Abstract: Foods of animal-products for the consumers have recently and mainly been from the non-ruminant animals (poultry). However, presence of anti-nutrients, in feeds is one of the constraints that impairing them from further productiveness. The study aimed to disclose both of the adverse effects of anti-nutrients and suggested solutions. Related findings of this topic were scientifically reviewed and summarized to being publicized. This review revealed that there are a range of anti-nutrients that are synthesized by plants as part of protections or as means to survive. Anti-nutrients that interfere with normal animals’ physiological activities could have anti-nutritional effects. Trypsin Inhibitor (TI) is one of the anti-nutrients and is almost nil in cereal grains; whereas highly concentrated in legumes, mainly in soybean grains (2-6 mg g–1). Kunitz-trypsin-inhibitors (KTI) and Bowman-Birk Inhibitors (BBI) are abundant of TI, containing 181 and 71 AA, respectively. The KTI and BBI contained 1.4 and 1.6 g kg–1 of seed, respectively. Although, TI in the diets of chicks, rats and mice caused for pancreatic hypertrophy and increased secretions, others including pigs, dogs or pre-ruminant calves were not experiencing these symptoms. A human endogenous-trypsin enzyme has also more resistance to TI as compared to other mammals. Although most anti-nutrients are suggested to be inactivated by heat treatments, over or under-heating affects those other important nutrients. The BBI, as part of TI always exhibits considerable resistance to heating. Other anti-nutrients, for example phytate is totally heat-stable. As TI contributes a major-proportion of sulfur-containing amino-acids (AA), soybean breeding for a low TI has limited application. When commercial soybean meal was replaced by raw soybeans (0-90 g kg–1 diet), the weights of pancreas and duodenum were proportionally increased by 67 and 21%, respectively. The TI in diets of poultry affects the pancreas, but TI could be reduced by either optimum heating or enzyme supplementations.
INTRODUCTION
Globally the non-ruminant animals have currently been taking the major parts in supplying and satisfying to the skyrocketing demands of protein source foods of animal origins. These are mainly originated from the farm animals. However, lack of proper utilization of the available feed resources, in developing countries is one of the constraints that are negatively affecting these animals from their further enhanced productivities. Due to many reasons, protein source feeds for these animals have recently and mainly been derived from the plants. Of which, legume grains, in particular are good sources of plant protein feeds. However, presence of anti-nutritional factors (ANF) or anti-nutrients, in such feed ingredients is one of the factors negatively affecting the feed quality.
These ANF cause for adverse physiological and functional effects of the animals when they consume the feeds containing the ANF that are more than the threshold levels1. As compared to other internal organs, many types of digestive enzymes are synthesized by the exocrine pancreas, stored in the zymogen granules and released into the duodenum2. However, ANF elicit their negative effects via different mechanisms, including binding to digestive enzymes and nutrients or increasing the gut viscosity3. In addition to the heat treatments, optional solutions to these problems may lie with the use of microbial feed enzymes3-5. This review paper attempted to review the findings on the topic and summarize them and then publicize to be used by the users.
DESCRIPTION OF ANTINUTRITIONAL FACTORS
Depending upon the structures of the individuals, a range of secondary metabolites (ANF) are always synthesized by plants as part of protection against the attacks by the other organisms or as a means to survive in adverse growing conditions. These are toxic compounds, with varying effects6 on animals when they consumed. These ANF are not only found in grains but also present in different edible leafy vegetables plants7 too.
Anti-nutritional factors can be classified mainly on three major categories, namely on their chemical descriptions, on their biological effects and on their ability to withstand heat treatments.
In the first place, when the ANFs are classified based on their chemical structures, they are grouped as: (a) Proteins (protease inhibitors and haemagglutinins (lectins), (b) Glycosides (glucosinolates, cyanogens, saponins and estrogenic factors), (c) Phenol (gossypol, tannins) and (d) Miscellaneous8. Secondly, when Francis et al.9 classified the ANF based on their biological effects, the ANF were grouped into three categories, such as (1) Protein utilization and digestion (i.e., protease inhibitors, tannins and haemagglutinins (lectins), (2) Mineral utilization (i.e., phytic acid) and (3) Anti-vitamins and miscellaneous (i.e., mimosine, cyanogens and estrogenic factors). Lastly, when the ANF are being described based on their heat-resistance9, they are grouped as heat-labile factors (i.e., protease inhibitors, haemagglutinins and anti-vitamins) and as heat-stable factors (e.i., saponins, non-starch polysaccharides, anti-genic proteins, estrogens and some phenolic).
