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. Submit your article to this journal Article views: 9 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gaan20 Download by: [UNIVERSITY OF ADELAIDE LIBRARIES] Date: 26 October 2017, At: 04:38 ARCHIVES OF ANIMAL NUTRITION, 2017 https://doi.org/10.1080/1745039X.2017.1390181 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 email@example.com © 2017 Informa UK Limited, trading as Taylor & Francis Group KEYWORDS Digestive tract; microbial ﬂora; performance; pigs; prebiotics; probiotics; synbiotics 2 P. NOWAK ET AL. Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 3 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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. Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 4 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. Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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. Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 *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. Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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] Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 04:38 26 October 2017 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 References Barszcz M, Taciak M, Skomiał J. 2011. A dose-response eﬀects of tannic acid and protein on growth performance, caecal fermentation, colon morphology, and β-glucuronidase activity of rats. J Anim Feed Sci. 20:613–625. Barszcz M, Taciak M, Skomiał J. 2016. The eﬀects of inulin, dried Jerusalem artichoke tuber and a multispecies probiotic preparation on microbiota ecology and immune status of the large intestine in young pigs. Arch Anim Nutr. 70:278–292. Barszcz M, Taciak M, Skomiał J. 2017. 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