Trypsin inhibitor activity of legume seeds (peas, chickling vetch, lentils, and soya beans) as affected by the technique of harvest

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1 Animal Feed Science and Technology 86 (2000) 261±265 Research note Trypsin inhibitor activity of legume seeds (peas, chickling vetch, lentils, and soya beans) as affected by the technique of harvest E. Pisulewska a, P.M. Pisulewski b,* a Department of Crop Production, Agricultural University of Cracow, al. Mickiewicza 21, Cracow, Poland b Department of Human Nutrition, Agricultural University of Cracow, al. Mickiewicza 21, Cracow, Poland Received 4 November 1999; received in revised form 3 May 2000; accepted 8 June 2000 Abstract The effect of two techniques of harvest (a direct versus two-phase) applied to ve legume plants (peas, chickling vetch, lentils, and soya beans) on gross chemical composition and trypsin inhibitor activity (TIA) of their seeds was studied in eld experiments, over a 3-year period. The chemical composition of legume seeds re ected only interspecies differences between the plants and was not affected by the treatments. In contrast, TIA of the seeds was signi cantly (P < 0:05) altered by the techniques of harvest. The two-phase technique reduced TIA of pea cultivars by 27 and 44%, chickling vetch cultivars by 25 and 18%, a lentil cultivar by 8%, and soybean cultivars by 4 and 2%, respectively. It is concluded that improvements in nutritional value resulting from this agricultural practice are limited to pea and chickling vetch cultivars, whereas in the case of lentils and soya beans, the relative reduction of TIA is too small to consider the proposed method feasible. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Legume seeds; Harvesting techniques; Trypsin inhibitor activity 1. Introduction Legume seeds are considered to be an important source of protein in animal and human nutrition. More recently, they have received particular attention as the only viable alternative to meat-based diets considered to have negative effects on human health (Bingham, 1999). However, the utilisation of legume seeds in animal and human nutrition * Corresponding author. Tel.: ; fax: address: rrpisule@cyf-kr.edu.pl (P.M. Pisulewski) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (00)00167-X

2 262 E. Pisulewska, P.M. Pisulewski / Animal Feed Science and Technology 86 (2000) 261±265 is still limited by the presence of antinutritional factors that decrease their nutritional value. Of these factors, trypsin inhibitors strongly inhibit trypsin activity, induce hypersecretion of pancreatic enzymes (trypsin), stimulate pancreatic hypertrophy, and nally reduce digestion and absorption of dietary protein. These negative alterations in digestive functions result in lower retention of nitrogen and sulphur, and impaired growth of experimental animals (Norton, 1991). At the same time, the interesting feature is anticarcingenic potential of trypsin inhibitors, addressed in relevant papers (Kennedy, 1998). The level of trypsin inhibitor activity (TIA) in legume seeds may be reduced by several small-scale processing methods of which thermal treatments such as soaking, boiling, and autoclaving (Vidal-Valverde et al., 1997), and more recently extrusion cooking (Alonoso et al., 2000), are the most common. However, these processing methods are not suitable for industrial production of legume seeds; the only exception being the large-scale industrial production (the oil extraction process) of heat-treated soya beans. The purpose of the present study was to compare the effect of two techniques of harvest (i.e. a direct versus two-phase) on gross chemical composition and TIA of pea, chickling vetch, lentil, and soybean seeds, obtained in eld experiments. In addition, the latter technique was considered to be a large-scale method which might be potentially used to supply substantial quantities of legume seeds with the reduced level of antitrypsin activity. 2. Materials and methods Samples of seeds of peas (cv. Tegma and cv. Granit), chickling vetch (cv. Derek and cv. Krab), lentils (cv. Nelka), and soya beans (cv. Aldana and cv. Nawiko), were obtained from 3-year eld experiments (1996±1998) carried out at the Prusy Experimental Station, located 20 km north-east of Cracow. The soil type was loess-derived chernozem. The experiments were two-factorial involving seven grain legume cultivars and two different harvest techniques (either a direct or two-phase technique) with three replications for each cultivar and harvest technique combination. Individual plots were 1:25 m 8 m each. Before sowing, all plots received the same fertiliser treatment, i.e. 30 kg N, 70 kg P 2 O 5, and 120 kg K 2 O per hectare. The seeds, treated with fungicide Funaben T, were sown at the end of March/the beginning of April (peas, chickling vetch, lentils) or at the end of April/the beginning of May (soya beans). The seeding rate was either 100 seeds/ m 2 (peas, chickling vetch, lentils) or 70 seeds/m 2 (soya beans). At the harvest, the plots were harvested either directly with a combine harvester or the plants were pulled manually and bundled together. Both threshed seeds and bundled plants (seeds in pods) were stored in the same conditions, in a well-aired experimental barn, for 6 weeks. This method of storage prevented the development of moulds during the drying period. At the end of storage, the legume plants were threshed using the combine harvester and the seeds were ground (1095 Knifetec Mill, Foss Tecator) before chemical analyses. Gross chemical composition of composite samples of seeds (three replicates) was determined using the standard AOAC (1995) methods. The same samples were used to determine TIA of seeds (Kakade et al., 1974).

