Review article
Exogenous enzymes in monogastric nutrition Ð their
current value and future bene®ts
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Michael R. Bedford
*Finnfeeds, Marlborough, Wiltshire SN8 1XN, UK
Accepted 18 May 2000
Abstract
Exogenous enzymes which, for the purpose of this paper, include carbohydrases and phytase, are now extensively used throughout the world as additives in non-ruminant diets. The chemical effects of these enzymes are well understood, but the manner in which their bene®ts to the animal are brought about is still under debate. Regardless, the overall effect of carbohydrase enzyme use is to reduce the variation between good and bad samples of a target ingredient substantially. The net bene®t is that the nutrient requirements of the animal are met more frequently, and with diets of lower nutrient concentration. Variation in animal performance from ¯ock to ¯ock is also reduced. Phytase, on the other hand, was originally used for one express purpose Ð to increase the availability of plant phytate phosphorus, which reduces phosphorus pollution and allows reductions in the amount of inorganic phosphate used. Further bene®ts of phytase utilisation on energy and amino acid availability have recently been identi®ed which will, with appropriate dietary modi®cations, allow for further improvements in resource utilisation. Current issues of concern for all enzymes include variability in response. Substrate variability and interactive factors signi®cantly in¯uence the response to exogenous enzymes. Currently, there are methods which take such factors into account and allow for prediction of optimum dose of carbohydrase enzymes in wheat and barley based diets and efforts are underway for maize based diets or for optimisation of the use of phytase. Future research in these areas will allow for more ef®cient use of the current enzymes and development of more ef®cient future products. Development of more thermotolerant enzymes will also allow their use in diets where they currently cannot be applied.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Xylanase;b-glucanase; Carbohydrase; Amylase; Phytase; Enzymes; Monogastric 86 (2000) 1±13
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This paper is based on the material presented at the BSAS Conference in Scarborough, UK, 22±24 March 1999.
*Tel.:44-1672-517-777; fax:44-1672-517-778. E-mail address: mike.bedford@danisco.com (M.R. Bedford)
1. Introduction
The use of enzymes in poultry diets in Europe is now almost universal. The reasons why they are used are manifold and include:
To increase the feeding value of raw materials. Many publications have demonstrated performance benefits of enzymes when added to barley (Classen et al., 1985; Elwinger and Saterby, 1987; Broz and Frigg, 1990; Brenes et al., 1993; Marquardt et al., 1994), wheat (Classen et al., 1995; Bedford and Morgan, 1996; Hughes and Zviedrans, 1999), and more recently maize based diets (Wyatt et al., 1997a, 1999; Steenfeldt et al., 1998). Phytases are routinely utilised particularly in environmentally sensitive areas of the world due to their ability to increase the phosphorous availability from vegetable ingredients (Simons et al., 1990; Jongbloed et al., 1997; Kornegay et al., 1997; Kemme et al., 1999). The consequences of such observations are two-fold, the targeted ingredient is used in greater abundance than would otherwise be the case, and secondly, the costs of diet manufacture are reduced due to decreased utilisation of scarce, high value ingredients such as fat and fishmeal.
To reduce the variation in nutrient quality of ingredients. The response to the use of enzymes is greatest on the poorest quality raw materials (Classen et al., 1995; Scott et al., 1995, 1998a; Bedford et al., 1998). As a result, variation in subsequent bird performance is reduced which results in a more uniform flock but also more uniform production from flock to flock. Such a benefit is considerable, given the losses incurred by producers when growing to set weights and by feed compounders when attempting to target a given diet nutrient density.
To reduce the incidence of wet litter. Feeding diets rich in barley, rye, oats, triticale and to a lesser extent wheat, often results in the production of a viscous, wet manure (Classen et al., 1985; Elwinger and Teglof, 1991; Newman et al., 1992; Carre et al., 1994; Bedford and Morgan, 1996).
