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Trypsin inhibitors in peas: Varietal effect and influence on digestibility of crude protein by growing pigs
Animal Feed Science and Technology, 29 (1990) 45-55 Elsevier Science Publishers BN., Amsterdam - - Printed in The Netherlands
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Trypsin Inhibitors in Peas: Varietal Effect and Influence on Digestibility of Crude Protein by Growing Pigs P. LETERME, Y. BECKERS and A. THEWIS
Facultd des Sciences agronomiques de l'Etat, Unitd de Zootechnie, Passage des Ddportds, 2, B-5800 Gembloux (Belgium) (Received 30 March 1989; accepted for publication 28 September 1989)
ABSTRACT Leterme, P., Beckers, Y. and Th~wis, A., 1990. Trypsin inhibitors in peas: varietal effect and influence on digestibility of crude protein by growing pigs. Anim. Feed Sci. Technol., 29: 45-55. The trypsin inhibitor activity (TIA) of 33 European spring pea varieties was determined. The TIA of the varieties Maro, Progreta, Consort and Radley ranged from 6.1 to 8.4 trypsin inhibited units per mg dry matter (TIU rag- ~DM), whereas all the other varieties had ~ 3 TIU rag- 1 DM. Maro, Progreta and Consort are British varieties of the 'marrowfat' type which have classically a high activity. The French winter varieties had a high TIA (mean, 9.1 TIU mg -1 DM), but the varieties arising from a cross between a winter and a spring variety, such as Fril~ne, Amac, Laser and Santon, had a lower activity. The results were compared with those obtained for six other legume seeds: faba beans, vetches, lentils, pigeonpeas, chickpeas and haricot beans. Finally, a comparison of the TIA of samples of the variety Finale originating from different fields showed that growth conditions influenced the pea TIA. The apparent digestibility of the main constituents of two spring pea varieties (Finale, 2.35 and Progreta, 7.48 TIU rag- 1 DM) was determined, by the difference method, using growing pigs with diets consisting of 40% peas. The apparent digestibility of organic matter, crude fibre and crude energy was similar for the two varieties, whereas the apparent digestibilityof the crude protein of Progreta was significantly lower (P < 0.05) than that of Finale. The influence of trypsin inhibitors on the apparent digestibility of crude protein was discussed and all other hypotheses which could explain this difference were considered.
INTRODUCTION
Peas used as grain are the legumes which contain the least anti-nutritional factors. Lectins, tannins and ~-galactosides are generally present in peas in quantities too small to disturb the growth of pigs, except that of piglets (H15dversson, 1987a, b: Bertrand et al., 1988; Bengala-Freire et al., 1989). In contrast, protease inhibitors such as trypsin and chymotrypsin inhibitors, are present in sufficient quantity, mostly in winter varieties, to decrease the per0377-8401/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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P. LETERME ET AL.
formance of growing pigs (Perez and Bourdon, 1982; Fekete et al., 1984; Grosjean et al., 1986). But if their depressive effect in animals is well known (Liener, 1979), it has never been possible to demonstrate their role in reducing the performance of pigs ingesting winter pea varieties (Bouard et al., 1980; Castaing and Leuillet, 1981; Qudmdrd et al., 1982; Fekete et al., 1984; Grosjean et al., 1989). The variability between varieties is important (Valdebouze et al., 1980) and certain spring varieties have inhibitor contents as high as winter varieties {Griffiths, 1984; Christiansen and Larsen, 1987; Johns, 1987). The intravarietal variability can also be important (Valdebouze and Gaborit, 1985). The first objective of this work was to measure the trypsin inhibitor activity of a number of pea varieties. As Belgium uses large quantities of non-European peas, we have also determined the trypsin inhibitor activity (TIA) of peas imported from other continents. These results are compared with those obtained for six other legume seeds. The second objective was to compare the digestibility of two pea varieties, varying mostly in trypsin inhibitor content value, in growing pigs. MATERIALSAND METHODS
Experiment I The trypsin inhibitor activity of 33 European spring varieties, 6 winter varieties, peas imported from Australia, Canada, China, Hungary, New Zealand and Poland and 6 other legume seeds was determined by the method of Valdebouze et al. (1980) using the synthetic substrate ~-N-benzoyl-DL-argininep-nitroanilide hydrochloride (BAPNA). The yellow coloration due to the formation of p-nitroanilide is measured at 410 nm. The results are expressed in trypsin inhibited units per mg dry matter (TIU mg -~ DM) which is defined as the quantity of enzyme which induces an increase of 0.01 in absorbance. The samples were ground with an impact grinder (Dangoumill 300, Prolabo, France). The size of all particles was < 150/tin. The samples originated from trial fields conducted at the Crop Husbandry Department of the Faculty of Gembloux in Belgium and from firms which produce seeds. The intravarietal variability was studied with the variety Finale with samples taken randomly in two fields situated in different regions, grown the same year and from the trial fields of the Faculty.
Experiment 2 The apparent digestibility in pigs was measured for two spring varieties by the difference method. The two varieties are essentially distinguished by their TIA, 2.35 TIU mg -1 DM for Finale and 7.48 TIU mg -1 DM for Progreta. The basal diet consisted of 65% wheat, 32% soya bean meal and 3% mineral and
TRYPSININHIBITORSIN PEAS
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TABLE 1
Chemical composition (% DM), gross energy value and trypsin inhibitor activity of the basal diet and of the two pea varieties studied Basal diet
Composition Dry matter (%) Organic matter Crude protein (Nx6.25) Ether extract Starch Crude fibre Cell wall (NDF) Lignocellulose (ADF) Ca P
Gross energy (MJ kg -1 DM) Trypsin inhibitor activity (TIU mg-1 DM)
89.8 93.6 25.1 1.8 43.6 3.3 9.2 3.9 1.0 0.6 18.4 0.68
Peas Finale
Progreta
89.9 96.7 26.7 1.4 41.8 6.1 9.6 5.5 0.1 0.5 18.5 2.35
89.1 96.7 26.0 1.5 42.9 7.2 11.2 6.5 0.1 0.5 18.5 7.48
ADF, acid detergent fibre; NDF, neutral detergent fibre.
