JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND Maataloustieteellinen Aikakauskirja 392 Voi 50:392-397, 1978 The use of cellulases for increasing the sugar content of AIV-silage Tarja Vaisto, Matti Heikonen and Matti Kreula Valio Laboratory, SF-00180 Helsinki 18, Finland Matti Linko Technical Research Centre of Finland, Biotechnical Laboratory SF-02150 Espoo 15, Finland Abstract. AIV silages were treated with cellulases. Five different enzyme prepa- rations were compared. The highest amount of reducing sugar in silage was 190 g/kg (dry basis) and the highest amount of glucose in the press juice 24 g/dmB. A combi- nation of two enzyme preparations produced more glucose than any enzyme tested alone. Glucose production was approximately proportional to the enzyme concentration. Because of lactic acid fermentation, cellulase alone could not maintain the sugar content at a high level, a preservative being required to inhibit the lactic acid bacteria. No differences were noted in the glucose contents in silages with different dry matter con- tents (24-29 %). Introduction In Finland silage is made mainly by the AIV method, in which the pH is lowered by AIV solutions. Nowadays most often AIV II solution, which contains 80 % formic acid and 2 % phosphoric acid, is used. In good silages made by the AIV method the lactic acid content is below 1 %, the ammonia content below 0.5 g 1 and the sugar content above 2 % (Heikonen et al. 1978). Rumen microbes need sufficient energy to synthesise protein from ammonia and other soluble nitrogen compounds in feed. In practice it may happen that silages are deficient in energy rather than in pro- tein. Therefore the production of more sugar in silage by cellulolytic enzymes was studied. In silage experiments cellulases have usually been used to produce sugars for fermentation by lactic acid bacteria. Sometimes the aim has been to improve the digestibility by lowering the fiber content. Only seldom have preservatives been used to stabilize the sugar content. In earlier investigations, enzymes produced by Aspergillus spp., which have hemicellulolytic and proteolytic activity in addition to cellulolytic ac- https://www.c-info.fi/en/info/?token=pWfbMx3He9A0Uzy6.LSk3PZKHL3niNaWvPWAE-A.9WlVa2Y96PbKF4cj6quOwI-IOffhDDF3KgXwS2cpj218SmBfqcgNFHXTt8FaYtYKJfgF8kh-rDjvvN5f47lzE_0WVi0x8l5zqRP7j6JmMD0ws1C9K_rU3cBcRoMAOY1B1h4YlhahN4fYfqSGW4xuS5aNdiYh5iFJ9R8X7SoWIi6D1suRWb4wKLzTWHSEJEdxIXJhyIT_EZLLo2LnsAaVQrSo0h3T 393 tivity, were used. The organoleptic properties of the treated silages were good, the lactic acid content was higher and the pH lower than in the control silages (Boiko et al. 1967, Ezdakov and Fesjun 1967, Konoplev and Stser- bakov 1970). Henderson and McDonald used formic acid as the preservative and added cellulases produced by A. niger (0.4 %) to the herbage. The amount of cellulose in the silage decreased markedly during a period of 61 days. They also compared cellulases produced by A. niger and Trichoderma reesei and found the latter more efficient. The highest sugar content produced was 153 g/kg DM. In alfalfa silage the hydrolysis of cellulose increased with successive addi- tions of enzyme (0.1—0.5 %). The maximum hydrolysis was 29 %. The titratable acidity and the content of reducing compounds were greater in enzyme-treated silages (Leatherwood et al. 1963). Autrey et al. (1975) added a fungal cellulase from T. reesei to whole maize. The cellulose content decreased significantly during one year’s ensilaging. There was some indication of improved digestibility by cows at the higher levels of cellulase addition, but the differences were not significant. Neither did Olson and Voelker (1961) succeed in improving the digestibility of maize by A. oryzae cellulases. By adding cellulase with CaC0 3 buffer to sor- ghum prior to ensiling, McCullough (1964) produced a silage with 20 % less cellulose than the untreated control silage. The digestibility of the former was slightly increased (treated 54.5 %, untreated 42.6 %). Experimental Materials and methods The timothy-clover mixture was obtained from the Viikki Experimental Farm of Helsinki University. Silage was made three times: June 15, July 26 and September 15. The dry matter contents of the silages were 24, 31 and 28 %, respectively. The freshly cut herbage was allowed to dry in the field for 2 5 hours before chopping. The preservative was sprayed onto the herb- age from a plastic spray bottle, and the dry enzyme preparation was spread onto the herbage. The silage was packed into polyethene bags, about 500 g per bag. The bags were put into plastic jars (two bags per jar) and a sandbag was placed on the top to compress the silage. The preservative used was AIV 11, which was first diluted 1: 