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Use of enzymes for improvement of feed
Animal Feed Science and Technology, 6 (1981) 105--114 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
105
USE O F E N Z Y M E S F O R I M P R O V E M E N T O F F E E D 1
B. REXEN
Biotechnical Institute, Holbergsvej 10, 6000 Kolding (Denmark) (Received 7 May 1979; accepted for publication 12 June 1980)
ABSTRACT Rexen, B., 1981. Use of enzymes for improvement of feed. Anim. Feed Sci. Technol., 6: 105--114. Investigations of the addition of enzymes to traditional feed-mixtures, for improvement of feed utilization and of growth of domestic animals, have been performed. A survey of the literature on the effects of enzyme addition is shown in tabular form. Experiments with rats fed on barley diets, with and without enzyme addition, showed no significant difference between the diets, but a tendency towards feed improvements by the addition of some of the enzymes, e.g., amylase, cellulase end protease. Some of the experiments with chickens fed on a barley-based diet, with the addition of either cellulase and/or peetinase or protease, showed increases in growth of up to 7% (P < 0.05), and improvements in feed utilization of 6% (P ~ 0.01). In one experiment on chickens fed on a barley of better quality than that in the above-mentioned experiments, no improvements were found by adding an enzyme mixture, consisting of cellulase, pectinase and protease, to the feed. The results thus suggest that a better feed-utilization by use of enzyme addition is obtainable only if the feed mixture is composed of less-digestible feed ingredients. The amount of added enzymes strongly influences the price of the resultant feed, and in the experiments showing feed improvement, the break-even price of the enzymes is less than 26 Dkr/kg enzyme. The use of enzymes for making slowly digested and less-expensive products (waste products) applicable as feed components will be examined in future work
INTRODUCTION In D e n m a r k , f e e d i n g costs a c c o u n t f o r 65% o f t h e t o t a l costs o f animal p r o d u c t i o n . T h e r e f o r e , it is i m p o r t a n t t h a t animals utilize t h e feed o p t i m a l l y . I t is possible t o i m p r o v e feed utilization in various w a y s , d e p e n d i n g o n feed q u a l i t y a n d species o f animal. One possibility is t h e a d d i t i o n o f substances s t i m u l a t i n g t h e digestive m e c h a n i s m or inhibiting u n w a n t e d microbial g r o w t h in t h e digestive s y s t e m . In t h e literature, t h e r e are references t o e n z y m e s , ' This investigation was financed by the Danish Technology Board. Two Danish enzyme producers (Novo A/S and Grindstedvmrket A/S) supplied the enzymes and served as consultants on the project. Some biomass and enzymes were obtained from U.S.A. (Worne Biochemicals) and Italy.
0377-8401/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
Silage (grain) Maize and rice hulls Silage (grass) Milk Wheat Maize
Ruminants Feed mixture
Pigs Traditional feed Traditional feed
(barley)
(maize)
(barley)
Pectolases, hemicellulase, cellulase and proteases Proteases, a-amylase and microorganisms Cellulase (fungi) Cellulase Lactobacilli and oxytetracycline Monensin Rumensin (Monensin sodium)
Proteases, amylases and ~-glucanase Proteolytic enzyme (Fradiase)
Proteolytic enzyme (bacterial) a-amylase and proteases ~-glucanase a-amylase a-amylase a-amylase (diastase) Fungal enzyme Monensin and penicillin
Poultry Feed mixture Barley/maize Feed mixture Barley Feed mixture Barley Feed mixture Starting feed
(maize)
Addition
Animal (feed)
-
+
17
7--12
10--20
2.4 10--25 4 14 3 6--12 15 +
Increased growth (%)
4 1 10 Saving of protein
--
13
-
1.4 -5 7 7 4--6 13 +
Improved feed utilization (%)
Improvements of feed value by different additives (according to the literature)
TABLE I
Millen and Harrison, 1970 Daniels and Hashim, 1977 Henderson and McDonald, 1977 Morril et al., 1977 Horn et al., 1977 Gill et al., 1977
Ezdakov, 1976
Ezdakov, 1976 Casteelsand Buy~e, 1976
Kasbaoui and Guillaume, 1976 Ezdakov, 1976 Gohl et al.,1978 Herstad and McNab, 1975 P~rk~ny-Gy~rf~s and T6th, 1978 Moss et al.,1977 Haskell et al.,1960 Luther et al.,1977
References
O
107
biomass, antibiotics,probiotics (preparations containing livingmicroorganisms, especially lactobaciUus preparations) and other preparations, such as Rumensin (monensin). Another possibilityis a pre-treatment ("pre
After a pre-period of 3 days, experiments were performed for 16 days with SPF rats, Wistar Af/Male/Mol (Male 67), a b o u t 4 weeks old. In one experiment, enzymes either cellulase, pectinase, amylase or protease -- were added to barley mixed with vitamins and minerals. Each mixture was given to four rats. -
-
108
In a n o t h e r e x p e r i m e n t , t h e s a m e e n z y m e s w e r e a d d e d , b u t t h e f e e d was t h e n s t e e p e d f o r six h o u r s w i t h 70% m o i s t u r e a n d f r e e z e - d r i e d p r i o r t o feeding. This f e e d was c o m p a r e d w i t h a c o n t r o l feed, also s t e e p e d f o r six h o u r s w i t h 70% m o i s t u r e a n d freeze-dried. G r o w t h a n d f e e d c o n s u m p t i o n w e r e m o n i t o r e d in b o t h e x p e r i m e n t s . E n z y m e b r a n d a n d a c t i v i t y are s h o w n in T a b l e II. T h e enz y m e s w e r e n o t all p u r i f i e d t o t h e s a m e degree. I n a n i m a l f e e d , c r u d e e n z y m e p r e p a r a t i o n s w o u l d b e p r e f e r r e d , p a r t l y b e c a u s e o f costs a n d p a r t l y b e c a u s e they provide a mixture of enzymes, which might be advantageous.
