Evolving enzyme technology: impact on commercial poultry nutrition
The use of exogenous enzymes to improve the nutritional value of poultry diets is a relatively new concept. The technology is advancing rapidly, with new enzymes, enzyme combinations, and applications being developed, although the pace of this progress is largely dependent on regulatory constraints. Most poultry nutrition researchers consider phytase to be the latest major advance in the use of enzymes in the feed industry.
However, extensive research is underway on the next generation of enzymes, focusing on raw material quality, prediction of reactions using least squares models, improvement of food safety, the effect of bird age, the effect of various side effects and enzyme dosage, increased net income, and reduced environmental pollution.
The aim of this review is to summarize the latest research in the field of feed enzymes for poultry and to anticipate future applications of enzymes and novel enzyme technologies that could be valuable to the poultry industry in the coming years.
Poultry: Enzymes: Nutrition: Digestibility: Microflora: Modeling
Endo-β-1,4-xylanases and β-1,3,1,4-glucanases have been used commercially in wheat and barley-based diets for about 20 years, and their return on investment has been such that in many “sticky grain” markets, the penetration of these enzymes in broilers is greater than 90% (Bedford, 2000a). Furthermore, the mechanism of action of these enzymes has been extensively studied and is now relatively well understood (Bedford, 2000a).
Although the use of enzymes to improve the nutritional value of wheat and barley has become common practice, the use of enzymes in corn-based diets is not as widespread. This is probably due to the common perception in the feed industry that corn is of high nutritional value and stable and therefore enzyme addition cannot be as beneficial as in diets based on sticky grains.
Undoubtedly, the typical improvement in feed conversion ratio (FCR) that can be expected to be achieved by using existing enzymes for wheat or barley-based diets (especially carbohydrases) is greater, or at least more stable, than the improvement expected in corn-based diets. For such a situation, least squares models can be very valuable, as they are able to predict to a large extent the likely response of a particular feedstock or diet to the addition of an exogenous enzyme (Rosen, 2001).
It is likely that in the future, the adoption of novel enzymes by the feed industry will largely depend on the ability of feed additive companies to demonstrate the efficacy of enzymes at different dosages in a predictable and consistent manner, which will prevent enzymes from being added to diets arbitrarily and without scientific support.
Therefore, the development of new enzyme systems or novel applications of existing enzymes should be carried out in parallel with research into the quality of feed ingredients.
Current research
Enzymes for wheat and barley-based diets
Two main mechanisms for the action of xylanase and β-glucanase in wheat or barley-based diets have been proposed:
Viscosity theory
Cage effect (Bedford, 2002)
The viscosity theory states that the performance improvement achieved by adding these enzymes to poultry diets is more than just explained by the nutritional value of the released sugars; Therefore, there must be an additional benefit of these enzymes for the digestibility of other dietary nutrients.
Soluble high molecular weight arabinoxylans and β-glucans strongly adsorb water and can cause a geometric increase in the viscosity of gastrointestinal (GI) contents (Bedford & Classen, 1992; Nilsson et al. 2000). Increased intestinal viscosity is negatively associated with animal performance, nutrient digestibility, and adverse changes in the microflora of the distal intestine. Therefore, reducing viscosity with the aid of exogenous enzymes provides a nutritional advantage to the animal (Bedford & Classen, 1992; van der Klis et al. 1993; Adeola & Bedford, 2005; Cowieson et al. 2005; Meng et al. 2005; Sieo et al. 2005).
The negative effects of viscosity on animal performance are so severe that the viscosity of feed ingredients (e.g. wheat and barley) has been suggested as a reliable indicator of the nutritional value of the ingredient and the effectiveness of exogenous pentosanases (Bedford et al. 1991; Bedford & Classen, 1993).
Furthermore, the effects of viscosity are more pronounced in younger animals, probably due to the maturity of the digestive tract in adult animals and their ability to cope with soluble polysaccharides (Yasar & Forbes, 1999, 2000).
The “caging” effect is related to the effects of carbohydrases on the cell wall, which reduces the strength of the wall and releases the trapped nutrients (Bedford, 2002). Both mechanisms are likely involved in the response to xylanase and β-glucanase in poultry diets, leading to positive changes in performance, nutrient retention, reduced incidence of sticky feces and wet litter, and favorable changes in the microbial flora of the lower gastrointestinal tract (Bedford, 2000b; Graham et al. 2003; Choct et al. 2004; Shakouri & Kermanshahi, 2005).