Evidently these bene®ts are realised by the poultry industry and the consumer. This paper will separate the enzymes currently being utilised into three distinctive categories:
1. Viscous grain targeted, i.e. rye, wheat, oats, triticale and barley. 2. Non-viscous grain targeted, i.e. corn and sorghum.
3. Phytase.
Categories 1 and 2 are generally carbohydrase based products and will be dealt with separately from phytase.
2. Carbohydrases
down the rate of digestion. The physical structure of the endosperm cell walls of these grains may also impede access to their contents by digestive enzymes. Addition of the appropriate enzyme diminishes these constraints and allows digestion to occur more rapidly and completely.
2. Non-viscous grain enzymes. Maize variability has recently been demonstrated to be as great as that observed for wheat and barley (Leeson et al., 1993; Collins et al., 1998). Whilst enzymes can reduce this variation and accelerate the rate of digestion of maize and sorghum based diets (Wyatt et al., 1997a,b, 1999; Pack et al., 1998a), the exact mechanism of action is yet to be con®rmed, although several are offered.
Taking both classes of cereals as one, regardless of mechanism of action, the result of enzyme use is an increase in the rate of nutrient digestibility. This is important since it moves the site of digestion and absorption of starch and protein to a more anterior site wherein the bird has a greater competitive edge over its resident micro¯ora. This is more the case as the bird ages and its intestinal tract matures and becomes more heavily populated, and is most signi®cant when antibiotics are not utilised. Fig. 1 illustrates the case in discussion.
As feed passes through the proventriculus/gizzard, it is largely sterilised by the extremes of pH and activity of pepsin. In addition, as it enters the duodenum it is exposed to a rapid and signi®cant pH shift towards neutral which further stresses any bacterial survivors of gastric transit. Large in¯uxes of digestive enzymes, bile acids, lecithin and lysozyme further test the surviving bacteria such that the duodenum is largely devoid of bacteria. In the upper regions of the gut, digestive ef®ciency is maximal due to the high concentrations of pancreatic enzymes and ef®cient and highly active absorptive enterocytes (Uni et al., 1999). As feed passes though the small intestine, there is a progressive decline in digestive enzyme and bile acid concentration as these are either
Fig. 1. Relationship between the rate of digestion of a diet and microbial population density. A rapidly digestible ration supports fewer microbes.
catabolised and/or absorbed (Campbell et al., 1983; Schneeman and Gallaher, 1985; Noy and Sklan, 1994; Raul and Schleiffer, 1996; Taranto et al., 1997). As a result, the environment of the small intestine becomes increasingly hospitable to bacterial colonisation.
If the diet being fed is highly digestible then the majority of nutrients are digested and absorbed prior to the establishment of an environment favourable to bacterial growth. As a result, the populations of the lower small intestine are kept to a minimum essentially through substrate limitation. With a poorly digested diet, however, nutrients evade digestion and absorption by the bird and as a result enter the mid-lower small intestine where the bacterial populations are able to make good use of such substrate, and ¯ourish as has been shown when comparing rye (poorly digested) with corn (well digested) based diets (Wagner and Thomas, 1987). In stimulation of bacterial growth, there are inevitably species which are able to colonise the anterior reaches of the intestine by production of enzymes which actively degrade the very antimicrobials the bird produces, such as bile acids (Christl et al., 1997; Taranto et al., 1997). Through deconjugation and dehydroxylation, these compounds lose their antibacterial effect and as a result the sensitive bacteria are able to thrive. Elimination of these active compounds also results in impaired fat digestion since bile acids are essential for ef®cient micelle formation (Campbell et al., 1983).
Evidently, the consequences of bacterial overgrowth are manifold, not least since the presence of a greater population will demand a greater energy and protein requirement from the diet which is ultimately taken at the expense of the host.