A wide variety of ANF, such as oligosaccharides, proteinaceous compounds (i.e., Trypsin Inhibitors (TI)), lectins, amylase inhibitors and phytic-acids are mainly found in legume grains10, such as soybean, peas, faba-bean, lupins, common vetch, grass pea and kidney bean, which are commonly used as feed ingredients for the non-ruminant animals. Protease inhibitors (i.e., TI), lectins and phytate are the best characterised ANF in soybeans11. In addition to negatively affecting the physiological and functionalities of internal organs, these ANF influence the bioavailability and absorption of nutrients to the animals when they are used as feeds. Guillamon et al.12 reported that the contents of TI ranged from negligible in cereals or Lupinus spp. to higher amounts in soybean cultivars (43-84 TIU mg–1) followed by common beans (21-25 TIU mg–1).
Heat treatment is generally considered as an effective approach to inactivate ANF but some of the ANF, e.g., phytate, oligosaccharides and antigenic proteins, cannot be reduced or decreased by heating3. Although, Campbell and Schone13 suggested that TI and lectins in soybeans could be inactivated by heating, Clemente et al.14 reported that Bowman-Birk Inhibitors (BBI), which are a group of TI, exhibit considerable resistance to the heat treatment.
Lectins are widely distributed in legumes and in some oilseeds23 but it can be effectively treated by heating, mainly in an aqueous medium (i.e., 100°C for 10-20 min). Limited numbers of ANF are shown in Table 1.
PHYSIOLOGICAL AND FUNCTIONAL EFFECTS OF ANTINUTRITIONAL FACTORS
If animals or humans consume the ANF in plants, these compounds may cause adverse physiological and functional effects. These ANF negatively affect the nutritive values of the leguminous grains through direct and indirect reactions. They inhibit proteins and carbohydrate digestibilities and then even they induce pathological changes in the intestine and liver tissues. Thus, ANF affects the metabolism and inhibits a number of enzymes and then bind nutrients, making them unavailable24.
| Table 1: | Selected anti-nutrients in both raw full-fat soybeans and commercial soybean meals |
| TI: Trypsin inhibitors, UA: Urease activity, phytate, TIU: Trypsin inhibitor units | |
Various scholars25-28 have reported that feeding raw soybean with high levels of TI and lectins negatively affects the pancreatic functions, the growth of birds and the feed efficiency. Mogridge et al.29 reported also that the consumption of raw beans increased the size of the pancreas and duodenum (0.80 vs. 0.37% and 1.35 vs. 1.06% of live weight, respectively) and reduced feed consumption and growth of the chicks (66 vs. 97 g/14 day).
Similarly, ASA30 reported that diets based on raw beans reduced the feed consumption and live weight. Availability of ANF within plant feedstuffs is the main factors that limit their use by the animals8,31. Emiola et al.32 studied the effect of feeding birds with raw and dehulled meals and then found an increased weight of pancreas, severe congestion of kidney, reduced weight of liver with its marked coagulative necrosis. Dietary ANFs have also adversely affected the digestibility of protein, bioavailability of Amino Acids (AA) and protein quality33. The TI in the diet of chicks, rats and mice caused pancreatic hypertrophy and increased pancreatic secretions. However, pigs, dogs or pre-ruminant calves were not experiencing these symptoms when consuming TI in their diets. One of the effects of the inactivation of digestive enzymes in the intestine is the stimulation of trypsin and chymotrypsin secretion from the pancreas, which can create an increased demand for the sulfur AA methionine and cysteine34.