vitamin supplement, and had almost the same chemical composition as that of the two pea varieties (Table 1 ). The proportion of the basal diet replaced by peas was 40%. The experimental design of the trial was a 3 X 3 Latin square. The three pigs (initial average weight, 44 + 2 kg) received daily 100 g fresh matter kg- 1 W0.75 in two meals (07:30 and 16:30 h) and mixed with an equal amount of water. The faeces were collected for 10 days, after a diet adaptation period of 8 days. Feed and faeces samples were taken daily and frozen. At the end of the trials, they were freeze-dried and analysed by methods previously described (Leterme et al., 1989). The statistical analysis of the data was performed with the ANOVA procedure of the Statistical Analysis Systems software (Statistical Analysis Systems, 1982 ). RESULTS
Experiment I The TIA of different types of peas and 6 other legume seeds is presented in Table 2. Most spring varieties had a very low TIA ( < 3 TIU rag- 1 DM). Only the British spring varieties Consort, Radley, Progreta and Maro had a much higher activity. The two garden varieties (Bruant and Tierce 210) had a TIA similar to the crop varieties. The French winter varieties had an activity corn-
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P. LETERMEET AL.
TABLE2 Trypsin inhibitor activity (TIU mg-1 DM) of various types of pea and of 6 other legume seeds Pea type
n
Mean
C.V.
Spring Amino Belinda Belman Birte Bodil Bruant Consort Countess Danto Filby Finale Katrin Maro Maxi Miranda Nike Progreta Radley Sentinel Solara Stehgolt Terese Tierce 210 CM 91 CM 93 CM 94 FR-57 FR-RAM LD 8954 MJASI 845 MJASI 893
11 6 2 5 3 1 6 6 1 2 15 3 2 4 5 2 8 1 2 14 3 2 1 1 1 1 1 1 1 1 1
2.13 2.36 2.51 2.44 2.29 2.53 6.08 3.25 1.94 2.46 2.02 2.00 8.40 1.96 2.59 2.08 8.40 7.46 1.90 2.12 2.15 2.30 2.03 1.85 2.33 2.11 1.88 1.71 2.17 2.27 3.09
19.5 23.8 12.7 19.8 18.5 19.1 18.3 0.3 20.4 13.8 20.4 19.5 20.0 12.6 12.7 15.3 13.8 18.8 3.7 -
Pea type NRPB 338 NRPB 412 Winter Frijaune Fril~ne Frisson Amac Laser Santon Imported peas Canada Unknown var. Trapper Century China Hungary Poland Fidelia Peluschken Australia and N. Zealand Dun Maple
n
Mean
C.V.
1 1
1.96 1.96
2 3 1 3 2 1
9.70 10.29 11.24 6.87 7.54 8.65
12.2 14.8 11.4 16.8 -
15 3 1 3 4
3.20 5.83 2.78 4.44 2.14
12.8 21.2 12.6 15.6
2 1
2.86 3.98
11.0 -
4 4
2.41 2.79
17.5 25.0
1 1 1 1 1 1
4.51 5.21 5.38 6.65 11.68 16.56
Other legumes
Viciafaba Vicia sativa Vicia lens Cajanus cajan Cicer arietinum Phaseolus vulgaris
C.V., coefficient of variation (%). p a r a b l e w i t h t h a t o f o t h e r w i n t e r v a r i e t i e s , e x c e p t for o n e s a m p l e o f t h e C a n a d i a n v a r i e t y T r a p p e r . T h e 15 C a n a d i a n s a m p l e s f r o m c a t t l e f e e d f i r m s h a d a low a v e r a g e T I A . T h e o t h e r l e g u m e s s t u d i e d all h a d a T I A h i g h e r t h a n t h a t of s p r i n g pea varieties. T h e T I A values o b s e r v e d in d i f f e r e n t s a m p l e s of t h e v a r i e t y F i n a l e f r o m two f i e l d s a n d 15 t r i a l p l o t s a r e p r e s e n t e d i n T a b l e 3. T h e d i f f e r e n c e b e t w e e n t h e t w o f i e l d s is h i g h , b u t t h e v a r i a t i o n w i t h i n e a c h f i e l d is v e r y low. T h e a v e r a g e o f t h e 15 p l o t s lies b e t w e e n t h a t o f t h e t w o fields, b u t t h e o b s e r v e d v a r i a t i o n s are in this case very i m p o r t a n t .
TRYPSIN INHIBITORSIN PEAS
49
TABLE 3 Trypsin inhibited activity ( T I U mg -1 D M ) of the pea variety Finale according to place of cultivation
Field 1 Field 2 Trial plots
n
Mean
Minimum
Maximum
C.V.
8 8 15
1.58 2.21 2.02
1.50 2.14 1.93
1.61 2.34 2.94
4.1 6.0 19.7
C.V., coefficient of variation. TABLE 4 Apparent digestibility ( % ) of the principal constituents and gross energy of the diets used and of the two pea varieties studied in pigs Diet
Organic matter Crude protein Crude fibre Cell wall Gross energy
Peas
Basal
Finale
Progreta
SEM
Finale
Progreta
SEM
89.2 87.3 a 43.3 51.6 87.4
90.0 89.0 b 57.1 57.0 88.2
89.1 85.0 c 59.7 63.0 87.2
0.2 0.7 3.5 2.0 0.3
91.0 91.0 a 58.4 64.9 89.4
88.9 81.8 b 69.9 81.2 86.8
0.7 2.7 5.0 4.4 1.0
a,b,cp < 0.05. SEM, standard error of the mean.
