Enzymes in Poultry Production

The following article is a special collaboration from AFMA (Animal Feed Manufacturers Association) www.afma.co.za
We thank their kind support.

Summary
Enzymes have been used in the feed industry for more than 30 years. Traditional applications have included improving the digestibility and bird performance of ingredients such as barley and wheat. As our understanding of the anti-nutritive factors that are associated with these and other feedstuffs has expanded, the applications for enzymes have also increased. Non-starch polysaccharides (NSP) can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance. NSP are also linked to other compounds such as peptides and proteins that can make the use of a purified enzyme designed to degrade NSP less effective than lower rates of NSP degrading enzyme combined with other enzymes such as proteases. Other anti-nutritional factors such as phytate can also adversely affect performance and the use of phytase enzyme has been shown to improve phosphorus utilization as well as cation minerals and protein.

Other enzyme applications include feedstuffs such as corn and soy which have historically not responded to enzyme use. Recent enzyme formulations have proven effective in corn/soy diets for poultry as well as other species. Other studies have examined the stability of enzymes to heat and pressures associated with feed processing and found beneficial responses to enzyme supplementation even at pelleting temperatures of 90oC.

INTRODUCTION
At the most basic level, feedstuffs consist of protein, starch, fat and fiber. In monogastric animals the fiber component has been considered to be wasted and in some instances, compounds called Non-starch polysaccharides (NSP) can exert anti-nutritive activity on the animal. The NSP of barley, wheat and rye has been the most intensively investigated. Beta glucan in concentrations ranging from 30-60 g/kg dry matter has been shown to depress production in broilers and cause sticky droppings (pasted vents). Wheat and rye contain relatively high levels of arabinoxylans or pentosans (50-80 g/kg dry matter for wheat; 100 g/kg dry matter for rye) which can also have a negative effect on broiler performance (Choct and Annison, 1990; Fengler and Marquardt, 1988). Ingestion of NSP by monogastrics results in increased viscosity of the digesta (Burnett, 1966; Antoniou and Marquardt, 1983). This increased viscosity reduces the passage rate of the feed leading to overall reductions in consumption and decreased performance, sticky droppings and dirty eggs (Classen and Bedford, 1991). The addition of enzymes to the diet to address NSP viscosity can improve feed efficiency, improve manure quality and increase the use of lower cost feed ingredients. Animal feed protein sources also have been shown to contain anti-nutritive factors which are listed in table 1.

Table 1. Anti-nutritional factors in commonly used feed protein sources.

Soybean meal	Trypsin inhibitors, lectins, saponins, raffinose, stachyose
Rapeseed meal	Glucosinolinates, tannins, phenolic acids, fiber
Sunflower meal	Fiber, tannins
Lupins	Alkaloids, fiber
Peas	Lectins, tannins, fiber, oligosaccharides

The use of enzymes in animal feeds is becoming more common. Reasons for this include; lower costs of commercial enzyme preparations, improved enzymes for animal feeds, and a better understanding of the composition of the anti-nutritive compounds. In order to obtain maximal benefits from enzyme inclusion in animal feeds, it is necessary to ensure that the enzymes are chosen on the basis of the feed composition. Simply put; the enzyme must be matched to the substrate. Enzyme cocktails containing more than one enzyme will often improve the response compared to pure, single enzymes, assuming that cost considerations are not ignored. This is due to the fact that feedstuffs are complex compounds containing protein, fat, fiber and other complex carbohydrates. Merely targeting a specific substrate such as Beta glucan may not provide maximal benefits since layers of other substrates may inherently protect some of the Beta glucan. For example, Beta glucans and arabinoxylans may be bound to peptide or protein moieties in the cell wall of the feedstuff. Therefore, enzymes capable of hydrolyzing protein may enhance the activity of pentosanases and beta glucanases. Methods commonly used to determine the effects of enzymes on feedstuffs include determination of the NSP content of the ingredients or by measuring changes in the viscosity of the feed with enzyme supplementation.

