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Isolation, separation and characterization of water soluble non-starch polysaccharides from wheat and rye
Food Hydrocolloids Vo1.6 no.3 pp.285-299, 1992
Isolation, separation and characterization of water soluble non-starch polysaccharides from wheat and rye Urban Girhammar and Baboo M.Nair Department of Applied Nutrition and Food Chemistry, Chemical Centre, University of Lund, Box 124, S-22100 Lund, Sweden Abstract. The water holding capacity of the dough and the freshness of the bread made of rye and wheat are much dependent upon their content of non-starch polysaccharides (NSP), most of which are polymers of xylose and arabinose, i.e. pentosans. The yield of water soluble pentosans from rye flour (1.76%) was higher than that of wheat flour (0.59%), and the xylose to arabinose ratio ofrye pentosans was 1:36 while it was 1:16 for wheat pentosans. The galactose content in wheat pentosans was 17% of the total monosaccharides. In rye pentosans the content of galactose was lower (3.5% of the total monosaccharides). The total content of monosaccharides in wheat pentosans (65%) was slightly lower than that in the rye pentosans (71.7%). The weight average molecular weight of rye pentosans (770000) was higher than that of the wheat pentosans (255 000). The number average molecular weight of the pentosans from rye was 90 000 while the corresponding figure for wheat was 61 000. The rye pentosans showed a higher degree of polydispersity in solution than the wheat pentosans. The separation of the pentosans on a DEAE-Sephadex column resulted in five fractions. The first two fractions contained mainly arabinose and xylose and the protein content in these fractions was -1 %. The protein content of the fractions increased with the increased concentration of the borate. The Xyl:Ara ratio was 2:23 and 2:39 in fractions I and II of wheat and 1:63 and 2:32 in fractions I and II of rye. Galactose appeared in fractions IV and V in wheat and fractions IV, V-A and V-B in rye along with some arabinose which suggests the presence of arabinogalactans in these fractions.
Introduction
Starch and protein are important components for the dough development and the baking quality of cereals like wheat and rye. The importance of non-starch polysaccharides has not yet been investigated in detail. The non-starch polysaccharides are found primarily in the plant cell walls, both as water soluble and as water insoluble components. So far the interest has been mostly concentrated on water soluble non-starch polysaccharides, probably because these are easily extracted with water from cereal flours. A major portion of the water soluble non-starch polysaccharides in wheat and rye flours are pentosans, i.e. polymers of pentoses. In other cereals, such as triticale, oats, and barley, they also contain appreciable amounts of mannose, galactose and glucose (1,2,3,4). There are two main types of water soluble non-starch polysaccharides in wheat and rye; the linear arabinoxylans and the highly branched arabinogalactans. Linear arabinoxylans contain D-xylose units linked 13-(1-4) glycosidically to form long xylan backbone chains. The linear structure of the arabinoxylans is expected to be responsible for the high viscosity of their solutions in water. The solubility of pentosans in water is caused mainly by the presence of single e-t-arabinofuranose side groups attached to the xylan chains. Arabinoxylans occur in grains bound to small amounts of protein. In the branched chains of arabinogalactans, arabinose units are attached as single, 285
U.Girhammar and B.M.Nair
terminal residues in the o-i.-arabinosyl configuration to a galactose in the /3-Dgalactopyranosyl configuration . Arabinogalactans are covalently linked to a polypeptide containing relatively large amounts of hydroxyproline (5,6) . Most of the water insoluble polysaccharides are found in the starch tailing fraction when wheat flour is washed to obtain gluten and starch . They consist mostly of arabinoxylans, arabinogalactans and varying amounts of soluble starch and water soluble proteins depending on the method of extraction. The water soluble pentosans are able to form highly viscous solutions in water and produce gels when oxidized . Wheat pentosans were estimated to bind 23% of the water in a wheat dough (7). The water soluble pentosans are reported to absorb 11 times their weight of water, and water insoluble pentosans absorb 10 times their weight of water. The pentosans of the soluble fraction have a greater effect on water absorption than the proteins. In some rye flours the water soluble pentosan and protein contents are so high that it is not possible to bake bread from these flours, because the dough is not able to retain its shape during baking . Pentosans have been found to retard staling (8,9) mainly due to their capacity to hold water in competition with starch. Pentosans are well suited as additives in the manufacture of gluten free breads (10). The present work is a part of an investigation on the water soluble pentosans from various cereals in order to evaluate their importance in baking and baking quality . It deals with the separation and characterization of pentosans isolated from wheat and rye . Future papers will contain data regarding the different physicochemical qualities of the pentosans and their effect on the rheological properties of the dough and the quality of bread.
Materials and methods
Isolation of pentosans Water soluble pentosans were isolated from whole grains of rye (var. Petkus) and wheat (var. Hildur) obtained from Svalof AB , Svalov, Sweden , as described by Antoniou et al. (11). Milled (1 mm screen) whole grain flour (200 g) was boiled with 80% ethanol (700 ml) for 1 h under reflux. The residue obtained by filtration was washed with 95% ethanol and dried at room temperature. The dried residue was then extracted with 4 volumes of distilled water for 15 min followed by centrifugation at 9000 g for 10 min. The pH of the supernatant was adjusted to 7.5 with NaOH before porcine pancreatin (Purum, Fluka AG, Buchs , Switzerland) was added to digest the protein and the starch. The suspension was incubated at 34°C for 24 h with 0.05% NaN 3 with continuous stirring. The solution was then centrifuged at 9000 g for 5 min. From the clear supernatant, pentosans were precipitated with ethanol, adjusting the final concentration to 80% . The pentosan precipitate was filtered (Munktell No. 0, Grycksbo pappersbruk AB , Grycksbo , Sweden) , washed with 95% ethanol, acetone and diethyl ether before it was dried at room temperature under a nitrogen atmosphere .
286
Non-starch polysaccharides in wheat and rye
Molecular weight determination The molecular weight was calculated using the Svedberg equation (12) as follows :
where So is the sedimentation coefficient , DO is the diffusion coefficient, V2 is the partial specific volume of the pol ymers , p is the solution density and M is the average molecular weight. The sedimentation coefficient was determined by analytical Ultra centrifugation (Centriscan 75, equipped with Schlieren optics, MSE, UK) with a rotor velocity of 58 100 r.p .rn. (250 00 g) at 20°C. The sedimentation coefficient values at different concentrations in 0.1 mol/drrr' NaCl solution were extrapolated to infinite dilution (Figure 1) and corrected to corresponding values in water to obtain the So 20,w' The diffusion coefficient was measured by using a laserlight scattering spectrometer (Model-Malvern argon laser, Spectraphysics-164, Mountain View, CA 94040 , USA) equipped with Loglin Correlator. The laser wavelength was 514,5 nm and the measurement angle 90°. The diffus ion coefficients of pentosans at different concentrations in 0.1 mol/drrr' NaCl were measured and extrapolated to infinite dilution at 21SC (Figure 2) and corrected to the corresponding values in water at 20°C to obtain D O20.w ' The partial specific volume was determined by using a digital densitometer (DMA-60, Anton Paar K,G ., Graz, Austria) at a temperature of 20°C. The density of the solution of the pentosans in water containing 0.1 rnol/drrr' NaCl was measured at a series of different concentrations and then the apparent partial specific volume was calculated .
Molecular weight distribution The molecular weight distribution of the soluble pentosans from wheat and rye were determined using high performance gel permeation chromatography (GPC). The separation was performed at 60°C on a liquid chromatograph equipped with a stainless steel column (50 em in length and 8 mm in diameter) packed with rigid microporous hydroxy polyester gel (Shodex OHpak B805, Showa Denko, Tokyo, Japan), a pump (modeI6000-A, Waters Associates Inc., Milford, MA, USA), an injector (U&K Waters), a refractive index detector (Sodex RI SE-II, Showa Denko) and a variable wavelength UV spectrophotometric detector (Uvidec 100-IV, Japan Spectroscopic, Tokyo , Japan). The flow rate of the mobile phase (0.05 mol/drrr' NaH 2P04 solution) was 1.0 mllmin . The response of the refractive index detector was recorded on a CRT HP 9826 computer (Hewlett Packard, Fort Collins, CO , USA). Mw was determined with reference to a calibration curve prepared with a series of standard dextrans of average molecular weight 500000, 110 000, 70000, 40000 and 10000 (Pharmacia Fine Chemicals , Uppsala, Sweden). The number average molecular 287
U.Girhammar and R.M.Nair
____ 0.6 Wheat, r = 0.986
'o
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§, 0.4
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r---
-
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Rye, r = 0.880
o
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0.5
2
1 1.5 Concentration (mg/ml)
Fig. 1. Effect of concentration on the sedimentation coefficient of the water soluble non-starch polysaccharides from wheat and rye .
• ......Ilt
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.
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3
Fig. 2. Effect of concentration on the diffusion coefficient on the water soluble non-starch polysaccharides from wheat and rye.
weight (M u ) and the weight average molecular weight (M w ) were calculated based on the results of duplicate analysis using the following equations:
where M, = exp (a + bVe + cVe2 + dVe 3 ) , hi = the height at each 2 s interval of the chromatogram peak, and a, b, c and d are calibration constants. The molecular weight distribution was calculated according to Ring et al. (13) assuming that Mw/Mn = exp(cr2 ) and the distribution function is approximated by the log-normal distribution.