Experiment 2 The apparent digestibility of the major components of the diets and of the two pea varieties calculated by difference is presented in Table 4. The observed differences between diets and the two pea varieties were significant only for crude protein. The very different coefficients observed for fibre fractions (cell wall and crude fibre) do not differ significantly, the variability between pigs being large. DISCUSSION
Experiment I The spring varietiesProgreta and Maro are included in the British classification in the 'marrowfat' class and are characterizedby an 'angular'seed. These peas are classicallyrich in trypsin inhibitors (Griffiths,1984; Christiansen and Larsen, 1987; Johns, 1987). Progreta and Consort are derived from Maro, which can explain their high activity.O n the other hand, the variety Radley is a cross-
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P. LETERME ET AL.
breed between a winter and a spring variety (F. Cors, personal communication, 1989). All winter varieties have a high activity. The old winter varieties such as Frisson are derived from a cross-breed between a variety from the sub-species hortense and one from the sub-species arvense. But the new varieties often arise from a cross-breed between a winter and a spring variety. Laser, Fril~ne, Amac and Santon are in this category. The genetics of the parents and the type of cross-breeding can probably explain the great variability between the different varieties. Peas cultivated in our area belong to the sub-species hortense, whereas peas cultivated extensively in countries such as Australia and New Zealand are field peas and belong to the sub-species arvense. In the samples analysed here, the peas Pelushken, Maple, Dun and the Chinese peas belong to the latter category. The seeds are coloured (brown or grey), which indicates the presence of tannins in the hulls. But the tannins also have the property of inhibiting protease enzymes (Griffiths, 1981 ). The TIA of these peas and of Canadian peas nevertheless remains moderate, being equal to or slightly higher than that of spring peas (Griffiths, 1984; Pisulewski et al., 1983). Winter peas have an activity higher than that of faba beans ( Vicia faba), vetches ( Vicia sativa ), lentils ( Vicia lens), and pigeonpeas ( Cajanus cajan ) , but equal to or slightly lower than that of chickpeas (Cicer arietinum), and considerably lower than that of haricot beans (Phaseolus vulgaris). The observed values for chickpeas are nevertheless exceptionally high (Saini, 1989). A single sample is not representative of one species but is sufficient to indicate the likely level of TIA. Growing conditions (soil, climate, sowing date) can influence the TIA of pea seeds (Valdebouze and Gaborit, 1985). This seems logical when one knows that the growing conditions and the degree of ripeness of the seeds influence their chemical composition (Holl and Vose, 1980).
Experiment 2 The difference in the digestibility of crude protein observed between the two pea varieties can partially be attributed to trypsin-inhibitor substances present in quantities three times greater in Progreta. The presence of protease inhibitors in the small intestine induces the formation of an inhibitor-enzyme complex which passes through the digestive tract and is excreted in the faeces. The decrease in enzyme concentration in the intestine induces a hypersecretory activity of the pancreas which leads to a hypertrophy of the pancreas. These two phenomena would thus contribute to an increase of the endogenous faecal nitrogen excretion (Liener, 1979). This would explain the lower apparent digestibility of the Progreta proteins. What is more, the content of chymotrypsin inhibitors is closely related to that of trypsin inhibitors, which means that the most important inhibitor has two sites of action (Griffiths, 1984; Christiansen
51
TRYPSIN INHIBITORS IN PEAS
and Larsen, 1987). This would also accentuate the endogenous faecal excretion. The resulting loss in endogenous amino acids would increase the deficiency in essential Amino acids of pea proteins and would explain the reduction in performance of pigs given peas rich in protease inhibitors. The greater TIA of Progreta cannot alone explain the great difference in protein digestibilityobserved between the two varieties. Several other factors could also explain this difference: (1) Most trials to explain the depressive effect of protease inhibitors (enzyme inactivation, pancreas hypertrophy) have been done on rats or chickens. But it is now known that pigs are considerably less sensitive to these substances than the two other species mentioned, and that their presence hardly affects pancreas secretions (Huisman et al.,1987; Huisman and van der Poel, 1989). The difference in TIA observed between the two diets containing peas cannot thus have induced such an important secretion of endogenous nitrogen. (2) The composition of proteins is very variable from one variety to another and even within batches of the same variety (Gueguen and Barbot, 1988). But Progreta has a less essential amino acid profile than the other varieties, in particular Finale (Christiansen and Larsen, 1987; Edwards et al.,1987) and this is reflected by a much lower biological value. (3) According to Bhatty and Christison (1980 ), it is essentially the glucidic fraction of peas which influences the digestibilityof their protein and this digestibilitydepends on the starch content of the ration (Fuller and Crofts, 1977 ) and on the rate of hydrolysis of the starch in the intestines; the latter varies considerably from one type of starch to another (Bengala-Freire et al.,1989). What is more, the c~-galactosldes also influence thin digestlblhty (Sere et al., 1989). Nevertheless, the starch and a-galactoside content is very variable from one variety to another (Cerning-Beroard and Filiatre-Verel, 1979). Progreta is a 'marrowfat' pea type, which means it is angular. Certain authors even classify it in the 'wrinkled pea' category (Griffiths, 1984). The starch of wrinkled peas is twice as rich in amylose as that of smooth peas, and amylose is less well digested by monogastrics than is amylopectin (Cerning-Beroard and Filiatre-Verel, 1979). (4) Progreta is richer in cell walls than Finale, and the depressive effect of fibre on protein digestibility is well known (Kass et al., 1980; Stanogias and Pearce, 1985; Hall et al., 1988). Stanogias and Pearce (1985) have shown in similar conditions to ours that an increase of 10 g of pea cellwalls kg-i of pig feed decreases protein digestibility by about one unit. This would partially explain the lower protein digestibilityof Progreta. (5) The apparent digestibilityof pea protein in the ileum is considerably lower than the faecal digestibilityobserved here (Moughan and Smith, 1985; Green, 1988). This means that the micro-organisms of the large intestine still break down an important part of the nitrogen products from the small intestine into ammonia. This ammonia then passes into the blood and is eliminated •
•
.
•
•
.
.