T A B L E II Enzymes used in the feed for experimental animals
Enzyme/biomass
Activity
Type and Manufacturer
Cellulase 1
C I ~ 500 units/g I
Cellulase 2
Cx ~5000 units/g 1 C~ 45 units/g I
sP 122, Novo A/S, 2880 Bagsv~erd, Denmark Batch PPTC -- 02 Onozuka SS, Yakult Biochemicals Co., Ltd., Japan
Pectinase 1
Cx 1652 units/g 1 Xylanase activity C 1 1110 units/g 1 C x 12400 units/g I Pectinase activity, 1000 (pH 5) ~
Pectinase 2
Glucanase activity, 400 (pH 5) ~ Pectolase activity, 24 units/g 3
CeUulase 3
Amylase 1 Protease 1 (Biomass)
Protease 2
a-amylase, 42,4 KNE/g (pH 5.7) 2 a-amylase, 22.5 FAE/g (pH 4.7) 2 Proteinase, 4.2 Ae/g (pH 7.5) 1 Proteinasg 2.2 Ae/g (pH 5.0) 1 a-amylase, 49.5 KNE/g x (pH 5.7) 1 27.1 FAE/gx) (pH 4.7) 1 ~-glucanase, 23 BGE/g Proteinase, 3.0 Ae/g (pH 7.5) 1 Proteinase, 1.3 Ae/g (pH 5.0) 1
G. Spalla, Italy Patent: 27237 A/70 Sigma, batch no. lot 55 C -0083 EC no. 3.2.1.15, 0.9 Pectolase, Grindstedvmrket A/S, 8220 Brabrand, DK S i g m a , batch no. lot 42 C - - 0130 Worne Biochemicals, Inc., Lyon Industrial Park, Route 73, Berlin, N e w Jersey, 08009, U S A
Papain, Merck, batch art. 7144
i Analyzed by Grindstedvserket A/S, Denmark. 2 Analyzed by Novo A/S, Denmark.
Experiments on chickens The experiments were p e r f o r m e d on day-old cockerels, which, after a p r e l i m i n a r y p e r i o d o f o n e w e e k , w e r e given e x p e r i m e n t a l f e e d f o r t h r e e weeks. G r o w t h a n d f e e d i n t a k e d u r i n g t h e e x p e r i m e n t a l p e r i o d w e r e r e c o r d e d . I n all, 3 9 6 c h i c k e n s w e r e involved in t h e e x p e r i m e n t s , w i t h f r o m 16 t o 48 birds p e r group. A n e x a m p l e o f feed c o m p o s i t i o n is given in T a b l e I I I . We a i m e d at
109
TABLE III Composition of the feed m i x t u r e for experiments on chickens Ingredient
Composition (kg/100 kg)
Soya grits (49% protein) Meat and bone meal (50% protein) Barley (~ 13% protein) ~ Maize oil Methionine (100%) Dicalcium phosphate Salt Limestone Vitamin mixture Digestible energy (kcal/kg) 2 Digestible crude protein (%)
28.47 4.00 62.17 2.45 0.057 1.42 0.380 0.541 0.500 2850 20
Different barley raw materials were used for the four mixtures. The content of barley varied between 60 and 62%, and the content of soya grits varied between 27 and 29%, according to the crude-protein content. 2 The composition of the feed mixture was chosen as the least cost solution to a linear computer program, the requirements of the feed mixture being 2850 kcal digestible energy/kg, 20% digestible crude protein, more than 0.04% methionine and about 1% calcium. Table values shown in Cirkul~re fra Statens Foderstofkontrol (1976) were used for optimizing the feed mixture.
a feed composition with normal contents of digestible energy and digestible protein. The content of crude fibre was higher than that of normal, maizebased chicken feed (4.2%, instead of 2.5%). No difficulties were experienced with barley feeding in these experiments, neither with regard to faeces-consistency, nor to the well-being of the chickens. The viscosity of the barley is shown in Table IV. The viscosity measurements were performed with a Brabender viscograph, according to the following procedure. Freshly milled barley ( ~ 1 5 5 g; 135 g dry matter) was made up to 450 g total weight by addition of 0.1 M citrate buffer (pH = 4.5) at 42°C, and the viscosity was measured at 37°C. (Cup speed, 75 rpm; maximum TABLE IV Viscosity of the barley used in the experiments on chickens
Barley sample
Brabenderuni~(cm x g)
Dynamic cP
1 2 3
259 329 280
700 850 75O
110
deflection, 700 cm × g.) Unfortunately, the figures are not comparable with those of Gohl et al. (1978) for barley viscosity. They measured viscosity on barley extract, and states figures below 10 cP. In addition to the effects of enzyme addition on growth and feed intake, the influence of pelleting and steeping of the feed mixture was examined. The results were subjected to analysis of variance. RESULTS
Addition of a-amylase to milled barley given to rats (Table V) showed a non-significant tendency towards better feed-utilization and weight gain. Steeping barley feed with enzymes for up to 6 h with 70% moisture did not show any improvement when a-amylase had been added, whereas the addition of cellulase 2 and protease 2 caused non-significant improvements in feed utilization and weight gain. No improvements were found by addition of pectinase 1 and cellulase 1 and 3. The differences between the experimental diets were not significant and the experiments should be repeated to see if the observed tendency is statistically significant. The results of the feeding experiments with chickens are shown in Tables VI--VIII, where growth and feed consumption/g of growth with different enzyme applications are shown. In two out of five comparisons, there was a significant improvement in growth rate and in three out of five comparisons, a significant improvement in feed utilization by the addition of enzymes. Application of cellulase 2 or pectinase 1 and 2 to feed mixtures, milled or pelleted, showed a feed saving of 4% per growth unit (P < 0.05) (Table VI). TABLE V E x p e r i m e n t s o n rats fed o n b a r l e y feed m i x t u r e s c o n t a i n i n g enzymes, given d i r e c t l y or a f t e r steeping
Additive
Concentration
Barley A '
(%)
Feed
Gain
(g)
(g)
135
39.1
3.47
137 134 131 134 125
39.6 38.5 36.9 41.5 38.8 ±1.6
3.47 3.49 3.56 3.25 3.49 ±0.13
ns
ns
None ( c o n t r o l ) Cellulase 1 Cellulase 2 Cellulase 3 Pectinase 1 Amylase 1 Protease 2 Standard e r r o r
Differences between diets
0.2 0.2 0.2 0.05 0.1 0.05
Barley B ~
Feed/gain
Feed
Gain
(g)
(g)
Feed/gain
154 154 154 152 151 147 154
37.9 37.9 39.4 37.0 35.9 36.2 40.2 ±1.1
4.08 4.06 3.92 4.11 4.22 4.08 3.84 ±0.11
ns
ns
' Direct f e e d i n g a f t e r a d d i t i o n o f enzyme. 2 S t e e p i n g f o r 6 h w i t h 70% m o i s t u r e , w i t h o r w i t h o u t e n z y m e a d d i t i o n p r i o r t o storage.