Currently, research on carbohydrases for wheat- and barley-based diets is focused on more detailed studies of the mechanism of action, the effects of the enzymes in species other than broilers, the assessment of the importance of enzyme inhibitors and resistant starches, as well as the effects of processing such as particle size, preparation temperature, and the use of whole grain.
One interesting and emerging area of research on xylanase in wheat-based diets is the role that the molecular structure of the enzyme plays in its affinity for soluble and insoluble fibers and susceptibility to inhibition.
For example, in a study by Fontes et al. (2004), it was shown that a xylanase containing a carbohydrate-binding module was approximately 5% more effective in wheat-based diets and 6% more effective in rye-based diets than a commercial enzyme without this substrate-binding ability, respectively.
The authors of this study concluded that the ability of enzymes to bind to substrates via binding modules increases the enzyme’s efficiency in vivo, particularly in relation to the hydrolysis of insoluble carbohydrates.
This finding is important because this mechanism of action appears to allow for greater hydrolysis of the cell wall, resulting in the release of encapsulated nutrients. This may be more important in diets with high concentrations of insoluble carbohydrates (e.g., growth and finishing diets in pigs).
The sensitivity of exogenous enzymes to inhibitors is also a matter of scientific debate, as the true significance of the concentration of xylanase inhibitors in wheat has not yet been fully elucidated. However, recent studies by Ponte et al. (2004) have concluded that the concentration of xylanase inhibitors present in wheat is a limiting factor in the efficiency of exogenous xylanase.
Ingelbrecht et al. (2000) also noted that the presence of xylanase inhibitors reduces the efficiency of exogenous xylanase enzymes and this is related to the rheological properties of the dough. Also, the difference in the effectiveness of different xylanases may be related to the difference in the degree of inhibition by the inhibitors.
Furthermore, the concentration of xylanase inhibitors in wheat is highly variable and this difference is due to the genetic origin, environmental conditions during plant growth, as well as harvesting and storage conditions (Gebruers et al. 2002; Bonnin et al. 2005). These factors can cause fluctuations in the apparent metabolic energy (AME) of wheat and also the range of responses to exogenous enzymes.
However, some researchers believe that most xylanase inhibitors use a competitive inhibition mechanism and may be rapidly saturated with the substrate (exogenous xylanase) (McLauchlan et al. 1999; Gebruers et al. 2004), and thus there may be sufficient active xylanase remaining to produce the desired physiological effects.
However, this assumption may not always be true; according to Bonnen et al. (2005), the concentration of xylanase inhibitor in wheat is about 30 mg/g, which is about 250 times higher in molar ratio than the amount of xylanase added. Therefore, the reason why complete xylanase inhibition is not observed under commercial conditions is probably due to the lack of direct contact between the enzyme and the inhibitor due to the low moisture content of the feed, rather than due to saturation of the inhibitor.
Whether xylanase inhibitors actually have a negative effect on the effectiveness of the exogenous enzyme in vivo is still not completely clear; Especially in conditions where the feed is conditioned at high temperatures, since the inhibitors are proteinaceous. However, there is evidence that these proteins are somewhat heat-resistant (Gebruers et al. 2004).
In any case, regardless of the actual effect of the inhibitors in practice, the presence of these compounds in cereals imposes a significant cost on feed enzyme producers, as fluctuations in the recovery of enzyme activity in the feed have a negative impact on the consumer’s perception of product quality and consistency.
The above studies indicate that in the development of a new generation of fiber-degrading enzymes for monogastric animals, the substrate affinity of the enzyme and the degree of enzyme inhibition should be considered as key factors.
In recent years, several papers have been published investigating the efficacy of non-starch polysaccharide (NSP) degrading enzymes in various non-broiler poultry species; probably in part this research was in response to the requirements of EU regulatory bodies for the registration of enzyme products. Table 1 summarizes some of the recent trials.
Adeola and Bedford (2005) recently demonstrated that the nutritional value of wheat-based diets for ducks can be improved by the addition of exogenous xylanase. In this study, weight gain and feed conversion ratio (FCR) were improved by approximately 12% when xylanase was added to a diet based on high-viscosity wheat. However, xylanase had no effect on the low-viscosity diet, indicating that diet viscosity plays an important role in the nutritional value of wheat for ducks.
Laboratory (–O–), pelleted feed; (–D–), pelleted feed with xylanase; (V), pre-pelleted feed; (S), pre-pelleted feed with xylanase. Values are mean values and their standard errors are shown by vertical bars. a,b Mean values with different letters are statistically significantly different (P < 0.05).
