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The effect of cellulase and hemicellulase plus pectinase on the aerobic stability and fibre analysis of peas and wheat silages
ANIMAL FEED SCIENCE AND TECHNOLOGY ELSEVIER
Animal Feed Science and Technology
55 (1995) 287-293
The effect of cellulase and hernicellulase plus pectinase on the aerobic stability and fibre analysis of peas and wheat silages ’ Z.G. Weinberg *, G. Ashbell, Yaira Hen, A. Azrieli Forage Preservation and By-Products Research Unit, Department of Stored Products, The Volcani Center. Bet Dagan 50250, Israel Received
13 September
1994; accepted 9 February1995
Abstract
The effect of applying increasing levels of cellulase and hemicellulase plus pectinase (CelluclastB and ViscozymeB, Novo, Denmark) to peas and wheat silages was studied under laboratory conditions. The enzymes were applied at 0.02.0.1 and 0.2% each, corresponding to 0.3, 1.5 and 3.0 NCU (Novo cellulase units) of Celluclast, and 0.024,O. 12 and 0.24 FBG (fungal P-glucanase units) of Viscozyme per gram of fresh crop. All treatments were enriched with a lactic acid bacteria inoculum, applied at I O4colony-forming units per gram of forage. The following parameters of final silages (45 days) had a significant (P < 0.05) linear regression on log (enzyme concentration + 1): pH, residual sugars, lactic acid, NDF (neutral detergent fibre) and ADF (acid detergent fibre). When enzyme levels increased from 0 to 0.2%, the NDF and ADF contents decreased from 355 and 317 to 303 and 255 g kg- ‘, respectively, in the pea silages, and they decreased from 568 and 357 to 522 and 340 g kg-‘, respectively, in the wheat silages. Enzyme treatments resulted in enhanced aerobic deterioration in both pea and wheat silages. This was evident from higher yeast and mould counts, more intensive CO2 production and visible mould growth during exposure to air for 5 days. Keywords: Cellulase; Hemicellulase;
, Pectinase; Silage; Aerobic stability; Fibre analysis
1. Introduction
A successful ensiling process requires a minimal concentration of fermentable sugars (3-5% in DM), mainly hexoses. However, the majority of carbohydrates in plants is in the * Corresponding author. ’ Contribution from the Agricultural E, 1994 Series.
Research Organization,
The Volcani Center, Bet Dagan, Israel, No. 1259-
0377.8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO377-8401(95)00785-7
288
Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
form of fibrous polymers that make up the cell wall and are not fermented by lactic acid bacteria (LAB). In order to obtain the necessary level of fermentable water-soluble carbohydrates (WSC) for the lactic fermentation in crops which are low in WSC, the use of cell-wall degrading enzymes has been suggested. Such enzymes comprise cellulases, hemicellulases and pectinases. The expected benefits attributed to enzymes are the release of fermentable sugars and a decrease in fibre content which is related to improved digestibility (Muck, 1993). Spoelstra ( 199 1) reviewed studies on the effect of enzymes on fermentation parameters in silages. Results of studies mentioned in this review indicate that such enzymes are more active in moist silages than in drier silages. Enzymes applied at commercial dosages were not effective in grass silage (Honig and Pahlow, 1990). Weinberg et al. (1990) showed that cellulase alone was not effective and a mixture which also contained hemicellulase plus pectinase was necessary to hydrolyse alfalfa cellwall. Such a mixture, applied at 0.025% of each enzyme preparation, resulted in a slight decrease in fibre content in direct-cut alfalfa silages, but not in wilted alfalfa silages (Tengerdy et al., 1991). Selmer-Olsen et al. ( 1993a,b) studied the effect on ryegrass silages of enzymes prepared from fungi. These enzymes caused a lower pH, lower ammonia-N and ADF contents, and increased residual WSC content. Similar trends were obtained with silages of moist grass*lover mixtures (Selmer-Olsen, 1994). In our laboratory, an enzyme mixture, comprising cellulases plus hemicellulases and pectinases, was added at 0.02% each, to pea, ryegrass and wheat silages at various stages of maturity and DM contents (Weinberg et al., 1993). The enzymes at this rate of application resulted in only a slight decrease in NDF and ADF contents in silages of young moist crops (%DM < 25)) and did not affect silages of more mature or dry crops. The purpose of the present work was to study the effect of these enzymes applied at higher rates on the fibre analysis and aerobic stability of pea and wheat silages.
