J. Sci. Fd Agric. 1978, 29, 293-297

Cellulase Methods for the Efficient Digestion of Grasses and Brassicas

Michael Allison and Ronald Borzucki

Scottish Plant Breeding Station, Pentlandfield, Roslin, Midlothian

(Manuscript received 2 June 1977)

A cellulase solubility method for the prediction of grass digestibility was modified by changing pepsin pretreatment conditions so that cellulase values were as high as those obtained using rumen liquor. Raising temperature from 40 to 50°C and acid concentration from 0.1 to 0 . 1 2 5 ~ during ‘pepsin’ treatments given before the cellulase stage, increased digestion by 3 to 10% depending on the type of grass, up to the level reached by rumen liquor. For samples of brassicas (digestibilities ranging from 66 to 84%) milder pretreatment conditions were sufficient to give cellulase digestion values as high as rumen liquor estimates.

1. Introduction

In 1963 Tilley and Terry1 demonstrated that the in vivo digestibility of grass could be reliably pre- dicted by a laboratory method. Their procedure consists of a two day digestion of the sample by rumen liquor (from fistulated sheep) followed by acid pepsin digestion for a further two days. Esti- mates of digestibility obtained by this means correlate highly (r > 0.9) with in-vivo measurements.

This is a relatively long procedure requiring at least six days from start to finish. It is also difficult to maintain a rumen liquor supply of consistent quality when the sheep diet, operators and the ani- mals themselves are changed. There is a need, therefore, for faster methods, particularly when selecting for digestibility in plant breeding programmes. One, successful, quicker method was developed by Jones and Hayward2 who used cellulase preparations from Trichoderma viride with an acidified pepsin stage, to digest forages. Estimates obtained from a regime of one day’s digestion in acidified pepsin followed by 2 days in cellulase, correlate well with in-vivo digestibilities. However the percentage digestions achieved by the cellulase procedure are usually about thirteen digestion units lower than the rumen values.

The present paper describes a modification to pretreatments in the cellulase method such that the percentages are increased to values equivalent to those obtained using rumen liquor. In addition it is shown that relatively mild, normal pretreatments are sufficient to raise the digestibility of varie- ties of Brassica oleracea to the level obtained using rumen liquor.

2. Experimental

The grass and brassica samples used in these experiments were grown either in small plots or trials at the Scottish Plant Breeding Station. Cellulases prepared from Trichoderma viride were purchased from Mayvil Chemicals Ltd. Pepsin was purchased from C . Zimmerman Co. Ltd. Rumen liquor was taken weekly from three fistulated sheep maintained on a controlled diet at the Hill Farming Research Organisation, Midlothian.

The in-vitro digestibility (IVD) method used was essentially that developed by Tilley and Terry1 with the modifications introduced by Alexander.3 Digestibility is expressed as per cent digestible organic matter in the dry weight (DOMD).

The cellulase solubility method is similar to that developed by Jones and Hayward2 except that the pH of the acidified pepsin given as the first digestion stage, was raised to 4.6 by adding approxi-

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294 M. Allison and R. Borzucki

mately 1.5 ml molar sodium carbonate to 25 ml of the acid pepsin. The volume of sodium carbonate varied according to the molarity of the pretreatment solution. Cellulases in acetate buffer (6 g litre-1) were added after adjustment of the pH to give a reaction volume of 65 ml per tube. Every value reported is the mean of duplicate measurements which are within 2 % of each other. Estimates more than 2% apart were repeated; in all cases the repeated estimates fell within the 2% limits. The standard errors of IVD and cellulase measurements on a wide range of forages were 2 1 % and f 0.7 x, respectively.

3. Results

While investigating the digestion of grass by cellulase, Jones and Hayward2 found that the correla- tion with in-vivo measurements improved if acid pepsin was added before, rather than after the cellu- lase stage. The problem of adjusting the pH from 1.2 (optimum for pepsin) to 4.6 (optimum for cellulase) was overcome by using filter sticks to remove acidified pepsin from grass material. When we used this method to remove acid from brassica digests, the loss of material via the filter sticks, resulted in unacceptably large errors. Alternative ways of changing the pH were therefore in- vestigated.

In a series of experiments, initially on grasses, the pH was raised to 4.6 after the pepsin stage by the addition of sodium carbonate (see section 2). Cellulase digestion then took place in solutions of high ionic strength. Other changes were made in pretreatment conditions in an attempt to increase digestion. Estimates made of digestibility of Poa grass samples by different methods are listed in Table 1.

