Effects of microbial enzymes and a complex of lactic acid bacteria and Saccharomyces boulardii on growth performance and total tract digestibility in weaned pigs

Hoang Huong Giang, Tran Quoc Viet*, J E Lindberg** and B Ogle**

Department of Livestock Production, Ministry of Agriculture and Rural Development,
2 Ngoc Ha Street, Ba Dinh District, Ha Noi City, Vietnam
* Department of Animal Nutrition, National Institute of Animal Science, Thuy Phuong Commune, Tu Liem District, Ha Noi City, Vietnam
** Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, P.O. Box 7024, SE-750 07 Uppsala, Sweden
Gianghoang97@yahoo.com

Abstract

A total of 96 weaned piglets [(Landrace × Yorkshire) × (Duroc × Pietrain)] (48 females and 48 males) from 12 litters (21-24 days of age, 7.4 ± 0.6 kg body weight [BW]) was divided into 4 treatment groups, balanced in sex, BW and litter origin (8 pigs per pen, 3 pens per group). There were four diets fed for 35 days: a basal diet without any antibiotics or probiotics (Diet C); the basal diet supplemented with 0.2% of a mixture of microbial enzymes (amylase, protease, cellulase, β-glucanase and xylanase) (Diet E); the basal diet supplemented with 0.2% of a mixture of lactic acid bacteria (LAB) (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7) and yeast (Saccharomyces boulardii Sb) (Diet LY); the basal diet supplemented with 0.2% of the LAB-yeast complex and 0.2% of the enzyme mixture (Diet LYE). Chromium oxide was used as a marker and added at 3 g/kg of basal diet to determine total tract digestibility.

Diet E improved (P<0.05) average daily gain (ADG) and feed conversion ratio (FCR), and apparent total tract digestibility (ATTD) of crude protein (CP), crude fibre (CF) and organic matter (OM) in the first 2 weeks post weaning (Per I), but not in the following 3-week period (Per II). Piglets fed diets LY and LYE had improved (P<0.05) ADG and FCR, and higher ATTD of CP, CF, OM in both Per I and II, except for ATTD of CF in Per II. There were no differences in performance or digestibility between diet LY and LYE (P>0.05).

Keywords: enzymes, lactobacilli, piglets, yeast

Introduction

In pig production, the weaning transition is the most difficult period that the piglets have to cope with, as many changes happen at the same time, such as separation from their dams, the transition from milk to solid feed, changes in their physical environment and even mixing with piglets from other litters. Weaning has also been found to cause gastrointestinal disturbances, for example, altering the balance of intestinal microflora, providing opportunities for coliform proliferation (Fuller 1989; Jensen 1998), or changes in villous and crypt architecture, resulting in a decreased capacity of digestion and absorption in the small intestine (Pluske 2001). In addition, weaning has also been found to decrease brush border enzyme (lactase and sucrase) activities, and pancreatic enzyme (trypsin, chymotrypsin, amylase and lipase) activities in piglets (Hampson and Kidder 1986; Lindemann et al 1986).

Manipulation of the post-weaning piglet diet without the use of antibiotic feed additives is still a challenge in the hot and humid climatic conditions of Vietnam. In developed countries, probiotics and feed enzymes have been widely promoted as alternatives to antibiotic feed additives in pig diets (Close 2000). Lactic acid bacteria (LAB) are normal inhabitants of the intestine, and probiotic LAB can reinforce or re-establish the gut microbial balance, produce antimicrobial substances, and increase the activities of useful enzymes (Fuller 1989; Nousiainen and Setälä 1998). Probiotic yeasts, such as Saccharomyces boulardii, which originates from plants, has also been found to be active against various pathogens and to stimulate enzyme activities in the animal gut (Czerucka and Rampal 2002). Supplementing enzymes (amylase, proteases, β-glucanase and cellulases) to early weaned piglet diets has been reported to improve performance (Dierick 1989).