•
•
J "
52
P. LETERME ET AL.
via the urine as urea (Zebrowska, 1973; Just et al., 1981 ). Nevertheless, when these bacteria have at their disposition a fermentable source of energy, they can use ammonia for their own synthesis. In this case, nitrogen is not absorbed by the large intestine but is excreted in the faeces as bacterial protein (Just et al., 1981; Misir and Sauer, 1982 ), thus increasing the faecal nitrogen output and decreasing the apparent digestibility of protein. Moreover, as suggested above, the starch of Progreta may be less well digested in the small intestine than that of Finale. What is more, pea fibres are very digestible for pigs (Lund and Hakansson, 1986; H15dversson, 1987a,b) and they contribute to the energy input in the large intestine. In our case, Progreta is richer in cell walls, and these are better digested than those of Finale. The greater digestion of the Progreta carbohydrates in the pig's large intestine should allow microbial multiplication and encourage the faecal excretion of microbial protein (Misir and Sauer, 1981, 1982; MalmlSf and Hakansson, 1984). It is thus preferable to determine the ileal protein digestibility of the two varieties. (6) Trials carried out on chickens, considered more sensitive than pigs, have shown that trypsin inhibitors had practically no effect on growth performance (Johns, 1987) and influenced only slightly the protein digestibility (Carr~ and Conan, 1989). What is more, Grosjean et al. (1989) have shown recently that, when one considers the digestible energy value and when one increases the addition of synthetic tryptophan and methionine to the diet, it is possible to obtain with diets containing 30% winter peas, a performance practically identical to that obtained with spring peas. Trials by Johns (1987) on chickens and by Grosjean et al. (1989) on pigs show that animal performance is essentially linked to the limiting amino acids content (tryptophan, S-amino acids) of peas and to the ileal digestibility. More recently, we have learned that the ileal digestibility of tryptophan and cystine in peas is particularly low in pigs (Moughan and Smith, 1985; Green, 1988; Buraczewska et al., 1989). CONCLUSIONS The TIA of peas is very variable from one type of pea to another and seems essentially to be a genetic problem. Its influence on protein digestibility by pigs has not been clearly demonstrated. Trials should be carried out on purified protease inhibitors, and measurements should be made of digestibility to the ileum. Meanwhile, in order to prevent poor growth from peas rich or poor in protease inhibitors, it is advisable to supplement the diet with synthetic tryptophan and methionine, the two limiting amino acids in pigs. ACKNOWLEDGEMENTS The authors wish to thank Mr. Cors from the Crop Husbandry Department of the Faculty, and all the seed breeders and the livestock food producers for
TRYPSININHIBITORSIN PEAS
53
the kind supply of the seeds. The expert technical assistance of Ch. Bodart, L. Givron and Th. Monmart is gratefully acknowledged. This study was supported by the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (IRSIA), Brussels, Belgium.
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des pois d'hiver et de printemps dans les aliments pour porcs charcutiers prdsentds en farine et en granulds. J. Rech. Porcine Fr., 21: 59-68. Gueguen, J. and Barbot, J., 1988. Quantitative and qualitative variability of pea (Pisum sativum L. ) protein composition. J. Sci. Food Agric., 42: 209-224. Hall, D.D., Fernandez, J.A., Jorgensen, H.J. and Boisen, S., 1988. Prediction of ileal and fecal digestibilityof crude protein and amino acids in pigs diets by NDF and NDF-nitrogen analyses. Wiss. Z. WPU, Rostock, N-Reihe, 37 (1): 54-55. H15dversson, R., 1987a. Comparison of the nutritional value of dark and white flowered cultivars of pea for growing finishing pigs. Swed. J. Agric. Res., 17: 97-101. H15dversson, R., 1987b. The nutritive value of white and dark flowered cultivars of pea for growing-finishing pigs. Anim. Feed Sci. Technol., 17: 245-255. Holl, F.B. and Vose, J.R., 1980. Carbohydrate and protein accumulation in the developing field pea seed. Can. J. Plant Sci., 60: 1109-1114. Huisman, J. and van der Poel, A.F.B., 1989. Comparison of effects of antinutritional factors (ANF) in different species. In: J. Huisman, T.F. van der Poel, and I.E. Liener (Editors), Recent Advances of Research in Antinutritional Factors in Legume Seeds. Pudoc, Wageningen, The Netherlands, pp. 317-327. Huisman, J., de Jong, J., van der Poel, A.F., ten Haaf, L.A. and Smiths, L.H., 1987. Species difference: different negative effects of antinutritional factors (ANf) in the Phaseolus vulgaris bean in young pigs and rats. Wiss. Z. WPU, Rostock, N-Reihe, 36 (10): 83-85. Johns, D.C., 1987. Influence of trypsin inhibitors in four varieties of peas (Pisum sativum) on the growth of chickens. N.Z.J. Agric. Res., 30: 169-175. Just, A., Jorgensen, H. and Fernandez, J.A., 1981. The digestive capacity of caecum-colon and the value of the nitrogen absorbed from the hindgut for protein synthesis in pigs. Br. J. Nutr., 46: 209-219. Kass, M.L., Van Soest, P.J., Pond, W.G., Lenis, B. and Mc Dowell, R.E., 1980. Utilisation of dietary fibre from alfalfa by growing swine. 1. Apparent digestibility of diet components in specific segments of the gastrointestinal tract. J. Anita. Sci., 50: 175-191. Leterme, P., Thielemans, M.-F., Bodart, C., Baudart, E. and ThSwis, A., 1989. Valeur nutritive et digestibilitd apparente ilSale et f~cale des acides amines du triticale, du froment et de l'escourgeon chez le porc. Rev. Agric. Brussels, 42: 305-316. Liener, I.E., 1979. The nutritional significance of plant protease inhibitors. Proc. Nutr. Soc., 38: 109-113. Lund, S. and Hakansson, J., 1986. Nutritional and growth studies with pea-crop meals and peas for growing-finishing pigs. Anim. Feed Sci. Technol., 16:119-128. MalmlSf, K. and Hakansson, J., 1984. The effect of dietary fibre level on the diurnal pattern of urinary nitrogen excretion in swine. Swed. J. Agric. Res., 14: 53-57. Misir, R. and Sauer, W.C., 1981. Nitrogen and amino acid metabolism in the hindgut of pigs fed barley or wheat diets as affected by the infusion of maize starch at the terminal ileum. Z. Tierphysiol. Tierernaehr. Futtermittelkd., 46: 221-233. Misir, R. and Sauer, W.C., 1982. Effect of starch infusion at the terminal ileum on nitrogen balance and apparent digestibilitiesof nitrogen and amino acids in pigs fed meat-and-bone and soybean meal diets. J. Anita. Sci., 55: 599-607. Moughan, P.J. and Smith, W.C., 1985. Determination and assessment of ileal amino acid digestibility coefficients for the growing pig. N.Z.J. Agric. Res., 28: 365-370. Perez, J.M. and Bourdon, D., 1982. Essai de remplacement total du tourteau de soja dans le rggime du porc en croissance: utilisation du pois suppl~ment~ en tryptophane ou associ~ ~ un concentrd de prot~ines de luzerne. J. Rech. Porcine Fr., 14: 283-296. Pisulewski, P., Pisulewska, E., Hanczakowski, P. and Ernest, T., 1983. The chemical composition and nutritive value of pea (Pisum sativum L. ) and field pea (Pisum arvense L. ) seeds. Rocz. Nauk Zoot., 10(2): 111-116.