The consequences of reduced diet digestibility, therefore, need to be assessed from two viewpoints if the bene®ts of cereal targeted enzymes are to be correctly assessed. The ®rst is the direct effects of a poorly digested diet on the nutrient assimilation rate of the host and the second is the rami®cations that such an increase in substrate delivery will have on micro¯oral populations inhabiting both the small intestine and the caeca. The former will of course limit the growth rate of the animal and the latter may result in a less ef®cient utilisation of digested and/or utilised nutrients through competition for substrates and interactions with the health status of the animal.
Enzymes have clearly been demonstrated to increase the digestibility of poorly digested cereals to a much greater extent than well digested cereals (Classen et al., 1995; Scott et al., 1998a,b). There are two consequences of such an effect of enzyme addition as far as the feed compounder is concerned:
1. Variation between the best and worst samples of a given grain is reduced.
2. In practice, the average nutrient content of the cereal is greater in the presence of enzyme than in the absence. As a result, addition of an enzyme allows feed formulation nutrient matrix values to be elevated.
not go into details here but refers the reader to the following references (Apajalahti et al., 1995; Apajalahti and Bedford, 1999). Essentially, the activity of the enzyme on viscous polymers and cell wall carbohydrates produces sugars and oligomers, which are utilised preferentially by certain ileal and caecal bacterial species. These ¯ourish at the expense of other, possibly detrimental species as far as optimal growth or health of the animal is concerned (Apajalahti and Bedford, 1999).
It is useful at this point to note that the consequences of a poorly digested diet, and hence the bene®ts of enzyme addition to such a diet, is more apparent in conventional compared with germ free chicks (Langhout, 1998; Schutte and Langhout, 1999). Since the micro¯ora increase in numbers as the animal progresses from essentially germ free to fully populated some 3 weeks later, it is likely that the bene®t of added enzymes is mediated through the micro¯oral route in older birds, whereas in young animals which have a poorly developed digestive system (Kirjavainen and Gibson, 1999), the effect is probably more direct. It is suggested that until the bird reaches 8 days of age, the output of pancreatic enzymes may well limit digestion (Lindemann et al., 1986; Krogdahl and Sell, 1989; Nitsan et al., 1991; Dunnington and Siegel, 1995). The addition of a relevant
Fig. 2. In¯uence of variety of barley on AME and subsequent response to enzyme addition (Scott et al., 1998b).
Fig. 3. In¯uence of enzyme addition on total ileal microbial counts (Apajalahti and Bedford, 1999). LAB: lactic acid bacteria.
exogenous enzyme is, therefore, likely to supplement the digestive capacity of the younger bird, and again the response to such will be more apparent with poorly digested compared with highly digestible diets. It is likely that, as birds age and their digestive ability increases, as does their micro¯oral population, the effect of exogenous enzymes is more and more mediated through the micro¯oral route.
The chemical identity of the viscous carbohydrates, the cell wall structures and the indigestible starch/protein complexes which are the focus of the enzymes discussed to this point is heterogenous in the extreme. The enzymes employed are, thus, wide ranging in their target substrate preference in the sense that the molecular weight of the substrate may vary several orders of magnitude as may the complexity and sugar identity of side chain adjuncts on the targeted backbone. As a result, bene®ts can be achieved by use of any one of a wide variety of enzymes, from divergent source organisms, which often have different mechanisms of action. As discussed later, this provides an avenue for improvements in ef®cacy as more is understood of the chemical nature of the most important antinutrients.
3. Phytase
There are many reviews recently published describing the use and effects of phytase in animal feeds. Phytate (Fig. 4), the target substrate of this enzyme, unlike that of the carbohydrases discussed in the previous section, is a ®xed chemical entity. It is presumed to be the plant storage form of phosphate which also happens to have considerable antinutritive effects for most animals. Phytate itself is not a good source of phosphorus for non-ruminants, particularly younger animals as they appear to lack a meaningful ability to utilise this compound, even though phytases have been isolated from the small intestines of broilers and laying hens (Maenz et al., 1997; Maenz and Classen, 1998). Phosphorus de®ciency would, therefore, often result if inorganic sources were not routinely added to most non-ruminant diets. As a result of uncertainty with regards to the availability of plant phosphorus sources, formulations routinely rely on added phosphates to supply most of the animals needs. Consequently, most non-ruminant diets contain far more phosphorus than the animal actually needs. The excess to requirement is simply excreted. Pressures to reduce phosphorus pollution levels in several parts of Europe and
more recently the USA has created a market opportunity for the introduction of exogenous phytases. The application of this enzyme allows the animal to access much of the plant phytate phosphorus, and thus, reduce reliance on inorganic phosphate sources. Phosphorus pollution as a result has been signi®cantly reduced.
