Contributions of Enzyme Technology to Poultry and Swine Nutrition

Introduction

The use of enzymes in modern non-ruminant production has become universal. This is due to the extensive benefits observed on the productivity of both poultry and swine with its use. Although most feed ingredients used in monogastric nutrition contain adequate nutrients, monogastric animals are not able to efficiently utilize all the nutrients present in these ingredients. This has been attributed to several factors including the presence of antinutritional factors (ANF) in several feed ingredients such as non-starch polysaccharides (NSP) in grains (wheat, barley, oats) and phytate in most oilseeds and cereals (Olukosi et al., 2007a). Similarly, pigs and poultry have inadequate endogenous enzymes required to hydrolyze certain components of feed ingredients including fiber, phytate-complexes and proteins thus, approximately 25% of feed passed through their intestinal tract remain undigested (Barletta et al., 2011). This has led to the loss of nutrients through manure resulting in environmental concerns such as eutrophication. Exogenous enzymes have been increasingly supplemented in feed to degrade naturally occurring ANF and increase the utilization of nutrients by animals. In addition, enzymes have been used to reduce the viscosity of digesta in the gut when cereals such as wheat or barley are fed to pigs and poultry (Le et al., 2013). Furthermore, due to the rising costs of corn and soybean, cheaper low-quality feed ingredients such as high-fiber feedstuffs and industrial by-products have seen increasing use in diets of pigs and poultry. This is partly due to the ability of enzymes to increase the bioavailability of nutrients in these ingredients to animals hereby raising their value as alternative feed materials. Lastly, the use of enzymes has proved effective in reducing the nutrient load of manure from pigs and poultry production thus reducing the negative impacts on the environment (Nahm, 2005).

Figure 1. Contribution of enzyme application to the feed cost. Pie chart represents the total production cost per pig sold and is based on the production level of 18.6 finished pigs sold/sow/year. (Adapted from Dhuyvetter et al., 2014).

Feed comprises about 60-70% of the total cost of commercial production with exogenous enzymes accounting for approximately 1% of that cost (Figure 1). Commercially available feed enzymes for pig and poultry include several phytases, carbohydrases, and proteases targeted at the phytate, fiber, and protein components of feeds, respectively. Although these enzymes have recorded varying degrees of success in improving the utilization of nutrients in feed and increasing the productivity of poultry and swine, they are not yet at optimum efficiency. There are ongoing concerted efforts targeted at improving the production, stability, storage, and efficiency of these enzymes through innovations and technology (Menezes-Blackburn and Greiner, 2014). Therefore, the purpose of this paper will be to briefly review some of the commercially important enzymes in monogastric nutrition and examine the technologies being employed to improve their efficacy and utilization in poultry and swine production.

Phytase

Phytase (myo-inositol hexakiphosphate phosphohydrolase) is one of the most commercially successful exogenous enzymes in the market. This is due to its proven ability in breaking down phytate and its complexes and releasing phosphorus (P) and other nutrients for use by pigs and poultry (Selle and Ravindran, 2007). Phytase was first examined in the 1900s and was discovered as naturally occurring in plants materials such as rice and wheat bran. They were also found endogenously in animals and could be extracted from microbes (Wodzinski and Ullah, 1996). Previously, microbial phytases were obtained from the fungus Aspergillus sp. However, current phytases in the market are obtained from the fermentation of bacteria species such as Escherichia coli, Buttiauxella sp, and Citrobacter braakii (Menezes-Blackburn and Greiner, 2014). These phytases are usually classified as 3- or 6-phytase, depending on the position of the carbon from which the hydrolysis of the phosphate group begins (Zyla et al. 2004). In addition, these phytases operate in the gut using the histidine acid phosphatase catalytic mechanism (Greiner and Konietzny, 2011).

