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An enzymic method for analysis of total mixed-linkage β-glucans in cereal grains
Journal of Cereal Science 3 (1985) 231-237
An Enzymic Method for Analysis of Total Mixed-Linkage ~-Glucans in Cereal Grains P. AMAN* and K. HESSELMAN
Swedish University of Agricultural Sciences, Department of Animal Nutrition and Management, S-750 07 Uppsala, Sweden Received 20 November 1984
An enzymic method for analysis of the total ~-glucan contents of cereals has been developed. The method involves complete degradation of starch using a thermostable ex-amylase and amyloglucosidase, precipitation of buffer-soluble ~-glucans with 80 % (vIv) ethanol and degradation of soluble and insoluble ~-glucans with a ~-glucanase preparation from Rhizomucor pusillus. Buffer-soluble polymers were precipitated with 80% ethanol, and mono- and oligo-saccharides formed from ~-glucans were isolated in the 80% ethanol extract. Isolated sugars were hydrolysed with acid and the content of total mixed-linkage ~-glucans was calculated from the glucose content, as determined by the glucose oxidase method. The different steps in the described method and its precision were investigated. Analytical results on some barley cultivars were compared with results obtained with two previously-described methods. Total ~-glucan contents of grain from different barley cultivars as well as wheat, rye, triticale and oats were analysed using the method described in this paper.
Introduction Barley is the major cereal crop grown in Sweden. The grain is mainly used as an animal feed, but a significant and economically important amount is used in the brewing industry. The current overproduction of cereals in Sweden has increased the interest in alternative uses for barley grain. Barley and oats contain mixed-linkage ~-(l ~ 3), (1 ~4)-D-glucans (referred to hereafter as ~-glucans), which are located mainly in the endosperm, but which also occur in aleurone cell walls-, Both soluble and insoluble ~-glucans are present, and the soluble ~-glucans may form viscous solutions that can cause problems during malting and brewing", The ~-glucans are also responsible for the low productivity value of barley and oats for broiler chickens": 4, mainly because of their effect on starch utilisation. A variety ofanalytical methods has been developed for the measurement ofthe ~-glucan contents of cereals". Major problems in most methods, however, are the incomplete extraction of Il-glucans and the inability to distinguish between the relatively low content of ~-glucans and starch, which is the major constituent in grain samples", Other methods, such as the use of Calcofluor, which binds specifically to ~-glucans, are dependent upon calibration using an independent methods- 7. Purified Il-glucanases have been used for
* To whom correspondence should be addressed. Abbreviations used: EU
= enzyme unit;
0733-5210/85/030231 +07 $03.00/0
OLe = gas-liquid chromatography; TFA
= trifluoroacetic acid.
© 1985 Academic Press Inc. (London) Limited 16-2
232
P. AMAN AND K. HESSELMAN
hydrolysis of J)-glucans, and the oligosaccharides released have been extracted and analysed, after acid hydrolysis", as glucose. Other p-glucanase preparations convert p-glucans to glucose, which can be analysed directly, without an acid hydrolysis step9-n. However, great caution must be exercised to ensure complete hydrolysis of p-glucans and to ensure the complete absence of other glucan-degrading enzymes, such as amylases and cellulases. Because of the technical importance of p-glucans in both the brewing and animal feed industries, there is a great need for an accurate and reliable method for p-glucan analysis. In the present paper a new enzymic method for total J)-glucan analysis is described. Experimental Materials Cereal cultivars were obtained from different locations in Sweden. In addition, 10 barley samples, which had been analysed for their contents of I)-glucan according to Anderson et al.8 , were obtained from Dr S. Aastrup, Carlsberg Research Center, Denmark, and pure barley endosperm, which had been prepared by a pearling procedure, was provided by Dr K. E. Bach Knudsen, National Institute of Animal Science, Denmark. A sample of purified I)-glucan from barley was obtained from Biocon Ltd, Cork, Ireland. Hydrozyme (Norsk Hydro, Oslo, Norway), is a partially-purified ~-glucanase preparation produced by fermentation of a selected strain of the filamentous fungus Rhizomucor pusillus and purified by chromatography on Sephadex 0-25 and SP-Sephadex C-25. The enzyme catalyses the hydrolysis of ~-glucans into oligosaccharides and, according to the manufacturer's information sheet, has a broad pH optimum (pH 2·5-5'5), a temperature optimum of about 70°C and very good storage stability. One ~-glucanase enzyme unit (ED) is defined as the amount of enzyme that increases the reciprocal specific viscosity by one unit/miu/ml when measured at 30 °C and pH 4 on a ~-glucan solution with an initial reciprocal specific viscosity of 0'130 (information sheet from Norsk Hydro).
