Maximising nutrient utilisation: from enzymes to nutraceuticals

Toward the end of last century a major lifestyle trend emerged in the developing nations emphasizing wellness and disease prevention in the human population rather than treatment therapies. This focus is expected to strengthen, rather than diminish, as the relatively affluent and well-educated ‘baby boomers’ of the 1950s and 1960s progressively reach middle age and begin to contemplate their retirement years. It has been calculated that in the US alone, and in the period from 1996 through to 2006, a ‘baby boomer’ (defined as someone born between 1946 and 1964) will turn 50 years of age every seven and a half seconds (Sloan, 1998). Such individuals are acutely aware of health and lifestyle issues.

A further significant driver of this trend, fuelled at government level, is the escalating cost of traditional disease treatment. Allied to this, there has been a growing realisation of the pivotal role of nutrition in disease prevention and the maintenance of human health. Epidemiological studies have exposed statistical correlations between the intake of certain dietary constituents and the development, or alternatively the prevention, of various non-communicable diseases. In many cases, controlled human intervention studies have supported the epidemiological observations; and a considerable body of knowledge and level of understanding has developed around the nexus of nutrition, health and longevity.

It has even been postulated (the Barker hypothesis, Barker, 1994; Barker, 1999), and considerable supportive evidence has been amassed, that a propensity to develop certain diseases in later life may be related to maternal nutrition and subsequently modified by the nutritional habits of the individual. Barker reports that the heavier the baby, the lower the incidence of coronary heart disease in later life. Barker found that a plump baby who gains weight well in the first year of life has a coronary infarct rate in later life one third that of lower weight babies. Barker has also found that blood pressure in the adult is clearly related to infant birth weight and placentalsize, with a large placenta and a low weight being highly associated with increased systolic pressures in adults, an association stronger than that linking salt consumption to blood pressure. These findings have far-reaching implications.

Nutraceuticals and functional foods

DEVELOPMENTS IN HUMAN NUTRITION

The food industry has been quick to capitalise upon the changes in societal attitudes and today pays far more attention to nutrient contents and bioavailability than was the case in the past. This growing emphasis on lifestyle has also influenced the emergence and vigorous marketing of a completely new category of food, the so-called ‘functional foods’ and‘nutraceuticals’. Functional foods are foods that when consumed as part of a normal diet elicit specific physiological effects on the consumer in addition to sustenance derived from the food nutrients.

Certain foods, when consumed on a sustained basis, may have quite subtle longer-term effects on aspects of physiological function, whereas yet other foods and natural food extracts may have acute pharmacological effects. It is in the latter case that the terms ‘nutraceutical’ or ‘pharmafood’ may be applied. Indeed, in the growing market of nutraceutical and functional foods, there is a merging of the traditional food, agribusiness and pharmaceutical industries. Moreover, and as the human genome project gains momentum, the very genetic basis of the so-called lifestyle diseases is being unravelled and described. This will soon allow the early identification (pre-symptomatic) of individuals prone to developing certain diseases, thus heralding specific dietary and lifestyle prescriptions.

Functional foods and their place in a balanced diet are going to become increasingly important. The functional foods revolution began in earnest in the early 1980s with the health-related benefits of materials such as plant fibre, fish oil, calcium and probiotics attaining respectability following publication of clinical studies in the medical literature and with physicians beginning to publicly promote their use.

Examples are diverse, including the use of ß-carotene in the prevention of cancer, pyridoxine to treat and prevent depression, tryptophan to induce sleepiness, garlic (allicin) to reduce artherosclerosis, cranberry juice to prevent urinary tract infections, the role of calcium in treating osteoporosis, phytoestrogens, lycopene, antioxidants, and many others. An example of one of the earlier and now well established functional foods is oat bran, whereby the soluble oat fibre acts in the alimentary canal to bring about a reduction in blood cholesterol. For hypercholestrolaemic individuals, the inclusion of oatbran in the diet (e.g. oatbran enriched cereals) can be used as part of an overall strategy to lower blood cholesterol, without resorting to prescription drugs. A further example of a cholesterol-lowering functional food, and one that has been enormously successful commercially, is that of phytosterol enriched margarines and spreads. Animal products may also be manipulated to produce unique functional foods, presenting the animal and feed industries with an opportunity to develop differentiated, branded food products. Examples here include omega-3 enriched hen’s eggs (‘heart-smart’) and high vitamin C/ vitamin E pork, with antioxidant potential, reduced drip loss and a superior shelf life.