The exocrine function of the pancreas is to produce and secrete several digestive enzymes, including trypsinogen, amylase, lipase and procarboxypeptidase, among others35. These enzymes are released into the ascending loop of the duodenum, certainly for digesting the nutrients36. As Shi et al.37 stated the activities of pancreatic trypsin and chymotrypsin increased sharply from hatching to day 14 and then decreased gradually until day 21. The recent findings also confirmed this fact that the adverse effects of protease inhibitors on internal organs, particularly pancreas is aggravated at early ages38-39. Hence, age must be considered as a factor while planning to replace the commercial SBM by raw soybean in the diets of on-ruminant animals, including chickens. Although, anti-nutrients/ANF, in plant are responsible for deleterious effects that related to the absorption of nutrients and micronutrients, some anti-nutrients, at some extent with low concentrations may exert beneficial health effects by reducing the blood glucose and insulin responses40.
Trypsin inhibitors: Trypsin Inhibitors (TI) are a type of serine protease inhibitor that reduces the biological activities of endogenous trypsin. Protease inhibitors are widely occurred across the plant kingdom and are protein-based molecules23. The concentration of TI is almost nil in cereal grains, otherwise highly concentrated in legumes, for example; TI are ranging between 2-6 mg g–1 in soybean9,41. The ANFs are found not only in soybeans but also in other legumes, including kidney beans42.
From various categories of plant protease inhibitors, the main inhibitors in legume seeds and cereals are the trypsin and chymotrypsin inhibitors43. In soybeans, there are two main groups of TI, such as the heat-labile, which is called Kunitz trypsin inhibitors (KTI). This type of inhibitors is blocking mainly the trypsin and the second one is BBI, which inhibits both trypsin and chymotrypsin9,23. Trypsin is an endogenous enzyme involved in the breaking down of many different proteins, including as part of digestion in humans and other animals. As a result, protease inhibitors that interfere with activities of endogenous trypsin can have anti-nutritional effects.
The most abundant TIs are the KTI and BBI, containing 181 and 71 AA, respectively. However, concertation of these two in a typical seed grain is a reciprocal to their number of AA contained. The concentration of KTI and BBI are 1.4 and 1.6 g kg–1 of the total seed contents, respectively44. The same authors reported that soybeans also contain between 6.8 and 17.5 g of phytic acids/kg. This is a ring form of phosphorus (P), which chelates with proteins and minerals to form a phytate not be readily digested within the gut of non-ruminants.
Campbell and Schone13 suggested that TI and lectins in soybeans could be inactivated by heating. Furthermore, moist heating (autoclaving at 121°C for 15-30 min) or aqueous heat treatment (100°C for 10 min) is preferably be recommended by other scholars46,47. However, Clemente et al.14 reported that BBI, which is a group of TI, exhibits a considerable resistance to heat treatment. On the other hand, researches into breeding for low TI cultivars currently has limited application as trypsin inhibitors contribute a major proportion of sulfur containing AA including the methionine and cysteine content of soybeans44.
Optionally, the scholars, for example Pettersson and Pontoppidan11 have differently suggested that by supplementing the diets, containing ANF with selected microbial feed enzymes, including protease and phytase could reduce the impacts of anti-nutritional proteins on the non-ruminant animals and thereby improve the digestibility of proteins.
The loss of endogenous protein has been found to be increased due to the activity of TI in the body consequently affecting the nitrogen balance44. Herkelman et al.48 also noted that the presence of TI in diets can cause digestive diseases in non-ruminant animals.
A high level of TI in diets, which are mostly from the soybean origins have also been found to correlate with a rapid feed passage, which leads to a greater nitrogen excretion and hence poor litter quality48,49. Extrusion of faba bean reduced the E. coli count in the small intestine and tended to decrease damage to intestinal villi50. Susmel et al.51 tested an in vitro degradation of proteinase inhibitors contained in raw soya bean, with rumen fluid and found that it was degraded at a much slower rate which suggested that the presence of the KTI leads to a higher residual TIA after rumen degradation and slightly lower microbial gas production.
EFFECTS OF TRYPSIN INHIBITORS ON PANCREASES
Commercial soybean meal (SBM) is considered to be the best protein source feeds for the non-ruminant animals52,. But, the nutritive value of raw full-fat soybean (RFSB) or that of poorly processed soybean is poor due to the presence of the ANF, especially the TI that adversely affecting the pancreas26,53. When the TI is there, the pancreas often tries to compensate the problems by increasing its size to synthesize and then to secret such sufficient digestive enzymes42. The linear increase in pancreas size with increasing soybean trypsin inhibitors intake suggested a dose dependent response of broiler chickens54, 55. Additionally, Rocha et al.56 reported that the pancreas of the broilers fed the diets containing RFSB was significantly heavier and the intestinal integrity, as well as structure was also impaired.
