By Roselina Angel Department of Animal and Avian Sciences University of Maryland College Park Md.Features Broilers Health Poultry Production Poultry Research Research
Enzymes could help improve digestibility
From a paper presented at the Midwest Poultry Conference
In poultry fed commercial type diets amino acids (AA) are obtained primarily through the consumption of protein in the feed as well as, in a lower proportion, synthetic AA (methionine (Met), lysine (Lys), threonine (Thr)). Ingested proteins are broken down through digestion, which typically involves denaturation of the protein through exposure to acid and hydrolysis by enzymes called proteases. In poultry, ingested protein undergoes a series of degradation processes, carried out by acid (hydrochloric acid secreted in the proventriculus but active both in the proventriculus and gizzard), and hydrolytic proteases in the proventriculus, gizzard and small intestine. The grinding that occurs in the proventriculus, reducing particle size, aids in the speed with which digestion proceeds and influences the extent to which proteins are digested.
Other enzymes, endogenous enzymes, primarily from the pancreas also can influence the extent of protein digestion but influencing the accessibility of proteases to proteins in feeds. The result of this degradation, and specifically of proteolysis, is a mixture of AA and small peptides that are rapidly absorbed by the enterocytes in the small intestine. Thus, availability of AA for utilization by the animal is dependent on the digestibility of protein and the absorption of AA and peptides.
Enzymes are proteins that catalyze, or accelerate, chemical reactions. Enzymes vary in their specificity working on only one specific reaction or several less specific chemical reactions.
Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. This is the case for several of the intestinal proteases. For example, enterokynase, a serine protease found in the small intestinal mucosa, must cleave the pro region from trypsinogen produced in the pancreas to form trypsin, the active proteolytic enzyme.
Protein Nutrition in Broilers
The progress that the broiler industry has made in the last decades is marked by impressive improvements in all areas of genetics, management, health and nutrition, yet it is genetics that is responsible for most of the improvements in live performance and meat yields that have been achieved. At least 85% of the improvements obtained in live performance between 1957 and 2001 can be attributed to genetic selection.1
Comparisons made between a strain of chicken that had not been selected since 1940 and a present 2009 commercial broiler (Ross 708) showed increases of 72% in body weight and 3.4 times more breast meat at 34 days of age.2 Two major physiological changes associated with how body tissues grow in proportion to one another are noticeably changed in the 2009 bird as compared to the non-selected 1940 bird. Breast muscles maintained their allometric growth (tissue as compared to body weight) after 14 days of age, not the case in the 1940s bird where rate of muscle growth was slower at later ages. After 14 days of age the allometric growth of breast muscles in the 2009 broiler was 1.25 while the 1940s birds grew breast muscle at a slower rate (allometric growth of 1.09). This continual faster growth of breast muscles into later ages in the modern broiler translates into a higher requirement for AA, especially Lys.
The rapid growth rate of the current broilers results in increased demands for AA and energy, but these demands are not increased in the same proportion. Requirements for AA increase proportionately faster than those for energy, thus a higher AA to energy ratio is required in faster growing strains of broiler.3 Morris and Njuru (1990) fed diets of increasing protein content to broilers and to laying-type cockerels.4 These authors reported that the concentration of protein needed, in laying-type cockerels, for maximal body weight gain and carcass protein content was less than that needed to maximize growth and carcass protein in broilers. Broilers continued to benefit from the additional dietary protein to later ages, and this was likely due to the continued tissue growth, especially of the breast muscles.
Providing dietary protein above levels considered adequate by the industry leads to improved feed conversion ratios and breast meat yields.5,6 In comparison to usual industry levels, benefits of increased AA density diets have been shown early in life as well as in finisher diets. Feeding diets containing high AA levels may result in greater economic return if implemented during periods when the birds’ feed intake is relatively low and growth rate is high, and also because at least some of the benefits obtained earlier can be carried through to market ages.7,8
Embryos hatch with myofibre numbers that are not expected to change after hatch. Growth of muscle after hatch occurs through increases in fibre size, which are accompanied by equal increases in the number of nuclei per myofibre.9,10 A group of myogenic precursor cells are found in between the muscle cell and its plasmalemma: the satellite cells. These myogenic precursor cells can multiply and later fuse with adjacent fibres aggregating more nuclei and therefore having a greater capacity for protein synthesis.11 It is estimated that 98% of the final DNA content of muscle results from this process.12 However, an actual increase in myofibre size can only be achieved by a concurrent and balanced supply of dietary AA. Halevi et al., (2000) showed that early starvation (hatch to two days of age) resulted in birds that were 7% and 9% lighter in body weight and breast muscle, respectively, at 41 days of age.9
Increasing Lys and other essential AA fed only in the finisher phase has also been shown to improve feed conversion ratio and breast meat yields.13 This last impact of high Lys and other essential AA in finisher diets can be partially explained by the higher proportional growth rate of the breast muscles compared to other body tissues at later ages in the modern broiler.2
Proteases perform a variety of roles in biology. These enzymes function in important physiological processes, including homeostasis, apoptosis, signal transduction, reproduction and immunity.14 In addition, proteases are involved in blood coagulation and wound healing. From a nutritionist’s perspective, the hydrolysis of proteins to individual AA and peptides in the intestinal tract is a key function for proteases. Several intestinal proteases exist, and comprise a protease system in the intestinal tract for the utilization of various dietary protein sources.