2. Materials and methods The crops used in these experiments were field peas (Pisum sutiuum) harvested at the podding stage (29.5% DM) , and wheat (Triticum vulgaris) harvested at the milk ripening stage (30.7% DM) . Whole plants were chopped to 1.5 cm (using a Wintersteigeti chopper, Austria), treated and ensiled in 1.5-1 glass jars equipped with a lid that enables gas release only (Week@, Germany). The jars were stored at 25 + 2°C. Three jars from each treatment were sampled for analysis on days 4 and 45 after ensiling. At the end of the ensiling period (after 45 days), the silages were subjected to an aerobic stability test lasting 5 days in a system developed by Ashbell et al. ( 1991) . In this system, number of yeasts and moulds, change in pH and the amount of CO1 produced during the test are indicators for aerobic deterioration. The whole batch of forage was treated before ensiling with 4 X lo4 colony-forming units -’ of lactic acid bacteria (H/M F inoculant No. 9927, Medipharm USA, containing g Pediococci acidilactici, Lactobacillus plantarum and Enterococcus faecium). The inoculated forage was used as control (no other additives); enzyme treatments comprised a mixture of Celluclast@ (cellulase) and Viscozyme@ (hemicellulase plus pectinase)
2.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
289
(Novo, Denmark). The enzymes were applied at 0.02,O.l and 0.2%, by spraying 1, 5 and 10 g of each enzyme solution, diluted to 20 ml with tap water, over 5 kg forage spread over 2 m*. The stated activities of the enzymes were 1500 NCU (novo cellulose units) ml-’ and 120 FBG (fungal Pglucanase units) ml- ‘, respectively. The former corresponded to 74 IU ml- ‘, (Weinberg et al., 1990). Thus 0.3, 1.5 and 3.0 NCU Celluclast@ and 0.024, 0.12 and 0.24 FBG ViscozymeG0, respectively, were applied per gram of chopped forage.
3. Analytical procedures DM was determined by oven drying for 48 h at 60°C. Ash was obtained after 3 h at 550°C. Crude protein was determined by the Kjeldahl method. WSC were determined by the phenol sulphuric acid method, according to Dubois et al. ( 1956). Lactic acid (LA) was determined by a spectrophotometric method according to Barker and Summerson ( 1941) The protein removal step was omitted in our laboratory to better fit LA determination in silages. Volatile fermentation end products (ethanol, acetic, propionic and butyric acids) were determined with a gas chromatograph using a Chromosorb 101 column over a temperature range of 140-210°C according to Theune (1978). The peaks were identified by comparison with standards which were run daily. Fibre analysis (neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) ) was performed according to Van Soest ( 1982). Yeasts and moulds were counted on spread plate malt extract agar (Difco) acidified to pH 4.0 with 10% LA. The plates were incubated at 30°C for 72 h. Statistical analysis included regression analysis of the various parameters on log (enzyme concentration + 1), performed using the GLM procedure of Statistical Analysis System (SAS, Cary, NC, USA).
4. Results The effect of the enzyme treatments on the chemical and microbiological compositions 1 Table 21 and 2, respectively. It can of the pea and wheat silages is given in TablesTable be seen that, in the pea silages, significant linear regressions were obtained for pH, WSC and LA after 45 days of ensiling, and for pH and WSC also after 4 days. This indicates that, in the peas, the enzymes were already active in the early stages of ensiling. In the wheat silages, significant regression for these parameters was obtained only after 45 days of ensiling. After 4 days of ensiling there were less WSC in the silages of the higher enzymes treatment. We can not explain this phenomenon, but only hypothesize that the sugars obtained from the enzymatic hydrolysis were used in the fermentation. This agrees with lower pH values of the enzyme-treated wheat silages on day 4 (the high concentration of WSC in the fresh wheat was probably not readily fermentable by LAB). The enzyme treatments did not markedly affect the numbers of LAB, yeast and moulds in the final silages. The fermentation end-products in the pea silages were acetic acid, ethanol and butyric acid (7-12,2-3 and 2-6 g kg- ‘, respectively) with no major differences between treatments. In the wheat silages, the major volatile products were ethanol (I l-24
290
Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