The exocrine pancreas is the organ with the highest level of protein synthesis in animals. Each day the pancreas produces liters of fluid filled with enzymes that are capable of breaking down nearly all organic substances54. The TI negatively affects the functions of the pancreas, in terms of synthesis and secretion of digestive enzymes, including proteases. As an optional solution, some geneticists and crop breeding companies have developed seeds that have very low anti-nutritional factors.
However, the TI are very rich in sulfur containing AA as they may account for about 40% of the total cysteine content of some legume protein46, which could show that breeding may not be strategic solution to solve the negative effects of ANF, particularly the TI in soybeans.
However, Pettersson and Pontoppidan11 reported that negative impact of TI in non-ruminant animal diets could be reduced by supplementing the diets with microbial protease. The adverse effects of raw soybeans was discovered and reported by Osborne and Mendel58, that it could not support the growth in rats unless the seeds are cooked.
Feeding raw soybeans resulted in growth inhibition and enlarged the pancreases. Following such a discovery, Chernick et al.59 found the reason why the chicks’ pancreases were enlarged in sizes was due to the presence of TI in raw soybeans. This led the pancreas to being enhanced producing more enzymes, pancreatic hypertrophy and hyperplasia as a mechanism withstand the problem. When an endogenous trypsin is inhibited by the TI, cholecystokinin production is also automatically enhanced resulting in an increased production of the pancreatic digestive enzymes.
As reviewed and reported by Liener and Kakade60, the consequences of TI on pancreases have been confirmed not only in chickens but also in rats, mice and young pigs. Grant61 added that dietary soybean or the TI do alter pancreatic secretion, which lead to pancreatic hypertrophy and hyperplasia occur, not only in chicks but also in the young of a number of species, proved that they are given the raw soybeans.
Furthermore, Batterham et al.62 reported that the growing pigs can tolerate dietary levels of at least 4.7 and 4.5 mg g–1 of trypsin and chymotrypsin inhibitors, respectively; otherwise the weights of the liver and pancreas were significantly affected. However, Struthers et al.63 reported that the TI neither from raw soy flour nor from any other soy products produced pancreatic enlargement in pigs or monkeys. Flavin64 suggested also that human trypsin is more resistant to inhibition than is the trypsin of other mammalian species. The effect of TI, from soybean does not appear to be a potential hazard on human trypsin.
Pancreatic hypertrophy, increased weight of small intestines and increment of the thicknesses of both villous and crypt of small intestine were found from the mice fed diet containing raw soya flour65. Hence, Baba et al.66 concluded that major resection of the pancreas leads to disorders of the endocrine and exocrine pancreas. An investigation was conducted to identify whether DNA concentration was proportionally increased in samples of pancreatic hypertrophy due to the TI from soybean. Then, the results showed that the concentration of DNA or the ratio of tissue protein to DNA was not affected28. Therefore, the increase in weight of the pancreas is due to hypertrophy than hyperplasia29,45,67. That means pancreatic hypertrophy may be due to cell sizes increments than increases of cell numbers.
When the supplementation of the raw soybean increased in diets, the weight of pancreas and other internal organs, including the duodenum, gizzard, proventriculus and small intestines were increased (Table 2-6). Although, microbial protease supplementation did not significantly reduce the weight, their effects were shown with improved BWG of the chickens across the trials45. When commercial SBM was increasingly (0-90 g kg–1 diet) replaced by raw soybean, the weight of pancreas and duodenum were increased by 67 and 21%, respectively39 at day 14. However, weight of pancreas was significantly reduced at day 24 when microbial protease was increased from 0-0.4 g kg–1 diet, containing 75 of raw soybean per kg of diet57. Moreover, weight of pancreas was significantly reduced at day 24 and tended at day 10 when increasing supplementation of microbial phytase from 0.1-0.3 g kg–1 diet (Table 6).