Phytase activity is not entirely predictable, however. The bene®t achieved is known to depend upon several factors, including the raw materials used, the source of the phytase, the age of the animals, dietary content of calcium, phosphorus and Vitamin D, and the level of phytase activity present in the ingredients used. Each will be discussed in more detail below.
Phytate contents of many ingredients varies considerably as shown in Table 1 and as reported recently; the availability of phytate to exogenous phytase hydrolysis varies from ingredient to ingredient (Ravindran et al., 1999). The consequence of such an observation is that for practical purposes, a signi®cant safety margin needs to be employed in estimation of the phosphorus contribution as a result of phytate hydrolysis. Indeed, the consequences of overestimating the bene®t of the enzyme are dramatic. Initial estimates of the P release as a result of use of phytase proved over-optimistic when translated into commercial terms with the result that savings are less than ®rst envisaged (van Tuijl, 1998). Nevertheless, the bene®ts in reducing pollution and feed formulation costs are clear and as a result phytase is now a ubiquitous feed additive.
Phytase delivers economic bene®ts through its ability to replace added inorganic phosphorus. One issue that needs consideration is that the amount of phytase required to replace 10 g added phosphate ranges from <400 u/kg for broilers between 21 and 42 days of age to nearly 1000 u/kg for broilers between 0 and 21 days of age (Kornegay et al., 1996; Kornegay et al., 1997). Such variation, whilst of academic interest, also results in greater safety margins being employed than is often needed. Reasons for such differences possibly relate to differences in feed passage rate, which tends to be slower in the older bird (Washburn, 1991; Almirall and Garcia, 1994), and presumably allow for greater opportunity for phytase activity, or greater use of the crop, the pH of which is better suited for Aspergillus derived commercial phytases (Zyla, 1992; Radcliffe et al., 1998).
The greater the calcium content of the diet the poorer the ef®ciency of phytase activity (Fisher, 1992; Lei et al., 1994; Sebastian et al., 1996). Calcium is thought not only to
Table 1
Phytate phosphorus content of various feed ingredients according to several sources
Ingredient Matthaus
Maize 0.265 0.196 0.20 0.2200.038
Wheat 0.235 0.255 0.24 0.2430.010
Barley 0.223 0.230 0.19 0.2140.021
Soybeans 1.200.03 1.2000.030
Rapeseed 1.350.10 1.3500.101
Soyabean meal 0.372 0.40 0.3060.020
Rapeseed meal 0.870 0.87 0.87
precipatite phytate but also to interact with the soluble substrate, in doing so reducing the susceptibility of the substrate to enzymatic attack (Sebastian et al., 1996). Use of chelators, such as citrate, which removes the calcium from soluble phytate complexes, are therefore, effective in increasing the apparent activity of phytase (Zyla et al., 1996; Boling et al., 1998; Maenz et al., 1999; Qian et al., 1999). Reduction in the level of dietary calcium also brings bene®ts in reduced dietary costs. It is not only the absolute calcium content but its ratio to phosphorus content in the ration which is seen to be of importance, the greater the ratio the poorer the response to phytase (Qian et al., 1997). Interactive with both dietary calcium and phosphorus levels is the dietary content of Vitamin D. Increasing the dietary level of Vitamin D whilst decreasing that of calcium increases phytate utilisation even in the absence of phytase addition (Edwards, 1992; Fisher, 1992).