Methods General methods. Dry matter was determined by drying for 5 h at 105 °C. All results are calculated on a dry matter basis. Starch was determined by an enzymic method, including treatment with a thermostable o-amylase'". Polysaccharides were hydrolysed with 2 M trifluoroacetic acid (TFA) for 2 h, and the resulting neutral sugars were converted to alditol acetates and analysed by gas-liquid chromatography (OLC) on an OV-225 capillary column's. Analysis of the total contents of f3-glucans in cereal grains. Step 1: Cereal grain (50 g) was ground in a Tecator Cyclotec sample mill to pass a 0'5 mm screen. Duplicate samples (200 mg) were weighed into 35 ml thick-walled Pyrex glass tubes with screw caps containing teflon liners. Step 2: Sodium acetate buffer (0'1 M, pH 5,0, 5·0 ml) and a thermostable a-amylase (E.C. 3.2.1. I, Termamyl 120L, Novo A/S, Denmark, 132 KNu/g*, 100 J.11) were added and the tubes capped tightly and the contents mixed vigorously using a vortex mixer. Immediately after mixing, the samples were incubated for 1 h in a boiling water bath and during this incubation the tubes were shaken on three occasions. After cooling the mixture to 40 °C, asuspension ofamyloglucosidase from Aspergillus niger (E.C. 3.2.1 .3, Boehringer Mannheim, 140 U /ml, 100 Ill) was added, the tubes were capped, shaken and incubated for 2 h at 60 °C in a waterbath with shaker. Step 3: Soluble polymers were precipitated with absolute ethanol (20 ml) at 4 °C overnight. The samples were then centrifuged (1500 g, 5 min.), the supernatant decanted and the pellet washed ,.. KNu = Enzyme units as defined by the suppliers.
~-GLUCAN ANALYSIS
233
twice with 80% (vIv) aqueous ethanol (25 ml) by centrifugation (1500 g, 5 min). After the last washing the test tubes were inverted on a filter paper for about 10 min. Step 4: Sodium acetate buffer (0'05 M, pH 4·8,5·0 ml) and ~-glucanase from R. pusillus (Norsk Hydro, 6·4 ED Iml, 50 Jll) were added to the insoluble residues. The capped tubes were shaken and incubated for 2 h at 50°C in a water bath with shaker. Step 5: Soluble polymers were precipitated by addition of absolute ethanol (20 ml) and the supernatant fraction was isolated by centrifugation (1500 g, 5 min). The residues were washed by resuspension in 80% (v/v) aqueous ethanol (20 ml) and centrifugation (1500~, 5 min). The combined supernatants were diluted to 50·0 ml with 80% (v/v) aqueous ethanol. Step 6: Aliquots of the diluted supernatant fractions (1,0 ml) were transferred to 8 ml pyrex glass tubes fitted with teflon-lined screw caps and evaporated to dryness in a stream of air at 40°C. Trifluoroacetic acid (2,0 M, 250 ul) was added and the carbohydrates were hydrolysed for 2 h at 125°C in the capped tubes. The samples were evaporated as above and, when no TFA remained, they were redissolved in water (4,0 ml). The glucose contents of the solutions were determined by the glucose oxidase method according to the supplier's manual (Kabi AB, Stockholm).
Results and Discussion
Development and verification of the method Of the six major steps in the ~-glucan analysis the first, grinding, ensures representative 200 mg samples for the analysis. In step 2, starch is completely degraded to glucose using the two enzymes, Termamyl and amyloglucosidasev, These enzymes have been shown previously not to degrade ~-glucans at elevated temperature-s- 14. In step 3, soluble ~-glucans are precipitated with 80 % (v Iv) ethanol and total ~-glucans, both buffer-soluble and insoluble, are isolated by centrifugation. Termamyl, by itself, completely degraded starch to compounds soluble in the acetate buffer, but some of these degradation products were precipitated with 80% ethanol during step 3. Further degradation of these soluble hydrolysis products from starch with amyloglucosidase ensured, however, that no degradation products from starch co-precipitated with soluble ~-glucans. Purified barley p-glucan (Biocon Ltd) was completely recovered in step 3, further demonstrating that the starch-degrading enzymes do not contain any p-glucanase activity and that soluble p-glucans precipitate with 80 % ethanol. During step 4 soluble and insoluble p-glucans were hydrolysed with a technical p-glucanase preparation from Norsk Hydro. The enzyme preparation was very low in cellulase activity as shown by failure to hydrolyse carboxymethyl-cellulose or cotton lint cellulose. Different concentrations of the enzyme preparation were incubated with freeze-dried, starch-free residues from step 3 for different periods of time (Fig. I). At a concentration of 6·4 ED/ml, complete degradation of ~-glucans to oligosaccharides, soluble in 80% (v Iv) ethanol, was obtained after about 1 h. At a concentration of 2·6 ED/ml the corresponding period oftime was approximately 2 h and, at a concentration of 1·6 ED/ml, about 4 h. Routinely a concentration of 6·4 ED Iml and an incubation time of 2 h was used. In step 5, buffer-soluble polymeric material that is resistant to p-glucanase was precipitated with 80% (v Iv) ethanol. Glucose oligomers, produced by hydrolysis of the p-glucans, remained soluble in 80% ethanol and could be separated into the supernatant fraction after centrifugation. These oligomers were quantified as total ~-glucans in step 6.
