The present invention concerns a procedure for improving the digestibility of animal feed raw material containing lignocellulose.

Furthermore the invention concerns feed pre¬ pared according to the procedure.

The feed of ruminants and onogastric animals like pigs and poultry consists mainly of material de¬ rived from plants and containing lignocellulose; such material is called lignocellulose-containing feed raw material in the following. Lignocellulose, and therefore lignocellulose-containing feed raw material, is composed of hemicellulose and cellulose fibres and of lignin binding the fibres together. The proportion of lignin and cellulose e.g. in wood is about fifty/fifty.

When ruminants are using lignocellulose-con¬ taining feed, their own enzymes are not able to deco - pose lignin nor the cellulose protected by the lignin, and the microbes in the rumen cause only partial hy¬ drolysis of the cellulose and hemicellulose fraction contained in the feed raw material. Monogastric animals are likewise unable to utilize the energy contained in the fibres to their growth, and fibres are traditionally avoided in feed for monogastric animals.

Attempts have recently been made to increase the digestibility of lignocellulose-containing feed with the aid of σellulolytic enzymes added to the feed. While significant improvements are achieved by adding cellulolytic enzymes, the greater part of the energy contained in the fibre is still unaccessible to the animal. One responsible cause is the lignin matrix, which by forming an indigestible lignin sheath around the cellulose and hemicellulose inhibits the contact between cellulolytic enzyme and cellulose.

The object of the present invention is to

eliminate the drawbacks mentioned.

The particular object of the invention is to provide a procedure by which the detriments caused by lignin in lignocellulose-containing feed raw material can be reduced and by which the digestibility and uti- lizability of lignocellulose-containing feed raw mate¬ rial can be improved.

The invention is characterized in that the lignocellulose-containing feed raw material is treated with an enzyme modifying lignin.

The invention is based on extensive research work that has been carried out, in which it was found that lignocellulose-containing feed raw material can be subjected on plant scale to treatment with lignin-modi- fying enzyme, which has a positive effect, and an ef¬ fect increasing the digestibility of the feed raw mate¬ rial, on lignocellulose-containing feed raw material and on feed therefrom prepared.

The positive effect of lignin-modifying enzyme is conceivably due to its action weakening the lignin and lignin matrix protecting the cellulose fibres, in such way that enzymes and microbes cleaving fibres gain access to the cellulose fibres, better than before.

It is known in the art through the reference Filamentous Fungi, Smith, J.E., Beny, D.R., Christian¬ sen, B.E.A., London 1983, Vol. 4, P. 284, to add lig- nin-decomposing microbe to feed raw material. However, the microbe will then grow in the feed raw material and use cellulose-containing material for its nutrition, cleaving cellulose and using carbohydrate. This natu¬ rally entails significant reduction of the food value of the feed. The microbe needs much time to cleave the lignin, e.g. several weeks.

The procedure of the invention differs from the procedure disclosed in the reference cited above in that treatment is effected directly with the aid of a lignin-modifying enzyme, such as the ligninase enzyme,

or of an enzyme preparation, that is, advantageously the microbe is not in contact with the feed raw mate¬ rial that is being treated and thereby will not decom¬ pose or cleave the cellulose therein contained. When the feed raw material is treated directly with the aid of a lignin-modifying enzyme, the treatment can be ear¬ ned out in controlled manner in plant conditions, and in exceedingly short time, in fact in a few minutes or, for instance in 1/2 hour. According to the invention, enzyme preparation is added to the feed raw material in the amount of 0.001 to 1% by weight, i.e., 50 to 10 ^"7 U/t, calculated on the total feed raw material quantity. The treatment is carried out at moisture content 1 to 90% by weight, suitably 20 to 80% by weight. If the feed to be treated consists of meadow grass, its moisture content will be e.g. 50 to 87% by weight. If the feed to be treated consists of dry feed, e.g. of industrially prepared dry feed, its moisture content may be comparatively low, w.g. on the order of 10 to 60% by weight, advantageous¬ ly 20 to 40% by weight.

