Maataloustieteellinen A ikakauskirja Vol. 57: 117—123, 1985 Leaf protein from green pulse crops and nutritive value of legume protein concentrates for poultry MATTI NASI and TUOMO KIISKINEN Department of Animal Husbandry University of Helsinki SF-00710 Helsinki Department of Animal Husbandry Agricultural Research Centre SF-31600 Jokioinen Abstract. Leaf protein concentrate (LPC) samples were prepared from four pulse crops, goat’s rue, pea, field bean and Persian clover. Their proximate composition was 42.5 — 53.4 % crude protein, 2.1 —7.9 % ether extract and 1.0—3.0 % crude fibre. The lysine con- tent was 4.1—4.8 g/16 g N, that of sulphur amino acids 2.0—2.8 g and that of threonine 4.5—4.8 g. The contents of tannins varied from 2.2 to 5.5 %. The nutritional values of the LPC samples were assesed in digestibility and balance trials with male chickens of 16—18 weeks, LPC composing 25 % of the diets. LPC from pea had the highest digestibilties of organic matter (70.3 %), crude protein (77.6 %) and carbohydrates (54.9 %), while LPC from Persian clover had the lowest (P < 0.01). The true digestibilities of crude protein for goat’s rue, pea, field bean and Persian clover were, respectively, 70.0, 82.2, 69,7 and 56.8 % (P < 0.01). The digestibilities of nutrients in the present LPC samples were rather low com- pared to their minimal crude fibre content. A reason for this may be the contents of tannins. The AMEn values for the LPC’s of goat’s rue, pea, field bean and Persian clover were, respectively, 10.79, 13.15,9.80 and 9.18 (P < 0.01). The gross energy metabolization ranged from 42.6 to 57.0 %. Introduction The increasing cost of nitrogenous fertil- izers and the need to develop domestic sources of protein for animal consumption have renewed interest in pulse crops in Fin- land. Peas and field beans have been found to be good sources of protein for pigs and poultry (Alaviuhkola 1979, Kiiskinen 1982). The use of legumes in silages has also been investigated, but the effluent losses have been marked (Syrjälä-Qvist et ai. 1984). These losses could be avoided and the pro- tein in plant tissues utilised for monogastrics by combining silage making with leaf protein production by extracting protein from the crop prior to ensiling. The true digestibility and biological value of the leaf protein con- centrates (LPC) obtained from some green crops have been found to be high when the Index words: Leaf protein, protein supplement, poultry nutrition, digestibility and metabolizable energy value 117 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=NhA2S_fOvznyzl5B.X7uIFkm9Y_IitwnZGTGyfw.hTBo0A8gWZoYHY6yCdimnHLgZpCHg5Ug2wdTrdx-5RxLq-qwOI4isIVg8clxz_bSgftdOm3WELK8T9T2TWgPs8jJcLupI5ZUcGfa-pMDQpKEKTP9pcj_UJI7ibClzaDotcRaAaF_QWLy17CGDwvxOhInm6SHAeHVZMYh2EacXZ0VRBG9CFC3wahoahw LPC was prepared under optimal conditions (Morris 1977, Pirie 1978). LPC from lucerne has given performance equal to that ob- tained with soybean meal with poultry (Kuz- micky and Kohler 1977) and pigs (Cheeke et al. 1977). In diets for poultry LPC has value as a source of pigment (Yoshida and Hoshii 1981), since its xanthophyll content is high. The objective of the present study was to investigate the leaf protein production of va- rious legumes and the nutritive value of leaf protein concentrate in poultry nutrition. Materials and methods Four green pulse crops were used as raw material for LPC production. Goat’s rue (Galega orientalis), pea (Pisum sativum), field bean {Vida faba), and Persian clover (Trifolium resupinatum) were cut with a harvester on experimental plots. Goat’s rue had been sowed two years before and the other crops in the same growing season. The fertilizer application was 500 kg trace PK per ha. Extraction involved pulping the green ma- terial, pressing out the juice, and separating the leaf protein from the juice by heat coagu- lation, the techniques being similar to those described by Näsi (1983 a). For the feed evaluation tests, the curd was dried in an oven at 65°C. The analytical methods were the same as used in previous leaf protein ex- periments (Näsi 1983). The feed evaluation was made with 36 WL male chickens, age 16—18 wk and initial weight 1610—1676 g, kept in individual cages. Each treatment comprised six cocks. Besides the basic and experimental groups, there was a fasting group, which was kept without feed for 1.5 days, after which collec- tion was performed for three days. The daily allowance of feed was 100 g and the feed in- take was measured daily. The basic diet (No 1) was composed of barley meal 96 % and premix 4 ®/o. The premix included dical- cium phosphate 39 %, calcium carbonate 20 %, sodium chloride 10 %, trace element and vitamin premixes 25.0 %, L-lysine 3 % and DL-methionine 3 %. The leaf protein concentrates were included in the basic diet at the level of 25 %. The birds were allowed to adapt to the diets for five days, after which the excreta were collected daily, for five days. Details of the procedures are presented by Kiiskinen and Huida (1984). The nutrient digestibilities of the test feeds and their ME values were calculated from the differences between the values obtained for the basic and test diets by the indirect method of total excreta collection. ME was corrected for the nitrogen balance MEN using 36.55 kJ/g retained nitrogen (Titus 1959). The results were evaluated statistically using the analysis of variance. The signifi- cance of differences between the test in- gredients was assessed by the t-test. Results and discussion The protein yields of the goat’s rue and pea crops were high, 760 —790 kg/ha while field bean and Persian clover gave 330— 500 kg protein/ha (Table 1). The dry matter and protein yields corresponded to the fig- ures given by Syrjälä-Qvist et ai. (1984). On average, 57 % juice was expressed from the green crop. The crude protein and true pro- tein extraction ratios averaged 37 and 33 %; the ratios of the pea crop exceeding the others (Table 1). The values were higher than the extractions for grass or clover (Näsi 1983 a). The crop moisture content has the greatest effect on the recovery of juice and its components (Houseman and Jones 1978). By heating the juice in leaf protein concentrate could be precipitated 58 % of the crude pro- tein and 85 % of the true protein of the juice. These values accord with those of previous experiments with clover and pea, exceeding the values of grass (Nasi 1983 a). The chemical composition and in vitro digestibilities of the crop and pulped pressed crop are compared in Table 2. The DM con- tent was almost as twice high in the pulped 118 Table 1. Outline of the experiments, extraction ratios of plant juice and its components from various crops and separation ratios of leaf protein concentrates and its components precipitated from plant juice. Goat's Pea Field Persian rue bean clover Outline of the experiments Cutting date 21. 6. 1982 20. 7. 1982 22. 7. 1982 28. 7. 