P. AMAN AND K. HESSELMAN
234
0 '4~-----------------,
E
0 -:3
c;
0
N
~
'" g ., -e.,
0 '2
'"
.c -0:
0 ')
o
2
4
6
8
Incubatian time ( h J
FIGURE 1. Hydrolysis of ~-glucans in a starch-free barley preparation using ~-glucanase (Hydrozyme) at 50°C. Hydrolysis was followed by measuring the formation of reducing groups by a colorimetric method lB . 0 , 6·4 EU/ml ; ., 2-6 EU/ml;o, 1'6 EUjml.
Comparison of enzymic and chemical methods for f3-glucan determination
In order to compare results from the enzymic method with those obtained by chemical analysis, a purified ~-glucan preparation from Biocon Ltd was hydrolysed with 2 M TFA and its content of neutral sugar residues was analysed by GLC. Glucose constituted 65 % by weight of the preparation and only traces of other sugar residues could be detected. The enzymic method for ~-glucan analysis also gave values of 65 ± 3 % of glucose in this p-glucan preparation. This shows that the ~-glucanase preparation, as used in the enzymic method, degrades soluble ~-glucans quantitatively to oligosaccharides that are soluble in 80% ethanol and which, after acid hydrolysis, can be analysed as glucose by the glucose oxidase method. Measurement of f3-glucans in barley endosperm
Cell walls of barley endosperm are known to contain about 70 % (wjw) of p-glucans and only small amounts of other glucans". An endosperm preparation from barley that had been prepared by pearIing and sifting was found to contain 0·9% (w jw, dry basis) of total p-glucans using the enzymic method described in this paper. The low value indicates that the preparation contained a low content of cell walls. In order to investigate whether the l3-glucanase had degraded the l3-glucans in the endosperm cell walls quantitatively, the insoluble residue isolated by centrifugation during step 5 was hydrolysed with 2 M TFA and analysed for its content of sugar residues. After correction for the glucose residues found in a blank determination, only 0·1 % of the barley endosperm content was due to glucan residues that were not hydrolysed by the p-glucanase. These residues constituted about 10% of the total p-glucans and may well originate from glucan polymers other than the p-glucans.
~-GLUCAN ANALYSIS
235
( b)
• •
5
• 4
•
• • •
3
• 2
3
4
5
6
'---;;-2---3:;-----4~----;!