Separation of pentosans on a D EAE-Sephadex column The pentosan preparation (150 mg) was dissolved in water (50 ml) and 288
Non-starch polysaccharides in wheat and rye
fractionated on a diethylaminoethyl (DEAE) A-25 Sephadex (Pharmacia Fine Chemicals) ion-exchange column (52 em in length and 5.3 cm in diameter) according to Kiindig et ai. (14) with minor modifications. Five fractions were collected by eluting first with distilled H 20 (fraction I), and then with solutions of 0.0025 mol/dnr' (fraction II), 0.025 rnol/dm? (fraction III), 0.125 mol/dnr' (fraction IV) disodium tetraborate (Na2B407) and 0.4 mol/drrr' sodium hydroxide (fraction V) in respective order. The pentose concentration in each fraction (10 ml) was determined by measuring the absorbance of the colour developed in an aliquot using an orcinol reagent (15). The fractions were pooled and concentrated using a Diaflo ultrafiltration apparatus (Amicon Corporation, Lexington, MA, USA) with membranes (PM 30) having a cut-off limit at 30 000 g/mol under nitrogen pressure, dialysed three times against 10 times its volume of water to remove the salts and then evaporated to dryness under reduced pressure. The amino acid content and monosaccharide composition of each of the fractions were also determined.
Determination of monosaccharides Determination of the monosaccharide content was done according to Theander and Aman (16). The polysaccharides were hydrolysed with sulphuric acid and the monosaccharides were converted to alditol acetates before they were separated in a gaschromatograph (model 3700, Varian, Palo Alto, CA, USA) equipped with a capillary column and a flame ionization detector using fucose or allose as internal standard. The monosaccharide content of the fractions obtained from the DEAESephadex column was determined by HPLC. After dispersion of the sample in 2 ml of sulphuric acid (1.0 mol/dm") and hydrolysis following dilution with 2 ml water, the hydrolysate was neutralized with excess of barium carbonate. The barium sulphate was removed by filtration and the solution containing monosaccharides was injected to a HPLC apparatus equipped with a solvent delivery system (model M-45, Waters Associates Inc.) and a 0.8 x 10 em Radial-Pak column (Waters RCM-100) with 4 J.1 spherical Silica-Pak HP. The monosaccharides were eluted with acetonitrile and water (80:20) containing 1% Silica Amine Modifier (Waters SAM reagent) and detected with a refractive index detector (model OPTILAB Multiref 902B, Tecator AB, Hoganas, Sweden). The monosaccharide content was expressed as per cent of the total monosaccharides.
Determination of nitrogen and amino acids The nitrogen content of the samples was determined by using the Kjeldahl method. A sample (-1 g) was digested (Digestion system 20, Tecator AB) with 12 ml concentrated H2S04 and a catalyst (CUS04' Kjeltabs) at 400°C for 60 minutes. After cooling the digested material was diluted with 75 ml of water. The mixture was distilled after addition of NaOH solution and the ammonia was absorbed into a 1% solution of boric acid. The amount of ammonia absorbed in the boric acid was determined by titration with HCI. 289
U.Girhammar and B.M.Nair
Table I. Yield and proximate composition of the water soluble non-starch polysaccharides from wheat and rye
Wheat (var. Hildur) Rye (var. Petkus)
Yield % n=7
Moisture % n= 5
X
SD
X
SD
0,59" 1.76"
0,06 0,19
9 7
0,9 1.7
Protein (N x 6,25) (mg/g TS) n=2 X SD
Sugars (mg/g TS) n=2
Ash (mg/g) n=2
X
SD
X
SD
178 86
650 717
9,7 13,7
40 39
1.8 1.4
45 13
"Significantly different (P < 0,05), SD: Standard deviation,
Determination of the amino acid content of the pentosans was done by GLC after acid hydrolysis as described by Nair (17) and the amino acid determination of the fractions from DEAE-Sephadex column was carried out by ion-exchange chromatographic method (Model LC 5001, BIOTRONIK amino acid analyser GmbH, Munich, Germany).
Determination of the ash content The samples were dry ashed at 550°C overnight and the ash content was determined by weighing. Results
Yield and proximate composition of the pentosans The yield of water soluble pentosans (Table I) obtained in this experiment from wheat flour (var. Hildur) was 0.59% and 1.76% from rye (var. Petkus) flour.
Proximate composition of the pentosans The composition of the soluble pentosan preparation from wheat and rye is given in Table 1. The protein content of the wheat pentosans was almost twice that in the rye preparation. The content of monosaccharides in wheat pentosans (65%) was slightly lower than that in rye pentosans (71.7%) while the content of ash was similar (4%).
Monosaccharide composition The results of the monosaccharide analyses are presented in Table II. The main components in both wheat and rye pentosans were xylose and arabinose. The xylose/arabinose ratio in wheat pentosans (1:16) was lower than that of the rye pentosans (1:36). Certain amounts of mannose, galactose and glucose were also present.
Amino acid composition The amino acid composition of the protein attached to the pentosans from wheat 290
Non-starch polysaccharides in wheat and rye
Table II. Monosaccharide composition of the water soluble non-starch polysaccharides from wheat and rye Monosaccharide
Wheat var. Hi/dur % of total
Rye var. Petkus % of total
so Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
0.2 34.3 39.9a 1.9 17.0b
±* ±0.8 ±4.3 ±* ±2.4 ±1.3
6.8 1.16
SD ±0.5 ±3.4 ±* ±0.7 ±1.4
36.5 49.7" 2.5 3.5 b 7.7 1.36
a,bSignificantly different (P < 0.05). SD: Standard deviation. ": Single observation. Table III. Amino acid composition of the water soluble non-starch polysaccharides from wheat and rye Amino acids (mg/g TS) Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Total
Wheat var. Hi/dur
Rye var. Petkus
20.8 9.0 13.5 18.5 7.5 10.6 9.7 10.0 2.0 7.9 9.5 5.6 10.5
11.3 3.9 5.2 10.0 4.0 6.2 4.5 5.0 0.7 3.7 4.9 2.4 4.3
135.2
66.1
Results are from single analysis of one sample. The standard deviation for this method determined with standard samples (all the amino acids taken together), is 5-10%.
and rye is shown in Table III. Aspartic acid and glutamic acid are the major amino acids (30% of the total) in both the rye and wheat preparations, while methionine was found in very low quantities (1-1.5% of the total). Molecular weight determination Sedimentation coefficient. The sedimentation coefficient (SO) for the water soluble pentosans from wheat was 2.2 S (±O.1 S) and for water soluble pentosans from rye it was 2.5 S (±0.2 S) (Figure 1).
291
U.Girhammar and 8.M.Nair
Diffusion coefficient. At 200 e the DO was determined to be 0.91 x 10- 7 cm2/s for wheat pentosans and 0.59 x 10- 7 cm2/s for pentosans from rye, with a standard deviation of 0.16 and 0.11 respectively (Figure 2). Partial specific volume. The apparent partial specific volume of the wheat pentosans was found to be 0.632 ml/g and that of rye pentosans was 0.633 ml/g. Molecular weight distribution relative to dextran standards. Figure 3 shows the gel permeation elution pattern of the water soluble pentosans and the calculated molecular weight distributions. The refractive index (RI) and the UV absorbance (220 nm) of each fraction was recorded simultaneously. The RI measurements show the presence of a peak with three major components in pentosans from wheat and two major components in pentosans from rye. Separation and characterization of pentosan fractions
Separation on a DEAE-Sephadex column gave five fractions of both wheat (Figure 4) and rye (Figure 5) pentosans designated I-V. The fifth peak of the rye sample was divided. The monosaccharide composition of the fractions is presented in Tables V and VII and the amino acid content is given in Tables VI and VIII, which are discussed in greater detail in relation to other observations in the following section. The first two fractions obtained by the separation of pentosans on the DEAE-Sephadex column according to Kundig et al. (25) in the present experiment contained mainly arabinoxylans. In the case of wheat pentosans fraction I and fraction II contained 28.4 and 3.1 % respectively of the sample applied to the column. The corresponding figures for rye were 57.6 and
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=
100 300 500 Molecular weight (M x 10E+3)
Fig. 3. Gelpermeation chromatography and molecular weight distribution relative to dextran standards of water soluble non-starch polysaccharides from wheat and rye.
292
Non-starch polysaccharides in wheat and rye
4.5% . The Xyl:Ara ratio in these fractions was 2:23 and 2:39 for fractions I and II of wheat and 1:63 and 2:32 for fractions I and II for rye. The content of protein in these two fractions as determined by amino acid analysis was -1 %. Galactose appeared in fractions IV and V in wheat and fractions IV , V-A and V-B in rye along with some arabinose which suggests the presence of arabinogalactans in these fraction s. The protein content of the fractions increased with the increased concentration of the borate . The amino acid pattern of the protein found in fraction V from wheat and rye samples shows that the predominant amino acids are glutamic acid, aspartic acid and serine in both cases.