The aim of our work was to investigate the impact on growth performance and nutrient digestibility in weaned piglets of a microbial enzyme mixture, a 3-LAB strain complex with a Saccharomyces strain alone or combined with the enzyme mixture. The 3-LAB strain complex was found in an earlier study (Giang et al 2010) to have probiotic properties in piglets in the first two weeks post-weaning, but not in the following three-week period. We hypothesized that the enzyme mixture would have positive effects on performance, and adding Saccharomyces alone or combined with the enzymes would have probiotic effects in weaned piglets during a longer period post-weaning than the 3-LAB complex only.

Materials and methods 

Animals and diets

Ninety-six healthy piglets [(Landrace × Yorkshire) × (Duroc × Pietrain)] (48 females and 48 males) from 12 litters in two 300-sow farms (located in Phu Long Commune, Nho Quan District, Ninh Binh Province, Vietnam) were weaned at 21-24 days of age, and piglets whose body weights were on average 7.4 ± 0.6 kg, were used in  a 35-day growth trial. The piglets were given free access to creep feed in meal form (24.1% crude protein in dry matter and 15.4 MJ metabolizable energy per kg dry matter, Table 1) without antibiotics or probiotics from 10 days of age. All piglets were vaccinated against pasteurellosis, parathyphoid, asthma and hog cholera.

At weaning, the piglets were randomly divided into four groups, balanced for sex, weight and litter origin, with 8 pigs per pen and 3 pens per treatment group. Each group was given one of four diets: a basal diet, in meal form, consisting  of extruded maize, extruded broken rice, soybean meal, soybean protein concentrate, milk replacer, sweet whey, and a vitamin-mineral premix, that  was formulated to meet NRC (1998) recommendations for starter piglets (Diet C) (Table 1); the basal diet supplemented with 0.2% of a mixture of microbial enzymes (amylase, protease, cellulase, β-glucanase, and xylanase) (Diet E); the basal diet supplemented with 0.2% of a mixture of lactic acid bacteria (LAB) (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7) and yeast (Saccharomyces boulardii Sb) (Diet LY); the basal diet supplemented with 0.2% of the LAB-yeast complex and 0.2% of the enzyme mixture (diet LYE). Chromium oxide was used as a marker and added at 3 g kg^-1 of the basal diet to determine apparent total tract digestibility.

Enzymes, lactic acid bacteria and yeast sources

LAB: Enterococcus faecium 6H2, Lactobacillus acidophilus C3 and Pediococcus pentosaceus D7 were isolated from the ileal digesta from healthy fattening pigs. The LAB strains were selected in previous tests, based on their resistance in vitro to heat, low pH, bile salts, and antagonism with pathogenic bacteria such as Salmonella and Escherichia coli (Viet et al 2006) and for their beneficial effect in weaned piglet trials (Viet et al 2009b; Giang et al 2010). Each LAB strain culture was mixed with a carrier (dextrin: whey powder, 1:1) and then was spray dried for 3 seconds (input temperature 160 ⁰C and output temperature 60 ⁰C).  The dry matter of the final products was around 96%.

Yeast: Saccharomyces boulardii Sb was obtained from the Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam. It was tested in vitro for resistance to heat, low pH, bile salts, and antagonism with the pathogenic bacteria Salmonella, Shigella and Escherichia coli (Viet et al 2009a).  The yeast strain culture was mixed with a carrier (1 dextrin : 1 whey powder ) and then was spray dried for  3 seconds (input temperature  180-200 ⁰C  and output temperature 80 ⁰C). The dry matter of the final product was around 96%.

The three LAB strains and yeast were mixed together with a ratio (weight) of 1:1:1:1. The mixture of LAB-yeast was tested for density of each strain, which contained Enterococcus faecium 6H2 at a concentration of 4.8 Í 10⁷ CFU/g, Lactobacillus acidophilus C3 at 5.2 Í 10⁷ CFU/g, Pediococcus pentosaceus D7 at 4.0 Í 10⁷ CFU/g, and Saccharomyces boulardii Sb at 2.6 Í 10⁸ CFU/g. The mixture was stored in a refrigerator at 4 ⁰C and mixed by hand to the diets LY and LYE every day at a level 0.2% in the diet.