Figure 2. Dietary viscosity in (cPs) of the final diets after in-vitro digestion. Pelleted feed, pelleted feed with xylanase, pre-pelleted feed; pre-pelleted feed with xylanase. The values presented are means and their standard errors are indicated by vertical bars. a,b,c,d,e,f: Means with different letters are statistically significantly different (P < 0.05).
However, conflicting results have also been reported; for example, in diets based on wheat, rye and triticale (Timmler and Rodehutscord, 2001 ) no significant effect of xylanase on performance indices was observed, although intestinal viscosity was reduced by the addition of the enzyme.
Mathlouthi et al. (2003a) recently demonstrated that enzyme supplementation of wheat- and barley-based diets for growing turkeys can also improve performance. They found that feed conversion ratio (FCR) improved by 2.5–5%, depending on the amount of wheat and barley used in the diet, and that diet viscosity was significantly reduced with enzyme supplementation.
It is worth noting that most of the beneficial effects of enzymes in turkey diets have been observed at younger ages (<10 weeks) and it is more difficult to demonstrate a statistically significant effect of enzymes in older turkeys.
Laying hens also benefit from exogenous enzymes; As Lazaro et al. (2003) showed, adding enzymes to wheat, barley and rye-based diets resulted in significant increases in egg production (2.1%) and feed efficiency (2.5%). They concluded that this improvement in performance was due to a reduction in diet viscosity by the addition of xylanase and glucanase.
These data are supported by the studies of Mathlouthi et al. (2003b) who showed that the performance of laying hens was improved by the addition of xylanase and glucanase to wheat and barley-based diets and that diet viscosity was reduced. The authors also found that the relative improvement associated with the addition of enzymes was greater in diets based on viscous cereals such as wheat, barley, rye and triticale than in diets based on corn. The use of xylanase and glucanase in wheat, barley, rye and triticale-based diets for poultry has been shown to improve various performance parameters. However, it can be concluded that these effects are not always consistent and are likely to depend on the quality of the feed ingredients (i.e. the apparent metabolizable energy inherent in the grain or more precisely the feed conversion ratio inherent in the grain), the age of the bird, the nature of the gut microbial community, the housing environment and/or the physical processing of the diet.
A recent study has shown the effect that high-temperature thermal processing can have on wheat-based diets for broilers (Cowieson et al., 2005). In this study, the performance improvements associated with the addition of xylanase were more significant in diets processed at 90°C than in diets processed at 80°C. Chemical characterization of the diets showed that heat treatment increased the viscosity and soluble arabinoxylan concentration of the diet (Figures 1 and 2). Furthermore, the addition of exogenous xylanase reduced both viscosity and the degree of polymerization of arabinoxylan-based carbohydrates.
Similar reactions have been observed previously, with the optimum processing temperature for wheat-based diets for broilers being around 85°C, with higher temperatures having negative effects (Silversides and Bedford, 1999).
Furthermore, recent data by Scott (2005) showed that similar effects were observed in Canadian wheat, with pelleting increasing the viscosity of the digestive tract contents by approximately 17%, but this was significantly reduced by enzyme addition. In addition to changes in diet properties associated with heat processing, the thermal stability of enzyme products should also be considered. Since enzymes are proteins, they are denatured to some extent (depending on their tertiary and quaternary structures) when exposed to feed processing conditions that involve the application of heat to the diet matrix (e.g., conditioning and pelleting).
Enzymes that have been made more thermally stable by means of targeted evolution processes or coating technology may have an advantage over less stable enzymes, as their yields after processing are usually higher. However, it should be noted that the optimum temperature for enzymes that are stable up to 85–90°C is often much higher than the internal body temperature of the bird (around 40°C), and therefore enzymes evolved with high intrinsic thermal stability may have lower performance at lower temperatures.
These data suggest that conditioning temperature should be considered as an important factor to achieve a more uniform response to exogenous xylanase.
Several recent papers have recommended the use of whole grains in poultry diets to improve performance, digestive health, and reduce disease incidence (Svihus et al., 2004; Bjerrum et al., 2005) presented data showing that feeding broilers whole wheat significantly reduced the number of Clostridium perfringens in the distal gastrointestinal tract and concluded that the gizzard is an important organ to prevent the establishment of undesirable bacteria in the ileum.
Favourable changes in the gut microbiota of chickens have been reported following the addition of whole grains to the diet (Apajalahti et al., 1998, 2001) (Figure 3). The interaction between xylanase and whole grain feeding has received less attention, but the articles that have addressed this issue have concluded that the effect of xylanase is largely independent of the presence of whole grains in the diet (Wurt al., 2004, Engberg et al., 2004, Jones and Taylor 2001).