1982 Fresh yield tn/ha 18.6 19.0 21.2 12.6 DM yield kg/ha 3704 3610 2967 2000 Protein yield kg/ha 760 786 499 329 Pressed amount, kg 435 651 531 302 Extraction ratios, % Juice 45.9 58.4 65.6 57.1 Dry matter 19.1 32.3 31.6 28.8 Ash 35.6 38.3 41.9 39.7 Crude protein 28.6 49.3 36.8 32.8 True protein 25.9 46.3 28.0 30.5 Separation ratios, % LPC 19.0 21.2 8.9 17.7 Dry matter 35.9 45.5 16.4 29.2 Ash 20.4 31.6 10.7 18.5 Crude protein 60.8 73.2 38.6 66.1 True protein 77.0 110.0 64.1 88.6 Table 2. Compositions and in vitro -digestibility of crop and pulped pressed crop (% of DM). DM Ash Crude True Ether Crude NFE Water Pepsine In vitro protein protein extract fibre soluble HCI __»._. . . ~ DOMDcarbo- soluble hydrate protein Goat's rue crop 19.9 8.2 20.5 15.6 2.3 23.5 45.4 7.3 71.8 71.3 pulp 30.3 5.8 18.0 14.0 2.1 30.2 43.9 4.7 68.5 66.4 Pea crop 15.1 9.4 21.8 13.1 3.2 22.8 42.8 19.5 87.2 77.8 pulp 27.3 8.0 16.5 10.9 3.1 30.4 42.0 9.1 80.5 73.1 Field bean crop 14.0 9.9 16.8 12.0 2.1 24.4 46.8 19.0 83.9 77.6 pulp 28.7 6.3 13.4 11.1 2.3 33.7 44.2 8.3 73.6 69.3 Persian clover crop 15.9 11.7 16.5 11.8 3.1 17.8 51.0 15.6 83.5 80.2 pulp 26.7 8.9 16.0 12.8 3.2 23.6 48.4 8.1 77.3 75.0 Mean Crop 16.2 9.8 18.9 13.1 2.7 22.1 46.5 15.4 81.6 76.7 Pulp 28.3 7.3 16.0 12.2 2.7 29.5 44.6 7.6 75.0 71.0 pressed crop and crude fibre one third higher, while the protein content was re- duced by only 15 °?o compared with the origi- nal crop. The pulp could be considered suitable material for ensiling and its use would eliminate effluent losses, which have been found to be rather high in the case of pea and field bean used directly after cutting (Syrjälä-Qvist et ai. 1984). The plant juices contained DM 6.8—8.3 % and the crude protein content was 31—33 % in goat's rue or pea and 19—20 % in bean and clover (Table 3). True protein composed on average 61 % of the crude protein in the juices. The sugar content of the juices was high. Näsi (1983 b) reported that pea juice deteriorated 119 120 Table 3. The composition of plant juices extracted from various legumes (in percentages). Goat's Pea Field Persian rue bean clover Dry matter 8.31 8.33 6.84 7.99 Ash 1.27 0.93 0.90 1.29 in DM 15.3 11.2 13.2 16.2 Crude protein 2.55 2.77 1.34 1.50 in DM 30.6 33.2 19.6 18.7 True protein 1.75 1.56 0.73 1.00 in DM 21.1 18.7 10.7 12.5 Water soluble carbohydrates 1.70 3.57 3.14 2.79 in DM 20.5 42.9 45.9 34.9 very rapidly, pulse crop juices thus being dif- ficult to use in animal feeding. Goat’s rue and pea LPC had a protein content of 52—53 °7o, while LPC derived from field bean or Persian clover had a lower value, 42—46 °7o of DM. The crude fat con- tent was found to be higher when the ether extraction was preceded by hydrolysis with HCI; Table 5 presents the values of the latter method. There were no great differences in amino acid composition between the sources of LPC. The contents of essential amino acids of the presents LPC samples were lower than the values reported for lucerne LPC (Connell and Foxell 1976, Hancza- kowski and Skraba 1984), but correspond to the values for alfalfa given by Cheeke et al. (1977). In all the samples in vitro digestibility and solubility in pepsin HCI were high, 84— 89 °7o. KuzMicKvand Kohler (1977) reported in vitro crude protein digestibilities for four lucerne LPC (Pro-Xan) samples ranging from 87.8 to 97.2 %. The chemical composition and gross ener- gy values of the leaf protein concentrates and the diets are shown in Table 5. GE was higher in pea LPC than in the other con- centrates. Diet no. 1, the basic diet, had a slightly lower energy content than the