THE MECHANISMS OF ANTINUTRITIONAL FACTORS IN AFFECTING PHYSIOLOGICAL FUNCTIONS
Anti-nutritional factors elicit their negative effects on animals via different mechanisms, including binding to digestive enzymes and nutrients or increasing gut viscosity3. Phytate, for example impedes nutrient digestion through electrostatic mechanisms, which reduces the solubility of proteins; consequently, increasing the loss of endogenous nutrients68.
| Table 2: | Effects of varyingly supplementing protease and raw full-fat soybean on the weights of visceral organs (g/100 g of body weight) of broilers at day 10 |
S. intestine: Small intestine, G+P: Gizzard plus proventriculus, SEM: Standard error of mean, RFSB: Raw full-fat soybean, NS: Non-significant, *p<0.05, **p<0.01, ***p<0.001, Source: Adopted from Erdaw et al.38 |
|
| Table 3: | Effects of varying both pelleting method and levels of raw full-fat soybean on the weight gains of the visceral organs (g/100 g of body weight) at day 14 |
G+P: Gizzard plus proventriculus, J+I: Jejunum plus ileum, SEM: Standard error of mean, RFSB: Raw full-fat soybean, NS: Non-significant, *p<0.05, **p<0.01, ***p<0.001, Source: Adopted from Erdaw et al.39 |
|
| Table 4: | Effects of varying the supplementation of both protease and raw-full-fat soybean on the weights of internal organs (g/ 100 g body weight) at day 24 |
| G+P: Gizzard plus proventriculus, SI: Small intestine, SEM: Standard error of mean, RFSB: Raw full-fat soybean, NS: Non-significant, *p<0.05, ** p<0.01, ***p<0.001, Source: Adopted from Erdaw et al.45 | |
| Table 5: | Effects of varyingly supplementing the raw full-fat soybean and protease on relative pancreas weight (g/100 g of the body weight) in relation to the body weight of broilers45 |
| RFSB: Raw full-fat soybean, SEM: Standard error of mean | |
| Table 6: | Effects of the extra-dosing of protease and phytase on the weight of the pancreas broilers (g/100 g of the body weight) at d 10, 24 or 35 when 25% of SBM was replaced by raw soybean57 |
| NS: Non-significant, *p<0.05, **p<0.01, ***p<0.001 | |
The response of chickens and rats to the TI requires the mediation of the gastrointestinal tract and is independent of vagal intervention to the pancreas69. Pusztai70 reported that since some lectins are heat stable and can survive going through the gut, their interaction with the gut surface epithelium can damage the gut at high dietary intakes and this may lead to digestive disorders/diseases in some instances. Many fish species have been affected by ANF, particularly the IT, which led to pancreas hypertrophy41. After a certain period however a compensation process stimulates trypsin secretion and it seems that with trypsin inhibitors levels below 5 mg g–1, most cultured fish are also be able to compensate9.
Because of approximately 65-80% of the total phosphorus in the soybean seed is bound to phytic acid or phytate, most of the minerals, particularly phosphorus (P) is biologically unavailable because poultry do not possess enough endogenous phytase to digest the complex71. proportional increment of pancreas weight, against to that of increasing levels of TI in diets might be an indicator of the physiological mechanism to coping the negative impacts of ANF on birds by enlarging the surface area of pancreas and thereby to produce more of indigenous enzymes, particularly protease57,45.
Implication of antinutritional factors on the productivity of animals: Protease (trypsin) inhibitors can interfere with the biological activity of endogenous protease and thereby reduce the digestion of nutrients, mainly proteins72. The loss of endogenous protein has been found to be increased due to the activity of TI in the body, consequently affecting the nitrogen balance73. Herkelman et al.48 noted that the presence of TI in diets can cause a digestive disease in non-ruminant animals. A high level of TI in diets, mostly of the soya origins, have also been found to correlate with a rapid feed passage, which leads to greater nitrogen excretion and resulted in poor litter quality50.
Phytate (phytic acid) is another ANF that is present at high level in soybeans. Phytate impedes nutrient digestions by forming protein-phytate or protein-phytate complexes and is resistant to normal digestions74. Phytic acids have a negative impact on amino acid digestibility and have recently been shown to increase the endogenous nutrient losses in pigs and poultry. Anti-nutritional factors cause depression in growth; performance and can negatively affects the health of the animals. Although tolerance depends on the age of the birds30, excessive levels of the ANF reduce the nutritional quality of the feeds. However, some of the ANF can be denatured by heating26.
