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Variation in the degree of D-Xylose substitution in arabinoxylans extracted from a European wheat flour
Journal of Cereal Science 22 (1995) 73-84
D-Xylose Substitution in Arabinoxylans Extracted from a European Wheat Flour
Variation in the Degree of
G. Cleemput*, M. van Oort*, M. Hessing*, M. E. F. BergmanslH. GruppenT, P.J. Grobet§ and J.A. Delcour§ *TNO Nutrition and Food ResearchInstitute, Division of Biochemistryand Gene Technology, P.O. Box 360, 3700 AJ Zeist, The Netherlands; t Wageningen Agricultural Universily, Departmentof Food Science, P.O. Box 8129, 6700 EV Wageningen, The Netherlands and §Katholieke Universiteit Leuven, ResearchUnit Food Chemistry, KardinaalMercierlaan 92, B-3001 Heverlee, Belgium Received4 July, 1994 ABSTRACT Total water-extractable arabinoxylan from flour of the European bread making wheat cultivar Camp Remy was fractionated by ethanol precipitation. Both LH-NMR spectroscopy and methylation analysis of the isolated arabinoxylan fractions showed wide variation in the degree of xylose substitution. More highly substituted arabinoxylan fractions were precipitated at higher ethanol concentrations. At ethanol concentrations of 10-30% (v/v) (1 ~ 3),(1 --*4)-13-o-glucanco-precipitated with arabinoxylan. Re-precipitation of the polymer mixture resulted in a partial separation of the arabinoxylan from the mixture. Arabinoxylans with wide structural variation were also isolated by extraction of the flour with aqueous ethanol solutions of decreasing concentations. By this isolation technique, fractions with the highest degree of substitution were extracted with the most concentrated (50%) ethanolic solutions. The range of structural variation in the fractions was quite similar for both isolation methods. A decrease in the proportion ofunsubstituted xylose residues occurred concomitantly with a decrease in the proportion of monosubstituted xylose units and with an increase in the proportion of disubstituted xylose units. An increase in the proportion of paired disubstituted xylose units as the arabinose to xylose ratio of the arabinoxylan fractions increased, and the presence of monosubstituted xylose residues next to disubstituted xylose residues in the highly substituted fractions, were illustrated by ~HN-MR spectroscopy. Methylation analysis indicated an increase in traces of side chains ofarabinose residues and in the levels of O-2 substituted xylose residues as the arabinose to xylose ratio increased. © 1995AcademicPress Limited
Ifevwords: arabinoxylans; wheat; flour; methylation; tH-NMR.
INTRODUCTION
v a r i o u s aspects o f the q u a l i t y o f w h e a t as a r a w m a t e r i a l for b r e a d m a k i n g . D e s p i t e n u m e r o u s earlier efforts, their i m p a c t has m o s t l y b e e n des c r i b e d in g e n e r a l terms, such as a n increase in viscosity ~'2, w a t e r b i n d i n g 3'4, oxidative g e l a t i o n 5 a n d steric h i n d r a n c e to g l u t e n n e t w o r k f o r m a t i o n 6' 7. T h e findings are r a t h e r c o n t r a d i c t o r y with r e g a r d to their c o n t r i b u t i o n to b r e a d v o l u m e 8-~3. R e c e n t l y , C l e e m p u t et.al. ~4 m e a s u r e d the p r o p o r t i o n o f un-, m o n o - a n d disubstituted xylose residues in the w a t e r - e x t r a c t a b l e a r a b i n o x y l a n s o f six E u r o p e a n w h e a t flours o f e q u a l p r o t e i n c o n t e n t
Nonstarch polysaccharides (NSP) have received c o n s i d e r a b l e a t t e n t i o n in literature d e a l i n g with
USED: A / X = a r a b i n o s e / x y l o s e ; A r a = arabinofuranosyl; FID = flame-ionisation detector; GC = g a s chromatography; m / e = m o l e c u l a r mass/ charge; MS = mass spectrometry; N M R = nuclear magnetic resonance; N S P = n o n s t a r c h polysaccharides; T W E A X = total water-extractable arabinoxylan; Xyl = xylopyranosyl. ABBREVIATIONS