The treatment time may vary within wide lim¬ its, and it is from 1 minute to 12 months. When the feed raw material is treated in plant conditions, the treatment time is e.g. 1 in. to 24 hrs, suitably 1 min. to 12 hrs. advantageously 1 min. to 5 hrs. par¬ ticularly 10 to 60 min., e.g. about 0.5 hrs. In the case of preserving silage, e.g. meadow grass, the treatment time is e.g. 2 weeks to 12 months.Treatment of the feed raw material may however equally be carried out e.g. in connection with preservation of the such as silage. There is then no need of swift reaction, in¬ stead which prevention of losses and good keeping qual¬ ity are the most important considerations. The treat- ent time may then be very long, even up to 12 months, e.g. 2 weeks to 12 months, e.g. 1 to 8 months. In that case very dilute enzyme quantities are suitably ap-

oiled, on the order of 0.001 to 1% by weight of the total feed quantity. The moisture percentage is then high, e-g- on the order of 60 to 90% by weight, advan¬ tageously about 80%. The treatment of the feed raw material with the aid of lignin-modifying enzyme is carried out at 0 to 60°C, suitably 5 to 50°C, advantageously 10 to 40°C. In plant conditions treatment at higher tempera¬ tures can be contemplated, e.g. up to 60°C; treatment taking place in connection with fodder preservation can be carried out at very low temperatures, e.g. down to 0°C.

In the procedure may be used any lignin-modi¬ fying enzyme known in itself in the art, for instance any ligninase enzyme or lignin-modifying enzyme with lignolytic activity secreted by any microbe, such as a fungus, as has been mentioned in the reference cited above (Smith, J.E. et al.), the ligninase enzymes and fungi secreting ligninase enzymes which have been men- tioned in said reference being incorporated in this application as part thereof. Further lignin modifying enzymes e.g. lignin peroxidases, mangenese peroxidaseε, phenol oxidases and/or reductases participating in lignin degradation may be contemplated.- All and any fungi secreting a lignin modifying and/or decomposing enzyme may be contemplated, for instance the lignin- modifying and/or ligninase enzymes secreted by any white rot fungus, brown rot fungus and/or soft rot fungus. The following may be mentioned as examples of lignin- modifying enzyme and ligninase enzyme secreting fungi: Phanerocaete, Polyporus, Polystictus, Coriolus, Trame- tes, Pleurotus, Trichoderma, Aspergillus, Rhizoctonia, Phlebia and Fo es. One may further contemplate microbe strains which produce lignin-modifying enzymes, as has been disclosed in the following references: Gilbertson, R.L., Mycologia 72(1980) 1-49; Kirk, T.K., Moore, E.W. , Wood Fiber 4(1972) 72-9; Trojanowski, A., Milstein, 0.,

Majcheiczyk, A., Haars, A., Huttermann, A., Lignin en- zymic and microbial degradation, Symposium Internation¬ al Paris, 23-24 avril 1987, Ed. INRA, Paris, pp. 223- 229; Flegel, T.W., Meevootisom, V., Kiatpapan, S.J., Ferment. Technol. 60(1982) 473-475; Haars, ., Hutter- an, A., Arch. Microbiol. 125(1980) 223-237; and Craw¬ ford, D.L., Crawford, R.L., Enzyme Microb. Technol. 2(1980) 11-22.

Treatment of the feed raw material may be made with any preparation containing lignin-modifying en¬ zyme, e.g. with a microbe culture solution from which the lespective microbe has been substantially remove _ or with a concentration of the respective enzyme, or with another preparation containing the respective en- sy e.