others. The group of cocks receiving diet no. 3, containing of pea LPC, consumed less (P < 0.05) feed mixture than the others (Table 6). The reason for this is uncertain, but the animals were a little lighter than tho- se in other treatments. The digestibility of the crude protein of the pea LPC was 77.6 %, which was significantly higher than in the other LPC products (P < 0.05). Carbohydrates and organic matter were also more digestible in pea LPC (P < 0.05). Persian clover LPC had the lowest nutrient digestibilities (P < 0.05). In the in field bean and Persian clover products the tannin con- tents were higher (5.5 —3.0 %) than in pea or goat’s rue LPC (2.7—2.2 %) and this is one probable reason for the differences in diges- tibility. With peas and field beans (tannins 1.8—2.3 % of DM) Lindgren (1975) found a strong correlation between the digestibility Table 4. The chemical composition and in vitro digesti- bility of leaf protein concentrates from various legumes. Goat's Pea Field Persian rue bean clover Chemical composition, % of DM Dry matter 15.7 15.0 12.7 13.2 Ash 8.7 7.8 8.6 10.2 Crude protein 52.0 53.4 46.1 42.5 True protein 45.2 45.3 41.7 38.0 Ether extract 4.6 7.3 2.1 7.9 Crude fibre 1.0 2.4 3.0 2.5 NFE 33.7 29.1 40.2 36.9 Tannins 2.2 2.7 5.5 3.0 Amino acids g/16 g N Alanine 5.1 5.2 5.3 5.6 Arginine 5.6 5.5 5.6 5.9 Aspartic acid 13.4 9.4 13.1 10.8 Cystine 0.9 1.2 1.0 1.1 Glutamic acid 10.4 10.0 10.6 10.7 Glycine 4.5 4.6 4.8 5.1 Histidine 1.9 2.1 2.1 2.3 Isoleucine 3.9 4.0 4.1 4.2 Leucine 7.9 8.3 8.5 8.9 Lysine 4.8 4.3 4.1 4.4 Methionine 1.1 1.6 1.5 1.2 Phenylalanine 4.8 5.1 5.2 5.6 Serine 4.4 4.0 4.1 4.3 Threonine 4.7 4.5 4.6 4.8 Tyrosine 3.4 3.9 3.8 4.0 Valine 5.2 5.2 5.0 5.5 In vitro digestibility Organic matter, % 89.5 83.5 86.4 87.2 Pepsin HCI soluble protein, %of CP 86.4 88.6 87.1 85.3 121 Table 5. The chemical composition of the LPC’s and diets used in the feed evaluation (% of DM). Dry Crude Crude Crude Ash GE matter protein fat fibre MJ/kg DM Leaf protein concentrate Goat's rue 97.5 53.9 8.1 0.6 9.0 22.33 Pea 93.2 54.6 10.7 1.0 8.0 23.08 Field bean 91.7 46.8 7.7 0.9 8.6 22.00 Persian clover 92.9 40.5 10.1 0.6 11.2 21.55 Experimental mixtures Mix no 1 (basic) 86.3 11.4 3.3 4.1 4.6 18.00 Mix no 2 89.3 23.2 4.7 2.9 6.2 19.16 Mix no 3 88.1 22.0 5.1 3.2 5.9 19.22 Mix no 4 87.8 20.4 4.5 3.2 6.7 18.84 Mix no 5 88.1 19.2 5.0 3.4 7.3 18.79 coefficient of crude protein and the percent- age of tannins in crude protein. Leguminous seeds have given protein digestibilities from 73.3 % (Kiiskinen and Huida 1984) to 80—88 % (Lindgren 1975, Askbrant and Hakansson 1984), but they have a higher Table 6. Dry matter intake, nitrogen balance, digestibilities of nutrients and calculated ME values (mean + s.d.). Goat's Pea Field Persian Basic rue bean clover diet Dry matter 393.3 a 292.9 b 395.1 a 376.4 a 385.8 intake, g/d 66.4 56.7 19.9 26.5 26.2 N balance 9.8 7.4 7.3 8.6 6.8 mg/kg W 1.8 1.3 2.1 0.9 1.1 Apparent digestibility, % Crude protein 68.0 e 77.6 d 67.6 e 54.0 f 67.1 1.6 4.6 3.5 3.5 2.6 Ether extract 70.5 72.2 68.0 66.6 57.3 2.9 2.9 4.5 4.0 1.2 Carbohydrates 38.6 b 54.9 a 39.7 b 43.5 b 81.2 6.5 6.2 7.2 4.3 0.73 Organic matter 58.8 e 70.3 d 56.4 e 50.9 f 77.0 2.1 4.2 3.4 3.1 0.44 True digestibility, % Crude protein 70.0 e 82.2 d 69.7 e 56.8 f 75.5 2.1 4.6 3.7 3.2 2.2 Metabolizable energy contents, MJ/kg DM Apparent AME class 11.42 e 13.48 d 10.12ef 9.72 f 13.68 0.09 0.62 0.61 0.45 0.07 AMEN 10.79 e 13.15 d 9.80ef 9.18 f 13.43 0.27 0.55 0.55 0.56 0.09 True TME 