Effect of multi-strain microbial fermented soybean meal on growth performance, serum profile and intestinal physiological status of weaned piglets

T Y Hung, S Y Lin*, C K Lin*, S C Liu* and J J Lu* 

Faculty of Land and Food Resources, University of Melbourne, Melbourne 3031, Australia

 t.hung@pgrad.unimelb.edu.au

^*Department of Animal Science, National Chiayi University, Chiayi 600, Taiwan 

Abstract 

The objective of this study was to use multi–strain microbial fermented soybean meal (FSM) as carrier of probiotics in order to investigate the effect of FSM on growth performance, serum profiles and intestinal physiological status of weaned piglets.

 

In Experiment 1, a total of sixty piglets was randomly allotted into five dietary treatments: Control treatment (basal diet); Probiotics 1 (P1), the basal diet + P1 FSM (fermented by Lactobacillus acidophilus, Bifidobacterium thermophilum, and Aspergillus oryzae); Probiotics2 (P2) the basal diet + P2 FSM (fermented by Streptococcus thermophilus, Bifidobacterium thermophilum and Bacillus subtilis); Probiotics3 (P3), the basal diet + P3 FSM (fermented by Lactobacillus acidophilus, Enterococcus faecium and Saccharomyces cerevisiae); and in Probiotics 4 (P4) the basal diet + P4 FSM (fermented by Enterococcus faecium, Saccharomyces cerevisiae and  Bacillus subtilis).

 

In Experiment 2, sixteen pigs were allocated into two treatments: Control and FSM (FSM fermented by Enterococcus faecium, Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium thermophilum). In Experiment 1 pigs fed P2 and P3 had higher average daily gain (ADG) overall. In Experiment 2 FSM increased total volatile fatty acid (VFA) and lactic acid concentration in caecum. Moreover, both aerobe and anaerobe microflora in duodenum as well as ileum increased through FSM administration. Piglets fed with FSM not only showed lower numbers of Escherichia coli in both jejunum and caecum, but also higher Lactobacillus in duodenum, jejunum and caecum. In conclusion, dietary FSM supplementation had a beneficial effect on growth performance and intestinal microflora regulation of weaned piglets. However, these effects may be dependent on probiotic combinations.

Key words: Blood traits, microflora, probiotics

Introduction

Soybean meal is a common component in farm animal diets. However, due to the antigenic activity and anti-nutritional factors of soybean meal, weanling farm animals are sensitive to soybean meal (Lalles 1993). Anti-nutritional factors, such as trypsin inhibitors in soybean meal interfere with digestion, absorption, and nutrients utilization of weaned piglets (Jiang et al 2000; Hong et al 2004).

 

Probiotics are defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Reid et al 2003). Earlier studies showed that soybean meal fermented by Bacillus subtillus, Aspergillus awamori, Aspergillus oryzae or Aspergillus usamii has benefits for farm animals (Feng et al 2007a; Feng et al 2007b). Fuller (1989) claimed that multi–strain microbials can be used in a broad spectrum and be expected to be active in several different species of host animal. Although a large number of studies have been made on soybean fermented by individual strain probiotics on farm animals, little is known about the effect of multi–strain microbial fermented soybean meal on weaned piglets. Moreover, most of previous studies were focused on protein source effects of FSM, rather than evaluating its probiotic effects. Thus, the aim of this study was to investigate the effects of multi–strain microbial fermented soybean meal on growth performance, blood traits and intestinal physiological status of the weaned piglet.   

 

Materials and methods 

Animals, housing and diets

 

The pigs were housed in slatted floor pens with a size of 1.5m × 3.2m. The temperature in the room for the weaned pigs was maintained at 30°C and water was freely available via nipple drinkers. Pigs were offered ad libitum access to feed for the entire experimental period. The experimental diet was formulated to meet the nutrient requirements of pigs according to the National Research Council (1998). The composition of the experimental diets is shown in Table 1.  