The term "lignocellulose-containing feed raw material" is understood to mean any plant-derived feed raw material containing lignocellulose which is used for raw material in preparing animal feed, e.g. plant parts such as grass, hay, straw, cereal husks, shells of oil plant seeds e.g. of sunflower, turnip rape or equivalent, cereals on the whole, bagasse, parts of ears, tops of root crops, wood or woody biomass, or other material or products made of these. The treatment may be given to compounded feed raw material, that is, to a feed raw material or feed mix containing lignocel¬ lulose-containing feed raw material. Treatment of ready-compounded complete fodder containing lignocel¬ lulose-containing feed raw material may also be contem- plated. Feed prepared by a procedure according to the invention is also within the sphere of the invention.

It has been found in tests that have been made that the procedure is suitable to be applied in the treatment of feed raw material and feed intended for feeding both ruminants and monogastrics. One may thus contemplate treatment of feed containing lignocellulose- containing feed raw material that is intended e.g. for

neat, such as dairy cattle, beef cattle and calves, for sheep, pigs, poultry, fur animals, fish, pets, horses, reindeer, and on the whole for any animals whatsoever. Through the invention the digestibility of feed containing lignocellulose-containing feed raw material has been found to improve. The animal is able to utilize the feed. The feed conversious efficiency (kg feed/by live weight, kg feed/by weight gain) has improved. The procedure of the invention is easily, sim¬ ply and advantageously implementable, and in the prepa¬ ration of silage fodder for instance the procedure can be implemented using conventional equipment, and in a feed factory e.g. using a conventional long-time con- ditioner.

The procedure further enables inexpensive feed raw materials to be used which the animal cannot util¬ ize in the absence of a separate treatment. The proce¬ dure is suitable for all and any lignocellulose-con- taining feed raw materials.

The invention is described in detail in the following with the aid of embodiment examples, which are intended to illustrate the invention.

Example 1: Increased rumen digestibility of hay

Fresh chopped hay, chaff length 1-4 cm, dry matter 18%, was made into silage on laboratory prem¬ ises. The enzymes were diluted in water 1;5 and added to the grass by spraying. To the samples of the control group (1) no enzymes were added, to those of the second control group (2) cellulases Multifect L 250, activity CMC-ase 2500 IU/ml, Finnish Sugar Co. Ltd., at 200 ml/tn and Multifect K, activity xylanase 10 000 IU/ml, Finnish Sugar Co. Ltd., at 200 ml/tn were added, and to the samples of test group (3) were added cellulases Multi¬ fect L 250 at 200 ml/tn and Multifect K at 200 ml/tn,

ind ligninase preparate B 8.5 ml/tn. The silos were tightly sealed and weighted, 200 kg/m ² , duration of silage process 28 days. The silos were then opened and the rumen digestibility of the feed was determined in sacco according to Mehrez and Orskov (J. Agric. Su, Camb. 88: 645-650) and analyses were made of pH, lac¬ tic acid, acetic acid, propionic acid, butyric acid and sugars.

The cellulase activity was determined as CMC- ase activity (Mandels M. , Weber J., Adv. Chem Ser. 95 (1969) 391-413). 1 ml Of suitable diluted enzyme solu¬ tion in acetate buffer (0.05 M NaAc, pH 4.8) and 1 ml of CMC substrate (1 % CMC, 0.05 M NaAc, pH 4.8) are mixed together. The solution is incubated for 10 min at 50 °C. The reaction is stopped by adding 3 ml of DNS reagent. One enzyme unit liberates 1 micromole of reduc¬ ing sugars calculated as glucose per one minute under assay conditons.

The hεmicellulase activity was determined as xylanase activity (Khan A.W. et al., Enzyme Microb. Technol. 8 (1986) 373-377). 1 ml Of a suitably diluted enzyme solution in acetate buffer (0.05 M NaAc, pH 5.3) is tempered at 50 °C. 1 ml of xylan substrate (1 % xylan, 0.05 M NaAc, pH 5.3) is added. The sample is incubated for 30 min at 50 °C. The reaction is stopped by adding 3 ml of DNS reagent (3,5-dinitrosalicylate) , and the colour is developed by cooking the sample mix¬ ture for 5 min. The absorbance is measured at 540 nm. One enzyeme unit liberates 1 micromole of reducing sugars per one minute under assay conditions calculated as glucose.