Table 1. Digestion of five Poa grass samples by different methods"

Treatments : Acid plus pepsin only Acid plus pepsin plus cellulase Rumen digestion

0.1N Sample Acid, number 40°C

0 . 1 2 5 ~ 0.1N 0.125N Acid, Acid, Acid, 40°C 50°C 50°C

1 . 43.5 2. 40.9 3. 43.2 4. 41.0 5. 43.6 X 42.4 -

42.9 44.2 45.1 42.1 45.3 43.7 45.4 45.9 46.9 40.2 41.5 45.0 44.4 45.6 46.8 43.1 44.5 45.6

0.1N 0.1253.1 0.1N 0 . 1 2 5 ~ Acid, Acid, Acid, Acid, 40°C 40°C 50°C 50°C

68.9 61.1 71.7 13.3 67.1 68.5 68.5 69.8 66.2 67.0 68.2 69.4 66.5 64.4 66.4 71.9 61.6 69.8 69.1 12.2 61.3 67.5 68.8 71.3

2 days rumen and 2 days pepsin 0 . 1 ~ acid, 40°C

72.4 69.3 70.3 71.1 72.4 71.1

a The results are expressed as percentage digestible organic matter in the dry weight.

It is evident from these results that cellulase activity is not adversely affected by solutions of high ionic strength. In fact, treatments with pepsin in 0.125~ acid at 50°C followed by cellulase, increase the digestibility up to the rumen Iiquor value. When the cellulase stage is omitted (columns 1 to 4 in Table 1) the stronger pretreatments increase digestion of these grasses by approximately 3 %. In studies on poor quality grasses, cellulase estimates with the usual mild pretreatments (0.1 normal acid at 40°C) can be 10 % or more below the corresponding rumen liquor estimates. To test whether stronger pretreatments could increase the digestion of poorer grasses or not, ryegrass samples of around 50 % DOMD were subjected to the modified cellulase method. The cellulase estimates are plotted against rumen liquor values in Figure 1. These results show that the modified treatments digest poor quality grass to the same extent as rumen liquor ( r= 0.95).

Our routine work is also concerned with the digestibility of Brassica plants which are mainly in the higher digestibility range (6844%). Varieties of Brassica oleracea covering the normal range of brassica digestibility were digested by the modified cellulase method. These results are shown graphically in Figure 2. As for grasses the results from cellulase digestions are equivalent to the

Digestion of grasses and brassicas 295

0 0 0

0 0

0 0

0

0

0

0 0

0

I I I 1 I I 48 50 52 54 56 58

% Drqestlbl l i ty (DOMD) by rumen

Figure 1. Digestion of ryegrass samples by rumen liquor and a modified cellulase method, In the regression equation, y is the rumen estimate and x is the cellulase estimate, r is the correlation coefficient and s.e. (est.) is the standard error of equation estimates: v=O.95; y = 1 . 0 4 ~ - 2 . 8 8 ; s.e. (est.) f 1.376.

rumen liquor values (r=0.94). Some of the brassica samples were digested with the milder pre- treatment conditions (0.1 normal acid at 40°C) and, again the results were not significantly different from those obtained using rumen liquor. In addition digestion regimes of 1 day in acid pepsin followed by 1 day in cellulase were sufficient to raise digestibility to the rumen liquor level. Similar results were obtained in digestions, using milder treatments, of kales and rapes. Thus brassicas differ from grasses in their response to relatively mild pretreatments and a shorter digestion by cellulases.

4. Discussion

The use of cellulases to predict digestibility is not only widely accepted as reliable, but also has con- tributed to our understanding of forage digestion. Only cellulases from certain fungi e.g. Tricho- derma viride give estimates of digestibility that correlate highly with in-vivo measurements. In addition to the hydrolytic cellulases, exo- and endo- /3 1,4 glucanases, T. viride also produces a cellulose solubilising enzyme4 and some hemicellulase and protease activity.2 Cellulases from other fungi that digest forages poorly lack the solubilising enzyme. Thus it seems that bringing cellulose into solution is an important step in forage digestion.