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QuSm~r~, P., Fekete, F. and Leuillet, M., 1982. Utilisation du pois prot~agineux par le porcelet sevr~ pr~cocement. Influence du taux d'incorporation et de la varietY. J. Rech. Porcine Fr., 14: 267-282. Statistical Analysis Systems, 1982. S.A.S. User's Guide: Basics. Statistical Analysis Systems Institute Inc., Cary, N.C. Saini, H.S., 1989. Activity and thermal inactivation of protease inhibitors in grain legumes. In: J. Huisman, T.F. van der Poel and I.E. Liener {Editors), Recent Advances of Research in Antinutritional Factors in Legume Seeds. Pudoc, Wageningen, The Netherlands, pp. 249-253. S~ve, B., Kerros, C., Lebreton, Y., Quemener, B., Gaborit, T. and Bouchez, P., 1989. Effect of the extraction of ~-galactosides from toasted or raw soybean meal on dietary nitrogen and fat utilization in the young pig. In: J. Huisman, T.F. van der Poel and I.E. Liener (Editors), Recent Advances of Research in Antinutritional Factors in Legume Seeds. Pudoc, Wageningen, The Netherlands, pp. 276-279. Stanogias, G. and Pearce, G.R., 1985. The digestion of fibre by pigs. 1. The effects of amount and type of fibre on apparent digestibility, nitrogen balance and rate of passage. Br. J. Nutr., 53: 513-530. Valdebouze, P., Bergeron, E., Gaborit, T. and Delort-Laval, J., 1980. Content and distribution of trypsin inhibitors and hemagglutinins in some legume seeds. Can. J. Plant Sci., 60: 695-701. Valdebouze, P. and Gaborit, T., 1985. Activit~ antitrypsique de graines de l~gumineuses. Revue de rAlimentation Animale, 11: 45-47. Zebrowska, T., 1973. Influence of dietary protein source on the rate of digestion in the small intestine of pigs. II. The rate of protein digestion and absorption of amino acids. Rocz. Nauk Roln., 95B (1): 135-155.
45
Trypsin Inhibitors in Peas: Varietal Effect and Influence on Digestibility of Crude Protein by Growing Pigs P. LETERME, Y. BECKERS and A. THEWIS
Facultd des Sciences agronomiques de l'Etat, Unitd de Zootechnie, Passage des Ddportds, 2, B-5800 Gembloux (Belgium) (Received 30 March 1989; accepted for publication 28 September 1989)
ABSTRACT Leterme, P., Beckers, Y. and Th~wis, A., 1990. Trypsin inhibitors in peas: varietal effect and influence on digestibility of crude protein by growing pigs. Anim. Feed Sci. Technol., 29: 45-55. The trypsin inhibitor activity (TIA) of 33 European spring pea varieties was determined. The TIA of the varieties Maro, Progreta, Consort and Radley ranged from 6.1 to 8.4 trypsin inhibited units per mg dry matter (TIU rag- ~DM), whereas all the other varieties had ~ 3 TIU rag- 1 DM. Maro, Progreta and Consort are British varieties of the 'marrowfat' type which have classically a high activity. The French winter varieties had a high TIA (mean, 9.1 TIU mg -1 DM), but the varieties arising from a cross between a winter and a spring variety, such as Fril~ne, Amac, Laser and Santon, had a lower activity. The results were compared with those obtained for six other legume seeds: faba beans, vetches, lentils, pigeonpeas, chickpeas and haricot beans. Finally, a comparison of the TIA of samples of the variety Finale originating from different fields showed that growth conditions influenced the pea TIA. The apparent digestibility of the main constituents of two spring pea varieties (Finale, 2.35 and Progreta, 7.48 TIU rag- 1 DM) was determined, by the difference method, using growing pigs with diets consisting of 40% peas. The apparent digestibility of organic matter, crude fibre and crude energy was similar for the two varieties, whereas the apparent digestibilityof the crude protein of Progreta was significantly lower (P < 0.05) than that of Finale. The influence of trypsin inhibitors on the apparent digestibility of crude protein was discussed and all other hypotheses which could explain this difference were considered.
INTRODUCTION
Peas used as grain are the legumes which contain the least anti-nutritional factors. Lectins, tannins and ~-galactosides are generally present in peas in quantities too small to disturb the growth of pigs, except that of piglets (H15dversson, 1987a, b: Bertrand et al., 1988; Bengala-Freire et al., 1989). In contrast, protease inhibitors such as trypsin and chymotrypsin inhibitors, are present in sufficient quantity, mostly in winter varieties, to decrease the per0377-8401/90/$03.50
© 1990 Elsevier Science Publishers B.V.