There are many other examples of functional foods and nutraceuticals and many more products can be expected to enter the market as product development and clinical testing increases apace. The increasing appreciation of the diverse physiological roles of food constituents is also having a profound influence on the contemporary view of nutritional science itself and of the very definition of ‘a nutrient’. In the past, for example, volatile fatty acids were viewed as products of the fermentative breakdown of fibre, acting as an energy source for the host animal, whereas now their role in the development and regulation of gut function is being emphasised (Sakata, 1987). Proteins were once viewed as simply supplying amino acids for body protein synthesis whereas now the distinct and diverse physiological effects of dietary peptides are being discovered and documented with an increasing frequency. Fatty acids, far from acting solely as sources of energy or as body energy stores, are now known to quite profoundly affect red blood cell membrane composition, with consequent physiological effects.

WHAT OF THE ANIMAL FEEDSTUFFS INDUSTRY?

The opportunities afforded by the often potent physiological effects of dietary constituents being exploited by the international foods industry are also open to the compound feed industry; and we have only seen the beginnings of biotechnological innovation in this area. The potential to manipulate animal health and performance through understanding the physiological roles of feed constituents is immense and will undoubtedly be a major focal point in research and development for the biotechnology industry in this century. In the following, two biotechnology products already available commercially (namely enzymes and peptides) will be discussed in detail as examples of how animal ‘function’ can be influenced by diet, other than through nutriment.

Bioactive peptides

A new technology offering considerable potential to the feed and animal industries is that of bioactive peptides. As mentioned above, dietary protein was once viewed as a source of amino acids, primarily acting as building blocks for body proteins. Now, however, it is understood that during the digestive process, peptides which are ‘hidden’ in an inactive state within the protein sequence may be released to act as physiological modulators, both locally in the gut and systemically. Both animal and vegetable proteins contain potentially bioactive sequences, though much of the research to date has been conducted with milk proteins.

An opioid activity of peptides derived from partial enzymatic digestion of milk proteins and wheat gluten was reported in the literature as early as 1979 (Brantl et al., 1979; Zioudrou et al., 1979). Following on from this discovery, much research into bioactive peptides has been conducted and many sequences and their physiological functions have been defined. In spite of this research effort, however, the area is still very much in its infancy and undoubtedly there is a great deal yet to be discovered. The potential for bioactive peptides in the development of functional foods for humans, farm animals and in the pet food industry is truly great. Bioactive peptides are now understood to have a wide range of physiological effects, some of which are listed in Table 1. Evidence (Lord, 1986; Webb et al., 1992; Gardner, 1998) that peptides can be released during digestion and absorbed to enter the portal blood intact provides a basis to explain the wide range of systemic activities so far discovered and documented. Moreover, the existence of bioactive peptides themselves offers an explanation for the often observed varying effect of diet on physiological response.

An array of bioactive peptides and protein hydrolysates can now be produced commercially, creating the opportunity for dietary addition. Indeed it is predicted that such materials will be used increasingly in the feed industry (Power and Murphy, 1999) and wider foods industry (Frokjaer, 1994). Casein derived peptides are already being used as food supplements (eg. phosphopeptides) and pharmaceutically (Meisel, 1997).

There have been a number of reports in the literature describing a role for bioactive peptides in regulating stomach emptying rate and gastrointestinal motility in mammals (Daniel et al., 1990, Kil and Froetschel, 1994; Froetschel, 1996), gut secretory and absorptive activity (Schlimme et al, 1988; Ben Mansour et al., 1988) and gut tissue growth (Birke et al., 1993). Such observations are consistent with findings from our own group working at Massey University, New Zealand, which has described a central role for diet-derived peptides in influencing gut protein dynamics. The following provides a brief summary of our studies to date and again highlights the importance of diet-derived peptides. The gut is a highly metabolic organ, accounting for a significant proportion of total energetic and protein costs during animal growth, and thus regulatory activities assume a disproportionate order of importance.

In our first set of studies we sought to determine the effect of nitrogenous alimentation per se (i.e. excluding effects of proteins and peptides) on the net effect of gut protein secretion and reabsorption (i.e. endogenous loss measured at the end of the small bowel). A semi-synthetic nitrogen-free diet (control) was formulated to mimic the effect of the non-protein component of a diet, along with a series of similar diets containing synthetic free amino acids as the sole source of nitrogen. By not including certain dietary non-essential amino acids in some of the diets and by also omitting certain dietary essential amino acids in others (but with accompanying intravenous infusion), we could directly and unambiguously measure endogenous amino acid loss at the terminal ileum.