Mechanisms of Enzymes in Poultry Production - Image 1

Enzymes for better feed utilization
Beta glucanase was one of the first enzymes used extensively in the feed industry. Dietary supplementation of barley with b -glucanase allows the inclusion of higher levels of barley to be used by hydrolyzing the b -glucan chains. This leads to a less viscous digesta and allows better nutrient utilization. Numerous studies confirm the efficacy of such enzymes when used in barley-based diets in poultry. One such trial examines a dose titration of a commercial b -glucanase (table 2). From this data it can be observed that maximal response was noted when 300 units/kg b -glucanase was used.

Table 2. Effect of b -glucanase on broiler performance fed a barley-based diet (to 39 days). Diet contains 50% barley containing 4.3% b -glucan.

Enzyme Level (BGU/kg of feed)	Feed Intake (g/bird)	Body Weight (g)	FCR
0	96.5^a	2163^a	1.742^a
100	95.0^ab	2193^ab	1.692^b
200	95.3^ab	2215^b	1.678^b
400	93.2^b	2160^a	1.685^a

Schutte, 1996

Wheat, rye and triticale contain a relatively high concentration of non-starch polysaccharide consisting mainly of arabinoxylans and some beta glucans. In order to overcome the adverse effects of arabinoxylans, a different enzyme must be used. Pentosanase (or xylanase) can overcome the deleterious effects of these NSP by aiding in the breakdown of the arabinoxylans. The beneficial effects of pentosanases in wheat-based rations are most noticeable in young birds as shown in table 3. From these results, it is evident that improvements in daily gain and feed efficiency can be obtained from proper enzyme inclusion.

Table 3. Effect of pentosanase on broiler chicks fed a wheat-based diet (to 21 days).

Parameter	Control	Pentosanase (1000 XU/kg)	SED	P Value
Daily gain (g)	34.0	36.6	0.475	0.001
Daily Intake (g)	54.0	54.8	0.641	0.05
Feed efficiency*	0.634	0.657	0.003	0.001

*Efficiency = body wt/feed consumed.
Tucker, 1992.

ENZYMES FOR OTHER NSPs AND PROTEASE ACTIVITY
Legume seeds such as soy contain NSP in the form of oligosaccharides, hemicellulose and pectin. Alpha galactosides are raffinose- and stachyose-based oligosaccharides that accumulate as the seed matures. Endogenous enzymes in monogastrics are specific for alpha-linked carbohydrates such as starch but have little or no effect on beta linked carbohydrates or galactose-containing oligosaccharides. Degradation of these galactosides is accomplished by the gut microflora yielding volatile fatty acids and gas production. The net result is less energy and gastric disturbances in many species. Enzymatic degradation of these compounds can produce monosaccharides and result in better energy and protein utilization. In poultry, improvements in gain and feed conversion have been noticed with an enzyme cocktail formulated for corn-soy diets (table 4). Although not shown in the table a dose titration yielded maximal response at a use rate equivalent to 3750 HUT/kg (protease) and 37.5 CMC/kg of feed (De Koning personal communication). Amino acid digestibility has also been improved with this enzyme (Pugh and Charlton, 1995; table 5).

Table 4. Effect of a soy-specific enzyme cocktail containing protease (7,500 HUT/g) and cellulase (75 CMC/g) on broilers fed a wheat, soy diet.

Parameter	Control	Enzyme Supplemented (500g/t)
Weight Gain 15 days (g)	549	569
Weight Gain 29 days (g)	1463	1500
FCR 15 days (kg/kg)	1.617^a	1.559^b
FCR 29 days (kg/kg)	1.733^a	1.707^b

^a,b Means in the same row with different superscripts differ significantly (P<0.05)

Table 5. Effect of a soy-specific enzyme cocktail containing protease (7,500 HUT/g) and cellulase (75 CMC/g) on true amino acid digestibility (%) of soybean meal (48%).

