Influence of Enterococcus faecium with and without xylanase to corn and soybean meal-based diet on growth performance, nutrient digestibility, blood profiles, and faecal microflora in growing pigs

ABSTRACT

A total of 120 pigs [Duroc × (Yorkshire × Landrace)], with an average BW of 25.22 ± 1.88 kg, were randomly allotted to 4 experimental diets based on initial BW and sex (six replicate pens/treatment; 5 pigs/pen) to determine the effects of Enterococcus faecium (EF) with and without xylanase in growing pigs. The experiment lasted for 6 weeks. Dietary treatments included CON, basal diet; EF1, CON + 0.002% (EF); EF2, CON + 0.005% EF; EX, CON + 0.002% EF + 0.01% Xylanase. During the overall study, pigs fed EX diet increased ADG and G:F compared to pigs fed CON and EF1 diets (P<0.05). Pigs fed EF2 and EX diets had a greater (P<0.05) energy digestibility and Lactobacillus counts than pigs fed CON diet. Pigs fed EX diet decreased creatinine concentrations in the blood and E. coli concentrations compared to pigs fed CON diet (P<0.05) at 6th week. In conclusion, supplementation of growing pigs diet with a combination of EF and xylanase improved performance, nutrient digestibility and faecal Lactobacillus counts and reduced faecal E. coli counts in growing pigs.

KEYWORDS:

• Digestibility
• growth performance
• pigs
• probiotics
• xylanase

Introduction

Feed cost is very high in animal production, by contributing from 60 to 70% of production cost. Prices of the main ingredients like corn and soybean meal have increased over years. This overprice has led to a change in feed formulation towards increased use of alternative feed ingredients. Consequently, diets may contain a higher level of anti-nutritional factors, including non-starch polysaccharides, and have a lower nutrient digestibility Dersjant-Li et al. (2015). Therefore, the use of enzymes to treat feeds, such as corn and soy bean meal, is a more recent concept. In corn and sorghum, with the grain-based diets, xylanase enzymes are effective in degrading the fibrous cell walls of feed grains and releasing nutrients previously inaccessible to the animals (Chuka 2014). It is reported that dietary xylanase supplementation could increase feed intake, bird performance, feed utilization, and nutritional diffusion (Wyatt et al. 2008).

The increased public concern on bacterial resistance to antibiotics jeopardizes antibiotic treatment of humans. As a consequence, the use of antibiotics in animal feed has been prohibited by many countries, including European Union, since 2006 as well as South Korea since 2011 (Nguyen et al. 2018). Therefore, the interest of discovering alternatives has been increasing day by day. The use of probiotics in animal feed has increased. Probiotic micro-organisms are responsible for the production of vitamin B complex and digestive enzymes which stimulate intestinal immunity and increase protection against toxins produced by pathogenic organisms (Rahman et al. 2014). Previous studies showed that probiotics have multiple beneficial effects, including the competitive exclusion of pathogenic bacteria (Vine et al. 2004; Balcázar et al. 2006), the enhancement of the immune system (Picchietti et al. 2009; Zhou et al. 2010), the stimulation of the digestive system (Suzer et al. 2008; Lazado et al. 2012), and the improvement of growth performance (Mountzouris et al. 2010; Zhang and Kim 2014). Enterococcus faecium (EF) is a lactic acid bacterium (LAB) which normally colonizes the gut (Samli et al. 2007; Park et al. 2016). It was reported that the supplementation of EF contributed to an improvement in the performance parameters of finishing pigs (Černauskienė et al. 2011), total tract nutrient digestibility and intestinal microbial balance of growing-finishing pigs (Yan and Kim 2013), and contributed towards excluding pathogen colonization in the host (Zhang and Kim 2015).

Probiotics are often combined with digestive enzymes to support optimal efficiency of the helpful bacteria. An interaction between multi-enzymes and direct-fed microbials (DFM) has been reported in the literature (Romero et al. 2013). Direct-fed microbials could improve microbial balance and intestinal health, and provide an environment that may stimulate the activity of enzymes (Dersjant-Li et al. 2015). Several studies have shown that such enzymes also reduce the colonization of the gut by pathogens (Vahjen et al. 1998; Danicke et al. 1999; Hübener et al. 2002). Hence, we hypothesized that there might be an interaction between the effects of probiotic and enzyme complex, especially in a diet based on corn and soybean meal with the enhancement of immunity system, and eventually increasing performance in pigs. In this regard, this study was conducted to examine the effects of EF with and without xylanase to corn and soybean meal-based diet on growth performance, nutrient digestibility, blood profiles, and faecal microflora in growing pigs.

