al. 2011). Phytobiotic can
also accelerate digestion and shorten the time of feed passage through the digestive tract
(Frankić et al. 2009). In this study, the phytobiotics generally did not affect the digesta
parameters but reduced DM concentration of ileal digesta, which could possibly
indicate faster digestion of feed or micro-organisms proliferation in the ileum. It is
also proven by a significantly lower number of yeasts and moulds in caecal digesta, yet
it had no effect on the coliform or Clostridium count. On the other hand, in this group
two cases of diarrhoea were noted, but because of low animal number in the group, this
data should be interpreted with some caution. In this study, no beneficial antibacterial
effect was found, which is probably due to a high specificity resulting from the chemical
composition of extracts or their inappropriate dose. Namkung et al. (2004) found
reduced coli group bacteria in pigs offered a mixture of thyme and oregano extracts,
whereas Oetting et al. (2006) found no effect of extracts from thyme, oregano and other
phytobiotics on intestinal microflora. According to Costa et al. (2013), active substances
present in thyme and oregano extracts can stabilise microflora and reduce potential
harmful microbes, but they tend to show the antioxidant properties, which is also
proven by the chemical composition of used phytobiotics.
4.2. Effect of the probiotic bacteria additive
The administration of probiotics is expected to stimulate the development of normal
microbiota in the gastrointestinal tract, reduce the pH of the intestinal contents and
enhance the digestion of feed, prevent or stop the spread of pathogenic microorganisms, improve the resistance of the organism and improve the integrity and proper
functioning of the gastrointestinal tissue. According to the literature, probiotic strains
should be non-pathogenic, acid and bile resistant, active against pathogenic strains,
stable, with good adhesion to intestinal mucosa, with a minimal dosage of 106 CFU/t
(Shim 2005; De Lange et al. 2010; Liu et al. 2014). The administered probiotic
preparation consisted of four bacteria strains isolated from domestic pigs. Assays
carried out in vitro showed their strong activity against pathogenic strains of E. coli
and C. perfringens, and resistance to the special conditions of the pigs’ digestive tract
(Grajek et al. 2016). The use of prepared probiotic mixtures had a positive effect on
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ARCHIVES OF ANIMAL NUTRITION
11
pigs’ growth in comparison with Groups Con and I, although no influence on FI or feed
utilisation was found. The highest activity of bacteria is generally observed in the
caecum. The lowest pH was observed in the caecal digesta of animals offered the
probiotic, which can suggest the best condition for lactic acid bacteria proliferation,
but it is not in the agreement with lower DM content and similar number of lactic acid
or coli group bacteria in Group Con. Tests in vivo also did not confirm the bactericidal
activity of the preparations against the Clostridium and E. coli organisms, for which the
in vitro study showed strong bactericidal activity against four types of C. perfringens
most common in Poland and related to the toxic E. coli strains. The good health of
piglets in the Control (Group Con) suggests that within the intestinal microbiota of the
examined animals, there were no bacteria (or there were very few) that were a potential
target for these preparations. Perhaps infectious tests and the use of more specific test
methods to monitor the number of pathogenic bacteria would allow a better verification
of the activity of preparations. The comparison of results with those of the other
authors is difficult due to a different composition of probiotics, dosage, feeding pattern
and environmental conditions. However, some authors failed to find any beneficial
effects of probiotic strains of Lactobacillus on growth, FI and feed efficiency or microbial population, diarrhoea score and growth performance in piglets (Xuan et al. 2001).
4.3. Effect of the prebiotic additive
Inulin is composed of linear chains of fructose monomers containing a terminal glucose
residue, with chain lengths varying between 3 and 65 monomers. Inulin is not digested
by the small intestinal enzymes of pigs but is fermented in the large intestine to produce
SCFA; therefore, bifidobacteria and possibly other genera are preferentially stimulated
to grow, causing changes in the gut microbiota composition, especially reducing the
number of potentially harmful species causing diarrhoea (Halas et al. 2009; Grela et al.
2013, 2014). In this study, the lowest incidences of diarrhoea was observed after single
inulin addition (Group I), which is comparable with results reported by Halas et al.
(2009). In the ileal digesta, no differences in the measured markers were found as
compared with Group Con, which confirms no significant role of inulin in the small
intestine. At the same time, a lower pH as well as yeast and moulds were found in the
caecal digesta, which may suggest that inulin fermented in the caecum. The microbiota
of the large intestine is involved in the conversion of procarcinogens, for example,
glucuronide conjugates and terminal non-reducing β–D-glucosides, into carcinogenic
or toxic substances. These reactions are catalysed by β-glucuronidase and β-glucosidase
(Desrouillères et al. 2015). Mucinase should be also taken into consideration because it
is used by pathogens to degrade the protective mucus layer and facilitates colonisation
of intestinal epithelium. Thus, the reduction of their activity can contribute to the
improvement of gut health and animal welfare (Barszcz et al. 2017). In this study, the
pH was higher, while the SCFA content, some bacterial enzymes activity and phenols
and indole production were lower in the colonic digesta in comparison with the Group
Con, although, the number of lactic acid bacteria and bacteria from the coli group and
Clostridium did not differ from Group Con. However, many studies found that
Lactobacillus populations significantly increased when inulin was added to the diet
(Tako et al. 2008; Grela et al. 2013, 2014). Both SCFA and polyamines can improve the
