Use of Combinations containing B. Subtilis, Xylanase, Amylase and Protease to Improve Broiler Performance

Gradual intensification of the poultry industry in the last half century has resulted in increasing challenges within the industry. With a growing world population the pressure has been on to grow bigger birds, faster and as cheaply as possible. Drivers for the industry have been increasing growth rates whilst maximising feed utilisation as feed is the biggest single cost for producers (Barletta, 2010). With these pressures the feed additive industry has evolved to meet the needs of the producers. Two key types of feed additives used in the poultry industry are enzymes and DFMs. Proven benefits of feed enzymes include improved feed efficiency, reduced feed costs, improved digestion and absorption of nutrients, improved uniformity within flocks and better maintenance of gut health (Barletta, 2010). Likewise DFMs have also been proven to improve bird performance and gut health, although unlike enzymes the mode of action is through improved diversity and stability of the gut microbiota, immunomodulation and production of anti-microbial agents (Lee et al., 2010). The exact mode of action of the DFM depends on the species of bacteria used in the product.

The purpose of this paper is to look at the benefits of multi-activity enzyme products and DFMs as individual feed additives and then to discuss the benefits seen when using the two product types together. 

Enzymes were first used in the poultry industry in the 1980s and are now widely used to improve bird performance and make feed cost savings. Most widely used are the phytases, whose mode of action has been extensively researched and is well understood. The penetration of products containing carbohydrase and protease activities is less extensive. With current high commodity prices, poultry producers are pressured to increase productivity without compromising food safety. Similarly, environmental pressures on waste output from animal production facilities have encouraged the increased use of enzymes, in particular phytase. High commodity prices can result in the inclusion of lower cost, cheaper raw materials in diets that increase the complexity of the diet and can result in more anti-nutritional factors, which can negatively affect bird performance. Increased use of such lower cost mixed diets has spurred use of carbohydrases (xylanase and/or amylase) as well as protease in poultry feed over the last 5 to 10 years. As enzymes are categorised according to the substrates that they target, complex or mixed poultry diets with their complexity of substrates may benefit from multiple enzyme activities.

One such combination enzyme is a mixture of distinct xylanase, amylase and protease (XAP) activities. Exogenous xylanase targets the soluble and insoluble arabinoxylans in cell walls (Barletta, 2010). These arabinoxylans can trap nutrients in the cell walls and the soluble fractions can also induce viscosity in the gut which results in reduced digestion of nutrients and a reduced passage rate of feed through the gut (Barletta, 2010). Use of exogenous xylanase has two well researched benefits, firstly releasing encapsulated nutrients such as starch and protein from the cells and secondly reducing the viscosity of the digesta, both leading to improvements in digestibility (Choct, 2006, Mirzaie et al., 2012). The breakdown of non-starch polysaccharides by xylanase can also have a beneficial effect on the gut microflora by creating conditions that encourage beneficial bacteria through reduction in viscosity and production of small oligomers that can be used by the beneficial bacteria in the lower gut (Bedford, 2000). Exogenous amylase is used to increase the hydrolysis of starch and improve starch digestibility which results in more energy being released for the bird (Gracia et al., 2003; Barletta, 2010). Exogenous proteases are used to increase the hydrolysis of proteins in the feed, including hydrolysis of proteinaceous anti-nutrients such as trypsin inhibitors (Caine et al., 1998), resulting in improved digestibility of protein and amino acids (Yu et al., 2007; Cowieson and Adeola, 2005).

Research on (XAP) have demonstrated improvements in the nutritive value of the diet. Several studies have demonstrated incremental improvements in energy digestibility when dietary carbohydrases (xylanase and amylase; XA) were used with or without protease (Cowieson and Ravindran., 2008, Romero et al., 2013a). Romero et al. (2013) analysed four digestibility studies which had compared the effects of XA or XAP in maize based diets with or without distillers dried grains with solubles (DDGs). A meta-analysis of the four studies showed that XA increased ileal digestible energy and AMEn by 77 and 67 kcal/kg respectively, whereas XAP increased them by 98 and 93 kcal/kg feed respectively. In addition the XAP was shown to increase protein and ileal amino acid digestibility. This increase in digestibility was between 12-13% of the ileal undigested amino acid fraction, irrespective of the amino acid concerned. Romero and Ravindran (2011a) also demonstrated significant improvements in crude protein as well as fat and starch digestibility with the XAP combination. The improvements in digestibility seen in trial work can be used when formulating diets with the XAP and can allow reductions in the amount of added fat in diets or addition of cheaper raw materials with a lower nutritional value to the diet. A lifecycle assessment has demonstrated that use of the XAP product in this way has benefits for the environment by reducing the green house gas emissions from poultry production by 5% (Bundgaard et al., 2012). The increases in digestibility seen with the XAP combination translate into improved growth and feed efficiency of the animal. Over eight trials the XAP combination was able to reduce the amount of feed required per kg broiler meat by between 3.8 and 8.7%, depending on the feed formulation (Bundgaard et al., 2012). 