Bacterial enzyme mixture: protease was produced from a Bacillus subtilis UL-15 fermentation, and amylase from Bacillus licheniformis VTCC8B-768 fermentation. β-glucanase and xylanase were produced from Rhodococcus fascian A2026 fermentation, and cellulase was from Aspergillus niger van tieghem var niger VN06-F0329 fermentation. All the bacteria were obtained from the Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam. The enzymes selected were mixed together with rice bran as a carrier, and then dried at 45 ⁰C until the dry matter reached 94%. The activity of each enzyme was tested and the results showed: amylase, 2200 IU/g; protease, 110 IU/g; cellulase, 1100 IU/g; β-glucanase, 200 IU/g, and xylanase, 1000 IU/g. The mixture was kept in a refrigerator at 4 ⁰C and mixed by hand into the diets E and LYE every day at a level of 0.2% in the diet.

To obtain a homogeneous distribution of the supplements in the diet, the supplements were mixed with the basal diet through several successive dilution steps, in a 1:1 weight ratio for each dilution. In diet LYE, the mixture of enzymes was mixed with the mixture of LAB and yeast before being added to the diet.

Measurements and data collection

The trial was divided into 2 periods: the first two weeks post weaning (Per I) and the following 3 weeks (Per II). The pigs were kept in pens which had concrete floors with no litter, and each had feeders and nipple drinkers. During the experimental period, feed and water were provided ad libitum.

The feed offered and refused was weighed daily, and the piglets were weighed at weaning, and on day 14 and day 35 post-weaning, to calculate average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) in each period and overall.

For the digestibility determinations, faeces were collected daily from each pen, as recommended by Kornegay et al (1995). Every day during 5 days before the end of each experimental period, the faecal samples from each of the 12 pens were collected, from 4 pens representing each of the 4 treatments at each sampling time (8.00 h, 12.00 h and 16.00 h) by 4 technicians in a 30-minute period. The faecal samples were taken around one hour after the cleaning the floor pens, from several faecal piles in each pen; each faecal sample was around 200-300 g. The collected faeces were stored in a refrigerator at 4 °C. At the end of the sampling period, the total faeces of each pen were pooled and sub-samples taken for analysis. The apparent total tract digestibility (ATTD) of crude protein (CP), crude fibre (CF) and organic matter (OM) was calculated according to the equation:

ATTD = [1 - (DC_F × I_D) / (DC_D × I_F)] × 100

Where

DC_F is the dietary component concentration in faeces (g/kg DM);
I_D is indicator concentration in the assay diet (g/kg DM);
DC_D is the dietary component concentration in diet (g/kg DM);
I_F is indicator concentration in faeces (g/kg DM).

Chemical analysis

Samples of feed and faeces were analyzed for dry matter, CP (N × 6.25; method 988.05), CF (method 978.10) and ash according to standard AOAC (1990) methods. The chromium content in feed and faeces was analyzed using an atomic absorption spectrophotometer (Spectr AA Perkin Elmer^TM, USA) (National Institute for Occupational Safety and Health 1994).

Statistical analysis

The data were analysed statistically using the GLM of Minitab Software Version 14.1. Treatment means which showed significant differences at P<0.05 were compared using Tukey’s pair-wise comparison procedure. The statistical model was as follows:

Y_ij  = M + B_i + e_ij

Where

Yij is growth performance or nutrient digestibility,
M is overall mean,
B_i is effect of treatment, e is random error,
i is treatment,
j is replicate within treatment.

Results 

Growth performance

Effects of microbial enzyme mixture, LAB and yeast on ADFI, ADG and FCR are presented in Table 2.

In the first two weeks post weaning, piglets fed diets E, LY and LYE had higher ADG (P<0.01) and lower FCR (P<0.05) compared to those fed the control diet, even though the ADFI was not affected by treatment (P>0.05). There were no differences in ADG and FCR among piglets fed diets E, LY and LYE (P>0.05).