The studies were undertaken with laboratory rats and pigs as generalised mammalian model animals. The animals consumed the amino acid-containing diets readily, grew normally and were in a positive body nitrogen balance. A comparison of endogenous ileal amino acid loss for animals receiving the synthetic amino acid-based diets and the protein-free control is given in Table 2. In spite of the treatment groups being in positive body nitrogen balance and receiving a gut luminal amino acid supply, the endogenous amino acid flows (the net result of overall gut secretion and reabsorption) were not higher than for the control (proteinfree) animals that were in negative body nitrogen balance and deprived of a direct dietary amino acid supply to the gut. The results of these studies indicated that gut protein dynamics do not appear to be influenced by nitrogenous alimentation per se.

The second series of experiments sought to determine whether feeding the animal protein rather than amino acids would have any effect. Here it was necessary to devise techniques to distinguish between undigested dietary protein and the endogenous protein flow. Isotopic markers may be used to make such a distinction, but their use is fraught with difficulties leading to inaccuracy. It occurred to us that if we could completely transform lysine in dietary proteins to an analogue such as homoarginine, then we could, by feeding animals the guanidinated protein, directly measure the endogenous loss of lysine.

Homoarginine is absorbed in a similar manner to lysine and is partially converted in the liver to lysine. In this case, animals would consume a protein-containing diet and would be in positive body nitrogen balance. We were successful in completely guanidinating proteins (Rutherfurd and Moughan, 1990) and subsequently several studies were conducted with the laboratory rat using this approach. We were also aware that there are naturally occurring proteins that are completely devoid of certain amino acids. An example of such a protein is zein, which can be isolated from the maize grain and is completely devoid of lysine. Accordingly, we prepared semi-synthetic zein-based diets and fed them to young pigs, which were simultaneously infused intravenously with lysine. Here again, the gut tissues were supplied with nitrogenous material and the animals were in positive body nitrogen balance. The ingestion of protein led to a dramatic almost doubling of the endogenous ileal lysine flow (Table 3).

The question now arose as to whether the pronounced effect of dietary protein on gut protein dynamics may be caused by peptides. To test this hypothesis, it was required to develop yet a further experimental technique.

Our resultant methodology, referred to as the enzyme-hydrolysed protein method, is now commonly employed in laboratories throughout the world to determine gut endogenous losses. In this approach an enzymic hydrolysate of protein is fed to the animal as the sole source of nitrogen in a purified diet. The size of the peptides in the hydrolysate is less than 5000 Daltons.

Ileal digesta are subsequently collected from the animals and the material is centrifuged and ultrafiltered (10,000 Dalton MW cut-off). Any unabsorbed dietary peptides are discarded in the ultrafiltrate, with the precipitate plus retentate being an estimate of gut endogenous loss. This estimate is slightly low due to the loss of a small amount of endogenous free amino acids and peptides in the ultrafiltrate. Our group has applied this technique widely in a number of studies, with the consistent result of a significant (P
Current research is investigating which of the peptides present in the complex hydrolysates are responsible for the effect, what are the mechanisms of action and what components of the endogenous milieu are affected. Clearly, given the quantitative importance of gut protein dynamics in overall body protein metabolism (Moughan, 1999), there are opportunities here to manipulate the gut processes. One can imagine, for example, enhanced digestive function arising from dietary peptide supplementation or significant increases in growth efficiency brought about by a reduction in endogenous amino acid loss consequent upon removing the effects of key bioactive peptides. This research field is fertile and the future exciting.

The observations described above regarding the role of bioactive peptides in regulating digestive function hopefully serve to underscore the biological importance of these components in production farm animals. Such peptides are potent and have wide-ranging effects. Industrial production of bioactive peptides heralds new opportunities for functional foods in the livestock and pet food industries.

Exogenous enzymes

The potential usefulness of enzyme preparations to improve performance in poultry has been known for many years (Hastings, 1946; Fry et al., 1957).

However, it has only been possible in the past decade to produce feed enzymes cheaply enough to warrant their use in commercial situations. This has been due primarily to a better understanding of target substrates and advances in microbiological technology. Enzymes may have their effect by directly altering the rate of hydrolysis of bonds thus releasing nutrients or by affecting physico-chemical properties (eg. viscosity of digesta). In either case, physiological mechanisms are being affected and enzymes can be considered as ‘functional’ additives.

The poultry industry is the largest user of feed enzymes. The highly integrated nature of the poultry sector has enabled the faster uptake of these new technologies, and exogenous enzymes are now well accepted as a feed enhancer to improve nutrient digestibility and efficiency of nutrient utilization in poultry. Progress into pig diets, on the other hand, has been slower due partly to the more fragmented nature of the pig industry and partly to species differences in production responses. However, interest within the pig industry, especially for weaner pigs, is gaining momentum.