Phytate (myo-inositol hexakiphosphate) is the main storage form of P in most cereals, legumes, and oilseeds (Babatunde et al. 2019a). Due to the negative charge on the phytate inositol structure, strong complexes are formed with positively charged ions such as calcium (Ca), zinc, magnesium, and amino acids (AA) in the gut, making them unavailable for use by pigs and poultry (Cowieson et al. 2016). Phytase hydrolyzes phytate-P in a stepwise manner to its lower inositol phosphate esters, orthophosphates, and then further into myo-inositol (Figure 2) with the support of endogenous phosphatases (Cowieson et al. 2016). Phytases can be categorized as acidic, neutral, or alkaline depending on the optimum pH of activity (Greiner and Konietzny, 2011). However, most microbial phytases used in monogastric nutrition are acidic and active in the upper section of the gastrointestinal tract (GIT).

Figure 2. Schematic action of phytase enzyme on phytate substrate

Previous studies have reported an increase in body weight gain, feed intake and feed efficiency when phytase was supplemented in diets of broiler chickens (Shirley and Edwards, 2003; Babatunde et al., 2020), ducks (Adeola, 2018), laying hens (Taylor et al., 2018) and pigs (Olukosi et al., 2007b). Similarly, several studies have reported increase in the utilization of P, Ca, nitrogen, AA and energy when phytase was supplemented in the diets of poultry (Selle and Ravindran, 2007; Babatunde et al. 2019a,b) and pigs (Dungelhoef et al., 1994; Adeola et al., 2006). Phytase has also been reported to improve the deposition of minerals on the bones of broiler chickens and pigs (Santos et al., 2014; Babatunde et al., 2020) and to reduce the P content of manure from pigs and poultry (Nahm, 2005). However, the effectiveness of phytase supplementation in monogastric nutrition could be affected by several factors including the intrinsic properties of feed ingredients, the Ca-P relationship in the diet and gut, particle size of Ca sources in the diet, age of animals, length of feeding, and species of animal (Menezes-Blackburn and Greiner, 2014; Babatunde et al., 2019a).

Carbohydrase

The term carbohydrase is a collective name for a group of enzymes which include xylanase, cellulase, glucanase, α-amylase, β-mannanase, α-galactosidase, and pectinase. Fibrous feed stuff such as wheat, rye or barley meal are made up of overlapping layers of different structural carbohydrates called NSP which must be unfolded in order for digestive enzymes to gain access to the trapped nutrients (Petry and Patience, 2020). Moreover, considerable amount of energy yielding nutrients and minerals are located in the aleurone layer, typically made up of NSP that include beta-glucans, pentosans, oligosaccharides, cellulose and lignin. However, they are protected from hydrolysis in the digestive tract since pigs and poultry do not secrete appropriate enzymes capable of degrading them hereby resulting in inefficient use of these feed stuff (Bach and Knudsen, 1997). High dietary NSP increases digesta viscosity, which reduces enzyme accessibility to substrates and slows down the rate of digestion.

Supplementing diets with carbohydrases reduces digesta viscosity via a decrease in feed polymerization and release of carbohydrate oligomers for improved nutrient utilization (Kalmendal and Tauson, 2012; Guo et al., 2014). The pig gut, compared to poultry, is better equipped to digest, and utilize a portion of the complex carbohydrates due to a relatively larger digestive tract, longer digesta residence time, and greater bacteria fermentation in the hind gut (Knudsen et al., 2012). However, changing composition of swine diet to include more fibrous ingredients and associated complexities of the target substrate creates an avenue for improvements in nutrient utilization by use of carbohydrase supplementation. Certain enzymes, like xylanase, mannanase and glucanase may be exogenously supplemented as feed additives to specifically target NSP hydrolysis. This unfolds the cell wall and grants other digestive enzymes like amylase access to nutritional constituents, such as protein, starch, lipids, and other minerals, trapped within the cell-wall matrix (Le et al., 2013). Some specific carbohydrases including their mode of action are discussed below.