(Caecum) of broiler chickens. The cecal microflora was analyzed using guanine plus cytosine (G+C) percentage profiling in three replicate chickens from two farms fed only standard commercial feed (A) and two farms fed this commercial feed supplemented with whole wheat (B). The mean values of bacterial abundance are based on G+C percentage and their standard errors are shown by vertical bars. The G+C profiling technique and statistical method are described in detail elsewhere (Apajalahti et al., 1998, 2001).
Enzymes for corn
The use of exogenous enzymes in corn-based diets has received considerable attention in the recent literature. This may be due to the large size of the global corn feed market, the realization that corn is a variable feedstuff, and the relatively low penetration of enzyme products in this feed segment, which has stimulated commercially sponsored research programs. A review has summarized the effects of enzyme supplementation in corn-based diets and suggested some mechanisms of action for the various enzymes (Cowieson, 2005). Therefore, this review will not be repeated. Another review by Summers, 2001 is comprehensive and summarizes the important factors in corn that determine its nutritional value for poultry and the effects of exogenous enzymes. The reader is referred to these sources for more detailed information.
Enzymes for plant protein meals
Research into enzyme supplementation of plant protein feeds for poultry diets is somewhat of a minefield, as knowledge about the substrates is very limited. For example, the carbohydrate composition of soybean meal, oilseed meal, lupin meal, and field bean meal is relatively complex compared to cereals, whose carbohydrates are mainly starch-based (Bach Knudsen, 1997; Choct, 1997; Leske & Coon, 1999; Graham et al., 2002). Furthermore, the carbohydrates themselves in vegetable protein meals may have antinutritional properties (Cowieson & Acamovic 2003). However, many studies have been reported in this area, showing diverse responses to a wide range of enzymes (Table 2).
A recent paper by Vahjen et al. (2005) showed that the most effective enzymes for soybean meal were 1,4-β-arabinogalactanases, but reported that when this enzyme was combined with galactomannanase, synergistic effects in the release of sugars were observed. However, these synergistic effects in vitro did not consistently improve the performance of broiler chickens in vivo. In fact, the combination of galactanase and mannanase caused a 22% reduction in body weight gain compared with chickens fed a control diet. The authors concluded that the disappointing effects of the enzyme on poultry performance may be due to the release of oligomers that increase intestinal osmotic pressure. Similar results have been reported for the use of a combination of polygalacturonase and pectin methylesterase in the in vitro digestion of lupin meal (Ali et al., 2005).
In this study, the combination of pectinases gave the highest hydrolysis of lupin polysaccharides. However, the authors concluded that although the combination of pectinases could be effective in hydrolyzing lupin polysaccharides, the correct dosages should be determined before testing these enzymes in vivo, as excessive use of pectin methylesterase may have detrimental effects on poultry performance.
Douglas et al. (2000) reported the effect of enzyme supplementation (xylanase, amylase, and protease) on the performance of broilers fed a diet containing twelve different soybean meal samples. In this study, it was found that the effect of the enzyme was dependent on the type of soybean meal batch, which may be due to the inherent digestible energy content of the meal. However, since the authors did not report trypsin inhibitor activity or lectin concentration in each meal sample, some of the variation in enzyme response may be due to changes in trypsin inhibitor activity, lectins, or the degree of damage to amino acids during processing.
Clark and Wiseman (2005) recently published data indicating that trypsin inhibitor concentration in soybean meal plays a very important role in determining its nutritional value. It is possible that the protease component in the enzyme composition of the Douglas et al. (2000) study could reduce the negative effects of trypsin inhibitors in soybean meal and improve digestible energy and prevent internal losses.
This hypothesis is supported by data published by Hu et al. (1993), who showed that exogenous protease could successfully denature protein antinutritional substances in soybean meal in vitro, potentially improving its nutritional value for poultry (Figure 4).
Ghazi et al. (2002) also found that protease was successful in improving the nutritional value of soybean meal for poultry and concluded that hydrolysis of antigenic proteins in the meal or improvement of the digestibility of heat-damaged proteins may be responsible for the increased performance.