This raises the question of whether other cellular components must be solubilised before digestion

296 M. Allison and R. Borzucki

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15 0

19 0

16 0

8 0

18 0

0 2 12 0

17 0 R

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II 6 0 0

14 0 10 0

6 "'0"

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70 75 8 0 85 90 65

% Digestlbdliy (DOMD) by rumen

Figure 2. Digestion of samples of Brassica olwacea varieties by rumen liquor and the modified cellulase method The samples are :

Whole Plant Stem Leaf Vremo Intr Brussel Sprout 1 2 5 Brunswick Cabbage 3 Thousand head Kale 21 20 4 Canson Kale 6 8 19 Best of All Savoy 7 Vulcan Kale 10 12 9 White Broccoli 11 & I 8 Greendale Kale 13 16 14 Purple Broccoli 15 Early Giant Cabbage 17 Darkmoor Brussel Sprout 22 Rearguard Savoy 23 R is a standard rape sample.

Regression equation: y = 1.05 x-3.5; r=0 .94; s.e. (est.) i 1.12.

can proceed to an advanced stage. In 1974 McQueen and Van Soests attempted to raise enzymic digestion to the rumen level by adding mixtures of heniicellulases from Aspergillus and T. viride cellulases to grasses and legumes. Various combinations of enzyme treatments did not, however, increase breakdown of the forages to the same extent as that achieved by the use of a rumen liquor. Therefore it seems that the hydrolytic activity is not limiting, but that the rumen may secrete en- zymes, not produced by fungi, capable of solubilising different cell polymers.

Digestion of grasses and brassicas 297

The results presented in this paper show that increases in acid concentration (from 0 . 1 ~ to 0.125~) and temperature (from 40 to 50°C) during pepsin treatments used before cellulase digestion, enhance the digestion of grasses, ranging in quality, up to the rumen values. These stronger pre- treatments may directly increase the efficiency of pepsin action. Alternatively, the stronger acidic treatments may enhance digestibility by solubilising certain polymers, allowing greater access of pepsin to protein substrates. Evidence against a direct effect on pepsin is provided by experiments6 in which digestion was increased by strong acidic pretreatments without pepsin, followed by cellulase digestion; then a pepsin post-treatment stage. There are, however, several ways in which the stronger acidic treatments may render polymers more accessible to hydrolytic attack. It has been suggested7 that acid molecules may penetrate fibrillar structures in the cell wall more readily than solubilising enzymes of high molecular weight. Acidic pretreatments may solubilise hemicellulose which becomes more soluble with increasing acidity in the lower pH range.* Cellulose-lignin complexes can limit digestion9 and acidic treatments may enhance the delignification of cellulose. It is also possible that the acid solubilises proteins rendering them more accessible to pepsin attack. In addition to inter- chain bonds, acids can also hydrolyse intrachain bonds, such as the j3 1,4 links in cellulose, thus directly hydrolysing polymers which have been brought into solution. Other possibilities include solubilisation of proteins, resistant to pepsin, but subsequently degraded by the proteases con- taminating the cellulase preparation.

In contrast to grasses, varieties of Brassica oleraceu are digested to a high degree (up to 84%) even when relatively mild pretreatments (0 .1~ acid at 40°C) are used. There is, therefore, a clear difference between grasses and brassicas in factors limiting digestibility.

The success of the modified cellulase methods in predicting in-vitro digestibility over the wide range of digestibilities reported here, suggests that such methods could be applied to other forages. It may be necessary to use different pretreatments for efficient digestion of different types of forage, but it seems likely that cellulase estimates will be equivalent to rumen values, if appropriate pre- treatments are used. Advantages of the cellulase methods over IVD include the greater consistency in quality of cellulases compared to rumen liquor and the increase in numbers processed because the cellulase method is shorter and sample filtration is faster, than in the IVD method. A possible disadvantage is that factors toxic to the rumen may not be detected in cellulase digestion. Further work on the factors limiting digestibility in forages is in progress.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Tilley, J. M. A.; Terry, R. A. J . Brit. Grass. Soc. 1963, 18, 104. Jones, D. I. H.; Hayward, M. V. J. Sci. Fd Agric. 1973, 24, 1419. Alexander, R. H. West of Scotland Agricultural College, Res. Bull. 1969, 42, 1. Li, L. H. ; Flora, R. M.; King, K. W. Arch. Biochem. Biophys. 1965, 111, 439. McQueen, T.; Van Soest, P. J. J. Dairy Sci. 1975, 58, 1482. Kellner, R. J.; Kirchgessner, M. Landwirtsch. Forsch. 1976, 29, 204. Ghose, T. K.; Pathak, A. N. Process Biochemistry 1973, 8, 20. Van Soest, P. J. Forage Economics-Quality 13, ASA, Wisconsin, 1968, p. 63. Bailey, R. W.; Jones, D. I. H. N.Z. J . of Agric. Res. 1971, 14, 847.