0733-5210/95/010073 + 12 $12.00/0
73
© 1995 Academic Press Limited
74
G. Cleemput et al.
with well established and greatly varying baking performances7 using the Westerlund et al. ~5 and Andersson 16 approach. Correlation coefficients found between the NSP contents and structures for these flours and their baking absorption and mixing time characteristics increased when the structural information was taken into account. This indicates that an increased knowledge of the arabinoxylan structure may lead to enhanced understanding of the role of NSP in bread making. Lack of such information in the past, has probably been one of the main reasons for the above cited conflicting views on the precise role of arabinoxylans in bread making. In the case of both rye ~7-~9and wheat 2°-22, evidence exists for variation in the structure of waterextractable arabinoxylan within a cultivar. Hoffmann et al. 2° fractionated arabinoxylans by ethanol precipitation of water extracts from the European puff pastry wheat cultivar Kadet. The arabinose to xylose (A/X) ratios of the resulting fractions ranged from 0"41 to 0"57. Further fractionation by gel filtration chromatography resulted in fractions with A / X ratios ranging from 0"39 to 0"89. Gruppen et al. ~l observed a wide variation in the proportion of un-, mono- and disubstituted xylose residues in the fractionated water-extractable and water-unextractable arabinoxylans with the European biscuit wheat variety Arminda. Izydorczyk and Biliaderis ~2 prepared different subfractions of water-extractable arabinoxylans from Canadian wheat flours by ammonium sulphate precipitation. They demonstrated that wheat endosperm arabinoxylans constitute a group of polysaccharides that are heterogeneous with respect to molecular mass, A / X ratio, ferulic acid content and arabinose substitution along the xylan backbone. In the case of rye, however, a virtually pure arabinoxylan with only un- and monosubstituted xylose residues was described ~9'23, whereas in the above-mentioned studies on wheat no such arabinoxylans were found. The present work was carried out to investigate the structural variation in the extractable arabinoxylans isolated from a European wheat cultivar of good bread making performance (cv. Camp Remy). In this work, arabinoxylans were fractionated by precipitation with increasing levels of ethanol and also by extraction with aqueous ethanol solutions of decreasing concentrations in order to investigate whether the distinct arabinoxylan structures reported for rye are also present in wheat flour.
EXPERIMENTAL
Flour Grain of the wheat cultivar Camp Remy (1992 harvest) was milled into white flour on a Miag Multomat KOMW0810 mill (MIAG, Germany). The extraction rate was 78% (0"49% ash, on a dry matter basis).
Fractionation of total water-extractable arabinoxylan
Isolation and purification of water-extractable arabinoxylan Water-extractable arabinoxylan was isolated from cv. Camp Remy flour by scaling up the method of Cleemput et al. 14. The isolation was carried out at room temperature unless indicated otherwise. Thus, flour (1"0 kg) that had been inactivated by heating at 130°C for 90 min was extracted with water (5:1 v/w; 15 min; 30°C) and centrifuged (6000g;, 15min). The supernatant (4"21) was heated to 90°C and residual starch was hydrolysed by addition o f alpha-amylase solution (2"0 ml, Type XII-A, from Bacillus licheniformis, A 3403, Sigma Chemical Co, St Louis, MO). The mixture was incubated at 90°C for 120 min. It was then allowed to cool to room temperature and centrifuged as above yielding 3"81 1ofarabinoxylan solution. The arabinoxylan was precipitated by stepwise addition of absolute ethanol to a final concentration of 65% (v/v). The mixture was stirred for 30 min, kept at 4°C overnight and recovered by centrifugation (3000g; 30min; 4°C). The precipitate was then dissolved in water (1"0 1), and ethanol was added to a final concentration of 60% (v/v). The mixture was then stirred as above and kept overnight at 4°C. The precipitate was recovered by centrifugation (6000g; 30 min; 4°C) and washed twice with 96% (v/v) ethanol (500 ml) and once with acetone (500ml) with intermediate stirring (120 min) and centrifugation (6000 g;, 30 min; 4°C). The final precipitate was dried for 24 h at 45°C in a drying oven. This fraction is referred to as total water-extractable arabinoxylan (TWEAX).