Materials and methods

The experimental protocol used in this study was approved by the Animal Care and Use Committee of Dankook University.

Source of Enterococcus faecium and Endo-1,4-xylanse

The probiotic product (Bonvital®) used in our study was provided by a commercial company (Schaumann Agri International GmbH, Pinneberg, Germany). This product is composed of spray-dried spore-forming EF, which is guaranteed to contain at least 1.0 × 10¹⁰ cfu g⁻¹ of live EF DSM 7134.

The enzyme preparation used in this study is Nutrase Xyla (Nutrex nv, Belgium) and the producing micro-organism is Bacillus subtilis. It’s a specific Endo-1,4-β-xylanase (IUB: EC 3.2.1.8; 9000 U/g) with an optimal activity at neutral pH.

Experimental design, animals, diets, and housing

A total of 120 growing pigs [(Yorkshire × Landrace) × Duroc], with an average body weight (BW) of 25.22 ± 1.88 kg, were randomly allotted to 4 experimental diets based on initial BW and sex (6 replicate pens per treatment; 2 gilts and 3 barrows/pen). The experiment lasted for 6 weeks. Dietary treatments included (1) CON, basal diet, (2) CON + 0.002% EF (EF1), (3) CON + 0.005% EF (EF2), and (4) CON + 0.002% EF + 0.01% Xylanase (EX). The diets were formulated to meet or exceed NRC (2012) nutrient requirements (Table 1). Pigs were housed in an environmentally controlled, slatted plastic floor facility in 24 adjacent pens (1.8 × 1.8 m each), and room temperature and humidity were maintained at approximately 25°C and 60%, respectively. Each pen was equipped with a self-feeder and nipple drinker to allow ad libitum access to feed and water throughout the experimental period.

Table 1. Composition of the basal diets (as-fed basis g kg⁻¹).

Ingredients
Corn	553.80
Soybean meal	334.30
Molasses	25.00
Animal fat	53.30
Defluorinated phosphate	19.30
Limestone	7.80
l-lysine HCl	1.70
Trace mineral premix^a	1.00
Vitamin premix^b	1.20
Salt	2.00
DL-methionine	0.30
Choline chloride	0.30
Chemical composition
ME (kcal/kg)	3360
Crude protein (%)	20.00
Lysine (%)	1.30
Calcium (%)	0.90
Phosphorus (%)	0.80
Analysed composition
GE (kcal/kg)	4112
Crude protein (%)	19.65
Calcium (%)	0.87
Phosphorus (%)	0.76

^a Provided per kg of complete diet: 12.5 mg Mn, 179 mg Zn, 140 mg Cu,0.5 mg I, and 0.4 mg Se.

^b Provided per kg of complete diet: 20,000 IU of vitamin A; 4000 IU of vitamin D3; 80 IU of vitamin E; 16 mg of vitamin K3; 4 mg of thiamine; 20 mg of riboflavin; 6 mg of pyridoxine; 0.08 mg of vitamin B12; 120 mg of niacin; 50 mg of Ca pantothenate; 2 mg of folic acid and 0.08 mg of biotin.

Sampling and measurements

For the growth assay, the individual pig weights and feed intake were recorded initially, at the end of week 3, and week 6 of the experimental period. Feed consumption was recorded on a pen basis during the experiment to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F). Chromium oxide was added to the diet as an indigestible marker at 0.20% of the diet for 7 days prior to faecal collection at the 6th week to calculate dry matter (DM), nitrogen (N), and energy digestibility. Faecal samples were collected randomly from at least two pigs (one barrow and one gilt) from each pen, mixed and pooled, and a representative sample was stored in a freezer at −20°C until analysed. The faecal samples were thawed and dried at 60°C for 72 h, after which were finely ground to pass through a 1-mm screen. The procedures used for the determination of DM, N, and energy digestibility were in accordance with the methods established by the AOAC (2002). Chromium concentrations were determined via UV absorption spectrophotometry (UV- 1201, Shimadzu, Kyoto, Japan) and the apparent total tract digestibility (ATTD) of DM, N, and energy was calculated using indirect methods, as described by Fenton and Fenton (1979).