MICROBIAL FEED ENZYMES IN REDUCING THE ADVERSE EFFECTS OF ANTINUTRITIONAL FACTORS
When animals lack specific enzymes to break down certain feed components, ANF interfere with the normal digestion process when they are present in the diet75. Birds fed on diets containing raw soybeans do not thrive. Their feed consumption rate is reduced by as much as 14%, their live weight gain drops by up to 15% and the feed conversion rate decreases by approximately 53%30.
The mechanism and effectiveness of the microbial protease and phytase on raw soya proteins are not yet clear but their relative effectiveness, in reducing the adverse effects of TI and phytates in RFSB. This is a confirmation of results previously obtained in in vitro and in vivo studies28,57,67. Supplementing the diets with microbial phytase however breaks the bonds of phytic acids to release the nutrients, such as minerals. Consequently, the physical parameters and mineral composition of tibia bones of broilers were improved when microbial phytase is added to broilers’ diets76.
Proteases are protein-digesting microbial enzymes that break down both stored proteins and proteinaceous anti-nutrients in vegetable protein75. Regardless of dietary protein or energy concentrations, protease supplementation has been shown to improve the feed efficiency and digestibility of CP and fat and thereby reduce the nitrogen excretion77. Protease supplementation in diets improves the digestibility of protein and reduces the impact of anti-nutritional proteins on non-ruminant animals11. Yadav and Sah4 added that the inclusion of dietary protease improves the digestibility of crude proteins in the diet and improves body weight gain in broilers. Furthermore, Rada et al.78 reported that supplementation of microbial protease enzymes improved the carcass yield of broilers. Protease also has the capacity to improve amino acid digestibility and thereby improve the feed: body gain ratio of birds77.
The digestibility of CP and starch increases in broilers fed diets supplemented with protease at most concentration79. However, each protease type has its own specificity and mode of action and hence the yield patterns of AA vary widely among the feedstuffs. Murugesan et al.80 noted also that an increase in nutrient utilization when broiler diets were supplemented with protease and phytase (cocktail).
However, Cowieson and Ravindran81 reported that there was no interaction between diets and enzyme products containing xylanase, amylase and protease in terms of ileal digestibility of nitrogen and Jiang et al.82 reported that endogenous pancreatic proteases, e.g., trypsin and lipase, were not affected by supplementing exogenous amylase; rather, their intestinal activity was improved. Because phytic acid and NSP are not heat-labile, unlike some protease inhibitors and lectins, supplementing diets with exogenous phytase is very necessary. However, the efficacy of supplementation in chicken feeds depends mainly on the rate of inclusion rate as well as the age and genotypes of the birds. Feed enzymes are used to assist the breaking down of ANF in many feed ingredients for poultry and pigs75 and generally, the pig and poultry industries are benefitting from the availability and use of phytase. Currently, around two-thirds of pig and poultry feeds contain supplemental phytase and this is yielding economic benefits75. Selle et al.83 suggested, however, that vegetable proteins and cereal sources are different in their response to phytase supplementation in terms of amino acid digestibility.
CONCLUSION
The concentration of TI is almost absent in cereal grains; whereas highly concentrated in soybeans. Although , most ANF are suggested to be inactivated by heating, over- or under-heating still affects the quality. Breeding for low TI soybean cultivars currently has limited application as TI contributes a major proportion of sulfur containing AA. Although, TI caused pancreatic hypertrophy and increased secretions for chicks, rats and mice, others like pigs, dogs or pre-ruminant calves were free of these symptoms. When a processed soybean meal was increasingly replaced by raw soybean, the weights of pancreas and duodenum were proportionally increased. Supplementation of selected microbial feed enzymes could reduce the impacts of ANF and thereby improves the digestibility of proteins. As a recommendation it would have been suggested to use an optimum combination of heat intensity and time durations for improved quality of products while heating the soybean grains. Supplementation of selected and appropriate microbial feed enzymes is always advised to reduce both the heat-stable ANF and residual ANF in diets of non-ruminant animals.