Fig. 4. Separation of water soluble non-starch polysaccharides isolated from whole grain wheat flour on a DEAE-Sephadex A-25 ion exchange column (52 em in length and 5.3 ern in diameter) . Pentose concentrations were measured with an orcinol reagent. Eluted with (I) water , (II) 0.0025 mol/drrr' Na2B.07, (III ) 0.025 mol/drrr' Na2B.07, (IV) 0.125 mol/drrr' Na2B.07, (V) 0.4 N NaOH respectively. ~
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Fig. 5. Separation of water soluble non-starch polysaccharides isolated from whole grain rye flour on a DEAE-Sephadex A-25 ion exchange column (52 cm in length and 5.3 cm in diameter). Pentose concentrations were measured with an orcinol reagent. Eluted with (I) water , (II) 0.0025 rnol/drrr' Na2B.07, (III) 0.025 mol/drn? Na2B.07. (IV) 0.125 rnol/dm? Na2B. 07, (V) 0.4 N NaOH respectively.
293
U.Girhammar and B.M.Nair
Table IV. Some physicochemical properties of water soluble non-starch polysaccharides from wheat and rye in water solutions Property
Wheat (SD)
Rye (SD)
Sedimentation coefficient S° 20.w (S)
2.2 (0.1) 0.91 x 10- 7 (0.16) 0.632 156000 361000 254630· (299) 60 544b (603)
2.5 (0.2) 0.59 x 10- 7 (0.11) 0.633 275000 563000 770460" (18555) 90 343b (2408)
Diffusion coefficient DOzo.w (crnvs)
Vz Partial specific volume (ml/g)
Mw (S° from main fraction) (sOmax = 5.0 estimated)
Mw Weight average molecular weight (from GPC analysis)
Mn Number average molecular weight (from GPC analysis) (from GPC analysis)
Mw/M n
8.54 c
4.21 C
(0.43) 560 158000
(0.05) 160 70200
[1]] Intrinsic viscosity (ml/g) M~ (from Scheraga-Mandelkern equation 13 = 2.5-106 ) 1\ Degree of hydration (gig TS) fifo Frictional ratio 1J Simha shape factor R H Hydrated radius of the eqv. sphere (A)
0.47 8.7 507 362
0.41 6.8 153 235
Significantly different: "(P < 0.001), b(p < 0.01), "P < 0.01). Table V. Monosaccharide composition (expressed as % of the total) of the various fractions of water soluble non-starch polysaccharides of wheat separated on a DEAE-Sephadex column Monosaccharide Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
Frac. I
Frac. II
Frac. III
Frac. IV
Frac. V
30.8 68.7
2.5 28.5 68.0
2.6 39.3 51.5
1.1 36.3 6.1
1.3 37.8 33.1
0.55
1.0
2.3 4.6
47.8 8.9
15.9 12.0
2:23
2:39
1:31
0:17
0:88
Results are from single analysis of one sample. The standard deviation for this method determined using standard sample was between 0.14 and 1.42 for all the monosaccharides taken together.
Discussion
The composition and properties of water soluble non-starch polysaccharides from cereal grains are dependent on the method used for their extraction and purification. One method of isolation (18) used by earlier workers is based on extraction of the flour with solutions of ammonium sulphate at various concentrations. After extraction the salt was removed by dialysis and the pentosans were precipitated by ethyl alcohol. As the solubility of the pen tosans is higher either at high or at low pH they have also been extracted with dilute acid or alkali before they are precipitated from the solution with addition of
294
Non-starch polysaccharides in wheat and rye
Table VI. Amino acid composinon of the various fractions of water soluble non-starch polysaccharides of wheat separated on a OEAE-Sephadex column Amino acid (mg/g TS)
Frac. I
Frac. II
Frac. III
Frac. IV
Frac. V
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.69 0.49 0.49 1.19 traces 0.61 0.71 0.48 0.24 0.86 0.20 0.29 0.36 0.19 0.34
1.68 0.70 1.13 1.86 traces 1.80 0.94 0.69 0.38 0.80 0.26 0.30 0.55 0.34 0.39
0.49 0.49 0.55 0.93 traces 1.05 0.54 0.47 0.08 2.08 0.19 0.20 0.26 0.07 0.10
2.68 2.88 5.13 7.10 traces 3.19 6.85 3.12 0.74 5.05 2.31 0.65 1.00 0.50 0.56
14.32 4.04 8.22 15.84 traces 7.92 5.48 4.08 2.35 3.24 1.94 1.35 1.84 1.09 0.90
Total
7.14
11.82
7.50
41.76
n.51
SO: see Table III. Table VII. Monosaccharide composition (expressed as % of the total) of the various fractions of water-soluble non-starch polysaccharides of rye separated on a OEAE-Sephadex column Monosaccharide
Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
Frac. I
0.3 37.8 61.5
Frac. II
0.6 29.2 67.6 1.4
Frac. III
Frac. IV
Frac. V A
B
4.5 52.1 36.6
1.4 39.1 26.8
2.9 38.2 29.7
1.3 34.3 32.1
12.4 20.2
11.7 17.6
5.9 20.0
0.51 0.64
1.2
1.6 5.3
1:63
2:32
0:70
0:69
0:78
0:94
SO: see Table V.
ethanol (19). Boiling of cereal flours with 80% ethanol under reflux cooking inactivates the endogeneous enzymes, coagulates the proteins and dissolves sugars and amino acids. Pentosans are then extracted with water and separated after precipitation with ethyl alcohol. More recently, higher yields of pentosans have been obtained by using amylolytic and proteolytic enzymes for efficient degradation of protein and starch as an intermediate step before the extraction of pentosans with water following precipitation with alcohol (11). Therefore we used this method in the present study. Antoniou et at. (11) determined the pentosan content in Canadian rye samples using a colorimetric method (orcinol) as well as a GLC method. Their results show that the content of water soluble pen tosans in a variety called Puma was 1.14% of the dry matter. Karlsson (3), who determined pentosan content in 11
295
U.Girhammar and B.M.Nair
Table VIII. Amino acid composition of the various fractions of water soluble non-starch polysaccharides of rye separated on a DEAE-Sephadex column Amino acid (mg/g TS)
Frac. I
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.41 0.22 0.17 0.57 0.36 0.32
Total
Frac. II
Frac. III
Frac. IV
Frac. V A
0.46 0.13 0.19 0.12
0.82 0.47 0.77 1.59 1.00 0.63 0.49 0.32 0.71 0.26 0.31 0.35
1.10 0.37 1.61 2.28 1.29 0.51 0.49 4.46 0.19 0.31 0.42
0.35 2.95
8.24
13.41
B
4.23 1.99 2.64 3.90 3.01 2.93 2.40 0.70 4.93 0.90 0.90 0.83 0.20 0.80
3.26 1.80 2.00 6.71 2.15 2.70 2.06 1.04 5.15 1.10 1.11 1.66 0.39 1.59
3.25 0.52 1.24 3.31 1.58 0.85 0.67 0.29 2.40 0.30 0.33 0.38 0.08 0.16
30.55
32.72
15.36
SD: see Table III.