In Experiment 1 a randomized complete block design was used, with in total sixty crossbred weaned piglets (Landrace × Yorkshire × Duroc) with average body weight of 8.58 kg randomly allotted into five treatments according their body weight. There were two pigs per replicate (pen) and six replicates per treatment. Pigs in the control treatment were fed the basal diet; In P1 pigs were fed the basal diet + P1 FSM, fermented by Lactobacillus acidophilus, Bifidobacterium thermophilum, and Aspergillus oryzae; P2 treatment were fed with basal diet + P2 FSM, fermented by Streptococcus thermophilus, Bifidobacterium thermophilum and Bacillus subtilis; P3 treatment were fed with basal diet + P3 FSM, fermented by Lactobacillus acidophilus, Enterococcus faecium and Saccharomyces cerevisiae; P4 treatment were fed with basal diet + P4 FSM, fermented by Enterococcus faecium, Saccharomyces cerevisiae and Bacillus subtilis. Experiment 1 was conducted for four weeks; the body weight and feed intake were measured on the 14^th day and 28^th day. The blood sample was collected at the end of the experiment. 

 

In the second experiment, 16 crossbred weaned pigs (Landrace × Yorkshire × Duroc) within average body weight of 8.2 kg were allotted to two treatments with four replicates, and two pigs per replicate. Pigs in the control treatment were fed the basal diet, and pigs in the FSM treatments were fed the basal diet + FSM, fermented by combinations of Enterococcus faecium, Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium thermophilum. The experiment was conducted for four weeks in order to investigate the intestinal physiological parameters. 

 

Source of microorganism cultures and maintenance

 

Cultures used in this study were obtained from the Food Industry Research Institute, Taiwan. The cultures Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium thermophilum were maintained by subculturing weekly using 1% lactobacilli MRS broth made from individual ingredients according to the manufacturer’s directions (Difco Laboratories, Detroit, MI). Aspergillus awamori, Saccharomyces cerevisiae, Corynebacterium acetoglutamicum, Bacillus licheniformis, Aspergillus oryzae, Aspergillus niger and Trichoderma koningi were subcultured in nutrient agar. All cultures were subcultured at least three times before use in the experiment.

 

Preparation of fermented soybean meal

 

Subcultured microorganisms were transferred into a premixed inoculated broth at 37⁰C for 12h. Soybean meal was sterilized by autoclave and cooled to room temperature for one hour, then supplemented with 17% premixed inoculated broth (3.2×10⁹ colony forming units (CFU) /ml) and fermented in a sealed 15 liters plastic bucket at temperature 30 ± 2 °C for three weeks. The same procedure was used for the FSM in the control diet, except 17% sterilized water was used instead of premixed inoculated broth. 

 

Sample collection  

 

In Experiment 1, blood samples were collected on day 28 through carotid arteries for serum profile determination. In Experiment 2, pigs were sacrificed on day 28 for intestinal physiology study. After slaughter, intestines were excised and immediately divided into duodenum, jejunum, ileum, colon, and caecum. Intestinal contents collected from duodenum, jejunum, ileum, colon and caecum were stored immediately at -20 °C.

 

Analytical procedures

 

Blood samples collected from each pig, were allowed to clot at room temperature for 1h and centrifuged at 1,500 × g, and at 4 °C, for 20 minutes, and then refrigerated at –20 ºC until analysis. Serum constituents, including blood urea nitrogen (BUN),  glutamic oxaloacetate transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-glutamyltransferase (γ-GTP), alkaline phosphatase (ALK-Pase), cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were measured by blood autoanalyzer (Vitro 950, Johnson & Johnson U. K.) with commercial kits (Vitros chemistry U. K.).

 

VFA were determined by the procedure of Hove and King (1979). Briefly, an acidified aliquot of each segment of intestinal contents was steam distilled in a micro-Kjeldahl still and the distillate titrated with 0.01 N NaOH to give total VFA. The lactic acid content of intestine segments was measured by blood autoanalyzer (Vitro 950 Johnson & Johnson, U.K.) and LAC slides (Vitros chemistry U.K.). The pH values of intestine contents were determined by pH meter (MP 220 Mettler Toledo, Switzerland).