5
.a-Glueen content (%) determined by the described method
FIGURE 2. Relationship between the described method and a fluorimetric" (a) and an enzymic" (b) method for analysis of total ~-gluean (%, w[w, of dry cereal grain). (a) y = -0,07 + 1'17x, r = 0·92. (b) y = 0·57 +0·92x, r = 0·78. TABLE 1. Precision of the described method for analysis of total (cultivars in parentheses)
~-glucan
in cereal grains
Barley Barley Oats Rye Triticale (Tellus) (Nordahl) (Sang) (Petkus II) (Lasko) Number of replicates Mean value (%, w/w, dry basis) Range of values (% ) Standard deviation Coefficient of variation (%)
Wheat (Holme)
19
21
8
8
8
8
3·8 3'5-3-9 0·12 3·2
3·7 3·5-4·0 0·14
3·0 2'7-3-1 0·18 5·9
1·3 1'2-1·5 0'08 5·8
0·53 0·46-0·59 0,04 7·7
0·54 0,47-0,57 0·04 6·9
3-8
Measurement of f3-glucans in various cereal grains
In order to investigate the precision of the enzymic method, two cultivars of barley and one cultivar each of oats, rye, triticale and wheat were investigated (Table I). All cereals contained ~-glucans and the mean values of total ~-glucans varied from 0.5% (wjw, dry basis) in the triticale and wheat samples to 3.8 % in one of the barley samples. The standard deviation was highest for oats, probably reflectingdifficultiesin obtaining a representative sample. These mean values are in a good agreement with those published previously", The figures for the coefficientsof variation varied between 3.2 and 7.7% with the highest figures for the triticale and wheat samples, probably reflecting the lowest mean values. Comparison of the described method with other published methods
The method for total ~-glucan analysis, as described in this paper, was compared with two published methods (Fig. 2). All analyses were carried out in duplicate. The
236
P. AMAN AND K. HESSELMAN
relationship between the described method and a fluorimetric method using Calcofluor" was close (Slope 1,17, r = 0'92), while that with an enzymic method using highly purified ~-glucanases8, and carried out at the Carlsberg Research Center, was weaker (Slope 0,92, r = 0,78). Measurement of f3-glucans in Swedish barley samples
The content of total f3-g1ucans was determined in Swedish barley samples (n = 32). The samples represented six different harvest years, 10cultivars and 10 localities (from Ojebyn in the north to Landskrona in the south). The content of total ~-glucans varied from 2·8 to 5-4% (wjw) with an average of 3·9% and a standard deviation of ±O·59.
General Discussion
With our facilities, 24 replicate samples can be analysed for total ~-glucans within two days. No sophisticated equipment is needed, and the method is accurate and has a relatively high precision. The removal of starch prior to ~-glucan degradation with f3-g1ucanase has the major advantage that commercially-available technical f3-g1ucanase preparations with low cellulase activity can be used, as contaminating amylases do not interfere with the analysis. Also, in this method, no transfers of solid material are required, thus minimising the risk of sample losses. The method described for total ~~glucan analysis can be used for calibration of more rapid viscometric-", spectrofluorimetric" or enzymic" methods. It is also possible to analyse both soluble and insoluble f3-glucans with the described method by analysing soluble f3-glucans from step 2 separately, and to use the extract from step 2 for a starch analysis, after a slight modification of the method according to Aman and Hesselman'>. A recent publication also describes a method for the determination of f3-glucans and starch using a simultaneous extraction with perchloric acid" Excellent technical assistance from A.-C. Tilly and M. Nilsson is gratefully acknowledged. Norsk Hydro, Oslo, Norway, is thanked for a sample of purified Hydrozyme. This work was supported by a grant from the Swedish Council for Forestry and Agricultural Research. References 1. Bade, A. and Stone, B. A. AUSf. J. Plant Physiol. 8 (1981) 475-495. 2. Barrett, J., Clapperton, J. F., Divers, D. M. and Rennie, H. J. Inst, Brew. 79 (1973) 407-413. 3. Hesselman, K. Ph.D. thesis, Swedish University of Agricultural Sciences, Department of Animal Husbandry, Report 112 (1983) pp. 1-32. 4. Hesselman, K. and Aman, P. in 'New Approaches to Research on Cereal Carbohydrates' CR. D. Hill and L. Munck, eds.) Elsevier, Amsterdam (1985) pp. 363-372. 5. Wood, P. J. and Weisz, J. Cereal Chem. 61 (1984) 73-75. 6. Jensen, S. A. and Aastrup, S. Carlsberg Res. Commun. 46 (1981) 87-95. 7. Jorgensen, K. G. Carlsberg Res. Commun. 48 (1983) 505-516. 8. Anderson, M. A., Cook, J. A. and Stone, B. A. J. Inst. Brew. 84 (1978) 233-239. 9. Prentice, N., Babler, S. and Faber, S. Cereal Chem. 57 (1980) 198-202.
P-GLUCAN ANALYSIS 10. 11. 12. 13. 14. 15. 16. 17. 18.
Martin, H. L. and Bamforth, C. W . J. Inst. Brew . 87 (1981) 88-91. Bamforth, C. W. J . Inst . Brew. 89 (1983) 391-392. Aman, P. and Hesselman, K. Swedish J. Agric. Res. 14 (1984) 135-139. Albersheim, P., Nevins, D. J., English, P. D. and Karr, A. Carbohydr, Res . 5 (1967) 340-345. Theander, O. and Arnan, P. Swedish J. Agric. Res. 9 (1979) 97-106. Bendelow , V. M. J . Insl . Brew. 81 (1975) 127-130. Henry, R. J. J. Inst . Brew . 90 (1984) 178-180. Ahluwalia, B. and Ellis, E. E. J. Inst, Brew. 90 (1984) 254--259. Somogyi , M. J. Bioi. Chem. 195 (1952) 19-23.