different varieties of rye grown in Sweden in three different years, found that the content of water soluble pentosans in these was 1.8% of the dry matter, i.e. very similar to our mean figure for rye. The average arabinoxylan content in the water soluble ethanol precipitated fractions from seven rye genotypes was 75.6% of the dry matter. The soluble pentosan content in 12 wheat genotypes grown in Sweden was 0.73% of the dry matter. The average arabinoxylan content in ethanol precipitated fractions from seven different varieties of wheat was 68.5% of the dry matter. Bengtsson and Aman (20) obtained water soluble crude arabinoxylan (0.74% of the dry matter) from whole rye grains of a Swedish variety cv.Kungs II, 72% of which on dry basis was constituted by arabinose and xylose. Delcour et at. (21) isolated a water soluble fraction by precipitating with ethanol from a water extract of rye and wheat flour. The yield of water soluble pentosans in rye was 2.18% of the dry matter of which 33% were arabinose and xylose residues. The corresponding figures for wheat were 0.18 and 32% respectively. In our experiments, arabinose and xylose together account for 74% of the total monosaccharides in the wheat NSP and 86% in the rye NSP. On a dry matter basis the arabinose and xylose content in the precipitate from wheat was 48% and 62% in rye. The amount of galactose in the precipitate obtained in the present study was higher in the wheat pentosans compared to that of the rye pentosans. This observation is in agreement with that of Karlsson (3). Most of the results which appear in earlier literature deal with wheat pentosans which are isolated in different ways. Thus, it is not surprising that there is a certain disagreement between results from different sources. The presence of higher amounts of galactose and arabinose in the wheat pentosans indicate that there is more arabinogalactan in wheat than in rye. 296
Non-starch polysaccharides in wheat and rye
A certain amount of minerals is obviously bound to the pentosans isolated in this experiment even after thorough washing with water, ethanol and diethyl ether. The polysaccharide itself may not bind any considerable amounts of minerals, while the protein to which the polysaccharide is found to be bound covalently may bind most of the minerals coprecipitated in this experiment. In addition it is also possible that a certain amount of phytate, which is known to bind minerals is present in this precipitate. The increased levels of most of the amino acids in wheat pentosans correspond to its higher protein content. The amino acid composition of the protein remaining bound to the water soluble pentosans isolated from wheat and rye appears to be very similar and showed hardly any resemblance to the amino acid composition of storage proteins in the respective grains. No hydroxyproline was detected either in the crude fraction or in the fractions obtained from the DEAE-Sephadex column. The UV absorbance of the eluate from the gel permeation chromatography shows the presence of protein attached mainly to low molecular weight fractions. It may also include some contribution from ferulic acid, which contains a phenolic group in its structure. Neukom (2) found ferulic acid only in certain parts of the arabinoxylans, most likely bound by ester linkages to the xylan chain. Yeh et al. (22) have also shown that ferulic acid is associated with the arabinoxylan fraction. Fincher and Stone (23) have determined the molecular size distribution of wheat endosperm arabinoxylan by gel filtration chromatography on Sepharose 4B using dextrans as standard. They observed an association of low molecular weight polysaccharides with protein while the high molecular weight polysaccharides were free of protein, which is in agreement with the results reported in the present paper. The xylose to arabinose ratio of the pentosans from wheat endosperm studied by Fincher and Stone (23) was 1:11 while the corresponding figure for the pentosans used in this study was 1:16 for wheat and 1:36 for rye. Karlsson (3) found no difference in the Xyl:Ara ratio (1:33 in both wheat and rye) in seven different wheat and rye varieties. The water soluble arabinoxylan from wheat flour studied by Andrewartha et al. (24) had a Xyl:Ara ratio above 2.0 and the solubility in water was drastically reduced when arabinose molecules were removed by treating the samples with a-L-arabinofuranosidase to alter the Xyl:Ara ratio to 2:3 and above. Kundig et al. (25) separated water soluble pentosans of wheat flour on an anion-exchange (DEAE-cellulose) column. The first fraction, which was eluted with water, contained a pure arabinoxylan while the next four fractions, eluted with 0.01, 0.1 mol/dm" and saturated Na2B407 and finally 0.5 N NaOH, contained various proportions of arabinose, xylose, galactose and protein. Morita et al. (26) separated water soluble pentosans from wheat on a DEAEcellulose column with water as Kiindig et al. (25). First of the four fractions separated by eluting fraction II successively with water to 0.01 mol/drrr' Na2B407 on the DEAE-cellulose column, consisted of 48.8% D-xylose, 41.4% L-arabinose, 1.8% D-galactose and 0.2% ferulic acid, but no protein. Lineback et al. (27) compared the carbohydrate composition of fractionated water soluble pentosans from different types of wheat flours on a DEAE297
U.Girhammar and B.M.Nair
cellulose column. The results agreed quite well with each other. The composition of fraction I, a pure arabinoxylan, was similar in all the samples. However, differences in galactose content of fraction II from different wheat pentosans were noted. In almost all earlier investigations the pentosan fraction IV contained the smallest amount of xylose and the largest amount of galactose, as was found by Kundig et al. (25) as well as in the present study. The values for Mw/M n (Table IV) of the wheat pentosans and the corresponding values for rye pentosans show that the wheat pentosans in solution had a lower degree of polydispersity compared to that of rye pentosans. However the rye pentosans contained a large proportion of molecules with a higher degree of polymerization (Figure 3). The partial specific volume of the samples of water soluble pentosans from rye and wheat show no significant difference between each other and agree well with the results obtained by Andrewartha et al. (24) and Cole,E.W. (28). The sedimentation coefficient of rye pentosans is slightly higher than that of the wheat pentosans which shows that the major part of the rye pentosan molecules are somewhat heavier than the wheat pentosan molecules. The diffusion coefficient of these two samples confirms the above observations to some extent as the value DO for the rye pentosans is lower than that of the wheat pentosans. The hydrated radius RH of the equivalent sphere of the molecules was calculated from the DO values according to the Stoke-Einstein equation (D = kTI6TjR H ) . The Simha shape factor II was calculated (u = [Tj]/(Vz + SVd from the values of partial specific volumes v. for water and Vz for solute, intrinsic viscosity [Tj] and degree of hydration S to estimate the difference in shape and size of the two pentosan molecules. The intrinsic viscosity was determined using an Ostwald viscometer and the degree of hydration was obtained as the amount of water unfrozen at - 53°C by using a differential scanning calorimeter (DSC) technique (U.Girhammar and B.M.Nair, in preparation). The Simha shape factor for the rye pentosan and the wheat pentosan shows that both of them have an extended and more or less rigid configuration in solution. The molecules of rye pentosans deviate more from the ideal spherical shape than the wheat pentosan molecules. The frictional ratio fIfo (calculated from fifo = (1 - VzIDZsOV z) of the two samples also appear to confirm the above conclusion. It is interesting to compare the values for molecular weights of the samples obtained by using Svedberg equation (MSODO) and by high performance gel permeation chromatography (Mw ) . The calculated from GPC shows higher values for rye pentosans and wheat pentosans compared to MsoDo calculated using So for the main fraction. However, MsoDo and Mw become similar when the estimated max So is used in the equation. The values of MsoDo, e; Mw and M"!) (calculated from SheragaMandelkern equation, (12) definitely show that the molecules of water soluble rye pentosans are larger than the molecules of the water soluble wheat pentosans as a result of a higher degree of polymerization. Rye pentosan molecules have a more extended and rigid configuration compared to that of the wheat pentosans in solution, which also causes the increase in viscosity. The lower Xyl:Ara ratio of the wheat pentosans is in agreement with their higher solubility in water.
«:
298
Non-starch polysaccharides in wheat and rye
Conclusion
The content of water soluble non-starch polysaccharides in rye grain is nearly three times as high as in wheat. The rye pen tosans contain more molecules of higher degree of polymerization compared to those isolated from wheat. There exist some arabinogalactan in wheat pentosans but not in rye pentosans. Rye pentosans produce more viscous solutions in water and the Xyl:Ara ratio of the pentosans from wheat and rye indicates that the solubility of wheat pentosans could be higher than that of rye pentosans. The first two fractions obtained by separation of pentosans on a DEAE-Sephadex column contained mainly arabinoxylans and their protein content was -1 %. The protein attached to the crude pentosans from wheat and rye is mainly associated with the low molecular weight fractions. Acknowledgements
Skilful technical assistance by Ingrid Jonsson was helpful in the execution of this project. We thank Professor Nils-Georg Asp for interesting discussions during the course of this study and for valuable suggestions which improved the scientific quality of this paper. This investigation was carried out with grants from the Swedish board for technical development and Cerealia foundation for research and development. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Podrazky.V. (1964) Chem. Indust., 2, 712-713. Neukorn.H. (1976) Lebensm. Wiss. u. Technol., 9,143-148. Karlsson,R. (1988) J. Swedish Seed Assoc., 98, 213-225. Saini,H.S. and Henry,R.J. (1989) Cereal Chem., 66,11-14. Meuser.F, and Suckow.P. (1986) In Blanshard,1.M.V., Frazier,P.J. and Galliard,T. (eds), Proceedings of an International Symposium on Chemistry and Physics of Baking (/985). Sutton Bennington, UK, pp. 42-61. Markwalder,H. (1975) Diss. Nr. 5497, E. T.H. Zurich. Bushuk,W. (1966) Bakers Digest, 40,38-40. Jankiewicz.M. and Michniewicz.J. (1987) Food Chem., 25, 241-249. D'Appolonia,B.L. (1980) J. Text. Stud., 10,201-216. Casier,J.P.J. (1975) Fermentation, 71, 117-134. Antoniou,T., Marquardt,R.R. and Cansfield,P.E. (1981) J. Agric. Food Chem., 29, 1240-1247. Cantor,e.R. and Schimmel,P.R. (1980) Biophys. Chem. Part II, 591-685. Ring,S.G., l'Anson,K.J. and Morris,V.J. (1985) Macromolecules, 18, 182-188. Kiindig,W., Neukom,H. and Deuel,H. (1961) He/v. Chim. Acta 44,823-829. Volkin,E. and Cohn,W.F. (1954) In Glick,D. (ed.), Meth. Biochem. Anal. I, Interscience, New York, pp. 287-303. Thenader.O, and Aman,P. (1979) Swedish J. Agric. Res., 9,97-106. Nair,B.M. (1977) J. Agric. Food Chem., 25, 614-620. Baker,J.e., Parker,H.K. and Mize,M.D. (1943) Cereal Chem., 20, 267-280. Simpson,F.J. (1955) Canad. J. of Microbiol., 1, 131-139. Bengtsson,S. and Aman,P. (1990) Carbohydr. Polym., 12, 267-277. Delcour,J.A., Vanhamel,S. and Hoseney,R.e. (1991) Cereal Chem., 68, 72-76. Yeh,Y.F., Hoseney,R.C, and Lineback,D.R. (1980) Cereal Chem., 57,144-148. Fincher,G.B. and Stone,B.A. (1974) Aust. J. Biol. Sci., 27,117-132. Andrewartha,K.A., Philips,D.R. and Stone,B.A. (1979) Carbohydr. Res., 77,191-204. Kiindig,W., Neukom,H. and Deuel.H. (1961) He/v. Chim. Acta, 44, 969-976. Morita,S.I., Ito,T. and Hirano.S. (1974) Int. J. Biochem., 5, 201-205. Lineback,D.R., Somnapan Kakuda,N. and Tsen,C.e. (1977) 1. Food Sci., 42, 461-467. Cole,E.W. (1967) Cereal Chem., 44, 411-416.