In the following three-week period, higher ADG (P<0.05) and lower FCR (P<0.05) were observed in piglets fed diets LY and LYE, while no differences (P<0.05) in ADG and FCR were found in piglets fed diet E compared to the control diet (C). Again, no dietary effect on ADFI was found among the treatments (P<0.05).

Overall, piglets fed diets LY and LYE had improved (P<0.05) ADG and FCR, but not diet E (P>0.05), compared to the piglets fed the control diet (C). No differences in ADG and FCR were found among piglets fed diets E, LY and LYE (P>0.05). There were no differences in ADFI among the four treatments (P>0.05).

Nutrient digestibility

Effects of enzymes, LAB and yeast on the ATTD are presented in Table 3.

In Per I, ATTD of CP, CF and OM were higher (P<0.001) in piglets fed diets E, LY and LYE compared to the piglets fed the control diet. There was no difference (P>0.05) in ATTD of CP, CF and OM among diets E, LY and LYE.

In Per II, the ATTD of CP in diet E was higher than in the control diet (P<005), but lower than in diet LY and LYE (P<0.05), while there was no difference between diet LY and LYE (P>0.05).  The ATTD of OM was improved in diets LY and LYE compared to the control diet and diet E (P<0.05), but was not different between diets LY and LYE (P>0.05), or between diets E and control (P>0.05). There was no dietary effect on ATTD of CF (P>0.05).

Discussion 

Effect of supplementation with enzyme mixture alone on piglet performance

Compared to the control, supplementation with a microbial enzyme mixture alone (diet E) improved (P<0.05) ADG (+11.4%) and FCR (+8.3%) in piglets in Per I, but no improvement in ADG and FCR was found in Per II. This was, in general, consistent with the results for nutrient digestibility. The ATTD of CP, CF and OM in diet E was improved (P<0.01) by 5.2%, 6.5% and 3.7%, respectively, in Per I, but in Per II only the digestibility of CP was improved (P<0.01), and only by 1.7%, and no improvement in the digestibility of CF and OM was observed. The proteolytic and amylolytic digestive system of pigs is not well developed until 4 or 6 weeks of age (Kidder and Manners 1978). In addition, lactase and sucrase activities were observed to decrease during the 11 days post weaning (Hampson and Kidder 1986), and pancreatic enzyme (trypsin, chymotrypsin, amylase and lipase) activities were found to decrease during the first week post-weaning (Lindemann et al 1986). Moreover, the microbiota in the gastrointestinal tract is not stable during the two or three weeks after weaning (Jensen 1998), meaning a lack of microbial capacity to degrade the non-starch polysaccharides (NPS) found in the current dietary ingredients (maize, rice and soybean meal). Thus, there is a poor digestive capability in the two-week period after weaning that in the current study could have provided opportunities for the microbial enzymes, containing amylase, proteases, cellulase, β-glucanase and xylanase to improve the nutrient digestibility of diet E, resulting in better growth performance. These results are generally in line with several previous studies. For example, Högberg et al (1983) (cited in Dierick 1989) found that dietary enzymes (amylase, protease and cellulase) improved live weight gain (+8.3%) and FCR (+8.4%) in piglets of 6-15 kg body weight. Another study reported that supplementation of feed enzymes (neutral proteases, α-amylase and β-glucanase) to a starter piglet diet improved ADG (+17.5%) and FCR (+18.4%) during 28 days post weaning (Collier and Hardy 1986). In the following period, the pig digestive capacity is more developed, and the microbiota in the gut is more stable and can fully develop its fermentative capacity (Jensen 1998). Digestive enzyme (maltase and sucrase) activities increase with age (Aumaitre and Corring 1978), and levels in pancreatic tissue of four enzymes (lipase, α-amylase, chymotrypsin and trypsin) were shown to increase considerably over the first two months of life (Manners 1976). In the current study, the basal diet and enzyme-supplemented diets had the same chemical composition, contained highly digestible ingredients and were formulated meet the nutrient recommendations of NRC (1998). In previous studies, enzyme supplementation has been found to be more effective in pigs offered diets based on ingredients known to present problems in digestibility (Johnson et al 1993; Officer 1995; Omogbenigun et al 2004). This can explain the lack of response to our enzyme mixture in diet E in Per II.