111
TABLE VI Experiments on chickens fed on milled or pelleted mixtures containing cellulase and/or pectinase. (Total of 32 chickens per treatment; asterisks indicate significance of difference from control.)
Additive None (control) Cellulase2 Pectinase Cellulase and pectinase Appropriate SE
Concentration (%)
Feed (g)
Gain (g)
Feed/gain
0.2 0.01 or 0.02 (as above)
1113 1118 1079 1124 +-17
541 566 546 580* -+12
2.06 1.98" 1.98" 1.94"* -+0.027
W h e n the above-mentioned enzymes were added simultaneously to the feed mixtures, milled or pelleted, growth rate increased by nearly 7% (P < 0.05), and feed/gain by 6 % per growth unit (P < 0.01). The resultsof the chicken experiment using feeds containing protease are shown in Table VII. The experiment was repeated using a pelleted feed mixture and a steeped feed-mixture. Growth rate was increased by about 6 % (P < 0.05) by using protease, and by about 5% feed per growth unit (P< 0.05 was saved. The results of an experiment on chickens, in which the enzymes cellulase, pectinase and protease were added simultaneously to the feed, are shown in Table VIII. N o differences were found between the diets in this experiment. This is surprising,considering the significantimprovements found in the previous experiments. However, in this experiment, the barley quality was probably superior, as shown by the better feed utilization (1.99 vs. 2.06 g per growth unit). The experiment revealed no difference in feed utilization or growth, by addition of enzymes prior to, or after, pelleting. Steeping the feed caused no improvements in the performance of the chickens.
TABLE VII Experiments on chickens fed on milled, pelleted or steeped feed mixtures, containing protease. (Total of 48 chickens per treatment; asterisks indicate significance of differences from control.) Additive
None (control) Protease 1 SE
Concentration Feed
Gain
(%)
(g)
(g)
0.05
1084 1094 +18
536 567* +10
Feed/gain
2.02 1.93" -+0.027
112 TABLE VIH Exper~,nents on chickens fed on milled or pelleted feed mixtures, containing cellulase, pectinase and protease. (Total of 80 chickens per treatment; no significant differences,) Additive
None (control) Cellulase 2 Pectinase 2 and Protease 1
SE
Concentration (%)
Feed (g)
Gain (g)
1096
551
1100 -+15
560 -+8.1
Feed/gain
1.99
0.2 0.02
0.05
1.97
+-0.017
DISCUSSION The investigation has shown that barley feed mixtures for chickens can be improved by addition of enzymes, but the improvement may depend upon the barley quality. With the improvements obtained, a barley-based feedmixture, with a digestible energy of 2800 kcal/kg, containing 20% digestible protein and costing 1.30 Dkr/kg, formed the basis of the following economic calculations. A saving of 4--6% feed per growth unit equals 4--6 kg/100 kg and corresponds to a saving of 5.2--7.8 Dkr. Therefore, the break-even price for the enzymes is ~100--150 Dkr/kg for protease added at 50 g/100 kg, 260--390 Dkr/kg for pectinase added at 10--20 g/100 kg, and 26--39 Dkr/kg for ceUulase added at 200 g/100 kg, if only one enzyme is added. Otherwise, the break-even price for the enzyme is less than 26 Dkr/kg enzyme. Where growth rate is also improved, the enzymes can be priced a little higher. From these calculations, it appears that the amount of enzyme added strongly influences the price, as far as standard feed is concerned. Therefore, optimizing quantities and addition methods is essential prior to final economic calculations. Improvement and use of by-products by enzyme treatment probably has wider economic significance than indicated in the present investigation. An investigation by P~rk~ny-GySxf~s and T6th (1978) using additions of 0.2-0.4% a-amylase to chicken feed containing 62--68% maize meal, showed improvements in feed utilization and growth. The feed saving was 3--4 times greater than indicated in the present investigation with barley feed, whereas the growth improvements were similar. Other authors have successfully used a-amylase in maize- and milo-feeds for chickens and hens (Gleaves and Dewan, 1970) and at 0.2% in barley feed (Herstad and McNab, 1975). Hennig (1972) mentions that there was no effect when proteases and a-amylases were added to barley for chickens, whereas ~-glucanases were very effective in improving the feed value. Gohl and Gohl (1977), Vohra (1972), Becket and Nehring {1967) and Burnett
113
1966) have also found ~-glucanase effective. However, a reduction of the viscosity of barley feed with cellulase containing.hemicellulase is also possible (Behnke and Deyoe, 1973). With a feed mixture composed of 60% barley, 2070 soya meal, 2070 digestible protein, and digestible energy 2900 kcal/kg, Ezdakov (1976) found improvements in growth of poultry from 10 to 2570 with enzyme-rich biomass or enzyme mixtures (0.05--0.170). The enzyme mixtures contained for example, pectinesterase and polyglucanase. Ezdakov (1976) described the application of enzyme-rich biomass or enzyme mixtures to the feeding of many animal species, and to the ensiling of many crops. To-day such additives are used routinely in animal husbandry in USSR. CONCLUSIONS
Experiments on chickens have shown that growth and feed utilizationcan be increased by nearly 7 and 670,respectively,by the addition of enzymes to barley. However, the improvements may depend on the quality of the barley. This suggeststhat feed ingredientsof low digestibility,and hence low cost, may be utilizedmore efficientlyby additionof enzymes.