237
An Enzymic Method for Analysis of Total Mixed-Linkage ~-Glucans in Cereal Grains P. AMAN* and K. HESSELMAN
Swedish University of Agricultural Sciences, Department of Animal Nutrition and Management, S-750 07 Uppsala, Sweden Received 20 November 1984
An enzymic method for analysis of the total ~-glucan contents of cereals has been developed. The method involves complete degradation of starch using a thermostable ex-amylase and amyloglucosidase, precipitation of buffer-soluble ~-glucans with 80 % (vIv) ethanol and degradation of soluble and insoluble ~-glucans with a ~-glucanase preparation from Rhizomucor pusillus. Buffer-soluble polymers were precipitated with 80% ethanol, and mono- and oligo-saccharides formed from ~-glucans were isolated in the 80% ethanol extract. Isolated sugars were hydrolysed with acid and the content of total mixed-linkage ~-glucans was calculated from the glucose content, as determined by the glucose oxidase method. The different steps in the described method and its precision were investigated. Analytical results on some barley cultivars were compared with results obtained with two previously-described methods. Total ~-glucan contents of grain from different barley cultivars as well as wheat, rye, triticale and oats were analysed using the method described in this paper.
Introduction Barley is the major cereal crop grown in Sweden. The grain is mainly used as an animal feed, but a significant and economically important amount is used in the brewing industry. The current overproduction of cereals in Sweden has increased the interest in alternative uses for barley grain. Barley and oats contain mixed-linkage ~-(l ~ 3), (1 ~4)-D-glucans (referred to hereafter as ~-glucans), which are located mainly in the endosperm, but which also occur in aleurone cell walls-, Both soluble and insoluble ~-glucans are present, and the soluble ~-glucans may form viscous solutions that can cause problems during malting and brewing", The ~-glucans are also responsible for the low productivity value of barley and oats for broiler chickens": 4, mainly because of their effect on starch utilisation. A variety ofanalytical methods has been developed for the measurement ofthe ~-glucan contents of cereals". Major problems in most methods, however, are the incomplete extraction of Il-glucans and the inability to distinguish between the relatively low content of ~-glucans and starch, which is the major constituent in grain samples", Other methods, such as the use of Calcofluor, which binds specifically to ~-glucans, are dependent upon calibration using an independent methods- 7. Purified Il-glucanases have been used for
* To whom correspondence should be addressed. Abbreviations used: EU
= enzyme unit;
0733-5210/85/030231 +07 $03.00/0
OLe = gas-liquid chromatography; TFA
= trifluoroacetic acid.
© 1985 Academic Press Inc. (London) Limited 16-2
232
P. AMAN AND K. HESSELMAN
hydrolysis of J)-glucans, and the oligosaccharides released have been extracted and analysed, after acid hydrolysis", as glucose. Other p-glucanase preparations convert p-glucans to glucose, which can be analysed directly, without an acid hydrolysis step9-n. However, great caution must be exercised to ensure complete hydrolysis of p-glucans and to ensure the complete absence of other glucan-degrading enzymes, such as amylases and cellulases. Because of the technical importance of p-glucans in both the brewing and animal feed industries, there is a great need for an accurate and reliable method for p-glucan analysis. In the present paper a new enzymic method for total J)-glucan analysis is described. Experimental Materials Cereal cultivars were obtained from different locations in Sweden. In addition, 10 barley samples, which had been analysed for their contents of I)-glucan according to Anderson et al.8 , were obtained from Dr S. Aastrup, Carlsberg Research Center, Denmark, and pure barley endosperm, which had been prepared by a pearling procedure, was provided by Dr K. E. Bach Knudsen, National Institute of Animal Science, Denmark. A sample of purified I)-glucan from barley was obtained from Biocon Ltd, Cork, Ireland. Hydrozyme (Norsk Hydro, Oslo, Norway), is a partially-purified ~-glucanase preparation produced by fermentation of a selected strain of the filamentous fungus Rhizomucor pusillus and purified by chromatography on Sephadex 0-25 and SP-Sephadex C-25. The enzyme catalyses the hydrolysis of ~-glucans into oligosaccharides and, according to the manufacturer's information sheet, has a broad pH optimum (pH 2·5-5'5), a temperature optimum of about 70°C and very good storage stability. One ~-glucanase enzyme unit (ED) is defined as the amount of enzyme that increases the reciprocal specific viscosity by one unit/miu/ml when measured at 30 °C and pH 4 on a ~-glucan solution with an initial reciprocal specific viscosity of 0'130 (information sheet from Norsk Hydro).