Spore forming bacteria such as Bacillus subtilis species have been used in human and animal nutrition for over five decades. The B. subtilis group traditionally comprises four species: B. amyloliquefaciens, B. licheniformis, B. pumilus and B. subtilis itself (Claus and Berkeley, 1986; Priest et al., 1988). Bacillus spp. are known for their good stability and resistance to digestion. Indeed, sporulated bacteria are resistant to extreme stresses such as UV, heat, high pressure, acidity etc. and have a demonstrated long shelf-life. These characteristics offer great versatility of use in different conditions. Their resistance to heat and pressure help them sustain during the hostile pelleting process. They can resist enzymatic digestion and acidity, which helps them in reaching sites in the lower intestinal tract, where they can exert their positive effects. This has made Bacillus spp. the direct fed microbial of choice in animal nutrition.

Many bacteria from Bacillus spp. are used in commercial production of bulk enzymes, such as proteases (from B. alcalophilus, B. amyloliquefaciens, B. lentus, B. licheniformis), α-amylases (from B. amyloliquefaciens, B. licheniformis, B. stearothermophilus) glucose isomerase (from B. coagulans) and pullulanase (from B. acidopullulyticus) (Arbidge 1993). This ability to produce enzymes may help animals in effective digestion and utilization of various feed ingredients and improve feed efficiency. This may be of help when animals are under stress, when endogenous enzyme production could be compromised or feed ingredients are of lower quality. Several research studies testing a three-strain B. subtilis combination DFM have shown improvements in FCR for broilers. The average improvement observed was 4.4% across all studies (Figure 1) (unpublished data). Other work has also noted an improvement in body weight (Romero and Ravindran, 2011b) or FCR (Amerah et al., 2013) when a B. subtilis product is added. Studies with laying hens have shown improvements in egg production performance and egg quality when fed dried B. subtilis culture (Xu et al., 2006). Xu et al., observed 2.8% improvement in number of eggs/hen/day, 1.2% improvement in egg weight, 2.4% improvement in egg mass/hen/day, and 5.9% improvement in FCRegg (Kg, feed intake/Kg, egg mass). In the same study, egg shell strength was improved by 10.6 %, Haugh unit by 3.1%, while yolk cholesterol was decreased by 12.2%. Santoso et al. (1999) reported significantly lower ammonia concentration in manure samples when laying hens are fed with dried B. subtilis culture, besides having significantly improved FCR. Effect of dried B. subtilis culture on lowering of ammonia concentration was bigger with length of exposure in feed (Figure 2).

 

Figure 1: Improvement in FCRc after addition of Bacillus subtilis to broiler diets

 

Figure 2: Ammonia concentration in manure during the period when dried Bacillus subtilis culture was fed. (adopted from Santoso et al., 1999)

 

Bacillus spp. are also known to have a positive effect on gut health. In a study with Eimeria vaccine challenge to broilers, Lee et al., (2010) showed that feeding a three strain B. Subtilis combination DFM restored the gut barrier structure by having significantly higher villus height compared to a control group. Internal studies conducted by authors also indicate that feeding a three strain B. subtilis combination DFM to broilers in necrotic enteritis challenge conditions can help sustain performance during challenge period. 

In animal nutrition, it is well known that maintaining gut health contributes significantly to maintenance cost. Protease can reduce indigestible N (Caine et al., 1998, which may decrease substrate available for pathogenic bacteria in the intestine. Xylanase can increase digestion of NSPs like arabinoxylans (Barletta, 2010), which could provide nutrients and/or substrates for beneficial bacteria (Bedford, 2000). Amylase can improve starch break-down and availability to the bird itself Gracia et al., 2003; Barletta, 2010. Thus logically, the combination of DFMs and enzymes may help maintain a better microflora balance as well as may help improve intestinal health and gut barrier function, something that may result in reduced gut maintenance cost and improved growth performance. Work carried out on the beneficial use of a combination of a DFM consisting of 3 strains of B. subtilis and an enzyme consisting of a combination of xylanase, amylase and protease (XAP) are summarized below.