Received December 3, 1991; accepted March 5, 1992
299
Isolation, separation and characterization of water soluble non-starch polysaccharides from wheat and rye Urban Girhammar and Baboo M.Nair Department of Applied Nutrition and Food Chemistry, Chemical Centre, University of Lund, Box 124, S-22100 Lund, Sweden Abstract. The water holding capacity of the dough and the freshness of the bread made of rye and wheat are much dependent upon their content of non-starch polysaccharides (NSP), most of which are polymers of xylose and arabinose, i.e. pentosans. The yield of water soluble pentosans from rye flour (1.76%) was higher than that of wheat flour (0.59%), and the xylose to arabinose ratio ofrye pentosans was 1:36 while it was 1:16 for wheat pentosans. The galactose content in wheat pentosans was 17% of the total monosaccharides. In rye pentosans the content of galactose was lower (3.5% of the total monosaccharides). The total content of monosaccharides in wheat pentosans (65%) was slightly lower than that in the rye pentosans (71.7%). The weight average molecular weight of rye pentosans (770000) was higher than that of the wheat pentosans (255 000). The number average molecular weight of the pentosans from rye was 90 000 while the corresponding figure for wheat was 61 000. The rye pentosans showed a higher degree of polydispersity in solution than the wheat pentosans. The separation of the pentosans on a DEAE-Sephadex column resulted in five fractions. The first two fractions contained mainly arabinose and xylose and the protein content in these fractions was -1 %. The protein content of the fractions increased with the increased concentration of the borate. The Xyl:Ara ratio was 2:23 and 2:39 in fractions I and II of wheat and 1:63 and 2:32 in fractions I and II of rye. Galactose appeared in fractions IV and V in wheat and fractions IV, V-A and V-B in rye along with some arabinose which suggests the presence of arabinogalactans in these fractions.
Introduction
Starch and protein are important components for the dough development and the baking quality of cereals like wheat and rye. The importance of non-starch polysaccharides has not yet been investigated in detail. The non-starch polysaccharides are found primarily in the plant cell walls, both as water soluble and as water insoluble components. So far the interest has been mostly concentrated on water soluble non-starch polysaccharides, probably because these are easily extracted with water from cereal flours. A major portion of the water soluble non-starch polysaccharides in wheat and rye flours are pentosans, i.e. polymers of pentoses. In other cereals, such as triticale, oats, and barley, they also contain appreciable amounts of mannose, galactose and glucose (1,2,3,4). There are two main types of water soluble non-starch polysaccharides in wheat and rye; the linear arabinoxylans and the highly branched arabinogalactans. Linear arabinoxylans contain D-xylose units linked 13-(1-4) glycosidically to form long xylan backbone chains. The linear structure of the arabinoxylans is expected to be responsible for the high viscosity of their solutions in water. The solubility of pentosans in water is caused mainly by the presence of single e-t-arabinofuranose side groups attached to the xylan chains. Arabinoxylans occur in grains bound to small amounts of protein. In the branched chains of arabinogalactans, arabinose units are attached as single, 285
U.Girhammar and B.M.Nair
terminal residues in the o-i.-arabinosyl configuration to a galactose in the /3-Dgalactopyranosyl configuration . Arabinogalactans are covalently linked to a polypeptide containing relatively large amounts of hydroxyproline (5,6) . Most of the water insoluble polysaccharides are found in the starch tailing fraction when wheat flour is washed to obtain gluten and starch . They consist mostly of arabinoxylans, arabinogalactans and varying amounts of soluble starch and water soluble proteins depending on the method of extraction. The water soluble pentosans are able to form highly viscous solutions in water and produce gels when oxidized . Wheat pentosans were estimated to bind 23% of the water in a wheat dough (7). The water soluble pentosans are reported to absorb 11 times their weight of water, and water insoluble pentosans absorb 10 times their weight of water. The pentosans of the soluble fraction have a greater effect on water absorption than the proteins. In some rye flours the water soluble pentosan and protein contents are so high that it is not possible to bake bread from these flours, because the dough is not able to retain its shape during baking . Pentosans have been found to retard staling (8,9) mainly due to their capacity to hold water in competition with starch. Pentosans are well suited as additives in the manufacture of gluten free breads (10). The present work is a part of an investigation on the water soluble pentosans from various cereals in order to evaluate their importance in baking and baking quality . It deals with the separation and characterization of pentosans isolated from wheat and rye . Future papers will contain data regarding the different physicochemical qualities of the pentosans and their effect on the rheological properties of the dough and the quality of bread.
Materials and methods
Isolation of pentosans Water soluble pentosans were isolated from whole grains of rye (var. Petkus) and wheat (var. Hildur) obtained from Svalof AB , Svalov, Sweden , as described by Antoniou et al. (11). Milled (1 mm screen) whole grain flour (200 g) was boiled with 80% ethanol (700 ml) for 1 h under reflux. The residue obtained by filtration was washed with 95% ethanol and dried at room temperature. The dried residue was then extracted with 4 volumes of distilled water for 15 min followed by centrifugation at 9000 g for 10 min. The pH of the supernatant was adjusted to 7.5 with NaOH before porcine pancreatin (Purum, Fluka AG, Buchs , Switzerland) was added to digest the protein and the starch. The suspension was incubated at 34°C for 24 h with 0.05% NaN 3 with continuous stirring. The solution was then centrifuged at 9000 g for 5 min. From the clear supernatant, pentosans were precipitated with ethanol, adjusting the final concentration to 80% . The pentosan precipitate was filtered (Munktell No. 0, Grycksbo pappersbruk AB , Grycksbo , Sweden) , washed with 95% ethanol, acetone and diethyl ether before it was dried at room temperature under a nitrogen atmosphere .
286
Non-starch polysaccharides in wheat and rye
Molecular weight determination The molecular weight was calculated using the Svedberg equation (12) as follows :
where So is the sedimentation coefficient , DO is the diffusion coefficient, V2 is the partial specific volume of the pol ymers , p is the solution density and M is the average molecular weight. The sedimentation coefficient was determined by analytical Ultra centrifugation (Centriscan 75, equipped with Schlieren optics, MSE, UK) with a rotor velocity of 58 100 r.p .rn. (250 00 g) at 20°C. The sedimentation coefficient values at different concentrations in 0.1 mol/drrr' NaCl solution were extrapolated to infinite dilution (Figure 1) and corrected to corresponding values in water to obtain the So 20,w' The diffusion coefficient was measured by using a laserlight scattering spectrometer (Model-Malvern argon laser, Spectraphysics-164, Mountain View, CA 94040 , USA) equipped with Loglin Correlator. The laser wavelength was 514,5 nm and the measurement angle 90°. The diffus ion coefficients of pentosans at different concentrations in 0.1 mol/drrr' NaCl were measured and extrapolated to infinite dilution at 21SC (Figure 2) and corrected to the corresponding values in water at 20°C to obtain D O20.w ' The partial specific volume was determined by using a digital densitometer (DMA-60, Anton Paar K,G ., Graz, Austria) at a temperature of 20°C. The density of the solution of the pentosans in water containing 0.1 rnol/drrr' NaCl was measured at a series of different concentrations and then the apparent partial specific volume was calculated .
Molecular weight distribution The molecular weight distribution of the soluble pentosans from wheat and rye were determined using high performance gel permeation chromatography (GPC). The separation was performed at 60°C on a liquid chromatograph equipped with a stainless steel column (50 em in length and 8 mm in diameter) packed with rigid microporous hydroxy polyester gel (Shodex OHpak B805, Showa Denko, Tokyo, Japan), a pump (modeI6000-A, Waters Associates Inc., Milford, MA, USA), an injector (U&K Waters), a refractive index detector (Sodex RI SE-II, Showa Denko) and a variable wavelength UV spectrophotometric detector (Uvidec 100-IV, Japan Spectroscopic, Tokyo , Japan). The flow rate of the mobile phase (0.05 mol/drrr' NaH 2P04 solution) was 1.0 mllmin . The response of the refractive index detector was recorded on a CRT HP 9826 computer (Hewlett Packard, Fort Collins, CO , USA). Mw was determined with reference to a calibration curve prepared with a series of standard dextrans of average molecular weight 500000, 110 000, 70000, 40000 and 10000 (Pharmacia Fine Chemicals , Uppsala, Sweden). The number average molecular 287
U.Girhammar and R.M.Nair
____ 0.6 Wheat, r = 0.986
'o
~ ~
§, 0.4
,._.__...__ .-_!-.~-- -
r---
-
•
~
Rye, r = 0.880
o
o
0.5
2
1 1.5 Concentration (mg/ml)
Fig. 1. Effect of concentration on the sedimentation coefficient of the water soluble non-starch polysaccharides from wheat and rye .
• ......Ilt
Wheat, r = -0.819
.
.
.. ~~
.~~~-
• ........
.
Rye, r = -0.906
o
I 2 Concentration (mg/ml)
3
Fig. 2. Effect of concentration on the diffusion coefficient on the water soluble non-starch polysaccharides from wheat and rye.
weight (M u ) and the weight average molecular weight (M w ) were calculated based on the results of duplicate analysis using the following equations:
where M, = exp (a + bVe + cVe2 + dVe 3 ) , hi = the height at each 2 s interval of the chromatogram peak, and a, b, c and d are calibration constants. The molecular weight distribution was calculated according to Ring et al. (13) assuming that Mw/Mn = exp(cr2 ) and the distribution function is approximated by the log-normal distribution.