The spesific gravities of the enzyme pre- parators were 1.0 - 1.3 kg/dm ³ .

Ligninase preparate B (phenol oxidase) was prepared by using the fungus Trametes hirsuta according to the following prodecure. Trametes hirsuta CBS 248.30 was kept at +4 °C YM Broth (Difco) with added lignin (5

g/1 indulin AT, Westwaco) and agar. Inoculum was grown in shake flasks using the same medium but without agar. The production medium contained 2.3 % glucose, 2.5 % yeast extract, 0.55 % birch lignin and 5 % mineral solution. The mineral solution contained 1 g/1 CaCl ₂ dihydrate 1 g/1 FeS0 ₄ heptahydrate, 0.1 g/1 ZnS0 ₄ hep- tahydrate, 0.16 g/1 CuS0 ₄ pentahydrate and 1.0 g/1 Titriplex III. The inoculum for the production fermenta¬ tion was prepared by homogenizing a mycelium containing Petri dish into the inoculum medium, and incubating the flasks in a shaker at +30 °C for two days. For the production fermentation about 12 % of inoculum from shake flasks was used. In the production fermentation temperature was kept at +30 °C, and dissolved oxygen was kept above 20 % by controlling the agitator speed automatically. The pH was kept between 5.2 and 5.4 by automatically adding aqueous ammonia. The duration of the production fermentation was 2.5 days. The phenol oxidase activity was analyzed from the culture filtrate at +25 °C by measuring the change in absorbance in one minute (at 460 nm) from a mixture which contained 4 ml of 2.8 mM guaiacol (pH 4.8 in 0.05 M citrate buffer) and 1 ml of suitable enzyme dilution. One activity unit increased the absorbance by one absorbance unit at the above mentioned conditions. The fermentation broth contained about 12 units/ml at the end of the fermenta¬ tion. The clear culture filtrate, which did not contain any live production organisms, was concentrated about 10-fold using conventional ultrafiltration. This con- centrate is ligninase preparate B.

For establishing the effect of the enzyme treatment the samples were subjected to the rumen de¬ composition test; the results are presented in Table 1.

Table 1: Rumen decomposability of silage, in per cent of dry matter, and (in brackets) improvement of rumen decomposability over the control samples.

Incubation period in rumen, hrs

treatment 0 hrs 2 hrs 8 hrs

(1) 29.6 33.1 46.7

(2) 31.6 36.8 49.7

(6.8%) (11.2%) (6.4%)

(3) 32.3 37.4 53.0

(9.1%) (13.0%) (13.5%

Tt is observable, in Table 1, that the diges¬ tibility improves after 8-hrε rumen incubation 13.5% relative to the control sample with ligninase and cel- lϋlase treatment, treatment (3); the corresponding im¬ provement with cellulase treatment alone was 6.4%. The result of the rumen digestibility test is supported by the measurements relating to quality of the test feeds; the results are presented in Table 2.

lable 2: Values describing the quality of the test feeds Dry Lactic pH HPLC matter acid, Acetic Prop. Butvric Glue.

I % fiesh acid acid acid

(15 23.71 1.54 4.2 0.40 0.0S 0.0 0.001

(2) 23.83 1.87 4.2 0.44 0.08 0.0 0.001 (3) 23.68 1.92 4.2 0.45 0.10 0.0 0.1

Xyl. Cellob. Arab. Fructose

(1) 0.002 0.001 0.001 0.06

(2) 0.025 0.004 0.0045 0.02 (3. 0.25 0.06 0.025 0.07

Table 2 reveals that pH and quantity of organic acids are on the same level in all groups, while tne quantity of glucose and xylose is about tenfold in the ligninase-treated feed, compared with the other treat¬ ments.