6 and then used at the rate of 42 ml/kg. The enzymes used, their manufacturers and activities are shown in Table 1. The /hglucosidase activity of cellulases was determined by the method of Norkrans (1957) and activity in the digestion of filter paper (FPU) by the method of Mandels et al. (1976). Silage quality was assayed by measuring the pH, determining lactic, propionic, acetic and butyric acids by paper chromatography (Miettinen and Virtanen 1951) and by measurement of ammonia. The hydrolysis of cellulose was followed by determining the reducing sugars (Nelson 1944 and Somogyi 1945) and glucose (GOD-Perid, Boehringer Mannheim GmbH, Mannheim, BRD). The dry matter was determined by drying the samples in aluminium dishes at 80° C overnight. Table 1. Enzymes used. Enzyme activity, xlO-3 FPU Supplier units Cellulase S 20 0.21 Society Rapidase, Sedin, France Hemicellulolytic complex 153 0.07 Socidtö Rapidase, Sedin, France Maxazyme 153 0.68 Gist-Brocades nv, Delft, Netherlands Meicelase 163 0.80 Meiji Seika Kaisha Ltd., Osaka, Japan Onozuka SS 165 0.22 All Japan Biochemicals, Co. Ltd., Shingikancho, Nishinomiya, Japan VTT cellulase 120 2.0 Technical Research Centre of Finland, Biotechnical Laboratory, Helsinki. Finland. Results Comparison of enzymes The silage was made on June 15. The enzymes used were: Maxazyme, Meicelase and VTT cellulase (0.05 %), Cellulase S and Hemicellulolytic com- plex (0.11 %). The greatest increase in the glucose content was obtained with VTT cel- lulase. The amount of glucose in the press juice of silage treated with it was 24 g/dm3 after six months of ensilaging (Fig. 1). The amount of reducing sugars was highest in silages treated with Maxazyme or VTT cellulase (Fig. 2). The amount of glucose was 30—65 % of the amount of reducing compounds in the press juice. Fig. 1. The concentration of glucose in the press juice of silage with the following cellulase preparations: Cel- lulase S V. Hemicellulolytic complex H, Maxazyme A, Meicelase A and VTT cellulase □ . Fig. 2. The content of reducing sugars in silage with Cellulase S V. Hemicel- lulolytic complex B Maxazyme A. Meicelase A. and VTT cellulase □ . 394 Combinations of enzymes The silage was made on June 15 and July 26. In addition to VTT cellulase, Onozuka (by weight 1:1, 1:3 and 3:1 June 15; 1.5:1, 3:1 and 6:1 July 26) and Hemicellulolytic complex (1:1 June 15; 1.5:1, 3:1 and 6:1 July 26) were used. The total enzyme concentration was 0.05 %. The combinations gave greater glucose concentrations than VTT cellulase alone. In the first series of experiments (June 15) only one silage treated with the combination of enzymes (VTT -f- Onozuka 1: 3) contained less glucose than the silage treated with VTT cellulase alone (Fig. 3). In the second series (July 26) the highest amounts of glucose were obtained with a combination of VTT cellulase and Hemicellulolytic complex. The highest glucose content in the press juice was 17 g/dm3 (Fig. 4). The lower glucose contents in the second series of experiments were probably due to the higher dry matter con- tent of the silage. Amount of enzyme The effect of the amount of enzyme was tested with VTT cellulase and Meicelase, using concentrations of 0.02 0.4 %. The silage with Meicelase was made on June 15 and with VTT cellulase on September 15. A control sample without added enzyme was included in each series. The amount of glucose formed in 56 days was proportional to the con- centration of enzyme, with both VTT cellulase and Meicelase (Fig. 5). The enzymes cannot be intercompared here because of the different raw material. The silages with highest enzyme concentrations (0.2 and 0.4 %) contained more free water than the control silages Fig. 3. The concentration of glucose in the press juice of silage, made on June 15, with VTT cellulase □ , Hemi- cellulolytic complex 9, VTT + Hemi- cellulolytic complex 1:1 VTT + Onozuka 1; 1 A. 1: 3 V and 3: 1 Fig. 4. The concentration of glucose in the press juice of silage, made on July 26, with VTT + Hemicellulolytic complex 1.5: 1 I, 3; 1 �, 6:1 T. VTT + Onozuka 1.5: 1 □ , 3: 1 A, 6: 1 V and no enzyme 0. 