This suggests that protease may be one of the candidate enzymes that can improve the nutritional value of soybean meal (and possibly other protein meals) by targeting protein antinutritional factors, antigenic proteins, and proteins damaged during processing. Kocher et al. (2002) reported that the addition of the enzyme to a corn and soybean meal-based diet increased the metabolizable energy of the diet by 1.6% (P < 0.05). The authors concluded that the beneficial effects of the enzyme on carbohydrate digestibility improved the apparent metabolizable energy of the meal. However, it should be noted that no significant improvement in performance (weight gain or feed conversion ratio) was observed in this study.
It may also be valuable to add enzymes to diets containing other protein meals. For example, research by Covizon et al. (2003) showed that the nutritional value of diets containing chickpea meal was improved by adding a combination of enzymes including xylanase, amylase and cellulase, with some of the beneficial effects being achieved through a reduction in endogenous mucus secretion.
One area that has received little attention to date but is likely to receive much attention in the coming years is the use of dried grains with solutions (DDGS) in monogastric animal feeding and the improvement of the energy value of DDGS by the use of appropriate exogenous enzymes. The nutrient profile of DDGS is relatively good, although it has a high fiber content that may limit its use in poultry diets (Table 3; Spiehs et al. 2002; Blia et al. 2004; Lumpkins et al. 2004). The phosphorus content of DDGS is about 0.9% (Spiehs et al. 2002) and is highly absorbable by poultry compared to other feedstuffs (54–100%; Martinez et al. 2004; Lumpkins and Batal 2005). This high absorbability suggests that phytase supplementation may not be necessary for DDGS. However, the significant fluctuations in phosphorus availability in DDGS are unacceptable to nutritionists, and phytase could therefore be a useful tool to reduce these fluctuations.
Due to the high fiber content of DDGS and the changes in its nutritional value (Cromwell et al. 1993), there is potential to improve the nutritional value by using NSP-degrading enzymes, especially enzymes with high affinity for insoluble fiber. Also, the amino acids in DDGS may have low absorbability due to damage during drying, which can be particularly severe for lysine (Goodson and Fontaine 2004; Lumpkins and Batall 2005).
Therefore, the nutritional value of DDGS may also be improved by adding exogenous proteases to the diet or enzymes capable of hydrolyzing glucosamine-type compounds. To the best of the authors’ knowledge, no articles have been published in peer-reviewed journals on the addition of enzymes to diets containing DDGS for poultry; this area requires further attention and research.
Phytase
Phytase is relatively unique as an enzyme for animal feed as it is used in both wheat or barley based diets and corn based diets, so the potential market for phytases is very large. For this reason, much research has been conducted on the effectiveness and mechanism of action of various phytases in monogastric animal diets. It can be concluded that phytase is effective in improving the retention of dietary phosphorus (P) (Simons et al., 1990) and may also improve the retention of dietary amino acids and energy (Selle et al., 2005).
One recent area of research on the mechanism of action of phytase concerns the effects of phytate and phytase on endogenous secretions and whether some of the beneficial effects of phytase may be due to favorable changes in endogenous secretions and mucocin production. Recent studies have shown that in the absence of feed, phytate (as an aqueous solution of inositol hexaphosphate) can increase the secretion of endogenous minerals, amino acids and mucin carbohydrates (Figure 5) from 6-week-old broiler chickens, and phytase can partially reduce these adverse effects (Cowieson et al., 2004). These results are also supported by Onyango et al. (2004), who showed that the presence of phytic acid increased the secretion of endogenous mucins in the intestines of ducks and broilers. Part of the beneficial effects of phytase in animal diets may be related to reducing the anti-nutritional effects of phytate and also to the release of nutrients from the feed. When the effect of phytase on amino acid digestion coefficients is examined, these effects are variable and dependent on the type of amino acid (Selle et al., 2000; Adeola & Sands 2003; Cowieson et al., 2006), such that the digestion of amino acids such as threonine, cysteine, arginine and serine is improved more by phytase than, for example, methionine (Biehl & Baker 1997; Kornegay et al., 1999; Namkung & Leeson et al 1999, Zhang et al., 1999).
Figure 5. Effect of phytase and phytic acid (inositol hexaphosphate; IP6) on sialic acid excretion in 6-week-old broiler chickens. Values are means and their standard errors are shown by vertical bars. The mean value was significantly different from the control group (P < 0.05) (adapted from (Cowieson et al., 2004).
It is important to note that the amino acids whose digestibility coefficients showed the greatest improvement with the addition of phytase were those found in the highest concentrations in endogenous enzymes and mucins (Forstner & Forstner, 1994).