T O

Example 2 : Increased rumen digestibility of hay

The digestibility of ground dry hay was deter¬ mined in vitro by the first step of Tillet and Terry's (1963) method (J. Org. Grass/Soc. 18: 104-111). 0.5 g of dry hay were weighed into centrifuge tubes and the enzymes and rumen fluid were added. The tubes were in¬ cubated in anaerobic conditions at 18°C during 12, 24 and 48 hours, whereafter the undigested residue was determined. For cellulase, Multifect L 250 was used at 0.1 ml per 0.5 g hay, for hemicellulase, Multifect K at 0.1 ml per 0.5 g hay and ligninase preparate B 0.7 ml/0.5 g; activities same as in example 1.

The test results are presented in Table 3.

Table 3: In vitro digestibility of ground hay with rumen fluid and enzyme treatments

Incubation time, hours

Treatment 12 r, 24 hrs 48 hrs ( (11)) 2255..4455 4 466,,4422 54.27

(2) 22.19 46.40 52.18

(3) 28.71 52.30 55.15

(4) 28.2δ 48.19 49.88

(5) 29.81 50.77 57.16

The samples of the control group were treated with rumen fluid only, the samples of the second con¬ trol group (2) were treated with cellulase, the samples of the first test group (3) were treated with cellulase and ligninase, the samples of the third control group (4) were treated with hemicellulase, and the samples of the second test group (5) were treated with hemicellu¬ lase and ligninase.

In Table 4 is presented the improvement of diges- tibility of hay compared with mere rumen fluid treat¬ ment, calculated from the results in Table 3.

Table 4: Hay digestibility improvements in vitro achieved with enzyme mixtures, compared with mere rumen fluid treatment

Incubation time, hours

Treatment 12 nr 24 hrs 48 hrs (2)/(l) -12.8% +0.0% -4.0%

(3)/(l) +12.8% 12.7% 1.6%

(4)/(l) 11.1% 3.8% -9.1%

(5)/(l) 17.1% 9.0% 5.3%

((33))// ((22)) 2299..44%% 12.7% 5.7%

(5)/ (4) 5.4% 5.4% 14.6%

! 4 )/ ( 2) 27.4% 3.9% -4.4%

( 5) / ( 3 ) 3.8-6 —2.9-6 3. D-6

Example 3; Improvement of young bull growth

The grass was cut and predried for 4 hrs, whereafter the grass was harvested with a high preci¬ sion chaff cutter, with simultaneous dosage of the en- silage substances. For ensilage substance was used (1) formic acid (AIV) at 4 1/tn of grass, (2) cellulase Multifect L 250 and hemicellulase Multifect K al¬ together 300 ml/tn of grass, (3) cellulase Multifect L 250 and hemicellulase Multifect K altogether 300 ml/tn of grass and ligninase preparate B 20 ml/tn. The silage period was 114 days, whereafter a feeding test was carried out with young bulls. The test comprised 18 animals, 6 animals per group, and duration of test was 57 days. The diets were 27% complete feed and the rest silage fodder ad libitum. The results of the feeding test are presented in Table 6.

Table 5: Feeding test performed with young bulls

Treat¬ Concentrate Silage Growth, Feed consump¬ ment consumption. consumption, g/day tion, kg dry kg dry kg dry matter per kg matter matter incr. growth

(1) 1.89 4.96 1006 6.82

(2) 1.90 5.09 1073 (6 .7%) 6.52

C3) 1.90 4.96 1111 (10.4%) 6.18

The results reveal that the improvement of growth by effect of ligninase and cellulase is 10.4%, while the a,mprovement of growth by effect of cellulases is only 6.7% compared with the control. The feed consump¬ tion per kg incremental growth was likewise reduced.

