Digestion of wheat aleurone by commercial polysaccharidases

Animal Feed Science and Technology, 32 (1991) 185-191 Elsevier Science Publishers B.V., Amsterdam

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Digestion of wheat aleurone by commercial polysaccharidases Marcel M. Mulder ~,3, Peter M. Hotten 2, Eoin Cowie 2, James A. L o m a x I and A n d r e w Chesson 2 t FOM Institute for Atomic and Molecular Physics, Unit for Mass Spectrometry of Macromolecular Systems, Kruislaan 407, 1098 SJ Amsterdam (The Netherlands) 2The Rowett Research Institute, Bucksburn, Aberdeen (Gt. Britain) 31nstituut voor Veevoedingsonderzoek, P.O. Box 160, 8200 AD Lelystad (The Netherlands)

ABSTRACT Mulder, M.M., Hotten, P.M., Cowie, E., Lomax, J.A. and Chesson, A., 1991. Digestion of wheat aleutone by commercial polysaccharidases. Anim. FeedScL Technol., 32: 185-191. Aleurone cells possess cell walls that cannot easily be digested by monogastric animals because they lack the appropriate enzymes. Therefore, pretreatment of aleurone cells with cell wall degrading enzymes may disclose the valuable protein within the cells. The cell walls, containing large quantities of glucans and arabinoxylans, were degraded to a larger extent by a commercial cellulase preparation than by a commercial glucanase preparation. The effects of the enzyme digestions were assessed by analysis of the reduced sugars and total sugars formed upon digestion, by methylation analysis and by pyrolysis mass spectrometry (PyMS). Methylation analysis showed that xylan was mainly degraded by the enzyme preparation. The PyMS results showed that pentosans were solubilisedfrom the residues.

INTRODUCTION

Plant cell walls consist of several components of which cellulose, hemicellulose, lignin and pectin are the most abundant (Armstrong, 1986). In general, the digestibility of these components in animals is not very good (Chesson, 1986). Digestion in ruminants differs from that in monogastrics in that the rich rumen microbial population provides ruminants with a better potential for plant cell wall degradation. Monogastrics lack the appropriate enzymes in the small intestine to digest plant material to any great extent. Cereals are used as a major feed for farm animals (Chesson, 1986). Starch (in the starchy endosperm) and proteins (mainly in the aleurone layer) are the compounds with the highest nutritive value in cereal grains. These compounds are surrounded by cell walls that limit their utilisation. The cell wall of aleurone tissue consists of arabinoxylans (65%), (1-3,1-4)-p-glucans 0377-8401/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

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M.M. MULDER ET AL.

(29%) and some ( l-3)-~glucans, glucomannan and cellulose (Fincher and Stone, 1986). In this paper the predigestion of the cell wall of wheat aleurone tissue with two commercial polysaccharidases, Genencor GC123 and Grinsted GV was studied, particularly in relation to their effect on the availability of intracellular protein bodies. MATERIALS AND METHODS

Wheat-aleurone tissue was prepared according to the method of Bacic and Stone ( 1981 ) with some minor adjustments. Two commercially available polysaccharidases were used in these studies, Genencor GC 123, a cellulase preparation and Grinsted GV, a glucanase preparation. The ability of each of these enzyme preparations to hydrolyse avicel, carboxymethylcellulose, starch, xylan, pectin and polygalacturonic acid was determined. The reducing sugars formed in the test were measured by the Nelson-Somogyi method (Somogyi, 1952 ). The same activities were also determined in Termamyl (Novo Industries, St Louis, MO), an amylase preparation and pancreatin (Sigma, Brussels), a preparation containing both amylase and protease activity. For control measurements, substrate without enzyme addition and enzyme without substrate addition were used. Digestion experiments were conducted in a 100-ml Eflenmeyer flask in a 40°C water bath. Aleurone tissue (1 g) was suspended in 37.35 ml 0.1 M NaAc-buffer (pH 4.5 ) and incubated for 24 h with 150/d Termamyl. Then, either 150/d Genencor GC123 or 345 Grinsted GC 123 was added to the suspension and incubated for 6 or 24 h. Subsequently, some incubations were treated with 100 mg pancreatin at pH 7 for 24 h to deproteinise the sample. The supernatant was separated from the residue by filtration using a GFA filter in a Biichner funnel. Supernatants were frozen immediately and were later analysed for total sugars by the phenol-sulphuric acid method (Dubois et al., 1956) and for reducing sugars by the Nelson-Somogyi method (Somogyi, 1952 ). Residues were freeze-dried and the weight difference before and after enzyme treatment was determined. A small portion of the residue was used for light-microscopic analysis. The residues were also subjected to methylation analysis according to the method described by Lomax et al. (1983, 1985) and to pyrolysis mass spectrometry (PyMS) (van der Kaaden et al., 1984) performed on the automatic FOM-PyMS. Data were subjected to discriminant analysis as described by Hoogerbrugge et al. (1983). RESULTS AND DISCUSSION