Pepsin is an acidic protease secreted in the stomach of most animals. Released as the non-active pepsinogen, it is activated in the presence of hydrochloric acid. It is active at low pH and inactive at pHs above six with variance across species.15 This protease hydrolyzes peptide bonds mainly between two hydrophobic AA. It falls within the group of carboxyl proteases.
The presence of hydrochloric acid, produced in the proventriculus, and of peptides, the products of initial protein digestion (hydrochloric acid denaturation as well as partial breakdown of protein by pepsin) from the gizzard into the duodenum stimulates the release of the hormones secretin and pancreozymin from S cells of the duodenum in the crypts of Lieberkühn.16 These hormones promote the secretion of pancreatic juice containing a number of enzymes and bicarbonate ions. The production of an alkaline solution quickly neutralizes the acid entering the duodenum.17 Small intestinal enzymes function best at pHs close to neutral or slightly below neutral, and thus, insufficient alkaline bile, lowers enzyme activity in the intestine.18
Pancreatic proteases as well as all known cellular proteases are synthesized as zymogens, or the inactive precursor, to prevent unwanted protein degradation at the point of origin.19 The conversion of the zymogens to the active protease requires low pH (autocatalysis) or limited proteolysis. Primary pancreatic zymogens are trypsinogen, chymotrypsinogens A and B, proelastase, and procarboxypeptidases A and B. Trypsin is activated after being cleaved by enterokinase, found in the brush border membrane. The active trypsin then hydrolyzes bonds in the other zymogens, releasing the active enzymes. Trypsin is the primary protease in the intestinal tract. Its active form hydrolyzes at the carboxyl side of Lys and arginine (Arg), except when followed by proline (Pro). It has an optimal operating pH of 8 or less.20 Chymotrypsin hydrolyzes peptide bonds in which the carboxyl groups come from one of the three aromatic AA (phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)). Elastase cleaves at the carboxyl end of the hydrophobic AA glycine (Gly),valine (Val) and alanine (Ala). These AA are common in connective tissue in muscle. Elastase, like trypsin and chymotrypsin, falls within the category of serine protease.
Carboxypeptidases A and B are exopeptidases also secreted by the pancreas. Carboxypeptidase A follows on the work of chymotrypsin and elastase that expose the carboxy-terminal aromatic or non- polar AA while carboxypeptidase B cleaves carboxy-terminal basic AA exposed by the activity of trypsin.21
Pancreatic proteases experience a progressive fall in activity as digesta passes through the small intestine.20 The decline in importance of these primary proteases is paralleled by a gain in brush border and cytosolic peptidase activity. It is estimated that 70-85% of all luminal AA are taken up from the small intestinal digesta as peptides.21 Yet, it is important to realize that approximately 85% of this quantity appears in hepatic portal blood as free AA because of intracellular hydrolysis.22
Ingredient Protein Digestibility
With the broad fluctuations in ingredient prices as well as historically high prices seen in the last four years, choice and quality of ingredients becomes more important. From a practical nutritionist’s standpoint, the value of ingredients as a protein source generally comes down to the content of digestible AA and the ratio of AA in the ingredient (aside from cost and ingredient accessibility). Numerous studies and tables on protein and AA availability (the digestibility, absorption and utilization by the animal) have been published. As can be seen in Table 1 there is high variability in AA content within an ingredient with content and variability being related, in part, to where the ingredient was harvested.
The common thread that runs through such summaries is that ingredient improvements can yet be made in digestibility of protein (Table 1 and Table 2). Digestibility of AA is lowest at younger ages.23 The AA digestibility was markedly better at 21 days of age as compared to five days of age in corn, DDGS and canola meal (Table 2) but this age effect was less marked with SBM.
In one large-scale study, apparent ileal digestibility was determined for various ingredients in broilers.24 For cereals, the overall AA digestibility coefficient for eight samples of corn was 0.81 (range 0.77 to 0.85). For SBM, the digestibility coefficient was 0.82 (range 0.81 to 0.83). In particular, the groups of meat and bone meal (average digestibility coefficient of 0.62) and meat meal samples (average digestibility coefficient 0.65) showed low AA digestibilities and marked variation. Similarly in work done at the University of Illinois on AA digestibility of ingredients, low values and high variation for meat and bone meals is often seen.25 More recent collaborative work with The Ohio State University, Purdue University and University of Illinois further emphasizes that the variability continues to be present in meat-based ingredients.23 Generally, however, digestibility data indicate that there is room for improving ingredient AA digestibility. This holds true for ingredients that generally are considered to have good digestibility, and certainly for ingredients such as animal byproduct meals and DDGS in which digestibility is generally lower.