Example 4: Broiler test 1

In the test, the test animals were fed (A) barley and oats treated with cellulase enzymes, (B) correspond¬ ing ligninase and cellulase enzyme-treated (EPR) barley and oats, and (C) as control, corresponding barley and oats (untreated). The test comprised altogether 1200 day-old ASA chicks, divided into three feeding groups. There were four replicates in each feed group. The chicks were fed at the age of 1 to 15 days, feeding period (1) with feeds which differed only regarding enzyme treatment of the barley and oats, during the age period of 16 to 37 days, feeding period (2), all feed composition were all changed in equivalent manner. Enzyme treatment (EPR) of barley and oats was carried out at 60°C, moisture 30%, time 30 min./ 60°C, moisture of treated barley and oats, respectively, less than 12% after treatment. During the treatment no substantial microbe growing was found. In the treatment of the cereals there was, per 1000 kg barley and oats, respec- tively, 256 kg water, 400 ml cellulase Multifect L 250 and 100 ml xylanase Multifect K., in the combined cel¬ lulase and ligninase enzyme treatment furthermore 85 ml

lignasε preparation B. The compositions and calculated values of the feeds are presented in Tables 6 and 7.

Table 6: Compositions of broiler feeds

5 Test feeds A B C

(1) (2) (1) (2) (1) (2)

Composition. %

Barley - - - - 33.3 36.0

Oats - - - - 20.0 20.0 0 EPR barley 33.3 36.0 33.3 36.0 - -

EPR oats 20.0 20.0 20.0 20.0 - -

Shelled oats 10.0 10.0 10.0 10.0 10.0 10.0

Soybean 21.0 21.0 21.0 21.0 21.0 21.0

Fish meal 8.0 4.5 8.0 4.5 8.0 4.5

Dicalciumphosph. 0.6 0.6 0.6 0.6 0.6 0.6

CaCOa 1.5 1.9 1.5 1.8 l .5 1.8

Na bicarbonate 0.2 0.2 0.2 0.2 0.2 0.2

Bonding agent 0.75 0.75 0.75 0.75 0.75 0.75

Coccidiostat 0.1 0.1 0.1 0.1 0.1 0.1 0 Lysine 0.1 0.1 0.1 0.1 0.1 0.1 ethionine 0.15 0.15 0.15 0.15 0.15 0.15

Fat 3.0 3.5 3.0 3.5 3.0 3.5

Vitamin and trace element premix 1.30 1.30 1.30 1.30 1.30 1.30

Table 7: Broiler :feeds, calculated ^, values

Test feeds A B C

(1) (2) (1) (2) (1) (2)

CalcuD ated values 0 ME, MJ/kg 11.7 11.7 11.7 11.7 11.7 11.7

Crude prot. % 22.0 22.0 22.0 22.0 22.0 22.0

Lysine 1.37 1.18 1.37 1.18 1.37 1.18

Methionine 0.56 0.50 0.56 0.50 0.56 0.50

5 The results of the raising test are presented in Table 8

Table 8: Results of the raising test

Feed Age A B C

Live weight, g (1) 15 302 311 307

(2) 37 1510 1515 1473

Ratio 102.5 102.9 100

Feed intake, (1) 1-15 388 383 395 g per broiler (2) 16-37 2330 2374 2292

Total 1-37 2757 2687

Ratio 101.7 103.6 100

Feed efficiency, (1) 1-15 1.28 1.23 1.29 kg/kg live weight (2) 1-37 1.81 1.83 1.82

Ratio 99.5 100.5 100

Feed efficiency, (1) 1-15 1.49 1.41 1.47 kg/kg incr. wt. (2) 16-37 1.94 1.99 1.96

1-37 1.86 1.88 1.87

There were no differences in the chicks' growth during feed (1). During feed (2) the EPR (A) and lig¬ ninase EPR groups (B) grew 2.5% and 2.9% better than the control group (C). The broilers in the ligninase EPR group (B) were 52 g heavier and those in the EPR group were 37 g heavier than the broilers of the control group. The chicks in group (A) ate 1.7% more and those of group (B) ate 3.6% more than the chicks of the con- trol group. The best feed efficiency value during feed (1) -as recorded in the ligninase EPR group (B) , dif¬ ference 4.7%.