REFERENCES Aumaitre, A. and Raynaud, J.P., 1978. Effectiveness of carbadox at 50 ppm on the performance of young pig: methodology and influence of the initial weight. Z. Tierphysiol., Tierernaehr. Futtermittelkd., 40: 67--74. Bailey, R.W., Monro, J.A., Pickmere, S.E. and Chesson, A., 1976. Herbage hemicellulose and its digestion by the ruminants. Miscellaneous papers. Landbouwhogesch., Wageningen, 12: 1--15. Baird, J.R., Cromwell, G.L. and Hays, V.W., 1976. Effects of enzyme supplementation and presoaking of diet on performance and nutrient digestibility in early-weaned pigs. J. Anita. Sci., 43: 249. Barrett, F.F., 1975. Enzyme uses in the milling and baking industries. In: G. Reed (Editor), Enzymes Food Process(2nd Edn.). Academic Press, New York, pp. 301--330. Becker, M. and Nehring, K., 1967. Handbuch der Futtermittel Bd. 3. Berlin, 419 pp. Behnke, K.C. and Deyoe, C.W., 1973. Using enzymes to reduce viscosity of a hemicellulose extract. Feedstuffs, 22. Boling, J.A., Bradley, N.W. and Campbell, L.D., 1977. Monensin levels for growing and finishing steers. J. Anita. Sci., 44: 867--871. Bossen, F., 1977. Probiotika - - e n landvinding i problematikken omkring grisediarreer og antibiotikaresistens. Dan. Veterinmr. Tidsskr., 60: 989---990. Bumett, G.S., 1966. Viscosity as the probable factor involved in the improvement of certain barleys for chickens by enzyme supplementation. Br. Poult. Sci., 7: 55--75. Casteels, M. and Buysse, F., 1976. Influence d'une enzyme proteolytique sur les presentations de porcelets. Rev. Agric. Belgium, 29: 1007--1019. Castell, A.G., 1977. Effects of Virginamycin on the performance of pigs fed barley diets supplemented with soybean meal or low-glucosinolate rapeseed meal. Can. J. Anim. Sci., 57: 313--320. Charles, O.W., 1976. The effect of antibiotic supplementation on broiler chick and laying hen performance. Poult. Sci., 55: 2019.
114 Cirkulaere fra Statens Foderstofkontrol (Denmark), 1976. Daniels, i~.B. and Hashim, R.B., 1977. Evaluation of fungal cellulase in rice hulls base diets for ruminants. J. Dairy Sci., 60: 1563--1567. Ezdakov, N.V., 1976. Anvendelse af enzymprmparater i husdyrproduktion. 224 sider ill., Moskva "Kolos". Gill, D.R., Owens, F.N., Martin, J.J., Williams, D.E. and Thornton, J.M., 1977. Protein levels and rumensin for feedlot cattle. Okla. Agric. Exp. Stn., Res. Rep.: 42--47. Gleaves, E.W. and Dewan, S., 1970. Influence of a fungal enzyme in corn and milo layer rationa Poult. Sci., 49: 596--598. Gohl, B., Alden, S., Elwinger, K. and Thomke, S., 1978. Influence of beta-glucanase on feeding value of barley for poultry and moisture content of excreta. Br. Poult. Sci., 19: 41--47. Gohl, B. and Gohl, I., 1977. The effect of viscous substances on the transit time of barley digesta in rats. J. Sci. Food Agric., 28: 991--1115. Griffiths, D.W. and Jones, D.I.H., 1977. Cellulase inhibition by tannins in the testa of field beans (Vicia faba). J. Sci. Food Agric., 28: 983--989. Han Young, W. and Andersen, A.W., 1975. Semisolid fermentation of ryegrass straw. Appl. Microbiol., 30: 930--934. Haskell, E.W., Leong, K.C., Jensen, L.S. and McGinnis, J., 1960. Influence of geographical area of production on response of different barley samples to enzyme supplements or water treatment. Poult. Sci., 39: 103--108. Henderson, A.R. and McDonald, P., 1977. The effect of cellulase preparations on the chemical change during the ensilage of grass in laboratory silos. J. Sci. Food Agric., 28: 486--490. Hennig, A~, 1972. Mineralstoffe, Vitamine und Ergotropika. Veb. Deutscher Landwirtschaft Verlag, Berlin, 636 pp. Herstad, O. and McNab, J_M., 1975. The effect of heat treatment and enzyme supplementations on the nutritive value of barley for broiler chicks. Br. Poult. Sci., 16: 1--8. Horn, G.W., Owens, F.N., Armbruster, S.L., Stevens, V.L. and Scott, M.L., 1977. Monensin for wheat pasture stockers. Okla. Agric. Exp. Stn., Res. Rep.: 42--47. Kasbaoui, M. and Guillaume, J., 1976. Essai d'un nouveau type d'additifs alimentaires les enzymes proteolytiques, chez le poulet de chair. Ind. Aliment. Anita., 21: 15--25. Luther, L.W., Thomas, M.C., Goatcher, W.D., Selwyn, M.R. and Colaianne, J.J., 1977. Dose-response of broilers fed penicillin plus monensin in supplemented diets. Poult. Sci., 55: 2058. Millen, H.M. and Harrison, D., 1970. Correlation of heifer feeding efficiency and the hydrolysis products of reconstituted grain sorghum. Feedstuffs, 12: 19. Moe, P.W. and Tyrrell, H.F., 1977. Effects of feed intake and physical form on energy value of corn in Timothy hay diets for lactating cows. J. Dairy Sci., 60: 752--758. Morril, J.L., Dayton, A.D. and Mickelsen, R., 1977. Cultured milk and antibiotics for young calves. J. Dairy Sci., 60: 1105--1109. Moss, B.R., Beeckler, A.F., Newman, C.W. and E1-Negoumy, A_M., 1977. Enzyme supplementation of broilers rations. Poult. Sci., 56: 1741. P~rk~ny-Gy~rf~s, A. and Tbth, M., 1978. Feed utilization efficiency of enzyme-containing feeds in livestock raising. Acta Aliment. Acad. Sci. Hung., 7: 111--120. Rasmussen, P. and Holm, F., 1978. Kvaliteten af foder til enmavedyr -- i relation til lagring og teknisk forarbejdning. Beretning nr. 86. Bioteknisk Institut, Kolding, 49 pp. Runnels, T.D., 1976. The influence of feed texture on broiler performance. Poult. Sci., 55: 1958--1961. Sellars, P.N., McGill, C.E.G. and Flannigan, B., 1976. Degradation of barley by Aspergillus fumigatus Fres. Proc. III Int. Biodegr. Symp., 1975: 635--643. Vabischevich, K.D., Margolin, S.E. and Kanashkova, E.F., 1977. Effects of colamine (Premix-517) on the productivity and pork properties of growing swine. S-KH-Biol. (Minsk), 12: 182--185. Vohra, P., 1972. Evaluation of metabolizable energy for poultry. World's Poult. Sci. J., 28: 204--214.