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12
P. NOWAK ET AL.
expression of intestinal tight junction proteins, which are the important constitutes of
the intestinal barrier, and help resist the invasion of pathogens (Han et al. 2016). In
comparison with Group Con, ADG in the group offered prebiotics were about 12%
lower and the final BW about 7% lower. Furthermore, feed consumption and utilisation
was worst in this group, which is difficult to explain. Grela et al. (2013) found a better
growth of pigs when 3% inulin was added to the diet, but some authors also highlighted
the important role of the degree of polymerisation of inulin in the mode of action,
digestibility, activity, caloric value, sweetening power, water binding capacity, etc.
(López-Molina et al. 2005; Van De Wiele et al. 2007). Frantz et al. (2003) found no
effect of inulin on weaning pigs’ growth but higher feed consumption and better feed
utilisation.
4.4. Effect of the multi-eubiotic preparation
Probiotic bacteria may be enhanced by the presence of other protective or stimulating
growth substances such as phytobiotics and prebiotics (Wang et al. 2016). However, the
method of connection and protection of these substances is important, as at right
circumstances resulting from both their characteristics, the mechanism of action and
concentration their mutual interaction can be positive or negative. The desired effect of
synergy occurs when the ingredients present in the supplement do not affect each other
competitively, or as a parasiticide, and act in different directions. In this study, two
different mixtures of ingredients were used, and to avoid a negative impact on the
components on others, they were dosed in the protected form. In the groups where
both mixtures were used, generally lower gains were found, especially in comparison
with the groups treated separately with phytobiotic and probiotic bacteria. In the ileal
digesta of Group PhP, a reduced pH and higher DM was found, which suggests
improved microflora proliferation. The PhP and PhPI diets increased the total bacteria
count and reduced the number of yeasts and moulds in comparison with the Group
Con. The colonal digesta from Groups PhP and PhPI were characterised by higher
propionate and lower isobutyrate contents, but the total SCFA concentration was
similar to the other groups. In this study, the observed effects were not synergetic but
rather antagonistic, especially as far as it concerns ADG and FCR. Although the FI was
satisfactory, it was utilised worse in both groups offered multicomponent additives. This
could be caused by the release of substances from granules and/or the antagonistic
action of phytobiotic for probiotic bacteria, although in the in vitro study, they showed
poor activity against isolated probiotic strains.
Similarly to this study, Zangeronimo et al. (2011) offering pigs a symbiotic mixture
combined with herbal extracts, did not found improved FCR, but there were differences
in the final BW, ADG and FI. These authors also did not observe differences in the
count of coliform or lactobacilli bacteria in the ileum and caecum, but higher phytobiotic contents in the diet changed the microflora composition in the digestive tract
more effectively. Grela et al. (2013), offering the pigs inulin (3%) or an inulin and garlic
extract (500 ml/l of water), found that the use of mixed additives reduced the FI but
improved pigs’ growth and feed utilisation in the total fattening period. Simultaneously,
the combined use of inulin and garlic extract in the finisher period decreased weight
gains of pigs as compared to those receiving inulin alone.
ARCHIVES OF ANIMAL NUTRITION
13
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5. Conclusion
The effectiveness of probiotics and phytobiotics administrated to growing pigs separately was better in comparison with the same additives administrated together, whereas
the inulin additive reduced pigs’ growth. So, the hypothesis that a multi-eubiotic
preparation containing a combination of probiotic bacteria strains, inulin and phytobiotics (watery extracts from thyme and oregano) at the dosage used would improve the
pigs’ performance, was not confirmed. All multicomponent additives stimulated the
microflora of the digestive tract, but the observed changes were not beneficial in
comparison with separately administered additives. Therefore, the formula and the
dose of multi-eubiotics should be determined. In subsequent studies, the changes in
the intestinal micro ecosystem of animals should be monitored in a more comprehensive manner, i.e. by using metagenomic analysis.
Acknowledgements
We would like to thank Martyna Tołpa and Łukasz Staśkiewicz for their great technical
assistance.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study has been carried out within the project: “Develop of eubiotic preparation for farm
animals”, financed by the National Research and Development Centre for Applied Research
Program [Grant No. PBS1/A8/10/2012].
ORCID
http://orcid.org/0000-0003-2753-4371
Anita Zaworska
http://orcid.org/0000-0001-5421-3165
Barbara Stefańska
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