46
P. LETERME ET AL.
formance of growing pigs (Perez and Bourdon, 1982; Fekete et al., 1984; Grosjean et al., 1986). But if their depressive effect in animals is well known (Liener, 1979), it has never been possible to demonstrate their role in reducing the performance of pigs ingesting winter pea varieties (Bouard et al., 1980; Castaing and Leuillet, 1981; Qudmdrd et al., 1982; Fekete et al., 1984; Grosjean et al., 1989). The variability between varieties is important (Valdebouze et al., 1980) and certain spring varieties have inhibitor contents as high as winter varieties {Griffiths, 1984; Christiansen and Larsen, 1987; Johns, 1987). The intravarietal variability can also be important (Valdebouze and Gaborit, 1985). The first objective of this work was to measure the trypsin inhibitor activity of a number of pea varieties. As Belgium uses large quantities of non-European peas, we have also determined the trypsin inhibitor activity (TIA) of peas imported from other continents. These results are compared with those obtained for six other legume seeds. The second objective was to compare the digestibility of two pea varieties, varying mostly in trypsin inhibitor content value, in growing pigs. MATERIALSAND METHODS
Experiment I The trypsin inhibitor activity of 33 European spring varieties, 6 winter varieties, peas imported from Australia, Canada, China, Hungary, New Zealand and Poland and 6 other legume seeds was determined by the method of Valdebouze et al. (1980) using the synthetic substrate ~-N-benzoyl-DL-argininep-nitroanilide hydrochloride (BAPNA). The yellow coloration due to the formation of p-nitroanilide is measured at 410 nm. The results are expressed in trypsin inhibited units per mg dry matter (TIU mg -~ DM) which is defined as the quantity of enzyme which induces an increase of 0.01 in absorbance. The samples were ground with an impact grinder (Dangoumill 300, Prolabo, France). The size of all particles was < 150/tin. The samples originated from trial fields conducted at the Crop Husbandry Department of the Faculty of Gembloux in Belgium and from firms which produce seeds. The intravarietal variability was studied with the variety Finale with samples taken randomly in two fields situated in different regions, grown the same year and from the trial fields of the Faculty.
Experiment 2 The apparent digestibility in pigs was measured for two spring varieties by the difference method. The two varieties are essentially distinguished by their TIA, 2.35 TIU mg -1 DM for Finale and 7.48 TIU mg -1 DM for Progreta. The basal diet consisted of 65% wheat, 32% soya bean meal and 3% mineral and
TRYPSININHIBITORSIN PEAS
47
TABLE 1
Chemical composition (% DM), gross energy value and trypsin inhibitor activity of the basal diet and of the two pea varieties studied Basal diet
Composition Dry matter (%) Organic matter Crude protein (Nx6.25) Ether extract Starch Crude fibre Cell wall (NDF) Lignocellulose (ADF) Ca P
Gross energy (MJ kg -1 DM) Trypsin inhibitor activity (TIU mg-1 DM)
89.8 93.6 25.1 1.8 43.6 3.3 9.2 3.9 1.0 0.6 18.4 0.68
Peas Finale
Progreta
89.9 96.7 26.7 1.4 41.8 6.1 9.6 5.5 0.1 0.5 18.5 2.35
89.1 96.7 26.0 1.5 42.9 7.2 11.2 6.5 0.1 0.5 18.5 7.48
ADF, acid detergent fibre; NDF, neutral detergent fibre.
vitamin supplement, and had almost the same chemical composition as that of the two pea varieties (Table 1 ). The proportion of the basal diet replaced by peas was 40%. The experimental design of the trial was a 3 X 3 Latin square. The three pigs (initial average weight, 44 + 2 kg) received daily 100 g fresh matter kg- 1 W0.75 in two meals (07:30 and 16:30 h) and mixed with an equal amount of water. The faeces were collected for 10 days, after a diet adaptation period of 8 days. Feed and faeces samples were taken daily and frozen. At the end of the trials, they were freeze-dried and analysed by methods previously described (Leterme et al., 1989). The statistical analysis of the data was performed with the ANOVA procedure of the Statistical Analysis Systems software (Statistical Analysis Systems, 1982 ). RESULTS
Experiment I The TIA of different types of peas and 6 other legume seeds is presented in Table 2. Most spring varieties had a very low TIA ( < 3 TIU rag- 1 DM). Only the British spring varieties Consort, Radley, Progreta and Maro had a much higher activity. The two garden varieties (Bruant and Tierce 210) had a TIA similar to the crop varieties. The French winter varieties had an activity corn-
48
P. LETERMEET AL.
TABLE2 Trypsin inhibitor activity (TIU mg-1 DM) of various types of pea and of 6 other legume seeds Pea type
n
Mean
C.V.
Spring Amino Belinda Belman Birte Bodil Bruant Consort Countess Danto Filby Finale Katrin Maro Maxi Miranda Nike Progreta Radley Sentinel Solara Stehgolt Terese Tierce 210 CM 91 CM 93 CM 94 FR-57 FR-RAM LD 8954 MJASI 845 MJASI 893
11 6 2 5 3 1 6 6 1 2 15 3 2 4 5 2 8 1 2 14 3 2 1 1 1 1 1 1 1 1 1
2.13 2.36 2.51 2.44 2.29 2.53 6.08 3.25 1.94 2.46 2.02 2.00 8.40 1.96 2.59 2.08 8.40 7.46 1.90 2.12 2.15 2.30 2.03 1.85 2.33 2.11 1.88 1.71 2.17 2.27 3.09
19.5 23.8 12.7 19.8 18.5 19.1 18.3 0.3 20.4 13.8 20.4 19.5 20.0 12.6 12.7 15.3 13.8 18.8 3.7 -
Pea type NRPB 338 NRPB 412 Winter Frijaune Fril~ne Frisson Amac Laser Santon Imported peas Canada Unknown var. Trapper Century China Hungary Poland Fidelia Peluschken Australia and N. Zealand Dun Maple
n
Mean
C.V.