Xylanase

Arabinoxylans are major constituents of the cell wall of plants like barley, oats, wheat, rye, and their by-products. This complex polysaccharide is mainly composed of two pentoses: arabinose and xylose and can affect nutrient utilization in pigs and poultry by forming viscous gels in the animal’s gut. Although majority of feed ingredients for livestock have high starch content, it is stored in granules within a matrix of storage proteins and enclosed by the thin cell walls of the starchy endosperm that mainly consist of arabinoxylans (Evers and Millar, 2002). This is referred to as the arabinoxylan cage, or the caging effect (Figure 3). There are several reports on improved feed digestibility in broiler chickens and pigs fed xylanase supplemented diets (Kalmendal and Tauson, 2012; Guo et al., 2014; Petry and Patience, 2020). It is broadly accepted that xylanases achieve this by two main functions: One is to decrease viscosity in the gut triggered by the watersoluble NSP and thereby facilitate the digestive and absorptive processes in the GIT. Another is to degrade the β-,4-glycosidic bonds of the xylan backbone of arabinoxylan to produce branched or non-substituted xylo-oligosaccharides that could potentially be used by the animal (Le et al., 2013). The disruption of the structural integrity of the cell walls opens up the “cage” for digestive enzyme access (Figure 3).

Figure 3. Microscopic visualization of the degradation of aleurone arabinoxylan cages present in milled wheat after incubation with Ronozyme WX xylanase (1 g of enzyme/kg of diet) at 30°C. Images depict a close-up of a cell wall structure containing ferulic acid, covalently bound to cell walls, and which fluoresces with an intense blue-green fluorescence. Panel A and B shows cell walls before and after addition of the xylanase, leading to the breakdown and disappearance of the cell wall architecture. (Adapted from Le et al., 2013)

Amylase

Starch is the major energy storage in plants, and is a polymeric carbohydrate consisting of numerous glucose units joined by glycosidic bonds. Generally, swine and poultry appear to secrete sufficient pancreatic amylase to cater for dietary starch intake. However, factors not directly related to starch itself, such as specific intestinal site may also affect its digestibility (Aderibigbe et al., 2020a). Similarly, the dynamics of starch digestion relative to intestinal efficiency and age may have considerable nutritional consequences (Aderibigbe et al., 2020a,b). Starch degradability is also affected by the proportion of amylose in feedstuff, its variability in corn and other cereal grains can significantly influence the ME content of feedstuffs to livestock (Tester et al., 2004). Some can escape enzymatic digestion in the GIT (also called resistant starch) and may only be fermented in the hindgut by microbes (Tiwari et al., 2019). However, fermentation in the distal gut is a much less efficient usage than the breakdown in the proximal regions of the GIT. Particularly in young chickens, exogenous amylases may augment the function of endogenous amylase which is limited by the maturation of the GIT (Croom et al., 1999), but this is not always true. The additional benefits from exogenous amylases in poultry and swine nutrition have been inconsistent largely due to the relatively high innate starch digestion capacity of the animal. Therefore, amylase supplementation as part of a carbohydrase mix containing xylanases or glucanases have been reported to improve energy utilization and the performance of non-ruminant animals (Olukosi and Adeola, 2008; Schramm et al., 2021).

Protease

Leguminous seeds such as peas and soybeans, contain large amounts of storage proteins essential for optimal animal growth and development. However, they also contain high concentrations of ANF (Table 1). Dietary protein is degraded by proteases produced in the GIT; the most important are pepsin, trypsin, and chymotrypsin. Anti-nutritional factors such as trypsin inhibitor (TI) contained in these seeds, inhibit protein digestion by blocking the action of endogenous trypsin critical to the activation of other digestive enzymes in the gut (Erdaw et al., 2017). This negatively affects nutrient utilization and animal performance. Furthermore, increased inefficiency of dietary protein utilization exacerbates the environmental impact of high nitrogen emissions (Leinonen and Williams, 2015). Although the inhibitors are heat labile and are commonly deactivated by heat treatment, excessive processing can negatively influence the nutritional quality of the feed (Newkirk, 2010).

Table 1. The trypsin inhibitor activity (TIA) of some legume seeds. (Adapted from Avilés‐Gaxiola et al., 2018).