12.04 e 14.83 d 10.67ef 10.39 f 14.27 0.46 0.54 0.58 0.43 0.08 TME N 11.41 e 14.50 d 10.35ef 9.89 f 14.03 0.29 0.54 0.52 0.50 0.10 AMEN %of GE 48.3 57.0 44.5 42.6 74.6 Means with different letters were significantly different, a—c (P < 0.05); d—f (P < 0.01). crude fibre content, 7 —9 % of DM as com- pared to I—31 —3 °7o in the LPC samples. The lower digestibility values of LPC pro- ducts compared with those of seeds of pulse crops are evidently connected with some pro- perties of the leaf proteins or some antinutri- tional substances in the products. From a survey of numerous protein quali- ty trials, Morris (1977) concluded that LPC had a satisfactory digestibility and gave bio- logical values and protein efficiency ratios which were consistent with its known amino- acid composition. From rat trials Saunders et al. (1973) and Bickoff et al. (1975) re- ported in vivo protein digestibilities in rat trials to ranging from 81—91 and 86 to 94 % for lucerne LPC and the true digestibility values obtained by Hanczakowski and Skra- ba (1984) for the crude protein of LPC samples prepared by different treatments ranged from 76 to 88 %. These data are in agreement with the values received for the present pea LPC but exceed the values for the other LPC products. Methionine supple- References Alaviuhkola, T. 1979. Herne ja härkäpapu lihasikojen rehuna. Sikatalouskoeaseman tiedote N:o 2. 13 p. Hyvinkää. Askabrant, S. & Hakansson J. 1984. The nutritive value of rapeseed meal, soya bean meal and peas for laying hens. Swedish J. agric. Res. 14: 107—110. Bickoff, E.M., Booth, A.N., de Fremery, D., Edwards, R.H., Knuckles, 8.E., Miller, R.E., Saunders, R.M. & Kohler, G.O. 1975. Nutritional evaluation of leaf protein concentrate. Protein nutri- tional quality of foods and feeds. Dekker, New York, pp. 319—340. Cheeke, P.R., Kinzell, J.H., de Fremerv, D. & Koh- ler, G.O. 1977. Freeze-dried and commercially -pre- pared alfalfa protein concentrate evaluation with rats and swine. J. Anim. Sci. 44: 772 —777. Connell, J. & Foxell, P.R. 1976. Green crop frac- tionation, the products and their utilization by cattle, pigs and poultry. Bienn. Rev. Natn. Res. Dairy 21: 21—41. Hanczakowski, P., Skraba, B. & Hanczakowska, E. 1981. Nutritive value of lupin-seed protein and leaf- mentation have improved the biological va- lue of LPC lucerne protein in a study with chicks (Hanczakowski et al. 1981). The metabolizable energy values for pea LPC exceeded those for the other LPC pro- ducts (P < 0.01; Table 6). The AMEN va- lues of the various pulse crop LPC’s, 9.2 13.2 MJ/kg DM, are sufficiently high to make these products suitable for poulty feeding. The values for pea LPC are promis- ing. The ME values reported for some legu- minous seeds are of the same size order as those for the present LPC products (Lind- gren 1975, Kiiskinen and Huida 1984, Ask- brant and Hakansson 1984). The results presented in this paper indicate that leaf protein from pulse crops can be utilized as a protein supplement in poultry rations, since the amino acid composition is rather promising and the digestibility and metabolization are fairly good, especially in the case of pea LPC. The protein yields and juice extraction from pea were higher than from the other crops. protein concentrates supplemented with various sulphur sources. Anim. Feed Sci. Technol. 6: 189— 195. & Skraba, B. 1984. The effect of different precipi- tating agents on quality of leaf protein concentrate from lucerne. Anim, Feed Sci. Technol. 