105
USE O F E N Z Y M E S F O R I M P R O V E M E N T O F F E E D 1
B. REXEN
Biotechnical Institute, Holbergsvej 10, 6000 Kolding (Denmark) (Received 7 May 1979; accepted for publication 12 June 1980)
ABSTRACT Rexen, B., 1981. Use of enzymes for improvement of feed. Anim. Feed Sci. Technol., 6: 105--114. Investigations of the addition of enzymes to traditional feed-mixtures, for improvement of feed utilization and of growth of domestic animals, have been performed. A survey of the literature on the effects of enzyme addition is shown in tabular form. Experiments with rats fed on barley diets, with and without enzyme addition, showed no significant difference between the diets, but a tendency towards feed improvements by the addition of some of the enzymes, e.g., amylase, cellulase end protease. Some of the experiments with chickens fed on a barley-based diet, with the addition of either cellulase and/or peetinase or protease, showed increases in growth of up to 7% (P < 0.05), and improvements in feed utilization of 6% (P ~ 0.01). In one experiment on chickens fed on a barley of better quality than that in the above-mentioned experiments, no improvements were found by adding an enzyme mixture, consisting of cellulase, pectinase and protease, to the feed. The results thus suggest that a better feed-utilization by use of enzyme addition is obtainable only if the feed mixture is composed of less-digestible feed ingredients. The amount of added enzymes strongly influences the price of the resultant feed, and in the experiments showing feed improvement, the break-even price of the enzymes is less than 26 Dkr/kg enzyme. The use of enzymes for making slowly digested and less-expensive products (waste products) applicable as feed components will be examined in future work
INTRODUCTION In D e n m a r k , f e e d i n g costs a c c o u n t f o r 65% o f t h e t o t a l costs o f animal p r o d u c t i o n . T h e r e f o r e , it is i m p o r t a n t t h a t animals utilize t h e feed o p t i m a l l y . I t is possible t o i m p r o v e feed utilization in various w a y s , d e p e n d i n g o n feed q u a l i t y a n d species o f animal. One possibility is t h e a d d i t i o n o f substances s t i m u l a t i n g t h e digestive m e c h a n i s m or inhibiting u n w a n t e d microbial g r o w t h in t h e digestive s y s t e m . In t h e literature, t h e r e are references t o e n z y m e s , ' This investigation was financed by the Danish Technology Board. Two Danish enzyme producers (Novo A/S and Grindstedvmrket A/S) supplied the enzymes and served as consultants on the project. Some biomass and enzymes were obtained from U.S.A. (Worne Biochemicals) and Italy.
0377-8401/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
Silage (grain) Maize and rice hulls Silage (grass) Milk Wheat Maize
Ruminants Feed mixture
Pigs Traditional feed Traditional feed
(barley)
(maize)
(barley)
Pectolases, hemicellulase, cellulase and proteases Proteases, a-amylase and microorganisms Cellulase (fungi) Cellulase Lactobacilli and oxytetracycline Monensin Rumensin (Monensin sodium)
Proteases, amylases and ~-glucanase Proteolytic enzyme (Fradiase)
Proteolytic enzyme (bacterial) a-amylase and proteases ~-glucanase a-amylase a-amylase a-amylase (diastase) Fungal enzyme Monensin and penicillin
Poultry Feed mixture Barley/maize Feed mixture Barley Feed mixture Barley Feed mixture Starting feed
(maize)
Addition
Animal (feed)
-
+
17
7--12
10--20
2.4 10--25 4 14 3 6--12 15 +
Increased growth (%)
4 1 10 Saving of protein
--
13
-
1.4 -5 7 7 4--6 13 +
Improved feed utilization (%)
Improvements of feed value by different additives (according to the literature)
TABLE I
Millen and Harrison, 1970 Daniels and Hashim, 1977 Henderson and McDonald, 1977 Morril et al., 1977 Horn et al., 1977 Gill et al., 1977
Ezdakov, 1976
Ezdakov, 1976 Casteelsand Buy~e, 1976
Kasbaoui and Guillaume, 1976 Ezdakov, 1976 Gohl et al.,1978 Herstad and McNab, 1975 P~rk~ny-Gy~rf~s and T6th, 1978 Moss et al.,1977 Haskell et al.,1960 Luther et al.,1977
References
O
107
biomass, antibiotics,probiotics (preparations containing livingmicroorganisms, especially lactobaciUus preparations) and other preparations, such as Rumensin (monensin). Another possibilityis a pre-treatment ("pre
After a pre-period of 3 days, experiments were performed for 16 days with SPF rats, Wistar Af/Male/Mol (Male 67), a b o u t 4 weeks old. In one experiment, enzymes either cellulase, pectinase, amylase or protease -- were added to barley mixed with vitamins and minerals. Each mixture was given to four rats. -
-
108
In a n o t h e r e x p e r i m e n t , t h e s a m e e n z y m e s w e r e a d d e d , b u t t h e f e e d was t h e n s t e e p e d f o r six h o u r s w i t h 70% m o i s t u r e a n d f r e e z e - d r i e d p r i o r t o feeding. This f e e d was c o m p a r e d w i t h a c o n t r o l feed, also s t e e p e d f o r six h o u r s w i t h 70% m o i s t u r e a n d freeze-dried. G r o w t h a n d f e e d c o n s u m p t i o n w e r e m o n i t o r e d in b o t h e x p e r i m e n t s . E n z y m e b r a n d a n d a c t i v i t y are s h o w n in T a b l e II. T h e enz y m e s w e r e n o t all p u r i f i e d t o t h e s a m e degree. I n a n i m a l f e e d , c r u d e e n z y m e p r e p a r a t i o n s w o u l d b e p r e f e r r e d , p a r t l y b e c a u s e o f costs a n d p a r t l y b e c a u s e they provide a mixture of enzymes, which might be advantageous.