The interest in proteases has increased in the last few years and new commercial proteases have entered the market recently. One of the new proteases to enter the market is an alkaline serine protease derived from Nocardiopsis prasina and the production strain Bacillus licheniformis (ProAct®, DSM).
Several research groups have presented ileal digestibility results with this protease, which is summarized in Figure 1.26-29 This work confirms the ability of this protease to improve ingredient protein digestibility. These researchers used the NFE diet or basal diet substitution methods to determine amino acid digestibility in different ingredients.
Across all ingredients tested – corn, soybean meal, full fat soybean meal and meat and bone meal – the protease improved AA digestibility. The improvement over the non-protease control ranged from about 2% to 14%.
A study with broilers and using the same commercial protease showed that apparent AA digestibilities were improved in a corn SBM diet in patterns similar to those previously reported for the single ingredients.27,29-31
Three studies were done to evaluate the true standardized amino acid (TAA) digestibility of individual ingredients for broilers, laying hens and turkeys. Straight run Ross 708 broilers and female Nicholas turkey poults (separate experiments done at the University of Maryland) were raised to 17 days in floor pens and assigned to battery pens in a completely randomized design of 12 treatments with eight replicates of seven birds per pen. In a third experiment Hyline W36 white Leghorn hens 56 wks of age (done at the University of Nebraska) were assigned to cages in a completely randomized design of 10 treatments (Trt) with six replicates of four birds per cage.
Diets were formulated and mixed and the same batch used in all three experiments. A nitrogen-free diet (NFD) was formulated with 0.3% titanium dioxide as a marker. The corn-starch, sucrose and solka flock in the NFD diet were replaced in part by the ingredients being tested such that all the protein in the diet came from the tested ingredient.
Ingredients were added to achieve 20% protein for the high protein ingredients or to a maximum of 96% of the diet for the low protein ingredients. Ingredient percentages tested in the final diets were: 42% soybean meal (SBM), 40% meat and bone meal (MBM), 75% corn distiller dried grains and solubles (DDGS), 96% corn and 96% bakery by-product meal (BPM). In the laying hen trial corn was not tested. Each Trt was supplemented with 0 or 200 ppm of a mono component serine protease (Ronozyme ProAct™, CT, DSM Nutritional Products, containing 75,000 protease units/g of enzyme product). Birds were fed the diets for four days. At 22 days broilers and turkeys were euthanized and the distal half of the ileal content collected, pooled by pen and freeze dried.
In the broilers trial, a main effect of protease on digestibility of Thr, Met, Cys, Lys, Arg, Ser, Val, Asp. Ile, and His was seen. There were no protease by ingredient interactions except for Cys. Addition of the protease improved (P<0.05) the digestibility of Thr, Cys, Met, Lys and Ser in SBM; Thr, Cys, Met, Ser and His in corn; Cys, Met, Arg, Ser, Val and His in DDGS; Cys, Met, Lys, Ser and His in MBM; and Met, Arg, and Ser in BPM. For SBM the TSAA digestibility was improved from 75.2 to 83.2% for Thr, 74.9 to 81.2% for Cys, 83.1 to 86.3% for Met, 83.8 to 87.1% for Lys, and 80.6 to 85.4% for Ser with the protease. Addition of the protease had similar impacts in laying hens and turkeys. Overall digestibilities of AA, in the presence or absence of the protease, from the same ingredients, were higher for laying hens and turkeys as compared to broilers.
There is no question that in today’s ingredient availability and cost environment, tools that allow for increasing the digestibility of AA would be welcomed. Given the digestibilities reported for the most commonly used ingredients and the higher variability in AA content and digestibility from locally grown ingredients, room exists for improvement. There is little information in the literature as to how proteases work in the intestinal tract, where in the tract they have the most impact and how they interact chemically with other exogenous enzymes as well as endogenous enzymes. It will be important as the role of proteases gains commercial application and importance that we better understand how these enzymes work and how they interact with endogenous and exogenous enzymes. We will also need to understand how to formulate proteases into diets.
The first step will be defining clearly and accurately what their impact is on AA digestibilities of ingredients, what the variability of this impact is on the same ingredient, possibly defining why this variability is occurring such that appropriate matix values can be used. Each exogenous protease, based on their specificity, will impact different AA differently and this impact will be ingredient related.
References for this article are available on our website, and by request (e-mail email@example.com).
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