Separation of pentosans on a D EAE-Sephadex column The pentosan preparation (150 mg) was dissolved in water (50 ml) and 288
Non-starch polysaccharides in wheat and rye
fractionated on a diethylaminoethyl (DEAE) A-25 Sephadex (Pharmacia Fine Chemicals) ion-exchange column (52 em in length and 5.3 cm in diameter) according to Kiindig et ai. (14) with minor modifications. Five fractions were collected by eluting first with distilled H 20 (fraction I), and then with solutions of 0.0025 mol/dnr' (fraction II), 0.025 rnol/dm? (fraction III), 0.125 mol/dnr' (fraction IV) disodium tetraborate (Na2B407) and 0.4 mol/drrr' sodium hydroxide (fraction V) in respective order. The pentose concentration in each fraction (10 ml) was determined by measuring the absorbance of the colour developed in an aliquot using an orcinol reagent (15). The fractions were pooled and concentrated using a Diaflo ultrafiltration apparatus (Amicon Corporation, Lexington, MA, USA) with membranes (PM 30) having a cut-off limit at 30 000 g/mol under nitrogen pressure, dialysed three times against 10 times its volume of water to remove the salts and then evaporated to dryness under reduced pressure. The amino acid content and monosaccharide composition of each of the fractions were also determined.
Determination of monosaccharides Determination of the monosaccharide content was done according to Theander and Aman (16). The polysaccharides were hydrolysed with sulphuric acid and the monosaccharides were converted to alditol acetates before they were separated in a gaschromatograph (model 3700, Varian, Palo Alto, CA, USA) equipped with a capillary column and a flame ionization detector using fucose or allose as internal standard. The monosaccharide content of the fractions obtained from the DEAESephadex column was determined by HPLC. After dispersion of the sample in 2 ml of sulphuric acid (1.0 mol/dm") and hydrolysis following dilution with 2 ml water, the hydrolysate was neutralized with excess of barium carbonate. The barium sulphate was removed by filtration and the solution containing monosaccharides was injected to a HPLC apparatus equipped with a solvent delivery system (model M-45, Waters Associates Inc.) and a 0.8 x 10 em Radial-Pak column (Waters RCM-100) with 4 J.1 spherical Silica-Pak HP. The monosaccharides were eluted with acetonitrile and water (80:20) containing 1% Silica Amine Modifier (Waters SAM reagent) and detected with a refractive index detector (model OPTILAB Multiref 902B, Tecator AB, Hoganas, Sweden). The monosaccharide content was expressed as per cent of the total monosaccharides.
Determination of nitrogen and amino acids The nitrogen content of the samples was determined by using the Kjeldahl method. A sample (-1 g) was digested (Digestion system 20, Tecator AB) with 12 ml concentrated H2S04 and a catalyst (CUS04' Kjeltabs) at 400°C for 60 minutes. After cooling the digested material was diluted with 75 ml of water. The mixture was distilled after addition of NaOH solution and the ammonia was absorbed into a 1% solution of boric acid. The amount of ammonia absorbed in the boric acid was determined by titration with HCI. 289
U.Girhammar and B.M.Nair
Table I. Yield and proximate composition of the water soluble non-starch polysaccharides from wheat and rye
Wheat (var. Hildur) Rye (var. Petkus)
Yield % n=7
Moisture % n= 5
X
SD
X
SD
0,59" 1.76"
0,06 0,19
9 7
0,9 1.7
Protein (N x 6,25) (mg/g TS) n=2 X SD
Sugars (mg/g TS) n=2
Ash (mg/g) n=2
X
SD
X
SD
178 86
650 717
9,7 13,7
40 39
1.8 1.4
45 13
"Significantly different (P < 0,05), SD: Standard deviation,
Determination of the amino acid content of the pentosans was done by GLC after acid hydrolysis as described by Nair (17) and the amino acid determination of the fractions from DEAE-Sephadex column was carried out by ion-exchange chromatographic method (Model LC 5001, BIOTRONIK amino acid analyser GmbH, Munich, Germany).
Determination of the ash content The samples were dry ashed at 550°C overnight and the ash content was determined by weighing. Results
Yield and proximate composition of the pentosans The yield of water soluble pentosans (Table I) obtained in this experiment from wheat flour (var. Hildur) was 0.59% and 1.76% from rye (var. Petkus) flour.
Proximate composition of the pentosans The composition of the soluble pentosan preparation from wheat and rye is given in Table 1. The protein content of the wheat pentosans was almost twice that in the rye preparation. The content of monosaccharides in wheat pentosans (65%) was slightly lower than that in rye pentosans (71.7%) while the content of ash was similar (4%).
Monosaccharide composition The results of the monosaccharide analyses are presented in Table II. The main components in both wheat and rye pentosans were xylose and arabinose. The xylose/arabinose ratio in wheat pentosans (1:16) was lower than that of the rye pentosans (1:36). Certain amounts of mannose, galactose and glucose were also present.
Amino acid composition The amino acid composition of the protein attached to the pentosans from wheat 290
Non-starch polysaccharides in wheat and rye
Table II. Monosaccharide composition of the water soluble non-starch polysaccharides from wheat and rye Monosaccharide
Wheat var. Hi/dur % of total
Rye var. Petkus % of total
so Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
0.2 34.3 39.9a 1.9 17.0b
±* ±0.8 ±4.3 ±* ±2.4 ±1.3
6.8 1.16
SD ±0.5 ±3.4 ±* ±0.7 ±1.4
36.5 49.7" 2.5 3.5 b 7.7 1.36
a,bSignificantly different (P < 0.05). SD: Standard deviation. ": Single observation. Table III. Amino acid composition of the water soluble non-starch polysaccharides from wheat and rye Amino acids (mg/g TS) Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Total
Wheat var. Hi/dur
Rye var. Petkus
20.8 9.0 13.5 18.5 7.5 10.6 9.7 10.0 2.0 7.9 9.5 5.6 10.5
11.3 3.9 5.2 10.0 4.0 6.2 4.5 5.0 0.7 3.7 4.9 2.4 4.3
135.2
66.1
Results are from single analysis of one sample. The standard deviation for this method determined with standard samples (all the amino acids taken together), is 5-10%.
and rye is shown in Table III. Aspartic acid and glutamic acid are the major amino acids (30% of the total) in both the rye and wheat preparations, while methionine was found in very low quantities (1-1.5% of the total). Molecular weight determination Sedimentation coefficient. The sedimentation coefficient (SO) for the water soluble pentosans from wheat was 2.2 S (±O.1 S) and for water soluble pentosans from rye it was 2.5 S (±0.2 S) (Figure 1).
291
U.Girhammar and 8.M.Nair
Diffusion coefficient. At 200 e the DO was determined to be 0.91 x 10- 7 cm2/s for wheat pentosans and 0.59 x 10- 7 cm2/s for pentosans from rye, with a standard deviation of 0.16 and 0.11 respectively (Figure 2). Partial specific volume. The apparent partial specific volume of the wheat pentosans was found to be 0.632 ml/g and that of rye pentosans was 0.633 ml/g. Molecular weight distribution relative to dextran standards. Figure 3 shows the gel permeation elution pattern of the water soluble pentosans and the calculated molecular weight distributions. The refractive index (RI) and the UV absorbance (220 nm) of each fraction was recorded simultaneously. The RI measurements show the presence of a peak with three major components in pentosans from wheat and two major components in pentosans from rye. Separation and characterization of pentosan fractions
Separation on a DEAE-Sephadex column gave five fractions of both wheat (Figure 4) and rye (Figure 5) pentosans designated I-V. The fifth peak of the rye sample was divided. The monosaccharide composition of the fractions is presented in Tables V and VII and the amino acid content is given in Tables VI and VIII, which are discussed in greater detail in relation to other observations in the following section. The first two fractions obtained by the separation of pentosans on the DEAE-Sephadex column according to Kundig et al. (25) in the present experiment contained mainly arabinoxylans. In the case of wheat pentosans fraction I and fraction II contained 28.4 and 3.1 % respectively of the sample applied to the column. The corresponding figures for rye were 57.6 and
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5
10
15
20
25 30 min
Mwl Mn = exp(cr2) Log-normal distribution: F(M) = (l/(O"M(2lt)'l2)) exp(-(In MlMo)2/2cr 2) 0" = Standard deviation Me Geometric mean molecular weight Me =(M n x Mw)1/2
=
100 300 500 Molecular weight (M x 10E+3)
Fig. 3. Gelpermeation chromatography and molecular weight distribution relative to dextran standards of water soluble non-starch polysaccharides from wheat and rye.
292
Non-starch polysaccharides in wheat and rye
4.5% . The Xyl:Ara ratio in these fractions was 2:23 and 2:39 for fractions I and II of wheat and 1:63 and 2:32 for fractions I and II for rye. The content of protein in these two fractions as determined by amino acid analysis was -1 %. Galactose appeared in fractions IV and V in wheat and fractions IV , V-A and V-B in rye along with some arabinose which suggests the presence of arabinogalactans in these fraction s. The protein content of the fractions increased with the increased concentration of the borate . The amino acid pattern of the protein found in fraction V from wheat and rye samples shows that the predominant amino acids are glutamic acid, aspartic acid and serine in both cases.
Fig. 4. Separation of water soluble non-starch polysaccharides isolated from whole grain wheat flour on a DEAE-Sephadex A-25 ion exchange column (52 em in length and 5.3 ern in diameter) . Pentose concentrations were measured with an orcinol reagent. Eluted with (I) water , (II) 0.0025 mol/drrr' Na2B.07, (III ) 0.025 mol/drrr' Na2B.07, (IV) 0.125 mol/drrr' Na2B.07, (V) 0.4 N NaOH respectively. ~
]
r-
bl)
~200
t:ll
0
Z
""
t::
.~
~ Q)
o
t::
0
u
....