Watson et al. (2005) have recently published data showing that the addition of phytase to nutritionally complete diets can lead to further increases in performance. Cowieson et al. (2006b,c) have also observed similar effects with the combination of carbohydrase and phytase enzymes in complete diets. These findings suggest that either the phosphorus requirement of broilers is greater than previously thought, or Phytase can improve the net nutritional value of foods by reducing maintenance requirements and thus increasing performance.
Another interesting study in this area concerns the proteolytic stability of phytase. Onyango et al. (2005) published data showing that different phytases (from Peniophora and Escherichia) have different resistances to hydrolysis in the digestive tract. This is important because the main site of phytate hydrolysis in the digestive tract is the gastric stage (when phytate is most soluble) and therefore loss of enzyme activity can have a significant impact on its bioavailability. The issue of enzyme stability in the digestive tract is important not only for phytase, but also for other enzyme systems and food additives such as essential oils that are claimed to be active in the distal part of the digestive tract, since they must first survive the initial part of the digestive tract (e.g., proteolysis, or emulsification and digestion by endogenous lipases).
It is clear that since exogenous phytase has a significant impact on the retention of exogenous nutrients and endogenous secretions, it is essential that the diet to which phytase is added is strategically designed to accommodate these changes in gut ecology and nutrient digestibility.
Experimental evidence suggests that water intake and excretion are increased when phytase is used, which may be related to changes in osmotic pressure in the gastrointestinal tract; this could be due to released ions or changes in sodium and potassium secretion (Cowieson et al. 2004).
Future research on phytase should focus on elucidating its effects on endogenous secretions, protein turnover, and the influence of acid-base balance and osmotic potential on phytase efficiency and bird performance.
Enzymes and endogenous secretions
During digestion, there is a net movement of nutrients from the lumen (inner space) of the gut into the body. However, at the same time, compounds They move from the gut-associated tissues into the lumen. These compounds mainly include mucins, endogenous enzymes, bile, electrolytes, bicarbonates, shed cells and other nitrogenous compounds such as uric acid (Fuller & Reeds, 1998). Although these endogenous secretions are essential for efficient digestion, they do have a nutritional cost to the animal, particularly when overproduction occurs; for example, when trypsin inhibitors are present in the diet (Clarke & Wiseman, 2005). However, this nutritional cost is of greater importance in terms of energy balance than amino acid requirements, as about 90% of the excreted nitrogen is retained by the animal (Krawielitzki et al. 1990, 1994). Therefore, it can be concluded that the true “cost” of endogenous secretions is probably greater for the net energy of the diet than its effect on amino acid requirements.
Diets and foods that are capable of stimulating increased endogenous secretions are nutritionally costly for animals. For example, phytate (Cowieson et al. 2004), tannins (Mansoori & Acamovic, 1998) and non-starch polysaccharides (NSP) (Larsen et al. 1993; Angkanaporn et al. 1994; Cowieson & Acamovic, 2003) have been shown to increase endogenous secretions and reduce performance in broiler chickens. This evidence leads to the clear conclusion that exogenous enzymes that hydrolyze these antinutritional compounds that stimulate endogenous secretions may exert (at least in part) their effects by reducing the secretion of these endogenous compounds.
Future research in this area should focus on the effects of exogenous enzymes on nutritional requirements, as this The field is still largely unknown.
Enzymes, immune competence and microbial flora
The concentration of microorganisms in the ileum and cecum of the chicken is very high and can reach numbers of 109 and 1011 per gram of digesta, respectively (Apajalahti & Bedford, 1998). These numbers are extremely high and such a concentration of metabolically active organisms can have a significant impact on the host’s ability to fight disease and metabolize nutrients. The bacterial community is highly dependent on the diet as a source of metabolism (Wagner & Thomas, 1977; Savory, 1992). Thus, changes in diet composition or nutrient density can have dramatic effects on the gut microbial populations (Gibson et al. 1996; Hillman, 1999; Reid & Hillman, 1999; Hopwood et al. 2002), which in turn can affect the animal’s ability to It helps in the digestion and absorption of nutrients.
Thus, exogenous enzymes can indirectly alter microbial populations in the digestive tract by affecting the carbon substrates that bacteria use (Choct et al. 1999; Bedford, 2000b; Cowieson et al. 2000). Since both transient and resident bacteria in the gut depend on the nutrients the bird consumes for their metabolism and growth (Savory, 1992; Lan et al. 2005), the type of material in the diet can have a significant impact on the microbial populations in the digestive tract (Wagner & Thomas, 1977).