 

For microbiological analysis, fermented soybean meal and intestinal content were serially diluted and plated in count agar. Aerobic and anaerobic microorganisms were cultured in count agar plate (MERCK 1.05463). Escherichia coli was cultured in a COLIFORM Agar (MERCK 1.10426). Lactobacillus strain was cultured in MRS-agar (MERCK 1.10660). Aerobes and Escherichia coli medium plates were placed in an incubator at 37 °C for 48h. Anaerobes and Lactobacillus medium plates were placed in Anaerobic jars (MERCK 1.16387) with an anaerobic gas pack system (Anaerocult^® MERCK 1.13829) at 37°C for 48h. The microflora enumerations were expressed as log₁₀ colony forming units (CFU) per gram.

 

Statistical analysis   

 

Data were analyzed by the general linear model procedure (GLM). Tukey’s studentized range tests were used to perform multiple comparisons between means in the first experiment and the t-test was used to compare differences between the control and FSM group means in the second experiment. The statistical analysis was done using the Statistical Analytical System (SAS) program (1999). Differences were considered to be significant at P < 0.05. The experiment design was according to the following model:
 

Y_ij =μ + T_i + B_j + E_ij

Where,

Y_ij is the dependent variable;

μ is general mean;
T_i is diet effect;
B_j is block by the initial body weight of pigs and
E_ij is experimental error.

 

Results and discussion

Effect of FSM on growth performance

 

Overall average daily gains (ADG) were higher in pigs given the P2 and P3 diets (Table 2), whereas no difference in average daily feed intake and feed:gain was found between treatments.

Previous studies (Xuan et al 2001; Van Heugten et al 2003) demonstrated that dietary probiotic supplementation has beneficial effects of growth performance in piglets. Although the P2 and P3 diets also showed a significantly improved ADG, the ability to improve growth performance may vary with different probiotic combinations. The complex design used in this study involved two lactic acid bacteria plus yeast or fungi strains in order to investigate the effects of different probiotic complexes in weaned piglets. The growth promoting effects may due to the improvement of nutritive value of FSM (Feng et al 2007a). In our study, however, the diets were supplemented with only 3 % FSM, and nutrient values did not differ among treatment diets. The growth performance of farm animals is an outcome of complex metabolic transformations, and the underlying mechanisms, including the interaction between intestinal bacteria as well as interactions between bacteria and intestinal physiological responses are likely to be complicated and multi-factorial. A previous study also showed that a diet supplemented with FSM can improve nutrient digestibility in growing pigs (Unpublished data). Therefore, it seems reasonable to conclude that FSM had growth promoting activity due to the probiotic effect. However, the growth promoting effect of FSM may depend on different probiotic combinations.     

 

Effect of FSM on serum profiles

 

The effect of dietary FSM supplementation on serum chemical composition is shown in Table 3. There were no statistically significant differences among treatments in BUN and creatinine.

In order to understand the effect of probiotics on hepatic function, serum GOT, GPT, γ-GTP and ALK-Pase were determined. GOT activities in P1 were higher than in P4 (P<0.05). Moreover, pigs fed the P3 diet had higher ALK-Pase activity with values of more than 300 IU/L. These values were slightly higher than the normal range of pig ALK-Pase content. Bai et al (1996) recommended that the normal activity of ALK-Pase of healthy pigs is between 92-294 IU/L. In a previous study, dietary 0.5 % Lactobacillus acidophilus and Bifidobacterium lactis had no effect on GOT and GPT (Hsu et al 2004). Furthermore, serum lipid, cholesterol, TG, LDL-C and HDL-C were not found to differ in this study. This result is also consistent with the study of Hsu et al (2004) who found that there were no differences in cholesterol, LDL and HDL in growing-finishing pigs fed with Lactobacillus acidophilus and Bifidobacterium lactis. Martin and colleagues (2008) indicated that probiotic exposure exerted microbiome modification and resulted in altered hepatic lipid metabolism coupled with lowered plasma lipoprotein levels. Gut Lactobacillus spp. are responsible for a significant proportion of bile acid deconjugation (Usman and Hosono 1999), and therefore stimulate cholesterol biosynthesis of liver. However, it is interesting that the level of serum lipid parameters were not significantly changed in our study, indicating a need for further research. 