Fractionation of water-extractable arabinoxylan by ethanol precipitation TWEAX was separated into 3 fractions by graded ethanol precipitation. A sample (2"5 g) was dissolved in water (1"01). Aliquots of ethanol were added over a 30rain period under continuous
o-Xylosesubstitutionin arabinoxylans stirring to a final concentration of 30% (v/v) ethanol. The mixture was stirred for an additional 30min and kept overnight at 4°C. The precipitated material was recovered by centrifugation (10 000 g;, 30 min; 4°C), and dried by solvent washing as described above. This material is referred to as F10-3o. The ethanol concentration of the supernatant was increased to 50% in a similar manner and an arabinoxylan fraction, referred to as F23o-50, was recovered. The second supernatant was brought to an ethanol concentration of 65% and resulted in the recovery of an arabinoxylan fraction termed F350_65. The fractions Flo_30 and F230-50 were further fractionated as follows. To a solution (1 "0 g of the fraction/500 ml water) ethanol was added to raise the concentration in a stepwise manner. For F10-30, the ethanol concentration was raised first to 20% and then to 30% (v/v) yielding fractions Flo-20 and F120-30, respectively. These fractions were solvent washed and dried as described above. In a similar way, fraction F230-s0 was sub-fractionated into two subfractions F23o-40 and F240-50 (i.e. precipitates were obtained at alcohol concentrations of 40% and 50% (v/v) subsequently). A separate fractionation of T W E A X was also performed to obtain a better fractionation in the low ethanol concentration range. The ethanol concentration of a solution of T W E A X (1-5 g) in water (1"0 1) was increased to 15% (v/v) and then to 20% (v/v). The fractions obtained are referred to as F40_~5 and F5~s-20, respectively.
Extraction of flour with aqueous ethanol solutions Inactivated f o u r (l'0kg) was mixed with 70% (v/v) ethanol (3"01), stirred for 30 min and centrifuged (10 000g; 30min; 10°C). The supernatant was removed and the residue was air-dried for 16 h. The residue was then extracted with 60% (v/v) ethanol (3:1 v/w), centrifuged and air-dried as above. The 60% and 70% ethanol supernatants were discarded since they contained gluten proteins. The resulting air dried residue was fractionated sequentially in a similar way with 50, 40, 30, 20, 10, and 0% (v/v) aqueous ethanol yielding six extracts. The ethanol in the supernatants was evaporated under reduced pressure by rotary evaporation (40°C) to reduce the extract volumes. The extracts were dialysed against distilled water (48 h; 4°C), treated with alpha-amylase (0"05 ml/
75
100 ml extract) as described above and centrifuged (6000g; 15 min). The arabinoxylans in the solutions were precipitated by stepwise addition of absolute ethanol to a final' concentration of 65% and further purified and dried as described for the isolation and purification of TWEAX, yielding six arabinoxylan fractions (F50, F40, F30, F20, F10, F0). The fraction F50 was further fractionated as follows. An aliquot (0"5g) was dissolved in water (500 ml) and ethanol was added to obtain a final concentration of 60 % (v/v). The mixture was stirred for 30 min and kept overnight at 4°C. The precipitated arabinoxylan was covered by centrifugation (10 000g; 30 min; 4°C) and dried with ethanol and acetone, yielding fraction F50,60.
Sugar analysis Samples (14"0-16"0mg) were hydrolysed with 2"0ra trifluoroacetic acid (5"0ml) for 60min at 110°C. Myo-inositol solution (1"0 ml, 2"0 mg/ml) was added. Derivatisation was performed by the method of Englyst and Cummings 34. The resulting alditol acetates were injected (2 gl) onto a Supelco SP-2330 column (30 m x 0"75 mm) fitted in a Hewlett Packard 5890 Series II gas chromatograph (GC). Injection and detection (flame-ionisation detector, FID) temperatures were 250°C. The separation was with two isothermal steps (190°C; 5 min and 210°C; 11 min) with an intermediate temperature increase (20°C/min), and a final linear heating step (15°C over 36 s) to a temperature of 225°C. After two min at 225°C, the run was terminated.