1 1
1.96 1.96
2 3 1 3 2 1
9.70 10.29 11.24 6.87 7.54 8.65
12.2 14.8 11.4 16.8 -
15 3 1 3 4
3.20 5.83 2.78 4.44 2.14
12.8 21.2 12.6 15.6
2 1
2.86 3.98
11.0 -
4 4
2.41 2.79
17.5 25.0
1 1 1 1 1 1
4.51 5.21 5.38 6.65 11.68 16.56
Other legumes
Viciafaba Vicia sativa Vicia lens Cajanus cajan Cicer arietinum Phaseolus vulgaris
C.V., coefficient of variation (%). p a r a b l e w i t h t h a t o f o t h e r w i n t e r v a r i e t i e s , e x c e p t for o n e s a m p l e o f t h e C a n a d i a n v a r i e t y T r a p p e r . T h e 15 C a n a d i a n s a m p l e s f r o m c a t t l e f e e d f i r m s h a d a low a v e r a g e T I A . T h e o t h e r l e g u m e s s t u d i e d all h a d a T I A h i g h e r t h a n t h a t of s p r i n g pea varieties. T h e T I A values o b s e r v e d in d i f f e r e n t s a m p l e s of t h e v a r i e t y F i n a l e f r o m two f i e l d s a n d 15 t r i a l p l o t s a r e p r e s e n t e d i n T a b l e 3. T h e d i f f e r e n c e b e t w e e n t h e t w o f i e l d s is h i g h , b u t t h e v a r i a t i o n w i t h i n e a c h f i e l d is v e r y low. T h e a v e r a g e o f t h e 15 p l o t s lies b e t w e e n t h a t o f t h e t w o fields, b u t t h e o b s e r v e d v a r i a t i o n s are in this case very i m p o r t a n t .
TRYPSIN INHIBITORSIN PEAS
49
TABLE 3 Trypsin inhibited activity ( T I U mg -1 D M ) of the pea variety Finale according to place of cultivation
Field 1 Field 2 Trial plots
n
Mean
Minimum
Maximum
C.V.
8 8 15
1.58 2.21 2.02
1.50 2.14 1.93
1.61 2.34 2.94
4.1 6.0 19.7
C.V., coefficient of variation. TABLE 4 Apparent digestibility ( % ) of the principal constituents and gross energy of the diets used and of the two pea varieties studied in pigs Diet
Organic matter Crude protein Crude fibre Cell wall Gross energy
Peas
Basal
Finale
Progreta
SEM
Finale
Progreta
SEM
89.2 87.3 a 43.3 51.6 87.4
90.0 89.0 b 57.1 57.0 88.2
89.1 85.0 c 59.7 63.0 87.2
0.2 0.7 3.5 2.0 0.3
91.0 91.0 a 58.4 64.9 89.4
88.9 81.8 b 69.9 81.2 86.8
0.7 2.7 5.0 4.4 1.0
a,b,cp < 0.05. SEM, standard error of the mean.
Experiment 2 The apparent digestibility of the major components of the diets and of the two pea varieties calculated by difference is presented in Table 4. The observed differences between diets and the two pea varieties were significant only for crude protein. The very different coefficients observed for fibre fractions (cell wall and crude fibre) do not differ significantly, the variability between pigs being large. DISCUSSION
Experiment I The spring varietiesProgreta and Maro are included in the British classification in the 'marrowfat' class and are characterizedby an 'angular'seed. These peas are classicallyrich in trypsin inhibitors (Griffiths,1984; Christiansen and Larsen, 1987; Johns, 1987). Progreta and Consort are derived from Maro, which can explain their high activity.O n the other hand, the variety Radley is a cross-
50
P. LETERME ET AL.
breed between a winter and a spring variety (F. Cors, personal communication, 1989). All winter varieties have a high activity. The old winter varieties such as Frisson are derived from a cross-breed between a variety from the sub-species hortense and one from the sub-species arvense. But the new varieties often arise from a cross-breed between a winter and a spring variety. Laser, Fril~ne, Amac and Santon are in this category. The genetics of the parents and the type of cross-breeding can probably explain the great variability between the different varieties. Peas cultivated in our area belong to the sub-species hortense, whereas peas cultivated extensively in countries such as Australia and New Zealand are field peas and belong to the sub-species arvense. In the samples analysed here, the peas Pelushken, Maple, Dun and the Chinese peas belong to the latter category. The seeds are coloured (brown or grey), which indicates the presence of tannins in the hulls. But the tannins also have the property of inhibiting protease enzymes (Griffiths, 1981 ). The TIA of these peas and of Canadian peas nevertheless remains moderate, being equal to or slightly higher than that of spring peas (Griffiths, 1984; Pisulewski et al., 1983). Winter peas have an activity higher than that of faba beans ( Vicia faba), vetches ( Vicia sativa ), lentils ( Vicia lens), and pigeonpeas ( Cajanus cajan ) , but equal to or slightly lower than that of chickpeas (Cicer arietinum), and considerably lower than that of haricot beans (Phaseolus vulgaris). The observed values for chickpeas are nevertheless exceptionally high (Saini, 1989). A single sample is not representative of one species but is sufficient to indicate the likely level of TIA. Growing conditions (soil, climate, sowing date) can influence the TIA of pea seeds (Valdebouze and Gaborit, 1985). This seems logical when one knows that the growing conditions and the degree of ripeness of the seeds influence their chemical composition (Holl and Vose, 1980).