Other factors such as the pH of the digestive tract, the rate of food passage, the size of the feed particles, the action of the gizzard, the presence of exogenous and endogenous enzymes, high oxygen pressure, and endogenous antimicrobial compounds such as bile salts help to limit microbial proliferation in the small intestine (Bedford, 2000b).
A well-functioning intestine must allow nutrients to pass between the intestinal lumen and the circulation, while at the same time preventing the entry of pathogens. Several defense mechanisms are active in the intestine, which can be divided into two categories: non-immunological (non-immunological) and immunological.
Examples of non-immunological defense mechanisms include acidity, bile secretions, and proteolytic enzymes that can degrade bacterial cell walls. Adhesion of microbes to the epithelium (lining) of the upper small intestine can be reduced by the mechanical movement of peristalsis (wavelike contractions of the intestine). Also, the mucus secreted by goblet cells protects the surface of the intestinal epithelium and at the same time creates a viscous matrix that can trap foreign antigens (Forstner & Forstner, 1994; King, 1998).
(Fernandez et al., 2000) It has been reported that the addition of the enzyme xylanase to the poultry diet affects the synthesis of mucin (mucous substance). The effect of the enzymes on mucin synthesis is not completely clear, but it is possible that xylanase increases mucin secretion by goblet cells by reducing the viscosity of the intestinal contents. Data also showed that xylanase modifies N-acetylglucosamine groups in the mucins of goblet cells in different parts of the poultry intestine.
The carbohydrate properties of intestinal mucins have been reported to inhibit the adhesion of pathogenic bacteria Salmonella typhimurium and Yersinia enterocolitica (Ensgraber et al. 1992). Addition of an enzyme cocktail containing xylanase, amylase and cellulase to a diet containing chickpea meal has been shown to reduce the secretion of endogenous mucins (Cowieson et al. 2003).
The function of enzymes in establishing a normal intestinal microbial flora (a flora that does not cause clinical or subclinical disease, grows in a controlled manner and provides energy to the host by producing metabolic products) may enhance host immunity. This enhancement may be associated with the stimulation of mucosal and systemic immune defense mechanisms, possibly by reducing the concentration of saprogenic compounds (spoiled substances).
Ketonen and Rautonen (2005) reported that the use of exogenous enzymes increases the uptake of nutrients by intestinal immune cells and contributes to improved immunity. These researchers observed an increase in IgA concentration in the intestinal contents when a mixture of xylanase, amylase and protease was added to the poultry diet.
The same authors (Kettunen et al. 2005) also recently reported that the combination of these enzymes with betaine promotes immune maturation, such that CD4+ cells in the ileal tissue increased and IgA levels in the intestinal contents increased.
Feng et al. (2004) also reported that immune-related responses such as natural killer cell activity and serum antibody titers were increased by the addition of exogenous enzymes to the diet.
The extent to which the ability to manipulate immune defense mechanisms through the use of exogenous enzymes depends on our understanding of the cellular and molecular mechanisms of the delicate balance between the normal microbiota, mucosal immune components and the effect of different types of enzymes on these processes.
Predictability
Ingredient Quality
The effect of exogenous enzymes is inextricably linked to their substrates and the subsequent impact on the intrinsic nutritional value of the food to which they are added. Therefore, knowledge of the quality of the raw materials is essential if enzymes are to be used fully and with measurable and consistent efficiency. Raw material quality, although a common term, does not have a simple definition. In general, the nutritional quality of a food is a function of the concentration and digestibility of nutrients in it, their dilution by non-nutritional components (such as cellulose), and their enhancement by components with negative nutritional value (such as trypsin inhibitors). When enzymes are added to this food matrix, they can be considered either as agents in increasing the digestibility of nutrients or diluents or as agents in reducing adverse anti-nutritional effects.
It is reasonable to assume that since enzymes have been shown to reduce fluctuations in, for example, the energy value of a feedstock, the energy value improvement will be greater for feedstocks with lower metabolizable energy than for feedstocks with inherently higher metabolizable energy. Indeed, this has been shown for both wheat and soybean meal (Douglas et al. 2000; Scott, 2005). Douglas et al. (2000) investigated the effect of adding exogenous enzymes to corn-soybean diets using 12 different batches of soybean meal. They found that the effect of the enzyme was independent of some of the variables measured, such as protein solubility or protein content or total energy of the meal,
but was more dependent on the base digestible energy content. Similarly, Scott (2005) reported that wheat samples with high intrinsic feed conversion ratios (FCR) responded well to added enzymes.
With improved knowledge of feed quality and greater understanding of substrates, the development of the next generation of feed enzymes may become easier.