0
... . 100 ~ B
t:ll""
~
Z
~
§'" c:::i
Z
~
~
'" S
'" ~
c:::i
~
~...
t-.
~""s-
0
~
~
II
ill
'0
>.
..r:::
~
U
0
0
50
150 200 100 Fraction number (10 ml/fr)
Fig. 5. Separation of water soluble non-starch polysaccharides isolated from whole grain rye flour on a DEAE-Sephadex A-25 ion exchange column (52 cm in length and 5.3 cm in diameter). Pentose concentrations were measured with an orcinol reagent. Eluted with (I) water , (II) 0.0025 rnol/drrr' Na2B.07, (III) 0.025 mol/drn? Na2B.07. (IV) 0.125 rnol/dm? Na2B. 07, (V) 0.4 N NaOH respectively.
293
U.Girhammar and B.M.Nair
Table IV. Some physicochemical properties of water soluble non-starch polysaccharides from wheat and rye in water solutions Property
Wheat (SD)
Rye (SD)
Sedimentation coefficient S° 20.w (S)
2.2 (0.1) 0.91 x 10- 7 (0.16) 0.632 156000 361000 254630· (299) 60 544b (603)
2.5 (0.2) 0.59 x 10- 7 (0.11) 0.633 275000 563000 770460" (18555) 90 343b (2408)
Diffusion coefficient DOzo.w (crnvs)
Vz Partial specific volume (ml/g)
Mw (S° from main fraction) (sOmax = 5.0 estimated)
Mw Weight average molecular weight (from GPC analysis)
Mn Number average molecular weight (from GPC analysis) (from GPC analysis)
Mw/M n
8.54 c
4.21 C
(0.43) 560 158000
(0.05) 160 70200
[1]] Intrinsic viscosity (ml/g) M~ (from Scheraga-Mandelkern equation 13 = 2.5-106 ) 1\ Degree of hydration (gig TS) fifo Frictional ratio 1J Simha shape factor R H Hydrated radius of the eqv. sphere (A)
0.47 8.7 507 362
0.41 6.8 153 235
Significantly different: "(P < 0.001), b(p < 0.01), "P < 0.01). Table V. Monosaccharide composition (expressed as % of the total) of the various fractions of water soluble non-starch polysaccharides of wheat separated on a DEAE-Sephadex column Monosaccharide Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
Frac. I
Frac. II
Frac. III
Frac. IV
Frac. V
30.8 68.7
2.5 28.5 68.0
2.6 39.3 51.5
1.1 36.3 6.1
1.3 37.8 33.1
0.55
1.0
2.3 4.6
47.8 8.9
15.9 12.0
2:23
2:39
1:31
0:17
0:88
Results are from single analysis of one sample. The standard deviation for this method determined using standard sample was between 0.14 and 1.42 for all the monosaccharides taken together.
Discussion
The composition and properties of water soluble non-starch polysaccharides from cereal grains are dependent on the method used for their extraction and purification. One method of isolation (18) used by earlier workers is based on extraction of the flour with solutions of ammonium sulphate at various concentrations. After extraction the salt was removed by dialysis and the pentosans were precipitated by ethyl alcohol. As the solubility of the pen tosans is higher either at high or at low pH they have also been extracted with dilute acid or alkali before they are precipitated from the solution with addition of
294
Non-starch polysaccharides in wheat and rye
Table VI. Amino acid composinon of the various fractions of water soluble non-starch polysaccharides of wheat separated on a OEAE-Sephadex column Amino acid (mg/g TS)
Frac. I
Frac. II
Frac. III
Frac. IV
Frac. V
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.69 0.49 0.49 1.19 traces 0.61 0.71 0.48 0.24 0.86 0.20 0.29 0.36 0.19 0.34
1.68 0.70 1.13 1.86 traces 1.80 0.94 0.69 0.38 0.80 0.26 0.30 0.55 0.34 0.39
0.49 0.49 0.55 0.93 traces 1.05 0.54 0.47 0.08 2.08 0.19 0.20 0.26 0.07 0.10
2.68 2.88 5.13 7.10 traces 3.19 6.85 3.12 0.74 5.05 2.31 0.65 1.00 0.50 0.56
14.32 4.04 8.22 15.84 traces 7.92 5.48 4.08 2.35 3.24 1.94 1.35 1.84 1.09 0.90
Total
7.14
11.82
7.50
41.76
n.51
SO: see Table III. Table VII. Monosaccharide composition (expressed as % of the total) of the various fractions of water-soluble non-starch polysaccharides of rye separated on a OEAE-Sephadex column Monosaccharide
Rhamnose Arabinose Xylose Mannose Galactose Glucose Xyl:Ara ratio
Frac. I
0.3 37.8 61.5
Frac. II
0.6 29.2 67.6 1.4
Frac. III
Frac. IV
Frac. V A
B
4.5 52.1 36.6
1.4 39.1 26.8
2.9 38.2 29.7
1.3 34.3 32.1
12.4 20.2
11.7 17.6
5.9 20.0
0.51 0.64
1.2
1.6 5.3
1:63
2:32
0:70
0:69
0:78
0:94
SO: see Table V.
ethanol (19). Boiling of cereal flours with 80% ethanol under reflux cooking inactivates the endogeneous enzymes, coagulates the proteins and dissolves sugars and amino acids. Pentosans are then extracted with water and separated after precipitation with ethyl alcohol. More recently, higher yields of pentosans have been obtained by using amylolytic and proteolytic enzymes for efficient degradation of protein and starch as an intermediate step before the extraction of pentosans with water following precipitation with alcohol (11). Therefore we used this method in the present study. Antoniou et at. (11) determined the pentosan content in Canadian rye samples using a colorimetric method (orcinol) as well as a GLC method. Their results show that the content of water soluble pen tosans in a variety called Puma was 1.14% of the dry matter. Karlsson (3), who determined pentosan content in 11
295
U.Girhammar and B.M.Nair
Table VIII. Amino acid composition of the various fractions of water soluble non-starch polysaccharides of rye separated on a DEAE-Sephadex column Amino acid (mg/g TS)
Frac. I
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.41 0.22 0.17 0.57 0.36 0.32
Total
Frac. II
Frac. III
Frac. IV
Frac. V A
0.46 0.13 0.19 0.12
0.82 0.47 0.77 1.59 1.00 0.63 0.49 0.32 0.71 0.26 0.31 0.35
1.10 0.37 1.61 2.28 1.29 0.51 0.49 4.46 0.19 0.31 0.42
0.35 2.95
8.24
13.41
B
4.23 1.99 2.64 3.90 3.01 2.93 2.40 0.70 4.93 0.90 0.90 0.83 0.20 0.80
3.26 1.80 2.00 6.71 2.15 2.70 2.06 1.04 5.15 1.10 1.11 1.66 0.39 1.59
3.25 0.52 1.24 3.31 1.58 0.85 0.67 0.29 2.40 0.30 0.33 0.38 0.08 0.16
30.55
32.72
15.36
SD: see Table III.