Table 1 Analysis of the pea silages treated with cell-wall hydrolysing enzymes (means f SD)
Day
4
Significance of regression 45
Treatment
pH
wsc
LA
FM
6.2 4.4 4.3 4.3 4.3 0.002 4.3 4.2 4.2 4.1 0.04
106*2 31*9 36*2 56*2 73*4 0.0001 27*0 41*11 97* 10 121+10 0.0001
87*4 101*14 88*4 99*8 0.6 78$-4 105*5 118*7 123*6 0.001
Control 0.02% 0.1% 0.2% Control 0.02% 0.1% 0.2%
Significance of regression
LAB
Yeasts
Moulds
5.9 -
7.0 -
5.6 _ -
7.7 7.4 6.6 6.4
NF 2.5 3.3 4.1
NF 2.5 NF 2.5
FM, fresh material; WSC, water-soluble carbohydrates; LA, lactic acid, in g kg-’ DM; LAB, lactobacilli; NF, not found. Microbiological data are given as log number of colony-forming units g-r DM. The enzymes used were Novo Celluclast@ and Viscozyme@ (cellulose and hemicellulase + pectinase, respectively). g kg-‘) and butyric acid. The control wheat silages contained more butyric acid than the enzyme-treated silages (22 vs. 8-l 1 g kg- ‘). The ash content of the fresh peas and wheat was 77 and 80 g kg- ’ DM, and the crude protein content was 173 and 76 g kg-’ DM, respectively. In the final silages, the mean ash values were 87 and 8 1, and the mean crude protein contents were 188 and 89 g kg- ’ DM for the peas and wheat, respectively. There were no major differences in ash and crude protein contents between treatments of each crop. The effect of the enzymes on the aerobic stability of the pea and wheat silages is shown in Table 3. The results indicate that, in both crops, enzyme treatments impaired the aerobic Table 2 Analysis of the wheat silages treated with cell-wall hydrolysing enzymes (means + SD) Day
4
Significance of regression 45
Significance of regression
Treatment
pH
wsc
LA
LAB
Yeasts
Moulds
FM Control 0.02% 0.1% 0.2%
6.3 5.9 4.9 4.8 4.7 0.06 4.3 4.1 4.1 4.0 0.01
112*2 133*1 124f6 108+13 94*8 0.01 91 f 15 104*6 119rt28 153f3 0.0003
_
2.6 -
5.8
-
5.8 -
-
8.1 8.2 8.4 8.1
4.5 4.7 3.1 4.9
4.4 4.9 2.0 3.5
Control 0.02% 0.1% 0.2%
11*1 lo&6 7*124*2 0.06 40*7 50*4 53*4 59*9 0.01
FM, fresh material; WSC, water-soluble carbohydrates; LA, lactic acid, in g kg - ’ DM; LAB, lactobacilli. Microbiological data are given as log number of colony-forming units g-’ DM. The enzymes used were Novo Celluclast@ and Viscozyme@ (cellulase and hemicellulase +pectinase, respectively).
ZG. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
Table 3 The effect of cell-wall hydrolysing
291
enzymes on the aerobic stability of pea and wheat silages
Silage
Treatment
Visible moulding
pH
COZ
Yeasts
Moulds
Pea
Control 0.02% 0.1% 0.2%
Very little Little Very much Very much
4.2 4.2 4.3 4.5
NF 8.6 9.2 9.5
5.4 6.6 1.5 1.5
Control 0.02% 0.1% 0.2%
None Very little Mouldy Mouldy
4.2 3.9 4.2 4.2
1.9j10.8 16.9 i 15.0 24.8 f 16.5 36.9 f 330 0.10 0 0 33.0f47.4 38.9k49.1 0.09
5.8 9.9 8.9 9.3
5.6 9.0 8.6 8.0
Significance Wheat
of regression
Significance
of regression
COZ is in g kg- ’ DM (means + SD). Microbiological data are given as log number of colony-forming units g- ’ DM. The enzymes used were Novo CelluclastB and Viscozyme@ (cellulase and hemicellulase + pectinase, respectively).
stability of the silages. This was apparent by visual appraisal, CO2 production and numbers of yeasts and moulds. Although statistical analysis did not indicate significant regression for CO, production (because of large standard deviations within treatments), the amounts of CO, increased with increased enzyme levels. All enzyme-treated silages also contained markedly higher numbers of yeasts and moulds after 5 days of exposure to air, as compared with the controls. The effects of enzymes on fibre analysis of the pea and wheat silages are shown in Table 4. The wheat contained more fibre than the peas. The NDF and ADF contents decreased with increasing enzyme level, more so in the pea silages than in the wheat. This is reflected in higher significance of regression for the pea silages. Table 4 The effect of cell-wall hydrolysing
enzymes on fibre analysis of pea and wheat silages (means f SD)
Silage
Treatment
NDF
ADF
ADL
Peas
FM Control 0.02% 0.1% 0.2%
366*4 355k8 345*5 312&4 303+8 0.000 1 586k2 568*6 532+4 523*2 522+2 0.04
308*3 317+8 292f4 264k5 255f2 0.0003 357k2 376+3 3481tl 341*4 340+1 0.10
51 58* 15 47f2 46&l 46*1 0.2 76fl 81*3 68*2 8Oi-5 88rfr 12 0.24
Significance Wheat
of regression
Significance
of regression
FM Control 0.02% 0.1% 0.2%
NDF, neutral detergent libre; ADF, acid detergent fibre; ADL, acid detergent lignin. The enzymes used were Novo CelluclastB and ViscozymeB (cellulase and hemicellulase tively).