Methylation analysis Methylation analysis was performed by the method of Hakomori 24 as modified by Sandford and Conrad ~6. After methylation, the samples were hydrolysed with 2"0 M trifluoroacetic acid, reduced with NaBD4 and converted to alditol acetates2k The samples were analysed by GC and GC-mass spectrometry (GC-MS) as described by Gruppen et al. 27. The amounts of the different methylated alditol acetates were calculated from the FID detector response according to their effective carbon responses 28, while identification of the derivates was confirmed by mass spectrometry. Since 2methylated xylitol (2-Me-Xyl) and 3-methylated xylitol (3-Me-Xyl) acetates were co-eluted, their
76
G.
Cleemputet al.
relative amounts were calculated from the relative abundance o f m / e (ratio molecular mass to charge) 118 and m / e 130 of the 2-Me-Xyl and 3-Me-Xyl peaks in GC-MS, respectively27'29. The relative amounts of 2,3- and 3,4-dimethylated xylitols (2, 3-Me2-Xyl and 3,4-Me2-Xyl), which were also coeluted, were estimated from the intensities of the peaks at m / e 118 and 117, respectively.
1H-NMR spectroscopy LH-NMR spectra were recorded on a Bruker 300MHz Fourier Transform-spectrometer at 85°C. The samples were dissolved in D20 (99%), stirred for 120 min and lyophilised. This step was repeated once and the resulting deuterium exchanged dry material was finally dissolved in D20 (1 "0 mg/ml). Pulse repetition was 2 s and number of scans varied from 1000 to 17 000. The proportions of unsubstituted, mono- and disubstituted xylose residues were calculated by combining the IH-NMR spectral data with the gas chromatography results as outlined before 15.
Gel permeation chromatography Aliquots of the isolated arabinoxylan fractions (5"0 mg) were solubilised in 1"0 ml of water and centrifuged (10 000 g; 15 min). Samples of the solutions (20 gl) were separated on a Shodex B-806 column (50 x 0"8 cm) (Showa Denko K.K., Tokyo, Japan) equipped with a pre-column (5 x 0"6 cm) by elution with water ( l ' 0 m l / h r at 30°C). The eluate was monitored by using a ECR-7510 (Erma Inc, Tokyo, Japan) refractive index detector. Molecular weight markers were Shodex standard P82 pullulan (1 "0 mg/ml) with molecular weights of 8"53x105 , 3"80x105 , 1"86x105 , 1"0x105 , 4"80 x 104, 2"37 x 104, 1"22 x 104 and 0"58 x 10~.
RESULTS Fractionation of total water-extractable arabinoxylan The isolation procedure yielded 0"42% (w/w on total flour) of TWEAX. Analysis of the monosaccharide composition showed (Table I) that apart from arabinose and xylose, a small amount of galactose but a substantial amount (10%) of glucose were present in this fraction. The glucose can be ascribed in part to the presence of [k)-glucan 14. The arabinose to xylose ratio (A/X) of 0"53 was
similar to that found in an earlier study t4. Graded ethanol precipitation of T W E A X yielded three arabinoxylan fractions (F10_30, F230-50 and F350-65). The yield and monosaccharide data of these fractions, representing 85% of TWEAX, are listed in Table I. These data show that fractions with varying A / X ratios, ranging from 0"43 to 0"82, were obtained and that the ratios increased with increasing ethanol concentration. F lo_3o and F230-5owere each further fractionated in two subfractions (F10-20, F120-30, F23o-40 and F24o_50). Fraction Flo_20 contained a large proportion of glucose, while fraction F12o_30appeared to consist of pure arabinoxylan. Both fractions had the same A / X ratio of 0"40, which was somewhat lower than the A / X ratio found for F10_30. The material in these two fractions corresponded to 47% of the F10_3o fraction. Fraction F10_20 contained only 33% arabinoxylan and was not characterised further. Fraction F230-50 could be fractionated in two fractions with obviously different arabinoxylan structures. Fraction F230~o was contaminated with [3-r)-glucan, as was demonstrated clearly by a broad doublet between 5 4"70 and 8 4"80 ppm in the tH-NMR spectrum (data not shown). The fraction of T W E A X that precipitated at 15% ethanol (F40_15) contained less than 5% arabinoxylan, and was not analysed further. Fraction F5L5-20 had the lowest A / X ratio of all fractions. Using the fractionation procedure described, fractions were obtained with varying A / X ratios, ranging from 0"36 to 0"82, indicating that there was a large variation in arabinoxylan structure between the different fractions.