T A B L E II Enzymes used in the feed for experimental animals
Enzyme/biomass
Activity
Type and Manufacturer
Cellulase 1
C I ~ 500 units/g I
Cellulase 2
Cx ~5000 units/g 1 C~ 45 units/g I
sP 122, Novo A/S, 2880 Bagsv~erd, Denmark Batch PPTC -- 02 Onozuka SS, Yakult Biochemicals Co., Ltd., Japan
Pectinase 1
Cx 1652 units/g 1 Xylanase activity C 1 1110 units/g 1 C x 12400 units/g I Pectinase activity, 1000 (pH 5) ~
Pectinase 2
Glucanase activity, 400 (pH 5) ~ Pectolase activity, 24 units/g 3
CeUulase 3
Amylase 1 Protease 1 (Biomass)
Protease 2
a-amylase, 42,4 KNE/g (pH 5.7) 2 a-amylase, 22.5 FAE/g (pH 4.7) 2 Proteinase, 4.2 Ae/g (pH 7.5) 1 Proteinasg 2.2 Ae/g (pH 5.0) 1 a-amylase, 49.5 KNE/g x (pH 5.7) 1 27.1 FAE/gx) (pH 4.7) 1 ~-glucanase, 23 BGE/g Proteinase, 3.0 Ae/g (pH 7.5) 1 Proteinase, 1.3 Ae/g (pH 5.0) 1
G. Spalla, Italy Patent: 27237 A/70 Sigma, batch no. lot 55 C -0083 EC no. 3.2.1.15, 0.9 Pectolase, Grindstedvmrket A/S, 8220 Brabrand, DK S i g m a , batch no. lot 42 C - - 0130 Worne Biochemicals, Inc., Lyon Industrial Park, Route 73, Berlin, N e w Jersey, 08009, U S A
Papain, Merck, batch art. 7144
i Analyzed by Grindstedvserket A/S, Denmark. 2 Analyzed by Novo A/S, Denmark.
Experiments on chickens The experiments were p e r f o r m e d on day-old cockerels, which, after a p r e l i m i n a r y p e r i o d o f o n e w e e k , w e r e given e x p e r i m e n t a l f e e d f o r t h r e e weeks. G r o w t h a n d f e e d i n t a k e d u r i n g t h e e x p e r i m e n t a l p e r i o d w e r e r e c o r d e d . I n all, 3 9 6 c h i c k e n s w e r e involved in t h e e x p e r i m e n t s , w i t h f r o m 16 t o 48 birds p e r group. A n e x a m p l e o f feed c o m p o s i t i o n is given in T a b l e I I I . We a i m e d at
109
TABLE III Composition of the feed m i x t u r e for experiments on chickens Ingredient
Composition (kg/100 kg)
Soya grits (49% protein) Meat and bone meal (50% protein) Barley (~ 13% protein) ~ Maize oil Methionine (100%) Dicalcium phosphate Salt Limestone Vitamin mixture Digestible energy (kcal/kg) 2 Digestible crude protein (%)
28.47 4.00 62.17 2.45 0.057 1.42 0.380 0.541 0.500 2850 20
Different barley raw materials were used for the four mixtures. The content of barley varied between 60 and 62%, and the content of soya grits varied between 27 and 29%, according to the crude-protein content. 2 The composition of the feed mixture was chosen as the least cost solution to a linear computer program, the requirements of the feed mixture being 2850 kcal digestible energy/kg, 20% digestible crude protein, more than 0.04% methionine and about 1% calcium. Table values shown in Cirkul~re fra Statens Foderstofkontrol (1976) were used for optimizing the feed mixture.