Methods General methods. Dry matter was determined by drying for 5 h at 105 °C. All results are calculated on a dry matter basis. Starch was determined by an enzymic method, including treatment with a thermostable o-amylase'". Polysaccharides were hydrolysed with 2 M trifluoroacetic acid (TFA) for 2 h, and the resulting neutral sugars were converted to alditol acetates and analysed by gas-liquid chromatography (OLC) on an OV-225 capillary column's. Analysis of the total contents of f3-glucans in cereal grains. Step 1: Cereal grain (50 g) was ground in a Tecator Cyclotec sample mill to pass a 0'5 mm screen. Duplicate samples (200 mg) were weighed into 35 ml thick-walled Pyrex glass tubes with screw caps containing teflon liners. Step 2: Sodium acetate buffer (0'1 M, pH 5,0, 5·0 ml) and a thermostable a-amylase (E.C. 3.2.1. I, Termamyl 120L, Novo A/S, Denmark, 132 KNu/g*, 100 J.11) were added and the tubes capped tightly and the contents mixed vigorously using a vortex mixer. Immediately after mixing, the samples were incubated for 1 h in a boiling water bath and during this incubation the tubes were shaken on three occasions. After cooling the mixture to 40 °C, asuspension ofamyloglucosidase from Aspergillus niger (E.C. 3.2.1 .3, Boehringer Mannheim, 140 U /ml, 100 Ill) was added, the tubes were capped, shaken and incubated for 2 h at 60 °C in a waterbath with shaker. Step 3: Soluble polymers were precipitated with absolute ethanol (20 ml) at 4 °C overnight. The samples were then centrifuged (1500 g, 5 min.), the supernatant decanted and the pellet washed ,.. KNu = Enzyme units as defined by the suppliers.
~-GLUCAN ANALYSIS
233
twice with 80% (vIv) aqueous ethanol (25 ml) by centrifugation (1500 g, 5 min). After the last washing the test tubes were inverted on a filter paper for about 10 min. Step 4: Sodium acetate buffer (0'05 M, pH 4·8,5·0 ml) and ~-glucanase from R. pusillus (Norsk Hydro, 6·4 ED Iml, 50 Jll) were added to the insoluble residues. The capped tubes were shaken and incubated for 2 h at 50°C in a water bath with shaker. Step 5: Soluble polymers were precipitated by addition of absolute ethanol (20 ml) and the supernatant fraction was isolated by centrifugation (1500 g, 5 min). The residues were washed by resuspension in 80% (v/v) aqueous ethanol (20 ml) and centrifugation (1500~, 5 min). The combined supernatants were diluted to 50·0 ml with 80% (v/v) aqueous ethanol. Step 6: Aliquots of the diluted supernatant fractions (1,0 ml) were transferred to 8 ml pyrex glass tubes fitted with teflon-lined screw caps and evaporated to dryness in a stream of air at 40°C. Trifluoroacetic acid (2,0 M, 250 ul) was added and the carbohydrates were hydrolysed for 2 h at 125°C in the capped tubes. The samples were evaporated as above and, when no TFA remained, they were redissolved in water (4,0 ml). The glucose contents of the solutions were determined by the glucose oxidase method according to the supplier's manual (Kabi AB, Stockholm).
Results and Discussion
Development and verification of the method Of the six major steps in the ~-glucan analysis the first, grinding, ensures representative 200 mg samples for the analysis. In step 2, starch is completely degraded to glucose using the two enzymes, Termamyl and amyloglucosidasev, These enzymes have been shown previously not to degrade ~-glucans at elevated temperature-s- 14. In step 3, soluble ~-glucans are precipitated with 80 % (v Iv) ethanol and total ~-glucans, both buffer-soluble and insoluble, are isolated by centrifugation. Termamyl, by itself, completely degraded starch to compounds soluble in the acetate buffer, but some of these degradation products were precipitated with 80% ethanol during step 3. Further degradation of these soluble hydrolysis products from starch with amyloglucosidase ensured, however, that no degradation products from starch co-precipitated with soluble ~-glucans. Purified barley p-glucan (Biocon Ltd) was completely recovered in step 3, further demonstrating that the starch-degrading enzymes do not contain any p-glucanase activity and that soluble p-glucans precipitate with 80 % ethanol. During step 4 soluble and insoluble p-glucans were hydrolysed with a technical p-glucanase preparation from Norsk Hydro. The enzyme preparation was very low in cellulase activity as shown by failure to hydrolyse carboxymethyl-cellulose or cotton lint cellulose. Different concentrations of the enzyme preparation were incubated with freeze-dried, starch-free residues from step 3 for different periods of time (Fig. I). At a concentration of 6·4 ED/ml, complete degradation of ~-glucans to oligosaccharides, soluble in 80% (v Iv) ethanol, was obtained after about 1 h. At a concentration of 2·6 ED/ml the corresponding period oftime was approximately 2 h and, at a concentration of 1·6 ED/ml, about 4 h. Routinely a concentration of 6·4 ED Iml and an incubation time of 2 h was used. In step 5, buffer-soluble polymeric material that is resistant to p-glucanase was precipitated with 80% (v Iv) ethanol. Glucose oligomers, produced by hydrolysis of the p-glucans, remained soluble in 80% ethanol and could be separated into the supernatant fraction after centrifugation. These oligomers were quantified as total ~-glucans in step 6.