1H-NMRanalysis The tH-NMR spectral data (protons on the anomeric carbon of the arabinose residues) of the fractions with largest variation in A / X ratios are shown in Figure 1. The relative intensity of the first peak (5 5-40 ppm), representing H-1 of a-Larabinofuranosyl (Ara) linked to 0 - 3 of xylopyranosyl (Xyl) residues, decreased with increasing A / X ratio, while the two peaks at 5 5"30 and 8 5"23 ppm, representing the anomeric protons of Ara linked to 0-2 and 0 - 3 of the same Xyl residue, became more pronounced. Table II lists the ratios of di- to monosubstituted Xyl residues obtained by the quantitative integration of the corresponding signals. Values range from 0"29 to 2"66. An increase in the amount of paired di-
D-Xylose substitution in arabinoxylans Table I
77
Yield, monosaccharide contents and compositions of arabinoxylan fractions obtained by ethanol precipitation Weight proportion (%)"
Fraction
ara
xyl
100
30"0
56"3
Flw30 F2~0-5o F35o-65
49 25 11
23"6 38"8 44"3
54"9 62"9 53"9
Flo-20 F12o-30
14 9
10'5 32"6
26'0 81"0
F23o-4o F24o_5o
3 13
22" 1 40'8
42"6 67-1
F4o-j5 F515_~0
7 2
2"8 28"2
2"6 78"4
TWEAX
Yield ~
man
gal
glc
"P~ (%)
A/X
0"7
9"7
75"9
0"53
1"4
14"0 6'9 4"3
69' 1 89'5 86"4
0'43 0'62 0"82
0"3
25'6
32-1 100-0
0"40 0"40
25"0 1"6
56'9 95'0
0'52 0'61
24'6 7'1
4'8 93"8
1"08 0"36
1" 1
"Abbreviations: ara, arabinose; xyl, xylose; man, mannose; gal, galactose; glc, glucose; AX, 0'88 (% a r a + % xyl); A / X , arabinose to xylose ratio. b Yield is expressed as weight percentage (as is basis) of TWEAX.
substituted Xyl units as the A / X ratio increased was shown by the intensity of the unresolved signals slightly downfield from the peaks at ~ 5"30 and 5 5" 23 ppm. L9.30The presence of a signal downfield from the 8 5"40 ppm peak in the spectra of fraction F350_65 results from a monosubstituted Xyl residue next to a disubstituted Xyl residue 3°. The proportions ofunsubstituted, mono- and disubstituted Xyl are given in Table II. A decrease in proportion of unsubstituted Xyl residues occurred concomitantly with a decrease in the proportion of monosubstituted Xyl units and an increase of disubstituted Xyl units.