12: 11—l7. Houseman, R.A. & Connell, J. 1976. The utilization of products of green-crop fractionation by pigs and ruminants. Proc. Nutr. Soc. 35: 213—220. Kiiskinen, T. 1982. Kotimaisten siemen- ja yksisoluval- kuaisrehujen ravintoarvo jakäyttömahdollisuudet sii- pikarjan ruokinnassa. Lisensiaattityö Helsingin yli- opisto, kotieläintieteen laitos. 145 p. & Huida, L. 1984. Metabolizable energy value and digestibility of some protein sources for poultry. Ann Agric. Fenn. 23: 26—38. Kuzmickv, D.D. & Kohler, G.O. 1977. Nutritional value of alfalfa leaf protein concentrate (ProXan) for broilers. Poult. Sci. 56: 1510—1516. Lindgren, E. 1975, The nutritive value of peas and field beans for hens. Swedish J. agric. Res. 5: 159 161. 122 Morris, T.R. 1977. Leaf-protein concentrate for non- ruminant farm animals. Occ. Symp. Br. Grassl. Soc. No 9: 67—82. Näsi, M. 1983 a. Extraction of leaf protein from green crops. Chemical composition and nutritive value of products of fractionation. J. Scient. Agric. Soc. Finl. 55: 143—154. - 1983 b. Preservation of grass juice and wet leaf pro- tein concentrate for animal feeds. J. Sclent. Agric. Soc. Finl. 55: 465—475. Pirie, N.W. 1978. Leaf protein and other aspects of fodder fractionation. 169 p. Camb. Univ. Press. London. Saunders, R.M., Connor, M.A., Booth, A.N., Bic- koff, E.M, & Kohler, G.O. 1973. Measurement of SELOSTUS Vihantapalkokasveisla saatu lehtivalkuaistii- viste siipikarjan rehuna Matti Näsi ja Tuomo Kiiskinen Helsingin yliopisto, kotieläinlieleen laitos, 00710 Helsinki Maatalouden tutkimuskeskus, kolieläinhoito-osaslo 31600 Jokioinen Tutkimuksessa selvitettiin lehtivalkuaisen eristämistä vuohenherneen, herneen, härkäpavun ja persianapilan vihantakasvustoista ja määritettiin saatujen palkokasvi- tiivisteiden sulavuus ja muuntokelpoinenenergia 16—18 viikon ikäisillä kukoilla kokonaiskeruumenetelmällä. Vesipitoisista vihermassoista saatiin keskimäärin 57 % mehua tuorepainosta. Palkokasvimehut sisälsivät kuiva-ainetta 7.9 % keskimäärin sekä kuiva-aineessa 25.5 % raaka-valkuaista ja 36.1 % sokereita. Lehtival- kuaisissa oli keskimäärin valkuaista 42.6 %, rasvaa 5.5 % ja raakakuitua 2.2 °/o. Lysiiniä oli 4.1—4,8, rikki- pitoisia aminohappoja 2.0—2.8 ja treoniiniä 4,5—4.8 g/16 g N. Valkuaisen pepsiini-HCI liukoisuus oli kor- digestibility by in vivo and in vitro methods. J. Nutr. 103: 530—535. Syrjälä-Qvist, L., Pekkarinen, E. & Setälä, J. 1984. Vihanta herne ja härkäpapu sekä niiden viljaseos säi- lörehun raaka-aineena. Kotieläintieteen laitoksen tie- dote No 4. 55 p. Titus, H.W., Mehring, A.L. Jr., Johnsson, D., Nes- bitt, L.L. & Tomas, T. 1959. An evaluation of M.C.F. (Micro-Cel-Fat), a new type of fat product. Poult. Sci. 38: 1114—1119. Yoshida, M. & Hoshii, H. 1981. Leaf protein con- centrates. Poult. Int. Nov. 106—110. Ms received January 22, 1985 kea,885.88.63 —88.6 %. Hernelehtivalkuaisen sulavuudet olivat merkitsevästi korkeampia kuin muiden, kun taas persianapilan lehtivalkuainen suli huonommin kuin toi- set, Raakaproteiinin todelliset sulavuudet olivat: 70.0 % vuohenherne, 82.2 % herne, 69.7 % härkäpapu ja 56.8 % persianapila. Tanniinipitoisuudellaoli vaikutus- ta sulavuuteen. Näennäiset muuntokelpoisen energian (AME n) arvot olivat 10.79, 13.15, 9.88 ja 9.18 MJ/kg ka., vastaavasti. Hernelehtivalkuaisen sulavuus ja ME -arvo ovat tasoltaan sitä luokkaa, että sen käyttö olisi mah- dollista siipikarjanrehuissa. Lehtivalkuaisen tuotanto- kustannukset ovat kuitenkin korkeammat kuin muiden nykyisin käytössä olevien proteiinirehujen. 123