The popularity of feed enzymes is a consequence of changing social and economic philosophies and emphasis on efficient animal production with minimal environmental damage. Consumers are demanding an all-natural feed supply. Feed enzymes, especially from non-genetically modified organisms, are acceptable alternatives because they are common in daily life.

The primary objective of adding enzymes to animal feeds is to improve the utilization of nutrients in raw materials. This is achieved by one or more of the following mechanisms: 1) degradation of specific bonds in ingredients not usually degraded by endogenous digestive enzymes, 2) degradation of anti-nutritive factors that lower the availability of nutrients, 3) increased accessibility of nutrients to endogenous digestive enzymes, and 4) supplementation of the enzyme capacity of young animals.

BEYOND CEREAL APPLICATIONS: THE POTENTIAL FOR PROTEINHYDROLYZING ENZYMES

Although the use of exogenous enzymes in animal nutrition has increased considerably in recent years, most of the use has been in cereal-based diets. The enzymes widely used by the industry are the glycanases (xylanases and ß-glucanases) that cleave the non-starch polysaccharides in some cereals, and more recently, microbial phytases that target the phytatecomplexes in plant-derived ingredients.

The use of exogenous glycanases in wheat- and barley-based diets is now widespread in many parts of the world. Excellent reviews on the influence of xylanases on animal performance and nutrient utilization and on their modes of action are available (Annison and Choct, 1991; Bedford and Schulze, 1998). The ecological benefits and production responses from the use of microbial phytase are current topics, but this will not be covered herein since a session of the symposium is devoted to this enzyme.

Several enzyme preparations, designed towards improving the utilization of protein, starch or lipids in specific ingredients, are also currently available.

In particular, proteases are of interest because protein is the most expensive item in animal diets. Poorly digested proteins not only lower the efficiency of nitrogen utilization, but also contribute to odour problems and ground water pollution. Whenever an ingredient or diet is utilized more efficiently, there is obviously a reduction in manure output per unit weight gain and the ecological benefits can be quite substantial. The use of proteases can be valuable in this context to improve the nutritive value of protein meals by hydrolyzing certain protein types that are resistant to digestive enzymes and/or by complementing the animal’s own digestive system.

Studies with Allzyme Vegpro in proteins for pig diets

Allzyme Vegpro, an enzyme complement with protease activity, has been proven to be successful in improving energy utilization, nutrient digestibility and growth performance of poultry and pigs fed on diets containing a variety of vegetable proteins.

At the Monogastric Research Centre at Massey University, we have evaluated the influence of Vegpro on the apparent digestible energy content and total tract nutrient digestibility of a number of vegetable proteins, including soyabean meal and canola meal (Pluske et al., 1999). In this study, growing pigs (35 to 45 kg) were housed individually and fed semi-purified assay diets containing the vegetable protein as the sole diets to determine digestibility. Following a seven day acclimatisation period, grab faecal samples were obtained daily for five days, pooled, processed and subsequently analysed for chromium, gross energy and nitrogen.

Although faecal digestibility is not an accurate measure of amino acid digestion in pigs due to the modifying effects of hindgut microorganisms, the trends observed demonstrate that the utilization of protein and energy were improved by the use of Vegpro (Table 6).

These improvements in nutrient digestibility have been confirmed and benefits in terms of growth responses seen in a subsequent trial with growing-finishing pigs fed barley-based diets containing soyabean meal or canola meal. The most marked responses to Vegpro were observed in a trial involving Australian sweet lupins (Lupinus angustifolius). Addition of Vegpro to a barley-based diet containing 300 g/kg lupins improved the digestible energy by 5.4%, growth rate by 8.4% and the feed efficiency by 8.0% (Table 7).

As a consequence of improved gain, pigs fed diets with Vegpro had a 4.5% heavier carcass. However, the carcass fat of pigs fed diets with Vegpro was 1.8 mm thicker (at P2). It appears that the additional energy released by the enzyme was responsible for the increase in fat thickness. Under these circumstances, a revised diet formulation taking into consideration the increments in nutrient digestibility is clearly necessary to maintain the carcass quality at slaughter. An additional benefit from the use of the enzyme is decreased nitrogen excretion. In the study, a 10% decrease in faecal nitrogen output was observed.

Increasing fat utilization: studies with Lipozyme

Full-fat rice bran, a by-product of white rice milling, is available abundantly in the Asian region. It is a potentially important feedstuff for broilers containing relatively high levels of crude protein (130-170 g/kg) and fat (130-200 g/kg). Early studies, funded by Alltech and conducted at Massey University, showed that rice bran also contains high levels of non-starch polysaccharides; but it does not appear that these are anti-nutritive for broiler chickens (Annison et al., 1995).