Four pigs (2 barrows and 2 gilts) were randomly selected from each treatment and blood samples (3 ml) were collected via jugular venipuncture with non-heparinized tubes to obtain serum at the 6th week of the experiment. The total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol in the serum samples were analysed with an auto analyser (Automatic Biochemical Analyzer, RA-1000; Bayer Corp., Tarrytown, NY) using colorimetric methods. Serum creatinine concentrations were determined using an Astra-8 Analyzer (Beckman Instruments, Inc., Brea, CA 92621). The blood urea nitrogen (BUN) was analysed using the Abbott Spectrum urea nitrogen test (Series II, Abbot Laboratories, Dallas, TX). Plasma glucose concentrations were determined by colorimetric assay (Glucose Procedure # 16-UV, Sigma Diagnostics, St. Louis, MO).

At the end of 6th week, faecal samples were collected directly by massaging the rectum of 2 pigs (1 gilt and 1 barrow) in each pen and then pooled and placed in the icebox for transportation to the laboratory where analysis was immediately performed. A calibrated, glass-electrode pH meter (WTW pH 340- A; WTH Measurement Systems Inc., Ft. Myers, FL) was used to measure the pH of the faecal samples, which were diluted with deionized water at a ratio of 1:7.5 (wt/wt). One gram of the composite faecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co.) and then homogenized. Viable counts of bacteria in the faecal samples were then conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, MI) and Lactobacilli medium III agar plates (Medium 638; DSMZ, Braunschweig, Germany) to isolate the Escherichia coli (E. coli) and Lactobacillus, respectively. The Lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. The E. coli and Lactobacillus colonies were counted immediately after removal from the incubator.

Statistical analyses

All experimental data were analysed using the Mixed procedure as a randomized complete block design (SAS Inst. Inc., Cary, NC). Each pen served as the experimental unit. The initial BW was used as a covariate for ADFI and ADG. Variability in data was expressed as standard error of means (SEM). Differences among the treatment means were determined by using the Duncan’s test. A probability level less than 0.05 was considered to be statistically significant.

Results

Growth performance

In the current study, pigs fed EF1, EF2, and EX diets increased (P<0.05) the BW at 3rd and 6th weeks compared to those of pigs fed CON diet (Table 2). In addition, the BW of pigs fed the EX diet increased (P<0.05) compared to pigs fed EF1 at the 3rd and 6th weeks. Pigs fed EF1, EF2, and EX diets had significantly higher ADG for week 1–3 and during the overall study than those fed the CON diet. The ADG of pigs fed the EX diet increased (P<0.05) compared to those of pigs fed the EF1 diet for week 1–3 and during the overall study than those fed the EF1 diet. The G:F of pigs fed EF2 and EX diets increased (P<0.05) compared to pigs fed CON and EF1 diets for week 1–3 and during the overall study than those fed EF1 diet. For week 3–6, pigs fed EF2 and EX diets increased (P<0.05) the ADG and G:F compared to pigs fed CON and EF1 diets. However, no significant differences (P>0.05) were observed in the ADFI among treatments for week 1–3, 3–6, and during the overall study.

Table 2. Effects of Enterococcus faecium with and without xylanase to corn and soybean meal-based diet on growth performance in growing pigs¹.

Items	CON	EF1	EF2	EX	SEM^2	P-value
Body weight (kg)
Initial	25.19	25.24	25.20	25.26	0.03	0.440
week 3	37.65^c	38.69^b	39.10^ab	39.29^a	0.18	<0.0001
Finish	52.19^c	53.81^b	54.76^ab	55.43^a	0.32	<0.0001
week 1–3
ADG (g)	593^c	642^b	660^ab	668^a	8.12	<0.0001
ADFI (g)	1481	1524	1469	1473	17.64	0.148
G:F	0.401^b	0.422^b	0.449^a	0.454^a	0.007	0.0004
week 3–6
ADG (g)	693^c	720^bc	745^ab	768^a	9.85	0.0004
ADFI (g)	1827	1821	1796	1818	27.64	0.863
G:F	0.379^b	0.395^ab	0.41^a	0.423^a	0.009	0.016
Overall
ADG (g)	643^c	681^b	703^ab	718^a	7.73	<0.0001
ADFI (g)	1654	1673	1632	1646	19.20	0.531
G:F	0.389^b	0.407^b	0.431^a	0.436^a	0.008	0.002

¹ Abbreviation: CON, basal diet; EF1, CON + 0.002% Enterococcus faecium; EF2, CON + 0.005% Enterococcus faecium; EX, CON + 0.002% Enterococcus faecium + 0.01% Xylanase.