Example 5: broiler test 2 The feeds used in this example were prepared as follows. Hydrothermally pretreated barley-oats (50/50) mixture (PR-treatment) was made by treating the mixture, which had been milled to a particle size of less than 1 mm, at +60 °C for 30 minutes. The moisture content of the feed was adjusted to 25 - 30 % by adding steam and water to the starting material. After the treatment the feed was dried to a moistur level of less than 12 % by

fluidized bed drying (temperature of incoming air +60 °C) . Hydrother ally and enzymatically pretreated feeds (EPR-treatment) were prepared as the PR-treated ma¬ terials, but the enzymes were added uniformly to the mixture by adding them with the added water used for increasing the moisture. The amounts of enzymes used in the various EPR-treatments are presented in table 9.

Table 9: Enzymes added to EPR-treated feeds (ml/kg)

Enzyme group (4) group (5) group (6)

Multifect L 250 0.4 - 0.4

Multifect K 0.4 - 0.4

Ligninase preparate A 10 2.5

Ligninase preparate A (lignin peroxidase, man¬ ganese peroxidase) was prepared by the fungus Phane- rochaete chrysosporium as described in Willershausen H., Jager A., and Graf H., Ligninase production of Phanerochaete chrysosporium by immobilization in bio- reactors, Journal of biotechnology, 6 (1987) pp. 239- 243 with following differences. The fermentation was limited by carbon instead of nitrogen using glucose as the carbon source, for aeration contiuous flow of pure oxygen was introduced and nylon acted as the carrier material for mycelium instead of silicon tubing. For activity measurement the method of Tien and Kirk (Proc Natl. Acad. Sci. USA 81 (1984) pp. 2280 - 2284) was used. After the activity reached a maximun (about 200 IU/1), the broth was recovered, and the clear broth, which did not contain any live production organisms, was concentrated about 200-fold by conventional ultra- filtration. The concentrate obtained as a result of this procedure is ligninase preparate A. In the test adult coccerels were starved 48 hours before the trial, during the time they received 50 ml glucose solution (containing 25 g of glucose) 16 and 40

hours before the trial. The test feeds were put by a stainless steel funnel to the crop of the animals. The feed dosage was 50 g. Excreta were collected during 48 hours after feeding. Endogenous energy loss (EEL) was measured by birds which received only 50 g of glucose during the trial. All the birds received 50 ml water 30 hours after feeding. Brutto energy and nitrogen were analyzed of the feeds and excreta.

The true metabolisable energy TME was determined from the results according to equation (I).

Feed Feed Excreta Exc.br. intake * br. - weight * energy + EEL g energy g kJ/g kg kJ/g

TME = (I)

Feed intake g

Nitrogen corrected TME _N was determined from equation (II) wherein

NR _r = nitrogen retention by the birds which received feed (g) NR _G = nitrogen retention by the birds which received glucose (g)

TME - ( R^ - NR _G ) * 0.034

TME _N = (II)

Feed intake g

The trial groups were following:

(1) 0-control

(2) barley-oats (50:50)

(3) hydrothermally pretreated barley-oats (PR-treatment)

(4) hydrothermally and entzymatically (hemicellulase + cellulase) pretreated barley-oats (EPR-treatment)

(5) EPR-barley-oats (ligninase)

(6) EPR-barley-oats (liginase + hemicellulase + sel-

lulase)

The effect of processing and enzymes on the TME* value of barley-oats mixture was following: (1)

(2) 100 (comparation number)

(3) +

(4) ++

(5) ++

(6) +++