Methylation analysis The above results were confirmed by methylation analysis of fractions TWEAX, F10-30, F23o_~oand F350-65 (Table III). The main residues in all fractions were terminal Ara (2,3,5-Me3-Ara) and unsubstituted Xyl units (2,3-Me2-Xyl). The relative amount of 2,3-Me2-Xyl to 3,4-Me2-Xyl was larger than 9/1 for all samples (data not shown) and unsubstituted Xyl was therefore further referred to as 2,3-Me2-Xyl. With higher ethanol concentration, more 2,3,5-trimethylated Ara (2,3,5Me3-Ara) was found, but also larger traces of substituted Ara (3,5-, 2,3-, and 2,5-Me~-Ara). In fraction F35o-65up to 5% of the Ara residues were substituted. Variation was also found in the degree ofxylose substitution (Table II). Apart from 2-MeXyl, small quantities of 3-Me-Xyl were detected, indicating the presence of Xyl residues mono-
substituted at the 2-position with Ara residues. Such Xyl units made up 0"5% of total Xyl units in TWEAX, but the level increased to 2"0% for the fraction with the highest level of substitution. As pointed out by Vinkx et al. 23, under our experimental conditions O-2 substitution of Xyl by Ara units cannot be detected by ~H-NMR. The values found by methylation analysis for the proportions ofdisubstituted Xyl were lower than those calculated from the combination of ~H-NMR and monosaccharide composition data. This difference was reflected in the di- to monosubstituted Xyl ratios, which were much lower than those found by ~H-NMR analysis (Table II). The overall trend of substitution, however, was the same for both methods. Terminal Xyl residues (2,3,4-Me3-Xyl, X,) increased from 1"9 to 4"0% with increasing degree of substitution. The detection of 2,3,6-, and 2,4,6-Me3-Glc in the fractions confirmed the presence of (1~3), (l~4)-[3-o-glucan as a contaminant. Small amounts of 2,4-Me2-Gal were detected in F23o_50and F350-65, which indicated the presence of arabinogalactan in these fractions.
Gel permeation analysis The gel permeation profiles of TWEAX, Fl0_30, F230-50 and F350-65 are shown in Figure 2. All four fractions contained components of the same molecular weight but the relative proportion of these components differed among the isolated fractions. The fraction precipitated at the highest ethanol concentration (65%) had a somewhat
78
G. Cleemput et al.
F515-2o
Fl2o-zo
A ~
_J
30--40
240-50
I
I
I
5.4
5.3 5
5.2
F i g u r e 1 kH-nuclear magnetic resonance spectra of the protons on the anomeric carbons of arabinose residues in arabinoxylan fractions of Camp Remy flour obtained by ethanol precipitation.
higher proportion of low molecular weight components compared with the fraction precipitated at lower ethanol concentrations. These results also imply that an increase in the A / X ratio of arabinoxylan occurs concomitant with a decrease of their molecular weight. These results are in agreement with the results of Izydorczyk and Biliaderis 22 for arabinoxylan fractions isolated by graded ammonium sulphate precipitation.
Extraction of flour with aqueous ethanol solutions The yields and monosaccharide compositions of the fractions are listed in Table IV. Extraction
of the resulting residue with 50% (v/v) ethanol, resulted in an arabinoxylan fraction containing small amounts of galactose. This galactose, originating from the presence of arabinogalactan, could be removed by precipitation of the material with 60% (v/v) ethanol. A pure arabinoxylan fraction with A / X ratio of 0"89 (F50,60) was then obtained. The fractions extracted with 40, 30, 20% (v/v) ethanol were almost free of galactose and the A / X ratio decreased with decreasing ethanol concentration. Fractions F10 and F0 had similar A / X ratios and both fractions contained mannose and glucose. These monosaccharides may have resulted from the presence of glucomannans L3.
1H-NMR analysis Figure 3 shows the tH-NMR spectral data for the six arabinoxylan fractions. The spectra confirmed the large variation in arabinoxylan structures present in the different fractions. The relative intensity of the peak at 5 5"40 ppm to that of the two major peaks at 5 5-30 and 5 5-23 ppm decreased with decreasing ethanol concentration (from 50 to 20% v/v). The variation in di- to monosubstituted Xyl residues is listed in Table V. Ratios ranging from 3"48 to 0"77 were found. All the spectra showed unresolved signals downfield from the signals at 5"30 and 5 5"23ppm. This indicates 3° that the arabinoxylan fractions had both isolated and paired disubstituted Xyl residues. Fraction F50 clearly contained higher levels of paired disubstituted Xyl residues than the other fractions. The presence of a monosubstituted Xyl residue next to a disubstituted Xyl residue was also clear 3° for this fraction. The signal at 5 5"26 ppm in F50 could be ascribed to terminal Ara residues in arabinogalactan. Table V lists the proportions of un-, mono- and disubstituted Xyl residues in the arabinoxylan fractions. The levels ofunsubstituted Xyl ranged from 50 to 67%. A higher degree of substitution (decrease in unsubstituted Xyl) was accompanied by a decrease in the proportion of monosubstituted Xyl and an increase in proportion of disubstituted Xyl residues.