² Standard error of means.

^a,b,c Means in the same row with different superscript differ significantly (P < 0.05).

Nutrient digestibility

Pigs fed EF2 and EX diets had increased (P<0.05) the ATTD of energy at 6th week compared to those of pigs fed the CON diet (Table 3). No significant differences (P>0.05) were observed in the ATTTD of DM and N among treatments at 6th week.

Table 3. Effects of Enterococcus faecium with and without xylanase to corn and soybean meal-based diet on nutrient digestibility in growing pigs.¹

Items	CON	EF1	EF2	EX	SEM^2	P-value
week 6
Glucose, mg/dL	76	83	87	87	5.45	0.431
Bun, Ug/dL	10	10	11	10	1.51	0.970
Creatinine, mg/dL	1.27^a	1.21^ab	0.90^b	1.07^ab	0.08	0.039
HDL/C, mg/dL	38	39	40	39	3.95	0.982
LDL/C, mg/dL	66	63	63	59	5.03	0.820
Total cholesterol, mg/dL	109	101	104	100	8.97	0.891

¹ Abbreviation: CON, basal diet; EF1, CON + 0.002% Enterococcus faecium; EF2, CON + 0.005% Enterococcus faecium; EX, CON + 0.002% Enterococcus faecium + 0.01% Xylanase.

² Standard error of means.

^a,b Means in the same row with different superscript differ significantly (P < 0.05).

Blood characteristics

Table 4 shows that pigs fed the EX diet decreased creatinine concentrations in the blood compared to the pigs fed the CON diet (P<0.05) at 6th week. However, no significant differences (P>0.05) were observed on glucose, BUN, creatinine, HDL, LDL, and total cholesterol among treatments at 6th week.

Table 4. Effects of Enterococcus faecium with and without xylanase to corn and soybean meal-based diet on blood profiles in growing pigs¹.

Items, %	CON	EF1	EF2	EX	SEM^2	P-value
week 6
Dry matter	74.16	75.13	76.10	76.38	0.70	0.136
Nitrogen	72.72	73.76	74.73	75.03	0.75	0.152
Energy	72.84^b	74.66^ab	75.48^a	75.73^a	0.69	0.022

¹ Abbreviation: CON, basal diet; EF1, CON + 0.002% Enterococcus faecium; EF2, CON + 0.005% Enterococcus faecium; EX, CON + 0.002% Enterococcus faecium + 0.01% Xylanase.

² Standard error of means.

^a,b Means in the same row with different superscript differ significantly (P < 0.05).

Faecal microflora

At 6th week, pigs fed the EF2 and EX diets had led to significant (P<0.05) higher counts of Lactobacillus compared to the pigs fed the CON diet (Table 5). In addition, pigs fed the EX diet increased (P<0.05) Lactobacillus counts compared to pigs fed the CON diet. Moreover, pigs fed the EX diet decreased (P<0.05) E. coli counts compared to those of pig fed the CON diet.

Table 5. Effects of Enterococcus faecium with and without xylanase to corn and soybean meal-based diet on faecal microbial in growing pigs¹.

Items, log10cfu/g	CON	EF1	EF2	EX	SEM^2	P-value
week 6
Lactobacillus	7.42^c	7.48^bc	7.55^ab	7.61^a	0.03	0.008
sE. coli	6.49^a	6.37^ab	6.29^ab	6.18^b	0.05	0.022

¹ Abbreviation: CON, basal diet; EF1, CON + 0.002% Enterococcus faecium; EF2, CON + 0.005% Enterococcus faecium; EX, CON + 0.002% Enterococcus faecium + 0.01% Xylanase.

² Standard error of means.

^a,b,c Means in the same row with different superscript differ significantly (P < 0.05).