+ pectinase, respec-
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Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
The component which was most strongly affected by the enzymes was cellulose (ADFADL) which decreased by about 15% in both silages. The hemicellulose content (NDF-ADF) in the pea silages increased as enzyme level increased, whereas in the wheat silages it decreased by about 8%. The ADL fraction was not affected, as expected from the nature of the enzymes used. 5. Discussion
These experiments were initiated following a previous study (Weinberg et al., 1993) in which the enzyme mixture (applied at 0.02% each) was tested on different crops with a wide range of DM content and chemical composition. In this study, the enzymes had only a minor effect when applied to young moist crops (flowering peas and wheat, and ryegrass) in combination with a LAB inoculant. In these crops, such a combination improved fermentation patterns and slightly reduced fibre content. Therefore, in the current study, we decided to apply increasing enzyme concentrations along with a LAB inoculant on moderately dry crops. The crops in these experiments, podded peas and wheat at the milk maturation stage, were similar to those used in the previous study for which 0.02% enzymes did not have a great effect. The DM content of the wheat was somewhat lower than that usually used for silage in Israel and, indeed, relatively low LA and high butyric acid levels were found in the control silages. The results indicate significant linear regression for the enzyme levels used and the contents of WSC, LA, NDF, ADF and pH values in the final silages. Spoelstra et al. ( 1992) and Selmer-Olsen et al. ( 1993a) also observed a dose response to similar enzymes applied to maize and ryegrass silages at about 350 and 200 g kg-’ DM, respectively, with regard to cell-wall hydrolysis. In the aerobic stability test, regression of CO* production on enzyme levels was not significant. This reflects large standard deviations within triplicates. However, increased numbers of yeasts and moulds, which usually precede CO, production, along with a tendency for more CO* production, served as indicators for the intensity of spoilage. The results reveal that enzyme treatments impaired the aerobic stability of the silages: the higher the enzyme level, the more intensive the spoilage. This is in agreement with the findings of Spoelstra et al. ( 1992) and with some of the results of Selmer-Olsen et al. ( 1993b). It may well be that the higher concentrations of residual WSC and LA associated with the higher enzyme treatments enhanced lactate-assimilating yeast and mould development upon exposure to air. In the control wheat silages, the high butyric acid concentration protected the silages against yeasts and moulds during the exposure to air. In conclusion, the addition of enzymes to silages at effective concentrations might have some beneficial effects on fermentation patterns, especially in difficult-to-ensile crops, and on reducing cell-wall contents. However, such silages might be much more susceptible to aerobic deterioration. In addition, the economical price should be considered. Acknowledgements
This study was supported by the Israeli Cattle Board. We wish to thank Dr. Avraham Genizi of the Department of Statistics and Experimental Design, The Volcani Center, for his help with the statistical analysis.
Z. G. Weinberg et (11./ Animal Feed Science and Technology 55 (1995) 287-293
293
References Ashbell, G., Weinberg, Z.G., Azrieli, A., Hen, Y. and Horev, B., 1991. A simple system to study the aerobic deterioration of silages. Can. Agric. Eng., 33: 391-393. Barker, S.B. and Summerson, W.H., 1941. The calorimetric determination of lactic acid in biological material. J. Biol. Chem., 138: 535-554. Dubois, M., Giles, K.A., Hamilton, J.K., Rebes, P.A. and Smith, F., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Honig, H. and Pahlow, G., 1990. The effect of an enzyme preparation on the fermentation of grass silage. In: Proc. 9th Silage Conf. 3-5 September 1990, Faculty of Agriculture, University of Newcastle-upon-Tyne, UK, pp. 18-19. Muck, R., 1993. Opportunities for silage research: preservation and management. In: P. O’Kiely, M. O’Connell and J. Murphy (Editors), Proc. 10th Int. Conf. Silage Res. 6-8 September 1993, Dublin City University, Ireland, pp. 1-3. Selmer-Olsen, I., Henderson, A.R., Robertson, S. and McGinn, R., 1993a. Cell wall degrading enzymes for silage. I.The fermentation of enzyme-treated ryegrass in laboratory silos. Grass Forage Sci., 48: 45-54. Selmer-Olsen, I., Henderson, A.R., Robertson, S. and M&inn, R., 1993b. Cell wall degrading enzymes for silage. II. Aerobic stability of enzyme-treated laboratory silages. Grass Forage Sci., 48: 55-63. Selmer-Olsen, l., 1994. Enzymes as silage additives for grass-clover mixtures. Grass Forage Sci., 49: 305-315. Spoelstra, S.F., 1991. Chemical and biological additives in forage conservation. In: G. Pahlow and H. Honig (Editors), Proceedings of a Conference on Forage Conservation towards 2000.23-25 January 1991, Braunschweig, Germany, pp. 48-70. Spoelstra, S.F., Van Wikselaar, P.G. and Harder, 9.. 1992. The effect of ensiling whole crop maize with a multienzyme preparation on the chemical composition of the resulting silages. J. Sci. Food Agric., 60: 223-228. Tengerdy, R.P., Weinberg, Z.G., Szakacs, G., Wu, M., Linden, J.C., Henk, L.L. and Johnson, D.E., 1991. Ensiling alfalfa with additives of lactic acid bacteria and enzymes. J. Sci. Food Agric., 55: 215-228. Theune, H.H., 1978. Gaschromatographische Bestimmung von kurzkettigen Fattsauren einschliesslich Athanol und Milchsaure in Garfutter. Ein Methodenvergleich. In: Landwirtschafiliche Forschung, Sonderheft 35, 90 VDLUFA Congress, 12-l 8 September 1978, Augsburg, Germany. Van Soest, P.J., 1982. Analytical systems for evaluation of feeds. In: P.J. Van Soest (Editor), Nutritional Ecology of the Ruminant. Cornell University Press, Ithaca, NY, Chapter 6, pp. 75-94. Weinberg, Z.G., Ashbell, G., Azrieli, A. and Brukental, I., 1993. Ensiling peas, ryegrass and wheat with additives of lactic acid bacteria (LAB) and cell wall degrading enzymes. Grass Forage Sci., 48: 70-78. Weinberg, Z.G., Szakacs, G., Linden, J.C. and Tengerdy, R.P., 1990. Recovery of protein and chlorophyll from alfalfa by simultaneous lactic acid fermentation and enzyme hydrolysis (ENLAC). Enzyme Microbial Technol.. 12: 921-925.
Animal Feed Science and Technology
55 (1995) 287-293
The effect of cellulase and hernicellulase plus pectinase on the aerobic stability and fibre analysis of peas and wheat silages ’ Z.G. Weinberg *, G. Ashbell, Yaira Hen, A. Azrieli Forage Preservation and By-Products Research Unit, Department of Stored Products, The Volcani Center. Bet Dagan 50250, Israel Received
13 September
1994; accepted 9 February1995
Abstract
The effect of applying increasing levels of cellulase and hemicellulase plus pectinase (CelluclastB and ViscozymeB, Novo, Denmark) to peas and wheat silages was studied under laboratory conditions. The enzymes were applied at 0.02.0.1 and 0.2% each, corresponding to 0.3, 1.5 and 3.0 NCU (Novo cellulase units) of Celluclast, and 0.024,O. 12 and 0.24 FBG (fungal P-glucanase units) of Viscozyme per gram of fresh crop. All treatments were enriched with a lactic acid bacteria inoculum, applied at I O4colony-forming units per gram of forage. The following parameters of final silages (45 days) had a significant (P < 0.05) linear regression on log (enzyme concentration + 1): pH, residual sugars, lactic acid, NDF (neutral detergent fibre) and ADF (acid detergent fibre). When enzyme levels increased from 0 to 0.2%, the NDF and ADF contents decreased from 355 and 317 to 303 and 255 g kg- ‘, respectively, in the pea silages, and they decreased from 568 and 357 to 522 and 340 g kg-‘, respectively, in the wheat silages. Enzyme treatments resulted in enhanced aerobic deterioration in both pea and wheat silages. This was evident from higher yeast and mould counts, more intensive CO2 production and visible mould growth during exposure to air for 5 days. Keywords: Cellulase; Hemicellulase;
, Pectinase; Silage; Aerobic stability; Fibre analysis
1. Introduction
A successful ensiling process requires a minimal concentration of fermentable sugars (3-5% in DM), mainly hexoses. However, the majority of carbohydrates in plants is in the * Corresponding author. ’ Contribution from the Agricultural E, 1994 Series.