Experiment 2 The difference in the digestibility of crude protein observed between the two pea varieties can partially be attributed to trypsin-inhibitor substances present in quantities three times greater in Progreta. The presence of protease inhibitors in the small intestine induces the formation of an inhibitor-enzyme complex which passes through the digestive tract and is excreted in the faeces. The decrease in enzyme concentration in the intestine induces a hypersecretory activity of the pancreas which leads to a hypertrophy of the pancreas. These two phenomena would thus contribute to an increase of the endogenous faecal nitrogen excretion (Liener, 1979). This would explain the lower apparent digestibility of the Progreta proteins. What is more, the content of chymotrypsin inhibitors is closely related to that of trypsin inhibitors, which means that the most important inhibitor has two sites of action (Griffiths, 1984; Christiansen
51
TRYPSIN INHIBITORS IN PEAS
and Larsen, 1987). This would also accentuate the endogenous faecal excretion. The resulting loss in endogenous amino acids would increase the deficiency in essential Amino acids of pea proteins and would explain the reduction in performance of pigs given peas rich in protease inhibitors. The greater TIA of Progreta cannot alone explain the great difference in protein digestibilityobserved between the two varieties. Several other factors could also explain this difference: (1) Most trials to explain the depressive effect of protease inhibitors (enzyme inactivation, pancreas hypertrophy) have been done on rats or chickens. But it is now known that pigs are considerably less sensitive to these substances than the two other species mentioned, and that their presence hardly affects pancreas secretions (Huisman et al.,1987; Huisman and van der Poel, 1989). The difference in TIA observed between the two diets containing peas cannot thus have induced such an important secretion of endogenous nitrogen. (2) The composition of proteins is very variable from one variety to another and even within batches of the same variety (Gueguen and Barbot, 1988). But Progreta has a less essential amino acid profile than the other varieties, in particular Finale (Christiansen and Larsen, 1987; Edwards et al.,1987) and this is reflected by a much lower biological value. (3) According to Bhatty and Christison (1980 ), it is essentially the glucidic fraction of peas which influences the digestibilityof their protein and this digestibilitydepends on the starch content of the ration (Fuller and Crofts, 1977 ) and on the rate of hydrolysis of the starch in the intestines; the latter varies considerably from one type of starch to another (Bengala-Freire et al.,1989). What is more, the c~-galactosldes also influence thin digestlblhty (Sere et al., 1989). Nevertheless, the starch and a-galactoside content is very variable from one variety to another (Cerning-Beroard and Filiatre-Verel, 1979). Progreta is a 'marrowfat' pea type, which means it is angular. Certain authors even classify it in the 'wrinkled pea' category (Griffiths, 1984). The starch of wrinkled peas is twice as rich in amylose as that of smooth peas, and amylose is less well digested by monogastrics than is amylopectin (Cerning-Beroard and Filiatre-Verel, 1979). (4) Progreta is richer in cell walls than Finale, and the depressive effect of fibre on protein digestibility is well known (Kass et al., 1980; Stanogias and Pearce, 1985; Hall et al., 1988). Stanogias and Pearce (1985) have shown in similar conditions to ours that an increase of 10 g of pea cellwalls kg-i of pig feed decreases protein digestibility by about one unit. This would partially explain the lower protein digestibilityof Progreta. (5) The apparent digestibilityof pea protein in the ileum is considerably lower than the faecal digestibilityobserved here (Moughan and Smith, 1985; Green, 1988). This means that the micro-organisms of the large intestine still break down an important part of the nitrogen products from the small intestine into ammonia. This ammonia then passes into the blood and is eliminated •
•
.
•
•
.
.
•
•
J "
52
P. LETERME ET AL.
via the urine as urea (Zebrowska, 1973; Just et al., 1981 ). Nevertheless, when these bacteria have at their disposition a fermentable source of energy, they can use ammonia for their own synthesis. In this case, nitrogen is not absorbed by the large intestine but is excreted in the faeces as bacterial protein (Just et al., 1981; Misir and Sauer, 1982 ), thus increasing the faecal nitrogen output and decreasing the apparent digestibility of protein. Moreover, as suggested above, the starch of Progreta may be less well digested in the small intestine than that of Finale. What is more, pea fibres are very digestible for pigs (Lund and Hakansson, 1986; H15dversson, 1987a,b) and they contribute to the energy input in the large intestine. In our case, Progreta is richer in cell walls, and these are better digested than those of Finale. The greater digestion of the Progreta carbohydrates in the pig's large intestine should allow microbial multiplication and encourage the faecal excretion of microbial protein (Misir and Sauer, 1981, 1982; MalmlSf and Hakansson, 1984). It is thus preferable to determine the ileal protein digestibility of the two varieties. (6) Trials carried out on chickens, considered more sensitive than pigs, have shown that trypsin inhibitors had practically no effect on growth performance (Johns, 1987) and influenced only slightly the protein digestibility (Carr~ and Conan, 1989). What is more, Grosjean et al. (1989) have shown recently that, when one considers the digestible energy value and when one increases the addition of synthetic tryptophan and methionine to the diet, it is possible to obtain with diets containing 30% winter peas, a performance practically identical to that obtained with spring peas. Trials by Johns (1987) on chickens and by Grosjean et al. (1989) on pigs show that animal performance is essentially linked to the limiting amino acids content (tryptophan, S-amino acids) of peas and to the ileal digestibility. More recently, we have learned that the ileal digestibility of tryptophan and cystine in peas is particularly low in pigs (Moughan and Smith, 1985; Green, 1988; Buraczewska et al., 1989). CONCLUSIONS The TIA of peas is very variable from one type of pea to another and seems essentially to be a genetic problem. Its influence on protein digestibility by pigs has not been clearly demonstrated. Trials should be carried out on purified protease inhibitors, and measurements should be made of digestibility to the ileum. Meanwhile, in order to prevent poor growth from peas rich or poor in protease inhibitors, it is advisable to supplement the diet with synthetic tryptophan and methionine, the two limiting amino acids in pigs. ACKNOWLEDGEMENTS The authors wish to thank Mr. Cors from the Crop Husbandry Department of the Faculty, and all the seed breeders and the livestock food producers for
TRYPSININHIBITORSIN PEAS
53
the kind supply of the seeds. The expert technical assistance of Ch. Bodart, L. Givron and Th. Monmart is gratefully acknowledged. This study was supported by the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (IRSIA), Brussels, Belgium.
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