Discussion

Growth performance and nutrient digestibility

The use of EF (Chen et al. 2005; Černauskienė et al. 2011; Mohana Devi and Kim 2014; Zhang et al. 2014) and exogenous digestive enzyme (xylanase) (Barrera et al. 2004; Zhang et al. 2014) as individual supplementation in pig diets has been previously reported. However, to the best of our knowledge, there are relatively limited data on the combined effects of probiotic and exogenous digestive enzyme on growing pigs. In this study, growing pigs were fed diet supplemented with probiotic in a different level or a combination of both the probiotic and enzyme. Given the potential complimentary modes of actions of exogenous digestive enzymes and probiotic, the two products (when used in combination) could offer more benefits than when used alone. Currently, there is little information on the influence of the combined effects of probiotic and exogenous digestive enzyme in pigs. In agreement with the current results, Nguyen et al. (2017) reported that a trend in interactive effect between EF and XY was observed for gaining feed ratio during weeks 6–9 in finishing pigs. Besides, it was also reported that broilers fed the diets with a combination of probiotics and enzymes significantly improved BW, feed conversion ratio (FCR), as well as nutrient digestibility compared to the control diets and the diets with single probiotic in broilers (Romero et al. 2013; Rahman et al. 2014). Murugesan and Persia (2013) has shown that the FCR was higher in broilers fed corn- and soybean meal-based diets with a combination of probiotic and enzyme compared to that of the additives used individually. In addition, Momtazan et al. (2011) found that there was a linear interaction in growth performance between the enzyme complex concentration and probiotic inclusion. Probiotic inclusion boosted the effect of enzyme complex on BW and FCR in broilers. Moreover, Černauskienė et al. (2011), Mohana Devi and Kim (2014), and Zhang et al. (2014) had suggested that weanling pigs fed diet with EF supplementation increased ADG and decreased FCR. Chen et al. (2005) has shown that the inclusion of EF linearly improved ADG compared to pigs fed CON diet in finishing pigs. Lee et al. (2009) and Zhang et al. (2014) also demonstrated that the energy digestibility was improved in pigs fed the dietary EF supplementation.

The enhanced growth performance could be attributed to the ability of probiotic to produce lactic acid to reduce the pH value of the intestinal content, and inhibit the development of invasive pathogens, which led to the improvement of nutrient digestibility by improving the efficiency of digestion and nutrient absorption processes and eventually increased growth performance in this study (Černauskienė et al. 2011) as well as the external xylanase capacity to increase the digestion of non-starch polysaccharides (NSPs) (e.g. arabinoxylans) which could provide substrates for probiotic action (Bedford 2000).

Faecal microflora

The presence of Lactobacillus in the gastrointestinal tract is believed to be beneficial for pigs. In the present study, the faecal Lactobacillus populations were increased, and the faecal E. coli counts were decreased by the application of the EF or a combination of EF and xylanase. In agreement with our results, Zhang et al. (2014) has shown that greater Lactobacilli counts were observed in the dietary EF inclusion. Mallo et al. (2010) and Zhang and Kim (2015) suggested that pigs fed the EF diets increased the ileal and faecal Lactobacilli counts and decreased E. coli counts in pigs. Dersjant-Li et al. (2015) reported that combination of multi-enzymes with DFM might have a positive effect on microbial balance and microbial metabolic end-products in the small intestine by stimulating beneficial bacteria population such as Lactobacilli (indicated by numerically higher lactic acid content in both ileal and cecal digesta) and reducing pathogen bacteria colonization such as C perfringens. This could be related to the increment total short-chain fatty acid and lactic acid production and might imply an improved intestinal health of pigs. A possible reason for the enhanced beneficial bacteria population in the current study may be a reduction of intestinal pathogen level: EF is a normal microorganism in swine intestine, which could produce lactic acid to reduce the pH value of the intestinal content, and inhibit the development of invasive pathogens (Černauskienė et al. 2011), and the use of exogenous enzymes that promote hydrolysis of NSP and can lead to increase the height of villi and the proportion height-to-depth of the crypts (Mathlouthi et al. 2002).

Blood profiles

The probiotic used in this study is designed to reduce creatinine concentrations in the blood. Creatinine is a waste product that forms when creatinine breaks down, which was normally eliminated from the body; therefore, higher levels of creatinine indicate a lower glomerular filtration rate and, as a result, a decreased capability of the kidneys to excrete waste products (George et al. 2016). However, currently, there is little information on the influence of EF supplementation on blood profiles; thus, no comparisons could be made with other studies. However, preliminary evaluation of the probiotic in cats showed reduction in creatinine (Palmquist 2006; McCain et al. 2011). Agboola et al. (2015) reported that EF had an immune-stimulating effect in pigs, which might be a reason to support for the decrease of creatinine. The reason for this is not clear. Therefore, further studies are necessary.

Conclusion

Supplementation of growing pigs’ diet with a combination of EF and xylanase improved growth performance, nutrient digestibility, faecal Lactobacillus counts, and reduced faecal E. coli counts and creatinine concentrations in blood. We suggest that the two products (when used in combination) could offer more benefits than when used alone.

Disclosure statement

No potential conflict of interest was reported by the author(s).