different varieties of rye grown in Sweden in three different years, found that the content of water soluble pentosans in these was 1.8% of the dry matter, i.e. very similar to our mean figure for rye. The average arabinoxylan content in the water soluble ethanol precipitated fractions from seven rye genotypes was 75.6% of the dry matter. The soluble pentosan content in 12 wheat genotypes grown in Sweden was 0.73% of the dry matter. The average arabinoxylan content in ethanol precipitated fractions from seven different varieties of wheat was 68.5% of the dry matter. Bengtsson and Aman (20) obtained water soluble crude arabinoxylan (0.74% of the dry matter) from whole rye grains of a Swedish variety cv.Kungs II, 72% of which on dry basis was constituted by arabinose and xylose. Delcour et at. (21) isolated a water soluble fraction by precipitating with ethanol from a water extract of rye and wheat flour. The yield of water soluble pentosans in rye was 2.18% of the dry matter of which 33% were arabinose and xylose residues. The corresponding figures for wheat were 0.18 and 32% respectively. In our experiments, arabinose and xylose together account for 74% of the total monosaccharides in the wheat NSP and 86% in the rye NSP. On a dry matter basis the arabinose and xylose content in the precipitate from wheat was 48% and 62% in rye. The amount of galactose in the precipitate obtained in the present study was higher in the wheat pentosans compared to that of the rye pentosans. This observation is in agreement with that of Karlsson (3). Most of the results which appear in earlier literature deal with wheat pentosans which are isolated in different ways. Thus, it is not surprising that there is a certain disagreement between results from different sources. The presence of higher amounts of galactose and arabinose in the wheat pentosans indicate that there is more arabinogalactan in wheat than in rye. 296
Non-starch polysaccharides in wheat and rye
A certain amount of minerals is obviously bound to the pentosans isolated in this experiment even after thorough washing with water, ethanol and diethyl ether. The polysaccharide itself may not bind any considerable amounts of minerals, while the protein to which the polysaccharide is found to be bound covalently may bind most of the minerals coprecipitated in this experiment. In addition it is also possible that a certain amount of phytate, which is known to bind minerals is present in this precipitate. The increased levels of most of the amino acids in wheat pentosans correspond to its higher protein content. The amino acid composition of the protein remaining bound to the water soluble pentosans isolated from wheat and rye appears to be very similar and showed hardly any resemblance to the amino acid composition of storage proteins in the respective grains. No hydroxyproline was detected either in the crude fraction or in the fractions obtained from the DEAE-Sephadex column. The UV absorbance of the eluate from the gel permeation chromatography shows the presence of protein attached mainly to low molecular weight fractions. It may also include some contribution from ferulic acid, which contains a phenolic group in its structure. Neukom (2) found ferulic acid only in certain parts of the arabinoxylans, most likely bound by ester linkages to the xylan chain. Yeh et al. (22) have also shown that ferulic acid is associated with the arabinoxylan fraction. Fincher and Stone (23) have determined the molecular size distribution of wheat endosperm arabinoxylan by gel filtration chromatography on Sepharose 4B using dextrans as standard. They observed an association of low molecular weight polysaccharides with protein while the high molecular weight polysaccharides were free of protein, which is in agreement with the results reported in the present paper. The xylose to arabinose ratio of the pentosans from wheat endosperm studied by Fincher and Stone (23) was 1:11 while the corresponding figure for the pentosans used in this study was 1:16 for wheat and 1:36 for rye. Karlsson (3) found no difference in the Xyl:Ara ratio (1:33 in both wheat and rye) in seven different wheat and rye varieties. The water soluble arabinoxylan from wheat flour studied by Andrewartha et al. (24) had a Xyl:Ara ratio above 2.0 and the solubility in water was drastically reduced when arabinose molecules were removed by treating the samples with a-L-arabinofuranosidase to alter the Xyl:Ara ratio to 2:3 and above. Kundig et al. (25) separated water soluble pentosans of wheat flour on an anion-exchange (DEAE-cellulose) column. The first fraction, which was eluted with water, contained a pure arabinoxylan while the next four fractions, eluted with 0.01, 0.1 mol/dm" and saturated Na2B407 and finally 0.5 N NaOH, contained various proportions of arabinose, xylose, galactose and protein. Morita et al. (26) separated water soluble pentosans from wheat on a DEAEcellulose column with water as Kiindig et al. (25). First of the four fractions separated by eluting fraction II successively with water to 0.01 mol/drrr' Na2B407 on the DEAE-cellulose column, consisted of 48.8% D-xylose, 41.4% L-arabinose, 1.8% D-galactose and 0.2% ferulic acid, but no protein. Lineback et al. (27) compared the carbohydrate composition of fractionated water soluble pentosans from different types of wheat flours on a DEAE297
U.Girhammar and B.M.Nair
cellulose column. The results agreed quite well with each other. The composition of fraction I, a pure arabinoxylan, was similar in all the samples. However, differences in galactose content of fraction II from different wheat pentosans were noted. In almost all earlier investigations the pentosan fraction IV contained the smallest amount of xylose and the largest amount of galactose, as was found by Kundig et al. (25) as well as in the present study. The values for Mw/M n (Table IV) of the wheat pentosans and the corresponding values for rye pentosans show that the wheat pentosans in solution had a lower degree of polydispersity compared to that of rye pentosans. However the rye pentosans contained a large proportion of molecules with a higher degree of polymerization (Figure 3). The partial specific volume of the samples of water soluble pentosans from rye and wheat show no significant difference between each other and agree well with the results obtained by Andrewartha et al. (24) and Cole,E.W. (28). The sedimentation coefficient of rye pentosans is slightly higher than that of the wheat pentosans which shows that the major part of the rye pentosan molecules are somewhat heavier than the wheat pentosan molecules. The diffusion coefficient of these two samples confirms the above observations to some extent as the value DO for the rye pentosans is lower than that of the wheat pentosans. The hydrated radius RH of the equivalent sphere of the molecules was calculated from the DO values according to the Stoke-Einstein equation (D = kTI6TjR H ) . The Simha shape factor II was calculated (u = [Tj]/(Vz + SVd from the values of partial specific volumes v. for water and Vz for solute, intrinsic viscosity [Tj] and degree of hydration S to estimate the difference in shape and size of the two pentosan molecules. The intrinsic viscosity was determined using an Ostwald viscometer and the degree of hydration was obtained as the amount of water unfrozen at - 53°C by using a differential scanning calorimeter (DSC) technique (U.Girhammar and B.M.Nair, in preparation). The Simha shape factor for the rye pentosan and the wheat pentosan shows that both of them have an extended and more or less rigid configuration in solution. The molecules of rye pentosans deviate more from the ideal spherical shape than the wheat pentosan molecules. The frictional ratio fIfo (calculated from fifo = (1 - VzIDZsOV z) of the two samples also appear to confirm the above conclusion. It is interesting to compare the values for molecular weights of the samples obtained by using Svedberg equation (MSODO) and by high performance gel permeation chromatography (Mw ) . The calculated from GPC shows higher values for rye pentosans and wheat pentosans compared to MsoDo calculated using So for the main fraction. However, MsoDo and Mw become similar when the estimated max So is used in the equation. The values of MsoDo, e; Mw and M"!) (calculated from SheragaMandelkern equation, (12) definitely show that the molecules of water soluble rye pentosans are larger than the molecules of the water soluble wheat pentosans as a result of a higher degree of polymerization. Rye pentosan molecules have a more extended and rigid configuration compared to that of the wheat pentosans in solution, which also causes the increase in viscosity. The lower Xyl:Ara ratio of the wheat pentosans is in agreement with their higher solubility in water.
«:
298
Non-starch polysaccharides in wheat and rye
Conclusion
The content of water soluble non-starch polysaccharides in rye grain is nearly three times as high as in wheat. The rye pen tosans contain more molecules of higher degree of polymerization compared to those isolated from wheat. There exist some arabinogalactan in wheat pentosans but not in rye pentosans. Rye pentosans produce more viscous solutions in water and the Xyl:Ara ratio of the pentosans from wheat and rye indicates that the solubility of wheat pentosans could be higher than that of rye pentosans. The first two fractions obtained by separation of pentosans on a DEAE-Sephadex column contained mainly arabinoxylans and their protein content was -1 %. The protein attached to the crude pentosans from wheat and rye is mainly associated with the low molecular weight fractions. Acknowledgements
Skilful technical assistance by Ingrid Jonsson was helpful in the execution of this project. We thank Professor Nils-Georg Asp for interesting discussions during the course of this study and for valuable suggestions which improved the scientific quality of this paper. This investigation was carried out with grants from the Swedish board for technical development and Cerealia foundation for research and development. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Podrazky.V. (1964) Chem. Indust., 2, 712-713. Neukorn.H. (1976) Lebensm. Wiss. u. Technol., 9,143-148. Karlsson,R. (1988) J. Swedish Seed Assoc., 98, 213-225. Saini,H.S. and Henry,R.J. (1989) Cereal Chem., 66,11-14. Meuser.F, and Suckow.P. (1986) In Blanshard,1.M.V., Frazier,P.J. and Galliard,T. (eds), Proceedings of an International Symposium on Chemistry and Physics of Baking (/985). Sutton Bennington, UK, pp. 42-61. Markwalder,H. (1975) Diss. Nr. 5497, E. T.H. Zurich. Bushuk,W. (1966) Bakers Digest, 40,38-40. Jankiewicz.M. and Michniewicz.J. (1987) Food Chem., 25, 241-249. D'Appolonia,B.L. (1980) J. Text. Stud., 10,201-216. Casier,J.P.J. (1975) Fermentation, 71, 117-134. Antoniou,T., Marquardt,R.R. and Cansfield,P.E. (1981) J. Agric. Food Chem., 29, 1240-1247. Cantor,e.R. and Schimmel,P.R. (1980) Biophys. Chem. Part II, 591-685. Ring,S.G., l'Anson,K.J. and Morris,V.J. (1985) Macromolecules, 18, 182-188. Kiindig,W., Neukom,H. and Deuel,H. (1961) He/v. Chim. Acta 44,823-829. Volkin,E. and Cohn,W.F. (1954) In Glick,D. (ed.), Meth. Biochem. Anal. I, Interscience, New York, pp. 287-303. Thenader.O, and Aman,P. (1979) Swedish J. Agric. Res., 9,97-106. Nair,B.M. (1977) J. Agric. Food Chem., 25, 614-620. Baker,J.e., Parker,H.K. and Mize,M.D. (1943) Cereal Chem., 20, 267-280. Simpson,F.J. (1955) Canad. J. of Microbiol., 1, 131-139. Bengtsson,S. and Aman,P. (1990) Carbohydr. Polym., 12, 267-277. Delcour,J.A., Vanhamel,S. and Hoseney,R.e. (1991) Cereal Chem., 68, 72-76. Yeh,Y.F., Hoseney,R.C, and Lineback,D.R. (1980) Cereal Chem., 57,144-148. Fincher,G.B. and Stone,B.A. (1974) Aust. J. Biol. Sci., 27,117-132. Andrewartha,K.A., Philips,D.R. and Stone,B.A. (1979) Carbohydr. Res., 77,191-204. Kiindig,W., Neukom,H. and Deuel.H. (1961) He/v. Chim. Acta, 44, 969-976. Morita,S.I., Ito,T. and Hirano.S. (1974) Int. J. Biochem., 5, 201-205. Lineback,D.R., Somnapan Kakuda,N. and Tsen,C.e. (1977) 1. Food Sci., 42, 461-467. Cole,E.W. (1967) Cereal Chem., 44, 411-416.
Received December 3, 1991; accepted March 5, 1992
299