Amino Acid	Control	Enzyme supplemented(1-kg/t)
Alanine	72.4	72.8
Aspartamine	67.0	69.6
Cystine	41.6	57.9
Glutamine	82.7	84.6
Histidine	54.7	68.5
Isoleucine	81.0	82.8
Leucine	80.6	82.0
Lysine	82.1	86.0
Methionine	65.4	69.0
Phenylalanine	86.3	88.9
Proline	70.0	77.4
Serine	78.6	83.9
Threonine	72.7	78.5
Tyrosine	74.3	77.1

Bernard and McNab, 1997.

Other corn/soy enzyme cocktails yield similar results. Zanella and coworkers (1999) found that an enzyme formulation containing protease (6,000 u/g), amylase (2,000 u/g), and xylanase (800 u/g) resulted in a 2.9% improvement in total protein digestibility and improvements in gain of approximately 50-g/bird, and feed conversion (about 4 points better with enzyme supplementation). Because of the improvements observed in protein digestibility it is tempting for the nutritionist to lower the overall protein and energy levels of the diet. However, because of the variation in individual amino acid digestibility, caution is advised in doing this in order to ensure adequate levels of limiting amino acids.

Phytase
The benefits of phytase have been known since the 1960s and a large number of studies have proven efficacy in poultry diets to lower the overall added inorganic phosphorus levels. This is due to the ability of the enzyme to degrade phytate phosphorus found in feedstuffs. Phytate has the ability to complex with protein, peptides or cations including calcium, magnesium, copper, zinc and iron. These compounds when complexed to phytate are less available and digestible. In addition, Singh and Krikorian (1982) found that phytate could also bind endogenous enzymes such as chymotrypsin and trypsin in the GI tract which could further inhibit protein digestibility. Supplemental phytase enzyme can therefore improve the availability of phosphorus, other minerals and improve digestibility of protein. Lowered costs of phytase production have allowed widespread use of the enzyme which was previously only used because of government mandates on lowering phosphorus emissions from livestock areas. Until recently phytase seemed like an oxymoron for the environmentalist. This is due to the fact that the predominant commercial phytase is derived from a genetically modified organism (GMO). However, current technologies in solid-state fermentation and traditional microorganism strain improvements have yielded non-GMO sources for this enzyme. Comparisons of GMO and non-GMO phytase sources have yielded similar results (Rowland et al., 2000; Sims et al., 1999) and led to a more competitive and lower cost phytase product. Additionally, recent studies have shown improvements in areas of nutrient utilization other than simply phosphorus and calcium absorption increasing the benefit to the producer (Namkung and Leeson, 1999).

Effects of Enzymes on the gastrointestinal environment
Microorganisms in the gastrointestinal tract utilize the digesta for energy in a similar manner to the host animal. Changes in rate of passage and the type of nutrients available to the microbes influence the different microbial populations in the GI tract. The end products of metabolism of many of the anaerobic bacteria found in the gut are volatile fatty acids which have been shown to be altered with enzyme supplementation (Choct, 1995). However, studies examining differences in specific microbial populations such as starch or xylan- degrading bacteria have yielded no significant effects (Persia, et al., 1999). This may be due to lack of technology to adequately examine these populations since it stands to reason that as the substrate changes so should the microorganisms that can use them. Gastrointestinal histology has also been shown to be affected by barley and wheat-based diets with reductions in villi height, increased diameter and damaged villi associated with wheat and barley diets (Viveros et al., 1994; Jaroni et al., 1999). Enzyme supplementation of these diets counteracted some of these effects with supplemented birds having a gut morphology more similar to birds receiving a corn/soy diet. This may also help explain reductions in mortality that is often seen in birds receiving enzyme supplementation. Damage to the GI tract may make the organ more susceptible to pathogenic bacterial invasion. In addition, enzyme supplemented birds had lower gut and pancreas weights. The strain of bird used also had a bearing on these results.