3 E. Pisulewska, P.M. Pisulewski / Animal Feed Science and Technology 86 (2000) 261± The data were analysed using ANOVA for a two-factorial design. A least signi cant difference (LSD 0.05 ) was used to test the effects of treatments when the F-test was statistically signi cant at P < 0: Results and discussion The growing use of plant proteins in animal and human nutrition results in high demand for legume seeds. However, the wider use of these seeds is still impaired by the presence of several antinutritional factors which may exert adverse effects on the animal/ human consumer. Indeed, even heat-treated legume seeds (e.g. soya beans) may still contain 20% of residual TIA, and thus, affect negatively pancreatic functions in both animals and humans, including the risk of carcinogenesis in rats (Friedman et al., 1991). Equally, extrusion cooking, considered to be the best method to inactivate trypsin and chymotrypsin inhibitors in legume seeds (Alonoso et al., 2000), was reported to leave residual activity of these inhibitors in processed soybean protein isolate (van Amerongen et al., 1998). Therefore, the need is for large quantities of legume seeds and large-scale processing methods reducing the content of antinutrients without affecting the nutritional value of these seeds. In this context, the present results suggest a two-phase technique of harvest of legume plants as the original solution to the above problem. First, this simple approach does not affect the chemical composition of seeds (Table 1). Although the data were not analysed statistically, gross chemical analysis of composite samples of peas, chickling vetch, lentils, and soya beans (Table 1), re ected only interspecies differences between the Table 1 Mean gross chemical composition (g/kg) of legume seeds obtained in 3-year eld experiments (1996±1998) as affected by legume species and techniques of harvest Species Cultivars Techniques of harvest a Dry matter Crude protein Ether extract Crude fibre Ash Peas Granit Tegma Chickling vetch Derek Krab Lentils Nelka Soya beans Aldana Nawiko a 1: A direct technique of harvest; 2: a two-phase technique of harvest.