395 Use of preservative The silage was made on June 15. AIV II solution was added at the rate of 0,5, 6 and 7 ml/kg herbage. The pH of the silage without preservative fell to 4 during a period of one month, while the amount of lactic acid rose to nearly 2 %. After using preservative to lower the pH the concentration of lactic acid remained below 0.2 % throughout the experiment. Without pre- servative the concentration of glucose fell quickly to below 0.5 %. Cellulases may have formed more sugar in the silage, but lactic acid bacteria fermented it immediately to lactic acid. Effect of dry matter content The herbage was dried in air before ensiling. The dry matter contents were 24 %, 27 % and 29 %. There were no significant differences in the amounts of glucose formed in these three silages. Discussion Henderson and McDonald reported that the amount of sugar in silage treated with T. reesei cellulases increased about 150 % during 175 days of ensilaging. In the experiments reported here the period of ensilaging was shorter; in 84 days the amount of reducing sugar increased about 90 % with the same amount of enzyme (Meicelase 0.4 %). The increase in the amount of glucose was greater, 170 %. The highest amounts of glucose were formed by cellulases from Trichoderma spp. (Maxazyme, Meicelase, VTT cellulase.) The use of cellulases to increase the sugar content of AIV silage made of low-sugar material would be advantageous if the sugar produced by the en- zymes were cheaper than that in the usual sugar-containing forages. Accord- ing to prices of whey powder and molasses the acceptable price for cellulases is 25—120 mk/kg, depending on the enzyme (enzyme protein 50—400 mk/kg). The effect of cellulases on the feeding value of AIV silage, and thus the eco- nomics of enzyme treatment, has not yet been evaluated. Recent progress in the cellulase production methods gives rise to the belief that the treatment of silage with enzymes will soon be economically feasible (Linko et ai. 1977). Fig.s. Glucose formed in 56 days with different concentrations of Meicelase (Y) and VTT cellulase (A). 396 397 REFERENCES Autrey, K. M., McCaskey, T. A. & Little, J. A. 1975. Cellulose digestibility of fibrous materials treated with Trichoderma reesei cellulase. J. Dairy Sci. 58:67 71. Boiko, I. 1., Zubrilin, A. A. & Berezovskii, A. A. 1967. Uluchshenie silosuemosti rastenij s pomoshch'yu fermentiykh preparatov. Zivotnovodstvo 29: 49 51. Edzakov, N. B. & Fesyun, A. P. 1967. Fermenty uskoryayut protsess silosovaniya kormov. Zivotnovodstvo 29: 51 53. Heikonen, M.. Moisio, T. & Kreula, M. 1978. AIV-rehun laatuperusteet. Karjatalous 54, 4: 14-19. Henderson, A. R. & McDonald, P. 1977. The effect of cellulase preparations on the chemical changes during the ensilage of grass in laboratory silos. J. Sci. Food Agric. 28: 486—490. Konoplev, E. G. & Shcherbakov, L. A. 1970. Vliyanie razlichnykh fermentnykh preparatov na mikrobiologicheskie i biokhimicheskie pokazateli silosa. Mikrobiologiya 34; 527 532. Leatherwood, J. M., Mochrie, R. D,, Stone, E. J. & Thomas, W. E. 1963. Cellulose deg- radation by enzymes added to ensiled forages. J. Dairy Sci. 46: 124 127. Linko, M., Markkanen, P., Bailey, M. & Leisola, M. 1977. Production of cellulases and hemicellulases by Trichoderma viirde. Proc. Bioconversion Symp. Indian Institute of Technology, Delhi, 329 350. Handels, M., Andreotti, R. & Roche, C. 1976. Measurement of saccharifying cellulase, Biotechnol. Bioeng. Symp. 6:21—23. McCullough, M. E. 1964. Influence of cellulase on silage fermentation. J. Dairy Sci. 47; 342. Miettinen, J. K. & Virtanen, A. I. 1951. A rapid method for determination of fatty acids and ammonia by means of paper chromatography. Ann. Acad. Sei. Fenn. A II 41. Nelson, N. 1944. A photometric adaptation of the Somoqyi method for the determination of glucose. J. Biol. Chem. 153:375 380. Norkrans, B. 1957. Studies of and cellulose splitting enzymes form Polyporus annosus Fr. Physiol. Plant. 10: 198 214. Olson, M. & Voelker, H. H. 1961. Effectiveness of enzyme and culture additions on the preservation and feeding value of alfalfa silage. J. Dairy Sci. 44; 1204. Somogyi, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160: 61 —75 Ms received September 29, 1978. SELOSTUS Sellulaasin käyttö AIV-rehun sokeripitoisuuden lisäämiseksi Tarja Vaisto, Matti Heikonen, Matti Kreula Valion laboratorio, 00180 Helsinki 18 Matti Linko Valtion teknillinen tutkimuskeskus, Biotekniikan laboratorio 02150 Espoo 15 Työssä vertailtiin eri sellulaasivalmisteiden vaikutusta selluloosan hydrolyysiin AlV- rehussa, haettiin tarvittava entsyymipitoisuus sekä tutkittiin säilöntäaineen määrän sekä raaka-aineen kuiva-ainepitoisuuden vaikutusta. Parhaiten rehun glukoosipitoisuutta lisäsi VTT:n selluinasi, puristenesteen glukoosipitoisuus oli lopuksi 24 g/dm3 . Pelkistävien sokeroi- den määrä kasvoi eniten Maxazymellä ja VTT:n sellulaasilla. VTT:n sellulaasin glukoosintuo- tantoa voitiin vielä lisätä käyttämällä sen kanssa yhdessä entsyymiä, jonka aktiivisuus oli suuri. Suurimmilla käytetyillä pitoisuuksilla rehun rakenne oli vetisempää ja hajonneempaa kuin kontrollirehujen rakenne. Käytettyjen kuiva-ainepitoisuuksien (24 29 %) välillä ei havaittu eroja syntyneiden sokeroiden määrissä. Ilman happoa säilötystä rehusta maitohappobakteerit käyttivät sokerin maitohapoksi, happoa lisättäessä sokeripitoi- suus pysyi korkeana.