Research Organization,
The Volcani Center, Bet Dagan, Israel, No. 1259-
0377.8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO377-8401(95)00785-7
288
Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
form of fibrous polymers that make up the cell wall and are not fermented by lactic acid bacteria (LAB). In order to obtain the necessary level of fermentable water-soluble carbohydrates (WSC) for the lactic fermentation in crops which are low in WSC, the use of cell-wall degrading enzymes has been suggested. Such enzymes comprise cellulases, hemicellulases and pectinases. The expected benefits attributed to enzymes are the release of fermentable sugars and a decrease in fibre content which is related to improved digestibility (Muck, 1993). Spoelstra ( 199 1) reviewed studies on the effect of enzymes on fermentation parameters in silages. Results of studies mentioned in this review indicate that such enzymes are more active in moist silages than in drier silages. Enzymes applied at commercial dosages were not effective in grass silage (Honig and Pahlow, 1990). Weinberg et al. (1990) showed that cellulase alone was not effective and a mixture which also contained hemicellulase plus pectinase was necessary to hydrolyse alfalfa cellwall. Such a mixture, applied at 0.025% of each enzyme preparation, resulted in a slight decrease in fibre content in direct-cut alfalfa silages, but not in wilted alfalfa silages (Tengerdy et al., 1991). Selmer-Olsen et al. ( 1993a,b) studied the effect on ryegrass silages of enzymes prepared from fungi. These enzymes caused a lower pH, lower ammonia-N and ADF contents, and increased residual WSC content. Similar trends were obtained with silages of moist grass*lover mixtures (Selmer-Olsen, 1994). In our laboratory, an enzyme mixture, comprising cellulases plus hemicellulases and pectinases, was added at 0.02% each, to pea, ryegrass and wheat silages at various stages of maturity and DM contents (Weinberg et al., 1993). The enzymes at this rate of application resulted in only a slight decrease in NDF and ADF contents in silages of young moist crops (%DM < 25)) and did not affect silages of more mature or dry crops. The purpose of the present work was to study the effect of these enzymes applied at higher rates on the fibre analysis and aerobic stability of pea and wheat silages.
2. Materials and methods The crops used in these experiments were field peas (Pisum sutiuum) harvested at the podding stage (29.5% DM) , and wheat (Triticum vulgaris) harvested at the milk ripening stage (30.7% DM) . Whole plants were chopped to 1.5 cm (using a Wintersteigeti chopper, Austria), treated and ensiled in 1.5-1 glass jars equipped with a lid that enables gas release only (Week@, Germany). The jars were stored at 25 + 2°C. Three jars from each treatment were sampled for analysis on days 4 and 45 after ensiling. At the end of the ensiling period (after 45 days), the silages were subjected to an aerobic stability test lasting 5 days in a system developed by Ashbell et al. ( 1991) . In this system, number of yeasts and moulds, change in pH and the amount of CO1 produced during the test are indicators for aerobic deterioration. The whole batch of forage was treated before ensiling with 4 X lo4 colony-forming units -’ of lactic acid bacteria (H/M F inoculant No. 9927, Medipharm USA, containing g Pediococci acidilactici, Lactobacillus plantarum and Enterococcus faecium). The inoculated forage was used as control (no other additives); enzyme treatments comprised a mixture of Celluclast@ (cellulase) and Viscozyme@ (hemicellulase plus pectinase)
2.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
289
(Novo, Denmark). The enzymes were applied at 0.02,O.l and 0.2%, by spraying 1, 5 and 10 g of each enzyme solution, diluted to 20 ml with tap water, over 5 kg forage spread over 2 m*. The stated activities of the enzymes were 1500 NCU (novo cellulose units) ml-’ and 120 FBG (fungal Pglucanase units) ml- ‘, respectively. The former corresponded to 74 IU ml- ‘, (Weinberg et al., 1990). Thus 0.3, 1.5 and 3.0 NCU Celluclast@ and 0.024, 0.12 and 0.24 FBG ViscozymeG0, respectively, were applied per gram of chopped forage.
3. Analytical procedures DM was determined by oven drying for 48 h at 60°C. Ash was obtained after 3 h at 550°C. Crude protein was determined by the Kjeldahl method. WSC were determined by the phenol sulphuric acid method, according to Dubois et al. ( 1956). Lactic acid (LA) was determined by a spectrophotometric method according to Barker and Summerson ( 1941) The protein removal step was omitted in our laboratory to better fit LA determination in silages. Volatile fermentation end products (ethanol, acetic, propionic and butyric acids) were determined with a gas chromatograph using a Chromosorb 101 column over a temperature range of 140-210°C according to Theune (1978). The peaks were identified by comparison with standards which were run daily. Fibre analysis (neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) ) was performed according to Van Soest ( 1982). Yeasts and moulds were counted on spread plate malt extract agar (Difco) acidified to pH 4.0 with 10% LA. The plates were incubated at 30°C for 72 h. Statistical analysis included regression analysis of the various parameters on log (enzyme concentration + 1), performed using the GLM procedure of Statistical Analysis System (SAS, Cary, NC, USA).