Effect of supplementation with LAB-yeast alone or combined with enzymes on piglet performance

Supplementation with the LAB-yeast complex alone (diet LY) or combined with the enzymes (diet LYE) improved ADG, FCR and nutrient digestibility in both experimental periods, except for CF digestibility in Per II. Earlier studies have shown positive effects on growth performance and feed efficiency in weaned piglets fed diets supplemented with LAB probiotics (Pollmann et al 1980; Huang et al 2004; Lessard and Brisson 1987), or with Saccharomyces (Mathew et al 1998; Bontempo et al 2006; Shen et al 2009). Both LAB and Saccharomyces probiotics were reported to have antimicrobial capacities, such as high production of organic acids resulting in lower pH and hydrogen peroxide (for LAB), and secretion of specific IgA, inhibition of the toxicity of the Escherichia coli surface endotoxins or secretion of a protease which digested Clostridum difficile toxins (for S. bourlardi) (Lidbeck and Nord 1993; Buts et al 1990; 2006; Castagliuolo et al 1999). Thus, a more healthy gut, as well as a lower gut pH in piglets fed the diets containing the LAB-yeast mixture should be expected in the current study. Low gut pH has been found to increase digestive enzyme activities, giving a beneficial effect on nutrient digestibility in weaned piglets (Canibe and Jensen 2003; Lyberg et al 2006). Similarly, Collington et al (1990) reported that a LAB probiotic increased lactase and sucrase activities in the small intestine mucosa of weaned piglets. In addition, Graham et al (1986) found that LAB can improve the digestion of β-D-glucans in fibres in pigs. Furthermore, a yeast culture was found to increase villus height and villus:crypt ratio, resulting in higher digestibility in weaned piglets (Shen et al 2009). Thus, the improvement in nutrient digestibility in diets LY and LYE in the present study, resulting in better ADG and FCR, was most likely due to an improved gut environment. Furthermore, the current results suggest that the combination of the 3-strain LAB complex and yeast in the current study was more effective with respect to performance and digestibility compared to the 3-strain LAB complex alone in our previous study, which showed improvements in performance and nutrient digestibility only in the first period post-weaning (Giang et al 2010). Timmerman et al (2004) reported that multi-strain probiotics have been found to have more effective and consistent functionality than mono-strain probiotics, and probiotics containing multi strains of more than one species are more efficient than those of multi strains of one species, as the mixed bacteria/strains can complement each other to act in the host’s gut environmental conditions.

However, adding the enzyme mixture in the LAB-yeast complex (diet LYE) did not show any additional effects, as no differences in ADG, FCR and nutrient digestibility between diet LY and LYE were observed during the experimental periods. These results suggest that, in addition to improving the gut environment, the dietary supplementation of LAB and yeast complex can improve the digestive capacity of piglets to the same extent as dietary supplementation of enzymes.

Conclusions 

• Starter piglet dietary supplementation with microbial enzymes improved the performance and diet digestibility only in the first two week post-weaning, while supplementation with LAB-yeast improved growth performance and digestibility of CP and OM in the five weeks post-weaning, and digestibility of CF in the first two week post-weaning. Adding the enzymes to the diet that contained the LAB-yeast complex did not result in any additional effects on performance and digestibility in piglets during the five weeks post-weaning.  We are currently conducting studies to determine the optimum techniques for preserving these products for commercial use.

Acknowledgments  

We thank the Sida-SAREC Mekong Basin Animal Research Network (MEKARN) for financial support; researchers at the Department of Animal Nutrition, and laboratory staff of the National Institute of Animal Sciences, Hanoi, Vietnam, for assistance in carrying out the study and chemical analyses; and also researchers and staff at the Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam, for assistance in preparing the microbial cultures.

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Received 19 July 2010; Accepted 29 August 2010; Published 1 October 2010

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