Methylation analysis Apart from terminal Ara units, 2 to 4% of the Ara units were present in longer side chains (3,5-Me2Ara, 2,3-Me2-Ara and 2,5-Me2-Ara; Table VI). While the proportion of 2-Me-Xyl increased in the series F50 to F30, the proportion of Xyl units methylated at position 3 was more constant for all
D-Xylose substitution in arabinoxylans
79
Table II Ratio of di- to monosubstituted xyloses and proportions (%) of unsubstituted, mono- and disubstituted xylosea in arabinoxylan fractions obtained by ethanol precipitation, calculated from ~H-NMR spectral data and methylation analysis data Fraction
Di/mono
X0
Xj X,(3)
X2
X,
X,(2)
H-NMR TWEAX
0"89
63"8
19-2
17"0
F 10-so F230-50 F350-~5
0"40 1' 19 2"66
66"5 60"0 52"4
24"0 18"3 13"0
9"5 21 '7 34-6
F120-3o
0"34
68'6
23-5
7-9
F2~0-40 F24o-s0
0"93 1"33
64"9 61 "4
18"2 16-6
16"9 22"0
F515-20
0"29
70"6
22"8
6"6
Methylation TWEAX
0"60
64"9
19"5
0"5
12"6
2"5
Flo-3o F230-50 F3s0-ts
0"37 0"48 1"99
72"5 66"3 50"0
18"4 19"6 13"5
0-3 1"3 1"9
6-9 10"0 30"6
1"9 2"8 4"0
Abbreviations: Di/mono, disubstituted/monosubstituted xylose; Xo, unsubstituted xylose; X~, monosubstituted xylose; X~(2), mono-substituted xylose on position 2; X~(3) monosubstituted xylose on position 3; X2, disubstituted xylose; X,, terminal xylose.
Table HI
Glycosidic linkage compositions of arabinoxylan fractions obtained by ethanol precipitation Amount (mol %)" TWEAX
Flo-3o
F2~o-so
F35~5
2,3,5-Me3-Ara b 3,5-Me2-Ara 2,3-Me2-Ara 2,5-Me2-Ara
36" 1 0"6
25"3 0"2 0"2
37"2 1"0
41 "7 1"5 0"3 0"3
2,3,4-Me3-Xyl 2,3-Me~-Xyl 2-Me-Xyl 3-Me-Xyl Xyl
1'4 36'2 10'9 0"3 7'0
1"2 46"9 11"9 0"2 4"5
1"6 37 '5 11" 1 0"7 5"7
2" 1 26"0 7"0 1"0 15"9
6"5 1"0
0" 1 8" 1 1"4
0" 1 5'0
0" 1 3"6
0' 1
0"5
Partial methylated acetates
2,3,4,6-Me4-Glc 2,3,6-Me3-Glc 2,4,6-Mea-Glc 2,4-Me2-Gal
Expressed as a proportion (mol/100 mol) of all partially methylated alditol acetates present. b 2,3,5-Me3-Ara,2,3,5-tri-O-methyl- 1,4-di-O-acetyl-arabinitol, etc.
fractions in c o m p a r i s o n w i t h the f r a c t i o n s o b t a i n e d by ethanol precipitation. More variation was found in the p r o p o r t i o n o f d i s u b s t i t u t e d X y l units, resulting in di- to m o n o s u b s t i t u t e d X y l ratios r a n g i n g
f r o m (3"05 to 0"66; T a b l e ' V ) . T h e v a r i a t i o n in the c o n c e n t r a t i o n o f t e r m i n a l X y l units w a s c o m p a r a b l e with t h a t o b t a i n e d for the fractions obtained by ethanol precipitation.