4 264 E. Pisulewska, P.M. Pisulewski / Animal Feed Science and Technology 86 (2000) 261±265 Table 2 Mean level of trypsin inhibitor activity (TIU g 1 ) in legume seeds obtained in 3-year eld experiments as affected by legume species and techniques of harvest a Species Cultivars Techniques of harvest b Means for cultivars 1 2 Peas Granit Tegma Chickling vetch Derek Krab Lentils Nelka Soya beans Aldana Nawiko Mean a LSD 0.05 for cultivars: 3051; LSD 0.05 for techniques of harvest: 299; LSD 0.05 for interaction: b 1: A direct technique of harvest; 2: a two-phase technique of harvest. plants and was typical for their seeds (Pisulewska et al., 1998). Second, TIA of legume seeds (Table 2) was signi cantly (P < 0:05) affected by both legume species and techniques of harvest. As expected, the highest TIA was detected in soya beans, and it was gradually decreasing, and in this order, in chickling vetch, peas, and lentils (Pisulewska et al., 1998). It was also found, that a small-seed soybean cv. Nawiko showed signi cantly (P < 0:05) higher antitrypsin activity than a large-seed soybean cv. Aldana. Of the methods of harvesting, the two-phase technique gave signi cantly (P < 0:05) lower TIA of seeds than the direct method, and this effect was consistent for all legume species under study. When compared with the activity determined in seeds harvested directly, the two-phase technique reduced TIA of pea cultivars by 27 and 44%, chickling vetch cultivars by 25 and 18%, a lentil cultivar by 8%, and soybean cultivars by 4 and 2%, respectively. Moreover, the two-phase technique was particularly effective (P < 0:05) when applied to chickling vetch, as indicated by the LSD 0.05 value for interaction. In spite of consistent reduction in TIA activity, the two-phase technique can be considered effective only for pea and chickling vetch cultivars, and not for lentils and soya beans. Besides, in the case of soya beans the traditional heat-treatment of these seeds (the oil extraction process) is assumed to be suf cient for reducing the content of trypsin inhibitors. The consistent reduction in TIA in legume seeds, as resulting from their prolonged storage in pods (e.g. for 6 weeks), has not been reported so far in available literature. Instead, trypsin inhibitors were demonstrated to be seed protective factors against endogenous proteolytic enzymes, and to play a defensive role against micro-organisms, fungi, and insects in legume seeds (Norton, 1991). In conclusion, a simple agricultural approach, i.e. a two-phase technique of harvest, seems to be a suitable method to produce large quantities of legume seeds with reduced, at least partly, TIA. At the same time, the practical application of this method is limited to pea and chickling vetch cultivars, whereas in the case of lentils and soya beans the

5 E. Pisulewska, P.M. Pisulewski / Animal Feed Science and Technology 86 (2000) 261± relative reduction of TIA, although signi cant (P < 0:05), has no practical meaning. Further studies on the effects of a two-phase technique of harvest on nutrient and antinutrient composition of legume seeds are needed. In addition, biological trials should be performed in order to correlate the analytical data with the subsequent nutritive value of these seeds. References Alonoso, R., Aguirre, A., Marzo, F., Effects of extrusion and traditional processing methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney beans. Food Chem. 68, 159± 165. AOAC, Of cial Methods of Analysis, 16th Edition. Association of Of cial Analytical Chemists, Arlington, VA. Bingham, S.A., High-meat diets and cancer risk. Proc. Nutr. Soc. 58, 243±248. Friedman, M., Brandon, D.L., Bates, A.H., Hymowitz, T., Effects of heat on the nutritional quality of soybean cultivars. In: Friedman, M. (Ed.), Nutritional and Toxicological Consequences of Food Processing. Plenum Press, New York, London, pp. 339±361. Kakade, M.L., Rakis, J.J., McGhee, J.E., Puski, C., Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem. 51, 376±382. Kennedy, A.R., The Bowman±Birk inhibitor from soya beans as an anticarcinogenic agent. Am. J. Clin. Nutr. 68, 1406S±1412S. Norton, G., Proteinase inhibitors. In: D'Mello, J.P.F., Duffus, C.M., Duffus, J.H. (Eds.), Toxic Substances in Crop Plants. The Royal Society of Chemistry, Cambridge, pp. 68±106. Pisulewska, E., Pisulewski, P., Hanczakowski, P., Szymczyk, B., Current methods of plant protein quality evaluation as applied to grain legumes. In: Proceedings of the Conference on Legume Seeds as Protein Sources. III. Lentils and Soya Beans. Lublin Agricultural University, Lublin, pp. 71±76 (in Polish). van Amerongen, A., Ostafe, V., Meijer, M.M.T., Gruppen, H., Meerdink, G., Berendsen, L.B.J.M., Koets, M., Wichers, J.H., Speci c-immuno-(chymo)trypsin-inhibitor-assays for determination of (residual) activity of Bowman-Birk or Kunitz soybean trypsin inhibitors. In: Jansman, A.J.M., Hill, G.D., Huisman, J., van der Poel, A.F.B. (Eds.), Recent Advances of Research in Antinutritional Factors in Legume Seeds and Rapeseed. Wageningen Pers, Wageningen, pp. 33±37. Vidal-Valverde, C., Frias, J., Diaz-Pollan, C., Fernandez, M., Lopez-Jurado, M., Urbano, G., In uence of processing on trypsin inhibitor activity of faba beans and its physiological effect. J. Agric. Food Chem. 45, 3559±3564.