P. AMAN AND K. HESSELMAN
234
0 '4~-----------------,
E
0 -:3
c;
0
N
~
'" g ., -e.,
0 '2
'"
.c -0:
0 ')
o
2
4
6
8
Incubatian time ( h J
FIGURE 1. Hydrolysis of ~-glucans in a starch-free barley preparation using ~-glucanase (Hydrozyme) at 50°C. Hydrolysis was followed by measuring the formation of reducing groups by a colorimetric method lB . 0 , 6·4 EU/ml ; ., 2-6 EU/ml;o, 1'6 EUjml.
Comparison of enzymic and chemical methods for f3-glucan determination
In order to compare results from the enzymic method with those obtained by chemical analysis, a purified ~-glucan preparation from Biocon Ltd was hydrolysed with 2 M TFA and its content of neutral sugar residues was analysed by GLC. Glucose constituted 65 % by weight of the preparation and only traces of other sugar residues could be detected. The enzymic method for ~-glucan analysis also gave values of 65 ± 3 % of glucose in this p-glucan preparation. This shows that the ~-glucanase preparation, as used in the enzymic method, degrades soluble ~-glucans quantitatively to oligosaccharides that are soluble in 80% ethanol and which, after acid hydrolysis, can be analysed as glucose by the glucose oxidase method. Measurement of f3-glucans in barley endosperm
Cell walls of barley endosperm are known to contain about 70 % (wjw) of p-glucans and only small amounts of other glucans". An endosperm preparation from barley that had been prepared by pearIing and sifting was found to contain 0·9% (w jw, dry basis) of total p-glucans using the enzymic method described in this paper. The low value indicates that the preparation contained a low content of cell walls. In order to investigate whether the l3-glucanase had degraded the l3-glucans in the endosperm cell walls quantitatively, the insoluble residue isolated by centrifugation during step 5 was hydrolysed with 2 M TFA and analysed for its content of sugar residues. After correction for the glucose residues found in a blank determination, only 0·1 % of the barley endosperm content was due to glucan residues that were not hydrolysed by the p-glucanase. These residues constituted about 10% of the total p-glucans and may well originate from glucan polymers other than the p-glucans.
~-GLUCAN ANALYSIS
235
( b)
• •
5
• 4
•
• • •
3
• 2
3
4
5
6
'---;;-2---3:;-----4~----;!
5
.a-Glueen content (%) determined by the described method
FIGURE 2. Relationship between the described method and a fluorimetric" (a) and an enzymic" (b) method for analysis of total ~-gluean (%, w[w, of dry cereal grain). (a) y = -0,07 + 1'17x, r = 0·92. (b) y = 0·57 +0·92x, r = 0·78. TABLE 1. Precision of the described method for analysis of total (cultivars in parentheses)
~-glucan
in cereal grains
Barley Barley Oats Rye Triticale (Tellus) (Nordahl) (Sang) (Petkus II) (Lasko) Number of replicates Mean value (%, w/w, dry basis) Range of values (% ) Standard deviation Coefficient of variation (%)
Wheat (Holme)
19
21
8
8
8
8
3·8 3'5-3-9 0·12 3·2
3·7 3·5-4·0 0·14
3·0 2'7-3-1 0·18 5·9
1·3 1'2-1·5 0'08 5·8
0·53 0·46-0·59 0,04 7·7
0·54 0,47-0,57 0·04 6·9
3-8
Measurement of f3-glucans in various cereal grains
In order to investigate the precision of the enzymic method, two cultivars of barley and one cultivar each of oats, rye, triticale and wheat were investigated (Table I). All cereals contained ~-glucans and the mean values of total ~-glucans varied from 0.5% (wjw, dry basis) in the triticale and wheat samples to 3.8 % in one of the barley samples. The standard deviation was highest for oats, probably reflectingdifficultiesin obtaining a representative sample. These mean values are in a good agreement with those published previously", The figures for the coefficientsof variation varied between 3.2 and 7.7% with the highest figures for the triticale and wheat samples, probably reflecting the lowest mean values. Comparison of the described method with other published methods
The method for total ~-glucan analysis, as described in this paper, was compared with two published methods (Fig. 2). All analyses were carried out in duplicate. The
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P. AMAN AND K. HESSELMAN
relationship between the described method and a fluorimetric method using Calcofluor" was close (Slope 1,17, r = 0'92), while that with an enzymic method using highly purified ~-glucanases8, and carried out at the Carlsberg Research Center, was weaker (Slope 0,92, r = 0,78). Measurement of f3-glucans in Swedish barley samples
The content of total f3-g1ucans was determined in Swedish barley samples (n = 32). The samples represented six different harvest years, 10cultivars and 10 localities (from Ojebyn in the north to Landskrona in the south). The content of total ~-glucans varied from 2·8 to 5-4% (wjw) with an average of 3·9% and a standard deviation of ±O·59.