Exogenous proteases can therefore be an option to improve performance of the animal while reducing dietary protein levels. This is achieved through enhanced protein solubilization and hydrolysis of dietary proteins (Zuo et al., 2015; Aderibigbe et al., 2020c). Microbial protease could also destroy or inactive the TI in soybean (Huo et al., 1993). Because TI are competitive substrate analogs of trypsin, hydrolysis of TI by exogenous proteases may allow for increased trypsin activity in the GIT This potentially improves the utilization of dietary protein and AA by pigs and poultry, and consequently improves animal performance (Zuo et al., 2015; Aderibigbe et al., 2020c). There are also observed responses that suggest extra-proteinous effect of protease, extending beyond a magnitude that is commensurate with increases in the digestibility of protein alone (Olukosi et al., 2015; Cowieson et al., 2017). This may be associated with the disruption of protein-starch matrix in the feed following proteolysis. However, the efficacy of exogenous proteases in poultry and swine nutrition is largely inconsistent and has been attributed to the different inherent characteristics of commercial proteases which may elicit divergent responses in the animal. This may also be related to compatibility with endogenous proteases, changing chemical gut environment during the digestive process, or perhaps modification of the seed protein during processing (Cowieson and Roos, 2016). Nevertheless, considerable opportunity exists to develop novel proteases which are more functional at low pH or which specifically target proteinaceous antinutrients such as lectins, TI or other antigenic proteins.

Table 2. Responses of non-ruminant animals to single or multi-enzyme supplementation in diets.

Single versus Multi-enzyme matrix

Conventionally, enzymes are administered as single additives in animal diets. In recent times, additional benefits have been reported for enzymes added to diets as part of a multi-enzyme cocktail or combined as stand-alone additive in the complete diet (Table 2). Although the potency of single enzymes is not in doubt, the use of multiple enzyme activities simultaneously targets multiple substrates i.e., attack different antinutritive compounds or break up nutrient complexes in feedstuffs to obtain the maximum benefit from the enzymes. Therefore, thorough knowledge of how enzymes work together to hydrolyze their respective substrate is essential to maximizing the efficacy of enzyme combinations. For instance, xylanase increases the permeability of the aleurone layer of wheat, which is also the site of phytic acid storage (Parkkonen et al., 1997). This suggest that supplementing animal diet with a combination of xylanase and phytase may be mutually beneficial to the animal. In contrast, there are reports that suggest that proteases contained in enzyme mixtures may degrade or limit the activity of other constituent enzymes (Saleh et al., 2004). Other works reported no additional benefit of using enzyme combinations compared with using enzymes individually (Wu et al., 2004; Olukosi et al., 2007a). This may be partly due to the complexity of the potential substrates, which also varies by cereal and diet formulation. Moreover, majority of the so-called ‘single-component’ enzyme products have ancillary side activities and thus interpretation of results using single enzymes should be treated with caution. Although it is possible that a multitude of enzyme activities is unnecessary or even detrimental, nevertheless there is substantial experimental data showing multi-enzymes to be effective in pigs and poultry (Olukosi et al., 2007a, b; Kalmandal and Tauson, 2012; Taylor et al., 2018; Cowieson et al., 2019).

Advances in Enzymes Technology

The use of enzymes in monogastric nutrition is not without challenges. One of which is the enhancement of enzyme properties for specific feed applications. Enzymatic properties such as thermostability, specific activity, storage stability, and resistance to the harsh environment of the GIT, are areas of interest in ongoing efforts to improve enzyme technology (Menezes-Blackburn and Greiner, 2014). New enzyme products with improved properties are being developed through the screening of extremophiles or other thermophilic organisms capable of living in hostile environments. Extremophiles have the capacity to thrive in areas with extreme temperatures, salinity, and pressure thus, isolation and production of this microbes may generate enzymes with similar properties (Iyer and Ananthanarayan, 2008). Another approach is the modification of existing microbes through modern biotechnological applications such as large-scale fermentation and recombinant DNA technology (Cherry and Fidantsef, 2003). This could result in the production of recombinant enzymes at maximal purity and with economic efficiency. Protein engineering, directed evolution, mutation, and screening of genes from current microbes may also improve specific enzyme properties such as thermostability and substrate specificity (Cherry and Fidantsef, 2003). A good example has been the construction of a ‘consensus phytase’. This is a combination and alignment of 13 phytic enzymes which has been reported to be more efficient and thermostable than the parent enzyme and has been regarded as a success in the phytase industry (Lehmann et al., 2002). Chemical modification of enzymes involving the alteration of AA side chain structures, could improve stability. However, the use of chemical cross-linking may not always result in the improved stability of the final enzyme product (Davis, 2003).

