Antinutritional effects of legume seeds in piglets, rats and chickens.

There is a growing interest in Europe to be self-supporting with regard to the protein supply for animal diets. Peas and beans growing well under European climatic conditions could provide alternatives to soya. However, these legume seeds contain the same classes of antinutritional factors (ANFs) as those found in raw soybeans. The use of such seeds in the raw state, therefore, is seriously hampered due to the presence of these ANFs. The role of ANFs in animal nutrition may become more important in the future. This is related to the expectation among zootechnicians that in the future farm animals will grow faster and deposit more body protein because of advances in animal breeding, health care and housing. It has been shown that the feed intake capacity has not increased in these fast growing animals, so they will therefore require relatively more highly digestible protein in the future. As a result, feedstuffs with a high protein content will become more important. However, most plant protein-rich seeds contain ANFs. The ANFs in peas, beans and soybeans have negative effects on digestibility and performance. In this respect it is necessary to find economically feasible inactivation processes which eliminate ANF activity. To achieve this, it is essential to know more about the way ANFs affect the digestion and absorption processes in animals. In this thesis, firstly a literature review (Chapter 1) was prepared on the occurrence and role of ANFs in peas ( <u>Pisum</u><u>sativum</u> ), common beans ( <u>Phaseolus</u><u>vulgaris</u> ) and soybeans ( <u>Glycine</u><u>max).</u> The main aspects to be considered were the state of the art concerning the action of ANFs in monogastric animals, the effect of ANFs on nutritional value, and the analytical methods for determining these ANFs. Also, recommendations for future research are given. The literature review (Chapter 1) shows that there are many unclear points related to the mode of action of ANFs in the animal. Major points being:- Most research into nutritional effects of ANFs in animals is carried out using small laboratory animals such rats, mice and chickens. An important question is whether results obtained in these animals are applicable to pigs.- Peas and beans always contain more than one ANF. In most studies whole ANF-containing seeds were fed to the animal. Information obtained in these studies gave no insight into the specific effects of separate ANFs. Only a limited amount of research has been done using isolated ANFs, and even then it was only carried out on small laboratory animals. To understand the relevance and the way ANFs act in the target animal, it is necessary to use isolated and purified ANFs in the investigations.- When a low apparent protein digestibility is measured it is not clear whether this is related exclusively to ANFs or whether the native protein itself may also be resistent to the hydrolysis by digestive enzymes.- Many analytical methods are not adequate. This has hampered the real identification of ANFs.- In the literature, lectin research is mainly focussed on the lectins present in Phaseolus vulgaris. Information about the mode of action of lectins in other seeds is limited.- There is insufficient information about the possibilities of eliminating ANF-activity.- There is insufficient information about the threshold levels, being the dietary levels of ANFs which can be tolerated without causing negative effects.In this thesis, aspects of the first three points were studied. The other points are being studied in related programmes.Animal species differences between piglets, rats and chickens were studied in three experiments. The results are described in Chapter 2. With common beans in the diet, performance was much more depressed in piglets than in rats or chickens. The piglets even lost weight. Weight loss in the piglets was also evident when extra protein was included in the diet. This indicates that a toxic factor must be associated with the reduced performance and not an insufficient amino acid supply. Protein digestibility was also markedly more depressed in piglets than in rats. The pancreas weight increased in the rats and chickens but not in the piglets. Increase in pancreas weight in rats and chickens may be related to the trypsin inhibitors present in the beans. Weights of the spleen and thymus were reduced in piglets but not in rats or chickens. With peas a reduction in weight gain was observed in piglets, but not in rats or chickens. Pancreas weight increased in the rats and chickens but not in the piglets. In all the animal species the weights of spleen and thymus were hardly affected by peas. Kidney and liver weights were not affected by either peas or beans. The results show that piglets are much more sensitive than rats or chickens to factors present in peas and beans. Some effects in piglets were the complete opposite of those found in rats and chickens. It is concluded, therefore, that ANF-research should be carried out using the target animals.In order to study which factor in peas caused the negative effects on protein digestibility, different fractions from peas were prepared: a pea protein isolate from which ANFs and carbohydrates were removed, a protein fraction with very high concentrations of ANFs and a fraction consisting of a mix of soluble and insoluble carbohydrates and free of protein and ANFs. Two pea varieties were involved, a summer variety with low trypsin inhibitor levels and a winter variety with relatively high levels of trypsin inhibitors. The fractions prepared from both varieties were applied in apparent ileal and faecal digestibility experiments with piglets. The results of these studies are described in Chapter 3. The apparent ileal protein digestibility of raw peas was with both varieties 14 units lower than in the pea protein isolate. Strikingly, the apparent ileal digestibility of some essential amino acids (S-containing amino acids, tryptophan and threonine) was very low at ileal level. The addition of pea carbohydrates to diets did not alter the apparent ileal protein digestibility. Small intestinal chyme flow increased due to pea carbohydrates. This effect could be related to a release of osmotic active components from the pea carbohydrates into the ileal chyme during the digestion process. The addition of pea-ANFs to a diet with pea protein isolate (low in ANFs) as the sole protein source, reduced the apparent ileal protein digestibility by about seven units. Weight gain of the piglets fed the diet enriched with ANFs was about 17% less compared with the control piglets. This demonstrates that ANFs are an important factor in explaining the reduced weight gain when more than 15-20% peas are included in the diets of piglets. The difference in apparent ileal protein digestibility between raw peas and pea protein isolate was 14 units. The other seven units which could not be attributed to ANFs could possibly be related to other factors such as antigenicity of the pea protein. True ileal and faecal protein digestibility of peas and common beans were measured using the <sup><font size="-1">15</font></SUP>N dilution technique. The results of this study are presented in Chapter 4. The apparent ileal protein digestibility of the raw summer and winter pea varieties were 79% and 74% respectively, the true protein digestibilities were between 93% and 95%. The apparent faecal protein digestibility was 85% for both varieties, the true faecal protein digestibility of both pea varieties was between 96% and 98%, respectively. These results indicate that native raw pea protein is highly digestible, and that digestion is nearly completed in the small intestine. The low apparent protein digestibility must be almost completely related to the secretion of endogenous protein. Common beans were studied in toasted form because the piglets refused the diets when raw Phaseolus beans were included. These beans were tested only for ileal and not for faecal digestibilities Apparent ileal protein digestibility of the toasted beans was about zero. The true protein digestibility was about 66%. The very low apparent ileal protein digestibility must therefore, be related to a very high secretion of endogenous protein. It was concluded that measurements of true protein digestibility are important for (bio)technologists. In order to improve protein digestibility it is necessary to know whether the treatments need to be focussed on the inactivation of ANFs and elimination of e.g antigenicity or to changes in protein structure. Our results show that with peas it is relevant to pay attention to factors causing an increased secretion of endogenous protein and not to the protein structure. With common beans, treatments should be directed to both: to factors causing an increased secretion of endogenous protein and to the protein itself. It was demonstrated (see GENERAL DISCUSSION), that when raw pea and soya protein are fed to piglets, guinea pigs and veal calves, trypsin activity in the small intestinal chyme and pancreatic activity was reduced and also protein digestibility was decreased. The lower pancreatic activity indicates that the low levels of trypsin inhibitors did not activate the negative feedback mechanism which in turn caused a hypersecretion of pancreatic enzymes. This negative feedback mechanism seems to be not present in pigs, veal calves or guinea pigs. This is in contrast to what is stated for rats. To elucidate which factor primarily is responsible for this observation, ANFs or possibly protein quality further research is required