The various activities of the enzymes as determined against different substrates at pH 4.5 (pancreatin at pH 7.0) are given in Table 1. The Genencor

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TABLE 1 E n z y m e activities against s t a n d a r d s u b s t r a t e s ( p m o l m l - ~ m i n - ~) Substrate

C a r b o x y m e t h y l cellulose Aviccl Starch Pectin Xylan Polygalacturonic acid

Genencor G C i 23

583 3 55 2

Grinsted GV

290 !5 260 111 1516

Termamyl

342 -

Pancreatin p H 4.5

p H 7.0

6 178 8 89

5 92 I 4 II 1(J

E n z y m e activities against different s t a n d a r d substrates in Mcillvains buffer ( p H 4.5; pancrcatin also at pH 7.0). Activities were m e a s u r e d as the a m o u n t o f reducing sugars that arose u p o n e n z y m e incubation by the N e l s o n - S o m o g y i m e t h o d (Somogyi. 1952 ).

GC 123 preparation showed a relatively high endocellulase activity whereas the activities against other substrates were quite low. The Grinsted enzyme preparation possessed activities against carboxymethylcellulose, avicel, pectin, polygalacturonic acid and xylan. Heat-treated Termamyl showed only amylase activity. Pancreatin surprisingly showed some ceUulolytic activity at pH 4.5 but at pH 7.0 an amylase activity was measured whereas only minor activities were measured against the various substrates. The observed cellulase activity in the pancreatin preparation is probably a result of contamination of the enzyme preparation. This preparation is derived from the small gut of pigs, which may be colonised by microorganisms. Because cellulase activity was considered to be important, the activities of the two preparations were normalised for the endocellulase activity by measuring the activity at various dilutions and plotting the activity against the logarithm of the dilution after the method of Galas et al. ( 1981 ). Thus, the Genencor GC 123 enzyme was calculated to be 2.3 times more active against CMC than Grinsted GV. In Table 2 results are shown of the amount of aleurone tissue solubilised after enzyme treatment. Grinsted treatment apparently does not degrade the tissue to any great extent as the amount solubilised equals the amount solubilised in the blank. In contrast, Genencor GC 123 treatment solubilised the cell wall to a large extent (49%). In the same table results are given of an experiment with wheat feed as substrate. A similar decrease in insoluble tissue was observed, suggesting that the enzymes exerted similar effects on the two substrates. The amount of soluble sugars formed upon enzyme treatment is also given in Table 2. Light-microscopic analysis of native wheat tissue and purified aleurone tissue (Fig. 1a) showed no differences in cell morphology, suggesting that the chemical purification of aleurone tissue from wheat grain has no, or only a

M.M. MULDERETAL.

188 TABLE 2 Enzyme treatment of wheat-aleurone and wheat-feed with polysaccharidases Aleurone

Wheat feed

Incubation time (h)

Enzyme m

Weight solubilised (rag (%))

Total sugar (mg)

6 6 6 24 24 24 6 6 6 24 24 24

Gn Gfi Gn Gn Gn Gfi Gn Gn

78 120 72 75 122 71 142 168 137 145 167 136

71 121 86 65 119 86 155 177 151 145 180 260

(31) (48) (28) (30) (49) (28) (57) (65) (54) (56) (66) (54)

mGn, Genencor GC123; Gri, Grinsted GV. After incubation with either of the polysaccharidases, pancreafin was added to solubilise the proteininclusion bodies. The total sugar values given are adjusted to take account of the sugar content of the enzyme preparations.