Other common and preferred approaches include the use of immobilization techniques in improving the thermo- and storage stability of enzymes (Iyer and Ananthanarayan, 2008). These techniques include adsorption, membrane confinement, entrapment, and covalent binding of which multipoint covalent attachment is the most effective in improving the thermostability of enzymes (Guisan et al., 1993). Coating of enzymes has been reported to improve stability and reduce the possibility of dustiness (Menezes-Blackburn and Greiner, 2014). In addition, the use of additives in enzyme preparations has proven reliable overtime in improving the storage stability of enzymes. Some effective additives include ligands, salts, polyols, sugars, and synthetic polymers (Iyer and Ananthanarayan, 2008). Similarly, feed processing conditions and raw material pretreatments could be means of improving efficacy of enzymes. High temperatures during pelleting of feed have been reported to negatively impact the activity of enzymes (Inborr and Bedford, 1994). However, post-pelleting applications of enzymes may ameliorate some of these effects. Developing pretreatment procedures for the hydrolysis of hemicellulose fraction cereals, high-fiber ingredients, and high-phytate feed materials may prove to be of economic importance as they will increase the efficacy of enzymes in nutrient extraction (Marquardt and Bedford, 2001). There have been efforts to determine the site of peak enzyme activity in the gut of pigs and poultry particularly with phytases (Rodehutscord and Rosenfelder, 2016). This information has helped produce enzymes with properties targeted at being most efficient in those sections of the GIT. Lastly, transgenic animals with the capacity to secrete enzymes in their saliva, effectively utilize nutrients in feed, and produce low-nutrient manure are being produced and investigated (Zhang et al., 2018). However, this technology faces challenges from consumer perspectives, ethical groups, and various legalities and will require time to become fully accepted.

Future Considerations

Research targeted at improving enzyme assays remains essential as there is no universal standard procedure for analyzing the quantity or quality of the different commercial enzyme products. Enzyme products generally have differing characteristics such as optimal temperature and pH, storage stability, and substrate specificity. Thus, determining the activity level in feeds and the comparison of various commercial products is challenging. Moreover, developing a standardized single enzyme assay will allow producers and purchasers of enzymes compare products based on relative activity, allow assessment of enzymes that survive feed processing procedures, and evaluate the survivability of enzymes in the GIT (Marquardt and Bedford, 2001). Further investigations into the relationship and interactions between the gut microbiome and existing enzymes may go a long way in explaining some of the inconsistencies observed with enzyme efficacies particularly with carbohydrases and proteases. Additionally, it may prove invaluable to further evaluate the exact structural conformation of the substrates to be degraded as this information will be important in developing the next generation of enzymes.

Conclusions

Enzyme technology has played a fundamental role in the increased productivity of monogastric animal production observed today. The ability of enzymes to improve the utilization of nutrients from feed ingredients, increase the production of pigs and poultry, while reducing their environmental impact has proven invaluable. Currently, the feed enzyme market is estimated to be worth $1.3 billion and it is projected to reach $1.9 billion by 2025. A large share of this market is dominated by the use of phytases in the diets of pigs and poultry followed closely by carbohydrases and proteases. To keep up with the projected growth in the world population, and the consequent increase in commercial non-ruminant production, efforts should be targeted at overcoming challenges with the inconsistencies in animal responses to exogenous enzymes. In addition, the development of economical and effective next-generation enzymes should be encouraged for supporting sustainable animal agriculture.

Presented at the 2021 Animal Nutrition Conference of Canada. For information on the next edition, click here.