There are various methods for evaluating the nutritional value of a feedstuff, including functional studies, metabolism studies, and in vitro laboratory experiments. However, it is neither practical nor ethical to use large numbers of animals to evaluate the nutritional value of all feedstuffs before formulating poultry diets. Therefore, it is desirable to have statistical models capable of evaluating the nutritional value of a specific batch of grains or legumes and their response to exogenous enzymes through in vitro experiments. This can be achieved by using holo-analysis of performance and metabolism data correlated with in vitro parameters (Rosen 2002a,b)
Animal Modeling
The use of animal models to predict the nutritional value of a feed or the response to a feed additive is not a new concept, but it has become more sophisticated in recent years, especially with the introduction of neural networks and fuzzy logic in animal growth modeling. Very large databases have been created using published data to provide best-fit algebraic models for responses to a variety of feed additives, from copper (Cu) to antimicrobial growth promoters and exogenous enzymes (Rosen, 2001). These models can be very useful to the feed industry, as they highlight important variables involved in the response to feed additives that are not apparent when examining the results of only one or two experiments.
However, models built solely on published data are inevitably biased toward positive results, since negative or unchanged responses to additives are rarely published. Also, the inherent variability in models based on published data makes it impossible to use smaller databases to achieve optimal results. Indeed, Rosen (2002a, b) suggests that a minimum of about 100 experiments are required to develop a usable model, a number that should be directly related to the variability in the database. For databases developed under more controlled conditions with less variance, the number of experiments required may be significantly lower. However, if the variance of the inputs is too low, the power of the database is weakened because there is not enough data to extract important variables based on environment, maintenance, etc.
Thus, there may be a trade-off between high variability, which may result in important real effects being overlooked, and a model that is too controlled and likely to appear artificial. It can be concluded that the use of least squares models based on animal performance data is a powerful tool for predicting animal response to a feed additive under specific feeding conditions.
It is likely that in the future the development of novel feed additives, including new enzymes, will be accompanied by models that are able to predict the magnitude and persistence of response. This is particularly true when combinations of feed additives are used, where responses may be subadditive, additive, or synergistic. Therefore, if new feed enzymes or other additives are developed without the assistance of statistical modeling, recommendations for optimal dosages will be at best educated guesses.
Future Developments
It is difficult to predict what the next big step in feed enzyme research will be. However, there are some clear gaps in knowledge that may lead to discoveries with important implications. The effect of enzymes on nutritional requirements and net energy and amino acid values is a largely unexplored area. For example, do exogenous (external) proteases have a sparing effect on endogenous (internal) protease production through a hormonal feedback mechanism? If so, supplemental enzymes may reduce the energy and amino acid requirements of birds. This possibility also applies to other enzymes such as amylase, maltase, isomaltase, and lipase. There is no reason for an animal to continue to produce its own enzymes if it is more expensive to produce than commercial fermentation processes or if it is exposed to excessive substrate under stressful or special conditions (e.g., neonatal, enteric disease, etc.).
Another area that remains largely unknown is the use of enzymes as direct antimicrobials to break down the binding polysaccharides or cell walls of bacteria and the effect of enzymes on the host’s immune capability.
The provision of substrates for “beneficial” bacteria by targeted enzymes and their possible impact on immune status is a highly promising area for future research. Finally, the influence of the age of the bird on enzyme dosage recommendations is not well understood. Younger animals are likely to have different enzyme requirements than older animals, so that the type of enzyme(s) and optimal dosage will change with age. Although there are many new avenues to explore in this area, the food enzyme industry also faces significant challenges. These include increasingly stringent regulatory requirements within and outside the European Union and possible future restrictions on the acceptability of using genetically modified (GM) organisms for enzyme production. In addition,
With the development of more “bioactive” and concentrated enzymes, it is crucial that producers of these products ensure that even at low additive concentrations, enzyme uniformity in the feed is maintained at an acceptable level. Despite these challenges, the use of exogenous enzymes to achieve nutritional goals in the poultry industry is likely to continue for the foreseeable future.
Conclusion
Exogenous enzymes are effective in improving the nutritional value of poultry diets and subsequent animal performance. However, their impact on net nutrient value, microbial flora, safety potential, and the extent to which substrate quality and variability affect enzyme bioavailability are not fully understood. By equipping decision-makers with products that are both effective and economical, and by providing them with the knowledge to maximize return on investment, rather than simply suggesting a desired dosage based on the lowest cost per ton of feed, feed enzyme companies will gain a strategic advantage in an increasingly competitive environment.