minor effect on the cell wall. After Genencor GC123 treatment it is shown that the cell wall was largely disrupted (Fig. I b). However, upon treatment with Grinsted GV no changes in aleurone cell walls were observed. Original material and residues obtained after various enzyme digestions were subjected to methylation analysis. The values obtained for total sugar recovery and the calculated polysaccharide composition of the aleurone tissue differ from those reported by Fincher and Stone (1986) using purified cell wall preparations. Only small changes were found in the pattern of methylation products even after treatments which solubilised over half of the material (Table 3). This is consistent with results of rumen incubation experiments with a range of plant materials which have shown that, for a single cell type, even after extensive degradation, no significant changes take place in the linkage composition of recovered indigestible material (Chesson, 1988). The reduced amount of 1,4-1inked glucose in the blank suggests that some breakdown is occurring (either chemically or enzymatically) during the prolonged incubation at pH 4.5. The increase in 1,4-1inked xylose on Grinsted treatment is probably caused by the carbohydrate fillers found in this enzyme preparation. Genencor selectively solubilises a linear xylan but does not seem to be able to degrade arabinoxylans as there is no change in the arabinose products and no increase in the terminal xylose value. Pancreatin was used in an attempt to digest the intracellular protein bodies after digestion with either of the cell wall degrading enzymes. Light-microscopical analysis showed no effect of pancreatin on the protein structures and

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Fig. I. Wheat-aleuronc tissueanalysed by light-microscopyat magnification of × 25. The dark bodies arc intracellularmaterial consisting (partly)of proteins,a, untreated alcurone tissue;b, aleurone tissuetreatedwith Termamyl and Gencncor.

micro-Kjeldahl analysis of the digestion supernatants did not show any increase in nitrogen content, suggesting that the intracellularprotein bodies arc not degraded by pancreatin even after extensive cellwall degradation.

M.M.MULDERETAL.

190 TABLE 3 Methylation analysis of original and enzyme-treated wheat-aleurone tissue Partially methylated sugar

Native (mg)

Blank (mg)

Gn (mg)

Gn + Pan (mg)

Gri (mg)

Gri+ Pan (mg)

2346 glucose 236 glucose 246 glucose 23 glucose 26 glucose 36 glucose 234 xylose 23 xylose 2 xylose Unmethylated xylose 236 mannose 235 arahinose 23 arabinose 25 arabinose 35 arabinose 5 arabinose

8 207 10 10 10 16 2 68 17 18 6 41 3 7 3 5

2 129 6 10 3 3 4 78 15 11 3 35 2 5 3 4

2 119 1 I1 4 3 5 9 8 13 4 27 3 7 4 7

2 103 1 9 2 2 3 13 7 13 3 23 3 6 3 6

2 82 2 8 2 2 3 112 15 8 3 26 2 3 2 2

4 116 2 10 3 2 4 143 17 13 4 38 3 5 3 4

Aleurone tissue was treated for 24 h with Termamyl, then with a polysaccharidase (Genencor GC 123 (Gn} or Grinsted GV (Gri)) and, subsequently, some samples were treated for a further 24 h with pancreatin (Pan). Methylation was performed on the residual fraction after enzyme digestion and all results are given for ! g of original material. The blank was obtained by treatment of aleurone tissue for 3×24 h with buffer. 2

B I

B

D D B

-2

-I C

C

I

2

C -I

E -2

Fig. 2. Scatter plot of the first two discriminant functions obtained from PyMS results. Samples B, C, D and E were treated with Termamyl for 24 h. Sample D was treated with Grinsted, Sample E with Genencor, and Sample C with buffer. Samples B, C, D and E were subsequently treated with pancreatin for another 24 h. Sample A was treated with buffer for 72 h.

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The results of PyMS are given in Fig. 2. In this scatter diagram the aleurone tissue (A) plots in the direction of pentosans and proteins. After Termamyl treatment no significant differences were observed (B) suggesting that the sample does not contain much starch. On pancreatin treatment after a 24 h treatment with buffer, the sample plots nearer to the origin (C), suggesting that the protein was (partly) digested. Samples D and E were treated with respectively, Grinsted and Genencor. Sample E moves in the direction of fatty acid products, the meaning of which is still unclear. Sample D moves in the direction of hexosans and lignin owing to a decrease in the amount of pentosans. ACKNOWLEDGEMENTS

The authors thank Jaap Boon for assistance with the interpretation of PyMS results. This work was conducted at the Rowett Research Institute funded by a fellowship to M.M. Mulder of the OECD under the OECD project on Food Production and Preservation.

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