a feed composition with normal contents of digestible energy and digestible protein. The content of crude fibre was higher than that of normal, maizebased chicken feed (4.2%, instead of 2.5%). No difficulties were experienced with barley feeding in these experiments, neither with regard to faeces-consistency, nor to the well-being of the chickens. The viscosity of the barley is shown in Table IV. The viscosity measurements were performed with a Brabender viscograph, according to the following procedure. Freshly milled barley ( ~ 1 5 5 g; 135 g dry matter) was made up to 450 g total weight by addition of 0.1 M citrate buffer (pH = 4.5) at 42°C, and the viscosity was measured at 37°C. (Cup speed, 75 rpm; maximum TABLE IV Viscosity of the barley used in the experiments on chickens
Barley sample
Brabenderuni~(cm x g)
Dynamic cP
1 2 3
259 329 280
700 850 75O
110
deflection, 700 cm × g.) Unfortunately, the figures are not comparable with those of Gohl et al. (1978) for barley viscosity. They measured viscosity on barley extract, and states figures below 10 cP. In addition to the effects of enzyme addition on growth and feed intake, the influence of pelleting and steeping of the feed mixture was examined. The results were subjected to analysis of variance. RESULTS
Addition of a-amylase to milled barley given to rats (Table V) showed a non-significant tendency towards better feed-utilization and weight gain. Steeping barley feed with enzymes for up to 6 h with 70% moisture did not show any improvement when a-amylase had been added, whereas the addition of cellulase 2 and protease 2 caused non-significant improvements in feed utilization and weight gain. No improvements were found by addition of pectinase 1 and cellulase 1 and 3. The differences between the experimental diets were not significant and the experiments should be repeated to see if the observed tendency is statistically significant. The results of the feeding experiments with chickens are shown in Tables VI--VIII, where growth and feed consumption/g of growth with different enzyme applications are shown. In two out of five comparisons, there was a significant improvement in growth rate and in three out of five comparisons, a significant improvement in feed utilization by the addition of enzymes. Application of cellulase 2 or pectinase 1 and 2 to feed mixtures, milled or pelleted, showed a feed saving of 4% per growth unit (P < 0.05) (Table VI). TABLE V E x p e r i m e n t s o n rats fed o n b a r l e y feed m i x t u r e s c o n t a i n i n g enzymes, given d i r e c t l y or a f t e r steeping
Additive
Concentration
Barley A '
(%)
Feed
Gain
(g)
(g)
135
39.1
3.47
137 134 131 134 125
39.6 38.5 36.9 41.5 38.8 ±1.6
3.47 3.49 3.56 3.25 3.49 ±0.13
ns
ns
None ( c o n t r o l ) Cellulase 1 Cellulase 2 Cellulase 3 Pectinase 1 Amylase 1 Protease 2 Standard e r r o r
Differences between diets
0.2 0.2 0.2 0.05 0.1 0.05
Barley B ~
Feed/gain
Feed
Gain
(g)
(g)
Feed/gain
154 154 154 152 151 147 154
37.9 37.9 39.4 37.0 35.9 36.2 40.2 ±1.1
4.08 4.06 3.92 4.11 4.22 4.08 3.84 ±0.11
ns
ns
' Direct f e e d i n g a f t e r a d d i t i o n o f enzyme. 2 S t e e p i n g f o r 6 h w i t h 70% m o i s t u r e , w i t h o r w i t h o u t e n z y m e a d d i t i o n p r i o r t o storage.
111
TABLE VI Experiments on chickens fed on milled or pelleted mixtures containing cellulase and/or pectinase. (Total of 32 chickens per treatment; asterisks indicate significance of difference from control.)
Additive None (control) Cellulase2 Pectinase Cellulase and pectinase Appropriate SE
Concentration (%)
Feed (g)
Gain (g)
Feed/gain
0.2 0.01 or 0.02 (as above)
1113 1118 1079 1124 +-17
541 566 546 580* -+12
2.06 1.98" 1.98" 1.94"* -+0.027
W h e n the above-mentioned enzymes were added simultaneously to the feed mixtures, milled or pelleted, growth rate increased by nearly 7% (P < 0.05), and feed/gain by 6 % per growth unit (P < 0.01). The resultsof the chicken experiment using feeds containing protease are shown in Table VII. The experiment was repeated using a pelleted feed mixture and a steeped feed-mixture. Growth rate was increased by about 6 % (P < 0.05) by using protease, and by about 5% feed per growth unit (P< 0.05 was saved. The results of an experiment on chickens, in which the enzymes cellulase, pectinase and protease were added simultaneously to the feed, are shown in Table VIII. N o differences were found between the diets in this experiment. This is surprising,considering the significantimprovements found in the previous experiments. However, in this experiment, the barley quality was probably superior, as shown by the better feed utilization (1.99 vs. 2.06 g per growth unit). The experiment revealed no difference in feed utilization or growth, by addition of enzymes prior to, or after, pelleting. Steeping the feed caused no improvements in the performance of the chickens.
TABLE VII Experiments on chickens fed on milled, pelleted or steeped feed mixtures, containing protease. (Total of 48 chickens per treatment; asterisks indicate significance of differences from control.) Additive
None (control) Protease 1 SE
Concentration Feed
Gain
(%)
(g)
(g)
0.05
1084 1094 +18
536 567* +10
Feed/gain
2.02 1.93" -+0.027
112 TABLE VIH Exper~,nents on chickens fed on milled or pelleted feed mixtures, containing cellulase, pectinase and protease. (Total of 80 chickens per treatment; no significant differences,) Additive
None (control) Cellulase 2 Pectinase 2 and Protease 1
SE
Concentration (%)
Feed (g)
Gain (g)
1096
551
1100 -+15
560 -+8.1
Feed/gain
1.99
0.2 0.02
0.05
1.97
+-0.017
DISCUSSION The investigation has shown that barley feed mixtures for chickens can be improved by addition of enzymes, but the improvement may depend upon the barley quality. With the improvements obtained, a barley-based feedmixture, with a digestible energy of 2800 kcal/kg, containing 20% digestible protein and costing 1.30 Dkr/kg, formed the basis of the following economic calculations. A saving of 4--6% feed per growth unit equals 4--6 kg/100 kg and corresponds to a saving of 5.2--7.8 Dkr. Therefore, the break-even price for the enzymes is ~100--150 Dkr/kg for protease added at 50 g/100 kg, 260--390 Dkr/kg for pectinase added at 10--20 g/100 kg, and 26--39 Dkr/kg for ceUulase added at 200 g/100 kg, if only one enzyme is added. Otherwise, the break-even price for the enzyme is less than 26 Dkr/kg enzyme. Where growth rate is also improved, the enzymes can be priced a little higher. From these calculations, it appears that the amount of enzyme added strongly influences the price, as far as standard feed is concerned. Therefore, optimizing quantities and addition methods is essential prior to final economic calculations. Improvement and use of by-products by enzyme treatment probably has wider economic significance than indicated in the present investigation. An investigation by P~rk~ny-GySxf~s and T6th (1978) using additions of 0.2-0.4% a-amylase to chicken feed containing 62--68% maize meal, showed improvements in feed utilization and growth. The feed saving was 3--4 times greater than indicated in the present investigation with barley feed, whereas the growth improvements were similar. Other authors have successfully used a-amylase in maize- and milo-feeds for chickens and hens (Gleaves and Dewan, 1970) and at 0.2% in barley feed (Herstad and McNab, 1975). Hennig (1972) mentions that there was no effect when proteases and a-amylases were added to barley for chickens, whereas ~-glucanases were very effective in improving the feed value. Gohl and Gohl (1977), Vohra (1972), Becket and Nehring {1967) and Burnett
113
1966) have also found ~-glucanase effective. However, a reduction of the viscosity of barley feed with cellulase containing.hemicellulase is also possible (Behnke and Deyoe, 1973). With a feed mixture composed of 60% barley, 2070 soya meal, 2070 digestible protein, and digestible energy 2900 kcal/kg, Ezdakov (1976) found improvements in growth of poultry from 10 to 2570 with enzyme-rich biomass or enzyme mixtures (0.05--0.170). The enzyme mixtures contained for example, pectinesterase and polyglucanase. Ezdakov (1976) described the application of enzyme-rich biomass or enzyme mixtures to the feeding of many animal species, and to the ensiling of many crops. To-day such additives are used routinely in animal husbandry in USSR. CONCLUSIONS
Experiments on chickens have shown that growth and feed utilizationcan be increased by nearly 7 and 670,respectively,by the addition of enzymes to barley. However, the improvements may depend on the quality of the barley. This suggeststhat feed ingredientsof low digestibility,and hence low cost, may be utilizedmore efficientlyby additionof enzymes.