Variation in response to added phytase and variation in phosphorus digestibility between ingredents is a result, in part to, the fact that many ingredients contain an endogenous phytase activity in their own right. For example, phytase activity has been determined in wheat and found to vary markedly between varieties. The P digestibility of those samples with higher endogenous activity were shown to be almost 50% greater than those with lower endogenous enzyme activities (Barrier-Guillot et al., 1996a,b) (Fig. 5). Addition of phytase to such samples would therefore be expected to yield a lower absolute response. The bene®t in exogenous phytase use is that variation in P retention between cereal samples is reduced.
Phytate itself is often present as a protein phytate complex, and when free in the intestine is thought to bind to proteins rendering them less susceptible to proteolysis and bind to digestive enzymes, rendering them less effective (Liebert et al., 1996; Sebastian et al., 1998). As a result, several authors have reported improvements, albeit to varying degrees, in amino acid digestibilities on use of phytase in poultry rations (Martin et al., 1998; Namkung and Leeson, 1999; Ravindran et al., 1999). The consistency of the amino acid response to phytase addition appears to depend very much on the ingredients used, however, since whilst responses were observed in an amino acid de®cient soybean meal based diet, no such bene®t was seen in a peanut meal based diet (Biehl and Baker, 1997).
As a result, it is dif®cult to predict the improvement in amino acid utilisation on use of a phytase if the ingredients of the diet change substantially.
The variation in response between ingredients on phytase addition may well relate to the respective storage sites of phytate. In maize it is concentrated in the germ, whilst in wheat it is in the aleurone layer. In soybeans it is associated with the protein bodies, whilst in peanuts it is concentrated in th crystalloids. Such `geographical' differences may well in¯uence the susceptibility of the native phytate to hydrolysis be exogenous phytase, and hence explain some of the variability in the results described to date.
4. Future bene®ts
It is clear that with both cereal targeted enzymes and with phytase, there have been considerable advances in the past 10 years. Nevertheless, there are three key areas in which signi®cant improvements can be made:
1. Substrate identi®cation. The substrate of interest is poorly de®ned. Even in the case of phytate where the chemical identity is known, the associations of this molecule with others in the typical feedstuffs used is poorly understood. A more precise understanding of the presentation of the substrate in feedstuffs will allow development of associative enzymes which will co-operate to degrade the target substrate. Use of cell wall degrading enzymes in association with phytase, for example, will more rapidly expose the phytate to hydrolysis, and thus, increase animal responses observed. This has already been demonstrated with the combination of xylanases and phytases in broilers (Ravindran et al., 2000). Analysis of both the target raw materials used and the dietary background in which that material is fed has allowed for optimisation of dose of enzymes in wheat and barley based diets (Pack and Bedford, 1998b), but such bene®ts are yet to be realised in maize based diets or those targetted with phytase.
2. Characteristics of the enzymes used. Enzymes in use today cannot withstand the rigours of the most aggressive feed processing techniques such as expansion or pelleting at temperatures in excess of 958C. Such conditions necessitate the use of post-pelleting application of liquid enzymes on such feeds. Not only is this dif®cult, requiring the use of expensive equipment, but may be impossible due to space constraints in existing feed mills. The development of much more thermotolerant enzymes will allow dry, powder enzymes to be used in high temperature processed diets with no need for such application equipment.
3. Product formation. It seems likely that the end products of the activity of the enzyme are just as important as the hydrolysis of the target substrate. Greater understanding of the role oligomeric sugars play in maintaining a favourable micro¯ora, for example, will lead to more effective products for the feed industry. This is of particular importance with the removal of most prophylactic antibiotics from animal feeds in Europe. This will increase the need to ensure that bene®cial bacteria more than ever are encouraged to grow and exclude pathogens and performance reducing organisms.
Nutrition and microbiology of the intestinal tract will become more and more inter-related subjects of study.
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