ENZYME STABILITY
Since enzymes are proteins, the structure of the enzyme is critical to its activity. PH, heat or certain organic solvents can alter enzyme structure. Changes in the structure of the protein can decrease or negate enzyme activity. The temperatures which feeds are exposed during the pelleting process can range from 60 to 90oC under normal conditions. These temperatures and pressures can therefore lead to loss of feed-borne and added enzyme activity (Rexen, 1981). Recent studies reveal that enzyme activity begins to decrease as pelleting temperatures reach 80oC. These data suggests that cellulase, fungal amylase, and pentosanase can be pelleted at temperatures up to 80oC and bacterial amylase up to 90oC without any considerable loss of activity (Spring et al., 1996). However, caution must be used in interpreting these data since substrates necessary for in-feed assay of enzyme activity may not reflect the actual components of the feed. In the case of cellulase activity, in vitro substrate activity was significantly diminished at 80oC while the viscosity of the feed was improved even at temperatures up to 90oC. Therefore, for the feed manufacturer, practical enzyme activity of cellulase was maintained up to 90oC. Subsequent work by Samarasinghe et al. demonstrates that, although cellulase activity found in the feed after pelleting at 90oC was reduced by 73%, the average growth rate of broilers increased by 11% when feed containing the enzyme was compared to feed with no enzyme (Samarasinghe et al., 2000). At first glance these results seem confusing. However, studies have shown that the viscosity of the feed increases with increasing pelleting temperatures (Nissinen, 1994; Spring et al., 1995; Vukic-Vranjes and Wenk, 1995). This is due to starch gelatinization and increased solubilization of fiber. The viscosity of feed pelleted a high temperatures has been shown to be negated by inclusion of enzymes in the feed prior to pelleting. In fact, enzyme inclusion lowered the extract viscosity of the feed by 11, 14 and 17% at 60, 75 and 90oC respectively compared to feed without enzyme inclusion (Samarasinghe et al., 2000). With this data in mind, it becomes apparent that one mechanism of enzymes may not be in the bird at all but rather the activity of the enzyme during the pelleting process which exposes the enzyme to moisture and heat giving the possibility of optimal conditions for enzyme activity prior to it’s inactivation by the excessive heat. In cases where temperatures are excessive for enzyme stability, post-pelleting application is commonly used.

Conclusions
Numerous studies over the past ten years have demonstrated improvements in feed utilization with enzyme supplementation. Use of b -glucanases in barley diets and pentosanases in feed ingredients high in NSP is now common practice. Reduction in viscosity of the digesta may enhance nutrient utilization by the bird by "normalizing" the histology of the gut when NSP diets are fed. Evidence also exists that enzyme cocktails containing proteases may enhance the beneficial effects associated with these enzymes. As enzyme technology improves we have also seen benefits in areas not traditionally associated with digestive inefficiencies such as energy and protein utilization from soy and other feed ingredients. Tradition tells us that the use of enzymes in corn/soy diets is not efficacious. However, recent advances indicate that this is not the case.

Future developments in enzyme technology will likely focus on more thermo-tolerant enzyme preparations, greater enzyme activity and enzymes which function optimally at low gastric pH values. Additionally, as more is known of the chemical nature of our feed ingredients, better methods of degrading these compounds may be found. In reporting studies on enzyme efficacy it is becoming increasingly important to ensure that the units of activity of the enzyme, as well as the use rate, be reported to improve our understanding of efficacy. Commercial enzymes can come from a variety of source microorganisms. The organisms produce enzymes that may have different pH and temperature optima. Because of this, different enzyme manufacturers may use other units of activity to describe an enzyme.

Kyle Newman, Ph.D.
Director of Laboratories, Venture Laboratories, Inc.
A044 ASTeCC Building
Lexington, KY 40506-0286
U.S.A.

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