However, the AME content of rice bran is lower than anticipated on the basis of its gross energy content (20-22 MJ/ kg dry matter); and this may be attributable in part to a low secretion of pancreatic lipase (Warren and Farrell, 1990). Supplementation with exogenous lipases may be beneficial in assisting birds to extract energy from diets containing rice bran. To this end, Allzyme Lipozyme has been tested in a series of trials at Massey University. Initial research, (summarised in Table 8), showed that supplementation with lipase led to a significant reduction in excreta energy concentration (16.57 vs. 15.78 MJ/kg dry matter), which translated to a higher apparent metabolisable energy (AME) value for rice bran (12.15 vs. 12.47 MJ/kg dry matter) for adult cockerels.

In subsequent studies (Tan et al., 2000), the use of Allzyme Lipozyme added to diets containing full-fat rice bran resulted in numerical improvements in the AME content of both the complete diet and the full-fat rice bran per se.

The responses were dependent upon the dietary level of rice bran, with greater response being observed at lower inclusion levels (Table 9).

However, observed responses could not be attributed to any improvement in lipid digestibility suggesting that other enzyme activities (i.e. protease, cellulase, and xylanase) present in the preparation may have worked singly or in combination. The overall results suggest that this enzyme preparation offers promise to improve the nutritive value of rice bran for broiler chickens.

LIQUID FEEDS: ENZYME APPLICATIONS

In pig production, liquid feeding systems are becoming increasingly popular.

Liquid feeding confers a number of benefits such as a reduction in food wastage, automatic (computerised) and accurate control of feed delivery, lowered dust levels, flexibility in the use of different diets and improved animal performance (Patterson, 1986). Moreover, the liquid medium offers significant opportunities for ‘biological’ processing of feeds using bacteria or exogenous enzymes. Liquid feeding systems offer a major opportunity for reacting mixed diets or particular feed ingredients with enzymes prefeeding.

Pigs differ from birds in that upon ingestion food directly enters the true stomach rather than a crop and is immediately subjected to a low pH.

With careful selection of the appropriate enzymes and taking advantage of the liquid medium, it may be possible to pre-react foods and deliver to the animals partially digested feedstuffs.

With this in mind, we undertook a pilot study (Hartley and Moughan, 1994) to evaluate the effects on pig growth of adding a standard enzyme cocktail to a barley-based diet steeped in water. The composition of the diet is given in Table 10.

Before feeding, the cereal component was steeped in water (3 parts water:1 part food, w/w). A crude preparation of carbohydrases was added to one half of the cereal. In a similar manner, the dietary protein component was steeped in water and to half the protein mixture a crude preparation of proteases was added. The animals were fed twice daily with the cereal protein mixture and vitamin/mineral component being mixed before feeding to the pigs. For the morning and evening meals, the cereal (with or without added enzyme) had been steeped for 5 hrs and the protein mixture for 2 hrs. The mean ambient temperature during the study was 16°C. The animals were given the liquid diet at a restricted level of feeding (0.09 x W0.75).

In spite of relatively low and unbalanced numbers of pigs per treatment, the effect of enzyme on food conversion ratio tended toward statistical significance and was significant at P=0.10 (Table 11). The absolute growth rates of both the male and female pigs were higher with the added enzymes.
Work in this area is continuing at our institute.

Enzyme preparations with higher activity at ambient air temperature should be developed. Enzymes used in conjunction with liquid feeding systems offer potentially increased efficiency in pig production. For a more comprehensive treatment of this topic the reader is referred to Brooks et al. (1999).

The above results also highlight the benefits of using enzyme cocktails in diets rather than preparations with single enzyme activity. Given the complexities of the substrates in raw materials, it is only logical to expect greater responses from a broad spectrum of exogenous enzyme activities. A combination of enzymes could facilitate each other’s activity by providing greater substrate access. The improvements observed with Vegpro, an enzyme with protease, "-galactosidase, ß-glucanase and endoxylanase activities, in pigs fed diets based on barley and lupin provides a good example of the multi-faceted effect of enzyme cocktails.

During the past decade, the feed enzymes have evolved from a virtually undefined entity to a well-accepted feed additive. The use of feed enzymes has steadily increased over this time and this trend is likely to continue in the future. The role of enzymes in improving precision and flexibility in least-cost feed formulations and, more importantly, narrowing within-flock or herd variability and ensuring more uniformity at market weight is well appreciated by the industry. Most currently available formulations, however, are effective only in specific situations. The challenge for the future is to develop and refine enzyme combinations that are cost effective with a wider applicability.

References