A trial study was carried out to determine the effect of DFMs, enzymes and their combination on digestibility of nutrients and growth performance of broilers. The treatments included: 1) negative control (NC) based on corn and soybean meal, 7% DDGs and 500 FTU/kg phytase; 2) NC + XAP; 3) NC + a three strain B. subtilis combination (DFM) and 4) NC + XAP and DFM combination (COMBO). It was observed that the COMBO treatment increased digestibility of fat, starch and energy, improved AMEn and feed efficiency in broilers compared to NC. Using XAP alone improved AMEn value by 84kcal/kg when compared to NC, addition of DFM further increased AMEn by 50kcal/kg on top of enzymes. Energy partitioning data showed that the improved AMEn was partially related to the improved digestibility of dietary NSPs (Romero et al., 2013b). Other work has reported that supplementation of the exogenous enzymes (containing xylanase, amylase and protease) and DFM (contained 3 different strains of Bacillus subtilis) combination increased energy digestibility, enhanced gut integrity, and reduced zoonotic pathogen load in first-cycle laying hens (Murugesan, 2013). In broilers, a significant interaction between enzymes and DFM was observed for apparent ileal digestibility of starch, crude protein, and amino acid, indicating that both additives increased the digestibility in a non-additive manner. An additive response was observed for AMEn when the enzymes were used in combination with DFM (Murugesan, 2013).

Necrotic enteritis (NE) is an infectious disease caused by Clostridium perfringens and is the most common bacterial disease in modern broiler flocks. It has been estimated that NE infection affects up to 40% of the commercial broiler flocks and is believed to cost the industry about 5¢ per broiler in the United States (McDevitt et al., 2006).

To evaluate the efficacy of a three-strain B. subtilis combination (DFM) and an XAP enzyme on the control of necrotic enteritis caused by C. perfringens in broiler chickens, three trial studies have been conducted with male broiler chickens (Cobb x Cobb 500). Necrotic enteritis was induced on Days 20, 21, and 22 by dosing with a broth culture of C. perfringens. In these trials, it was observed that the challenged control group had significantly reduced growth performance, increased mortality and lesion score when compared to unchallenged control. Both DFM and XAP were effective in improving the performance of broilers compared to challenged control. However, when DFM was combined with XAP, the performance of broilers recovered to the level of unchallenged control, implying the combination can be beneficial despite the presences of necrotic enteritis challenge. The combination reduced significantly NE caused mortality compared to challenged control. Table 1 shows the results from one of these trials (Mathis et al., 2013). 

Table 1: Effect of DFMs and enzymes combination on performance of broilers under NE challenge*

Feed antibiotics growth promoters (AGPs) have been banned in Europe, however, AGPs are still commonly used in animal feed in North and South America. Due to the awareness of antibiotic resistance in humans, feed producers are searching for alternatives to antibiotic growth promoters (AGPs) in animal feed. Studies indicate that the combination of DFM with enzymes can have a role in antibiotics growth promoter free broiler feeding programs. In a field study, performance of birds fed a basal diet that contained mixed enzymes (XAP) and phytase with antibiotics or with DFM (no antibiotics) was measured. When compared for cost effectiveness of usage, based on current price of live weight of chickens and feed cost, it was estimated that DFM treatment resulted in 2.5% higher gross profit when compared to AGP treatment (Danisco research report).

The benefits of using the combination of DFM and XAP in broiler production have been demonstrated in multiple studies. By meta-analysis of 7 broiler trials, it was observed that the combination of DFM with enzymes significantly improved growth performance and significantly reduced feed conversion ratio compared to negative control. The improvement was more pronounced at severe challenged situations (feed conversion ratio reduced by 3 and 14% at low and necrotic enteritis challenged situation respectively).

In a trial conducted at an experimental farm in the Netherlands, it was observed that the combination of DFM with enzymes (COMBO) resulted in significantly (P<0.05) higher final BW (2475 vs 2370 g), BWG (2433 vs 2328 g), feed intake (4166 vs 4063 g) and 2% lower FCR (1.71 vs 1.745) compared to the control group. In this study, COMBO treatment reduced foot pad lesion score at 42 days (P<0.05) (Danisco research report). Based on the data from this study, the production efficiency factor (PEF) was calculated as 315 and 296 respectively for the test and control treatment, indicating a 6.4% improvement in production efficiency when COMBO is used in the diet. 

These studies indicate that use exogenous enzymes in combination with direct fed microbials can potentiate the efficacy of enzymes alone through a synergistic effect. However, possible mechanisms involving the ability of: i) DFMs to produce fiber degrading enzymes that may complement enzymes or ii) exogenous enzymes to increase the availability of suitable substrate for direct fed microbials and/or other beneficial bacteria need to be further researched. Authors would like to conclude based on the observations mentioned in this review that combination of feed enzymes such as xylanase, amylase and protease along with direct fed microbials will offer a better solution in terms of healthy nutrition and performance of broilers. 

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