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114 Cirkulaere fra Statens Foderstofkontrol (Denmark), 1976. Daniels, i~.B. and Hashim, R.B., 1977. Evaluation of fungal cellulase in rice hulls base diets for ruminants. J. Dairy Sci., 60: 1563--1567. Ezdakov, N.V., 1976. Anvendelse af enzymprmparater i husdyrproduktion. 224 sider ill., Moskva "Kolos". Gill, D.R., Owens, F.N., Martin, J.J., Williams, D.E. and Thornton, J.M., 1977. Protein levels and rumensin for feedlot cattle. Okla. Agric. Exp. Stn., Res. Rep.: 42--47. Gleaves, E.W. and Dewan, S., 1970. Influence of a fungal enzyme in corn and milo layer rationa Poult. Sci., 49: 596--598. Gohl, B., Alden, S., Elwinger, K. and Thomke, S., 1978. Influence of beta-glucanase on feeding value of barley for poultry and moisture content of excreta. Br. Poult. Sci., 19: 41--47. Gohl, B. and Gohl, I., 1977. The effect of viscous substances on the transit time of barley digesta in rats. J. Sci. Food Agric., 28: 991--1115. Griffiths, D.W. and Jones, D.I.H., 1977. Cellulase inhibition by tannins in the testa of field beans (Vicia faba). J. Sci. Food Agric., 28: 983--989. Han Young, W. and Andersen, A.W., 1975. Semisolid fermentation of ryegrass straw. Appl. Microbiol., 30: 930--934. Haskell, E.W., Leong, K.C., Jensen, L.S. and McGinnis, J., 1960. Influence of geographical area of production on response of different barley samples to enzyme supplements or water treatment. Poult. Sci., 39: 103--108. Henderson, A.R. and McDonald, P., 1977. The effect of cellulase preparations on the chemical change during the ensilage of grass in laboratory silos. J. Sci. Food Agric., 28: 486--490. Hennig, A~, 1972. Mineralstoffe, Vitamine und Ergotropika. Veb. Deutscher Landwirtschaft Verlag, Berlin, 636 pp. Herstad, O. and McNab, J_M., 1975. The effect of heat treatment and enzyme supplementations on the nutritive value of barley for broiler chicks. Br. Poult. Sci., 16: 1--8. Horn, G.W., Owens, F.N., Armbruster, S.L., Stevens, V.L. and Scott, M.L., 1977. Monensin for wheat pasture stockers. Okla. Agric. Exp. Stn., Res. Rep.: 42--47. Kasbaoui, M. and Guillaume, J., 1976. Essai d'un nouveau type d'additifs alimentaires les enzymes proteolytiques, chez le poulet de chair. Ind. Aliment. Anita., 21: 15--25. Luther, L.W., Thomas, M.C., Goatcher, W.D., Selwyn, M.R. and Colaianne, J.J., 1977. Dose-response of broilers fed penicillin plus monensin in supplemented diets. Poult. Sci., 55: 2058. Millen, H.M. and Harrison, D., 1970. Correlation of heifer feeding efficiency and the hydrolysis products of reconstituted grain sorghum. Feedstuffs, 12: 19. Moe, P.W. and Tyrrell, H.F., 1977. Effects of feed intake and physical form on energy value of corn in Timothy hay diets for lactating cows. J. Dairy Sci., 60: 752--758. Morril, J.L., Dayton, A.D. and Mickelsen, R., 1977. Cultured milk and antibiotics for young calves. J. Dairy Sci., 60: 1105--1109. Moss, B.R., Beeckler, A.F., Newman, C.W. and E1-Negoumy, A_M., 1977. Enzyme supplementation of broilers rations. Poult. Sci., 56: 1741. P~rk~ny-Gy~rf~s, A. and Tbth, M., 1978. Feed utilization efficiency of enzyme-containing feeds in livestock raising. Acta Aliment. Acad. Sci. Hung., 7: 111--120. Rasmussen, P. and Holm, F., 1978. Kvaliteten af foder til enmavedyr -- i relation til lagring og teknisk forarbejdning. Beretning nr. 86. Bioteknisk Institut, Kolding, 49 pp. Runnels, T.D., 1976. The influence of feed texture on broiler performance. Poult. Sci., 55: 1958--1961. Sellars, P.N., McGill, C.E.G. and Flannigan, B., 1976. Degradation of barley by Aspergillus fumigatus Fres. Proc. III Int. Biodegr. Symp., 1975: 635--643. Vabischevich, K.D., Margolin, S.E. and Kanashkova, E.F., 1977. Effects of colamine (Premix-517) on the productivity and pork properties of growing swine. S-KH-Biol. (Minsk), 12: 182--185. Vohra, P., 1972. Evaluation of metabolizable energy for poultry. World's Poult. Sci. J., 28: 204--214.