4. Results The effect of the enzyme treatments on the chemical and microbiological compositions 1 Table 21 and 2, respectively. It can of the pea and wheat silages is given in TablesTable be seen that, in the pea silages, significant linear regressions were obtained for pH, WSC and LA after 45 days of ensiling, and for pH and WSC also after 4 days. This indicates that, in the peas, the enzymes were already active in the early stages of ensiling. In the wheat silages, significant regression for these parameters was obtained only after 45 days of ensiling. After 4 days of ensiling there were less WSC in the silages of the higher enzymes treatment. We can not explain this phenomenon, but only hypothesize that the sugars obtained from the enzymatic hydrolysis were used in the fermentation. This agrees with lower pH values of the enzyme-treated wheat silages on day 4 (the high concentration of WSC in the fresh wheat was probably not readily fermentable by LAB). The enzyme treatments did not markedly affect the numbers of LAB, yeast and moulds in the final silages. The fermentation end-products in the pea silages were acetic acid, ethanol and butyric acid (7-12,2-3 and 2-6 g kg- ‘, respectively) with no major differences between treatments. In the wheat silages, the major volatile products were ethanol (I l-24
290
Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
Table 1 Analysis of the pea silages treated with cell-wall hydrolysing enzymes (means f SD)
Day
4
Significance of regression 45
Treatment
pH
wsc
LA
FM
6.2 4.4 4.3 4.3 4.3 0.002 4.3 4.2 4.2 4.1 0.04
106*2 31*9 36*2 56*2 73*4 0.0001 27*0 41*11 97* 10 121+10 0.0001
87*4 101*14 88*4 99*8 0.6 78$-4 105*5 118*7 123*6 0.001
Control 0.02% 0.1% 0.2% Control 0.02% 0.1% 0.2%
Significance of regression
LAB
Yeasts
Moulds
5.9 -
7.0 -
5.6 _ -
7.7 7.4 6.6 6.4
NF 2.5 3.3 4.1
NF 2.5 NF 2.5
FM, fresh material; WSC, water-soluble carbohydrates; LA, lactic acid, in g kg-’ DM; LAB, lactobacilli; NF, not found. Microbiological data are given as log number of colony-forming units g-r DM. The enzymes used were Novo Celluclast@ and Viscozyme@ (cellulose and hemicellulase + pectinase, respectively). g kg-‘) and butyric acid. The control wheat silages contained more butyric acid than the enzyme-treated silages (22 vs. 8-l 1 g kg- ‘). The ash content of the fresh peas and wheat was 77 and 80 g kg- ’ DM, and the crude protein content was 173 and 76 g kg-’ DM, respectively. In the final silages, the mean ash values were 87 and 8 1, and the mean crude protein contents were 188 and 89 g kg- ’ DM for the peas and wheat, respectively. There were no major differences in ash and crude protein contents between treatments of each crop. The effect of the enzymes on the aerobic stability of the pea and wheat silages is shown in Table 3. The results indicate that, in both crops, enzyme treatments impaired the aerobic Table 2 Analysis of the wheat silages treated with cell-wall hydrolysing enzymes (means + SD) Day
4
Significance of regression 45
Significance of regression
Treatment
pH
wsc
LA
LAB
Yeasts
Moulds
FM Control 0.02% 0.1% 0.2%
6.3 5.9 4.9 4.8 4.7 0.06 4.3 4.1 4.1 4.0 0.01
112*2 133*1 124f6 108+13 94*8 0.01 91 f 15 104*6 119rt28 153f3 0.0003
_
2.6 -
5.8
-
5.8 -
-
8.1 8.2 8.4 8.1
4.5 4.7 3.1 4.9
4.4 4.9 2.0 3.5
Control 0.02% 0.1% 0.2%
11*1 lo&6 7*124*2 0.06 40*7 50*4 53*4 59*9 0.01
FM, fresh material; WSC, water-soluble carbohydrates; LA, lactic acid, in g kg - ’ DM; LAB, lactobacilli. Microbiological data are given as log number of colony-forming units g-’ DM. The enzymes used were Novo Celluclast@ and Viscozyme@ (cellulase and hemicellulase +pectinase, respectively).
ZG. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
Table 3 The effect of cell-wall hydrolysing
291
enzymes on the aerobic stability of pea and wheat silages
Silage
Treatment
Visible moulding
pH
COZ
Yeasts
Moulds
Pea
Control 0.02% 0.1% 0.2%
Very little Little Very much Very much
4.2 4.2 4.3 4.5
NF 8.6 9.2 9.5
5.4 6.6 1.5 1.5
Control 0.02% 0.1% 0.2%
None Very little Mouldy Mouldy
4.2 3.9 4.2 4.2
1.9j10.8 16.9 i 15.0 24.8 f 16.5 36.9 f 330 0.10 0 0 33.0f47.4 38.9k49.1 0.09
5.8 9.9 8.9 9.3
5.6 9.0 8.6 8.0
Significance Wheat
of regression
Significance
of regression
COZ is in g kg- ’ DM (means + SD). Microbiological data are given as log number of colony-forming units g- ’ DM. The enzymes used were Novo CelluclastB and Viscozyme@ (cellulase and hemicellulase + pectinase, respectively).