General Discussion
With our facilities, 24 replicate samples can be analysed for total ~-glucans within two days. No sophisticated equipment is needed, and the method is accurate and has a relatively high precision. The removal of starch prior to ~-glucan degradation with f3-g1ucanase has the major advantage that commercially-available technical f3-g1ucanase preparations with low cellulase activity can be used, as contaminating amylases do not interfere with the analysis. Also, in this method, no transfers of solid material are required, thus minimising the risk of sample losses. The method described for total ~~glucan analysis can be used for calibration of more rapid viscometric-", spectrofluorimetric" or enzymic" methods. It is also possible to analyse both soluble and insoluble f3-glucans with the described method by analysing soluble f3-glucans from step 2 separately, and to use the extract from step 2 for a starch analysis, after a slight modification of the method according to Aman and Hesselman'>. A recent publication also describes a method for the determination of f3-glucans and starch using a simultaneous extraction with perchloric acid" Excellent technical assistance from A.-C. Tilly and M. Nilsson is gratefully acknowledged. Norsk Hydro, Oslo, Norway, is thanked for a sample of purified Hydrozyme. This work was supported by a grant from the Swedish Council for Forestry and Agricultural Research. References 1. Bade, A. and Stone, B. A. AUSf. J. Plant Physiol. 8 (1981) 475-495. 2. Barrett, J., Clapperton, J. F., Divers, D. M. and Rennie, H. J. Inst, Brew. 79 (1973) 407-413. 3. Hesselman, K. Ph.D. thesis, Swedish University of Agricultural Sciences, Department of Animal Husbandry, Report 112 (1983) pp. 1-32. 4. Hesselman, K. and Aman, P. in 'New Approaches to Research on Cereal Carbohydrates' CR. D. Hill and L. Munck, eds.) Elsevier, Amsterdam (1985) pp. 363-372. 5. Wood, P. J. and Weisz, J. Cereal Chem. 61 (1984) 73-75. 6. Jensen, S. A. and Aastrup, S. Carlsberg Res. Commun. 46 (1981) 87-95. 7. Jorgensen, K. G. Carlsberg Res. Commun. 48 (1983) 505-516. 8. Anderson, M. A., Cook, J. A. and Stone, B. A. J. Inst. Brew. 84 (1978) 233-239. 9. Prentice, N., Babler, S. and Faber, S. Cereal Chem. 57 (1980) 198-202.
P-GLUCAN ANALYSIS 10. 11. 12. 13. 14. 15. 16. 17. 18.
Martin, H. L. and Bamforth, C. W . J. Inst. Brew . 87 (1981) 88-91. Bamforth, C. W. J . Inst . Brew. 89 (1983) 391-392. Aman, P. and Hesselman, K. Swedish J. Agric. Res. 14 (1984) 135-139. Albersheim, P., Nevins, D. J., English, P. D. and Karr, A. Carbohydr, Res . 5 (1967) 340-345. Theander, O. and Arnan, P. Swedish J. Agric. Res. 9 (1979) 97-106. Bendelow , V. M. J . Insl . Brew. 81 (1975) 127-130. Henry, R. J. J. Inst . Brew . 90 (1984) 178-180. Ahluwalia, B. and Ellis, E. E. J. Inst, Brew. 90 (1984) 254--259. Somogyi , M. J. Bioi. Chem. 195 (1952) 19-23.
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