 

Effect of FSM on VFA and lactic acid concentration of caecum contents

 

Due to the improvement in growth performance found in the first experiment, it was concluded that the P2 and P3 treatments had growth promoting activity. The probiotic complex used in the second experiment was a mixture of  P2 and P3 in order to develop more efficient growth promoting activity. 

 

Total VFA and lactic acid concentration of caecum content are indicators of the metabolism of gut microorganisms (Barnes et al 1980). The FSM treatment had significantly higher total VFA concentration than the control treatment (P<0.01) (Table 4).

Short-chain fatty acids (SCFAs), such as acetic acid, propionic acid and butyric acid, are the products of carbohydrate and protein fermented by microorganisms in the intestine (Macfarlane and Gibson 1995). More than 95% of SCFAs can be absorbed by the host (Cummings 1995) and supply approximately 9 % of the energy requirement of the host (Hume 1995). Szylit et al’s research (1988) also showed that diets supplemented with Lactobacillus acidophilus and Veillonella alcalescens can enhance the amount of caecal VFA. This may explain, in the first experiment, why P2 and P3 could improve ADG. Moreover, FSM treatments had higher lactic acid concentrations than the control treatment (P<0.001). Lactic acid is the product of lactic acid bacteria via anaerobic fermentation. In the current study, FSM treatment showed a higher amount of lactobacillus in the gut. Therefore, it is reasonable to conclude that caecal lactic acid level and the population of caecal lactobacillus had a positive correlation.

 

Effect of FSM on intestinal pH value

 

It is clear from Table 5 that pigs given the diets supplemented with FSM generally had lower intestinal pH values.

The gut pH could have been reduced through stimulation of the lactic acid producing microflora (Langhendries et al 1995).  Although only slight changes in intestinal pH occurred in pigs fed with FSM, FSM tended to lower gut pH. However, pervious research by White et al (2002) reported that dietary supplementation of yeast had no effect on fecal pH. The pH value of intestinal contents might alter with age (Mathew et al 1993) and food source (White et al 2002). With regard to pH values and intestinal bacteria distribution, it is also noteworthy that the higher the lactobacillus counts in intestine contents the lower the pH value.          

 

Effect of FSM on intestinal microbial population

 

The results for intestinal microflora are shown in Figure 1.

Both aerobe and anaerobe microflora in duodenum as well as ileum increased significantly through FSM administration (P<0.05). Mortality and morbidity associated with Escherichia coli cause economic losses in pig production. Compared with the control treatment, pigs fed with FSM had reduced populations of Escherichia coli in jejunum and caecum (P<0.05). In stark contrast, the FSM treatment showed a significantly higher population of lactobacillus in duodenum, jejunum and caecum (P<0.05). Yu et al (2007) indicated that dietary probiotic supplementation enhances the lactobacillus counts in the crop, ileum, and cecum of broilers. A study by Dharmawan et al (2006) showed that lactic acid bacteria supplementation could inhibit the adhesion of Escherichia coli O157:H7 in the human intestine. Gonzalez et al (1995) used a mixture lactobacillus species as bacteriotherapy against infantile diarrhea caused by Escherichia coli and Salmonella. Although the stressful physiological and environmental conditions around weanling pigs often promote the proliferation of pathogens in the digestive tract, resulting in diarrhea and reduced daily weight gain, dietary supplementation with probiotics can potentially alter gut microflora by selectively stimulating the growth of beneficial bacteria while suppressing the growth of pathogenic bacteria (Van Heugten et al 2003). In our study, dietary supplementation of 3% FSM had beneficial effects on the balance of the gut ecosystem, improving the health status of weaned piglets

 

Implications 

• This study was conducted to investigate the effect of different FSM on growth performance and intestinal parameters of weaned pigs.

  
  

• A dietary supplement with 3% FSM showed benefits with respect to growth performance and intestinal physiological status.

  
  

• These finding demonstrate that FSM is an effective carrier of probiotics, used as functional ingredients in weaned piglet feed.

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