stability of the silages. This was apparent by visual appraisal, CO2 production and numbers of yeasts and moulds. Although statistical analysis did not indicate significant regression for CO, production (because of large standard deviations within treatments), the amounts of CO, increased with increased enzyme levels. All enzyme-treated silages also contained markedly higher numbers of yeasts and moulds after 5 days of exposure to air, as compared with the controls. The effects of enzymes on fibre analysis of the pea and wheat silages are shown in Table 4. The wheat contained more fibre than the peas. The NDF and ADF contents decreased with increasing enzyme level, more so in the pea silages than in the wheat. This is reflected in higher significance of regression for the pea silages. Table 4 The effect of cell-wall hydrolysing
enzymes on fibre analysis of pea and wheat silages (means f SD)
Silage
Treatment
NDF
ADF
ADL
Peas
FM Control 0.02% 0.1% 0.2%
366*4 355k8 345*5 312&4 303+8 0.000 1 586k2 568*6 532+4 523*2 522+2 0.04
308*3 317+8 292f4 264k5 255f2 0.0003 357k2 376+3 3481tl 341*4 340+1 0.10
51 58* 15 47f2 46&l 46*1 0.2 76fl 81*3 68*2 8Oi-5 88rfr 12 0.24
Significance Wheat
of regression
Significance
of regression
FM Control 0.02% 0.1% 0.2%
NDF, neutral detergent libre; ADF, acid detergent fibre; ADL, acid detergent lignin. The enzymes used were Novo CelluclastB and ViscozymeB (cellulase and hemicellulase tively).
+ pectinase, respec-
292
Z.G. Weinberg et al. /Animal Feed Science and Technology 55 (1995) 287-293
The component which was most strongly affected by the enzymes was cellulose (ADFADL) which decreased by about 15% in both silages. The hemicellulose content (NDF-ADF) in the pea silages increased as enzyme level increased, whereas in the wheat silages it decreased by about 8%. The ADL fraction was not affected, as expected from the nature of the enzymes used. 5. Discussion
These experiments were initiated following a previous study (Weinberg et al., 1993) in which the enzyme mixture (applied at 0.02% each) was tested on different crops with a wide range of DM content and chemical composition. In this study, the enzymes had only a minor effect when applied to young moist crops (flowering peas and wheat, and ryegrass) in combination with a LAB inoculant. In these crops, such a combination improved fermentation patterns and slightly reduced fibre content. Therefore, in the current study, we decided to apply increasing enzyme concentrations along with a LAB inoculant on moderately dry crops. The crops in these experiments, podded peas and wheat at the milk maturation stage, were similar to those used in the previous study for which 0.02% enzymes did not have a great effect. The DM content of the wheat was somewhat lower than that usually used for silage in Israel and, indeed, relatively low LA and high butyric acid levels were found in the control silages. The results indicate significant linear regression for the enzyme levels used and the contents of WSC, LA, NDF, ADF and pH values in the final silages. Spoelstra et al. ( 1992) and Selmer-Olsen et al. ( 1993a) also observed a dose response to similar enzymes applied to maize and ryegrass silages at about 350 and 200 g kg-’ DM, respectively, with regard to cell-wall hydrolysis. In the aerobic stability test, regression of CO* production on enzyme levels was not significant. This reflects large standard deviations within triplicates. However, increased numbers of yeasts and moulds, which usually precede CO, production, along with a tendency for more CO* production, served as indicators for the intensity of spoilage. The results reveal that enzyme treatments impaired the aerobic stability of the silages: the higher the enzyme level, the more intensive the spoilage. This is in agreement with the findings of Spoelstra et al. ( 1992) and with some of the results of Selmer-Olsen et al. ( 1993b). It may well be that the higher concentrations of residual WSC and LA associated with the higher enzyme treatments enhanced lactate-assimilating yeast and mould development upon exposure to air. In the control wheat silages, the high butyric acid concentration protected the silages against yeasts and moulds during the exposure to air. In conclusion, the addition of enzymes to silages at effective concentrations might have some beneficial effects on fermentation patterns, especially in difficult-to-ensile crops, and on reducing cell-wall contents. However, such silages might be much more susceptible to aerobic deterioration. In addition, the economical price should be considered. Acknowledgements
This study was supported by the Israeli Cattle Board. We wish to thank Dr. Avraham Genizi of the Department of Statistics and Experimental Design, The Volcani Center, for his help with the statistical analysis.
Z. G. Weinberg et (11./ Animal Feed Science and Technology 55 (1995) 287-293
293
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