80
G. Cleemputet al.
The presence of arabinogalactan in fraction F50 and glucomannan in fraction F20, F10 and F0 was indicated by the occurrence of 2,4Me~-Gal and 2,3,6-Me3-Glc and 2,3,6-Me3-Man respectively.
DISCUSSION Graded ethanol precipitation versus extraction with aqueous ethanol solutions
A
~]~
TWEAX
Flo-3o
F23o-5o
F35o-65
I
I
I
10
15 ml
20
F i g u r e 2 Gel permeation profiles ofarabinoxylan fractions obtained by ethanol precipitation. Elution volumes of the puUulan standards of molecular weight 8"53 x 105, 3'80 x 105, 1"86x105 , l ' 0 x l 0 ~, 4'80x104 . 2-37x104 , 1'22x104 , 0"55 x 104 (1 through 8, respectively) are indicated.
In this work two techniques, graded ethanol precipitation and extraction with ethanol solutions of different concentration, were used to fractionate the water-extractable arabinoxylans from the European bread making wheat cultivar Camp Remy. Both fractionation methods yielded arabinoxylan fractions with variation in their chemical structures as demonstrated by ~H-NMR spectroscopy and methylation analysis. With gradual ethanol precipitation, more highly substituted arabinoxylan fractions were precipitated at higher ethanol concentrations. The same observation was made by Gruppen et al. 2', who also used ethanol as the precipitant to fractionate the arabinoxylan from the biscuit wheat cultivar Arminda, as well as by Izydorczyk and . . . . ~2 Bahadens", who used gradual ammonium sulphate precipitation as the fractionation technique for Canadian wheat flours. The solubility of the extracted arabinoxylans from wheat is therefore very dependent on the degree of substitution of the polymer. At low ethanol concentrations (1~3), (l~4)-~-D-glucan was co-precipitated with arabinoxylan. Reprecipitation of the polymer mixture resulted in a partial separation of the arabinoxylan from the (1 ~3),(1--*4)-[3-D-glucan, however. This observation may be of interest in the purification of cereal cell wall polysaccharides in general. Structural analysis of the fractions obtained by aqueous ethanol extraction of the flour indicated that this fractionation technique is equally effective in yielding arabinoxylan fractions with varying degree of D-xylose substitution. The fraction with the highest degree of disubstitution was extracted from the flour with 50% (v/v) ethanol. Fractions extracted with decreasing ethanol concentrations (down to 20% v/v) had lower A / X ratios and lower levels of disubstitution. This indicates not only that the solubility of extracted arabinoxylans but also that the extractability itself depends on the level of substitution. The range of structural variation in the fractions
o-Xylose substilution in arabinoxylans Table IV
81
Yield, monosaccharide contents and compositions of arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions Weight proportion (%)°
Fraction F50 F40 F30 F20 FI0 F0
Yield b
ara
xyl
0"07 0" 18 0" 14 0"07 0"03 0-02
43'3 45-0 35"6 29"2 29"8 28'2
48-4 68"7 69-6 61"5 54-1 49"8
51"5
58"0
F50,6o
man
0"5 7-5 12'8
gal
glc
AX (%)
A/X
3"0 0" I
l'0 2-2 1"0 1-8 1-7 10"2
80'7 100"0 92'6 79'8 73'8 68"9
0"89 0"66 0'51 0-47 0"55 0-56
I'0
96"4
0"89
0"2 0" I
"Abbreviations: ara, arabinose; xyl, xylose; man, mannose; gal, galactose; glc, glucose; AX, 0"88 (% ara + % xyl); A/X, arabinose to xylose ratio. b Yield is expressed as weight percentage (as is basis) of flour.
Table V Ratio of di- to monosubstituted xyloses and proportions (%) of unsubstituted, mono- and disubstituted xylose" in arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions, calculated from ~H-NMR spectral data and methylation analysis data Fraction
Di/mono
Xo
X~
x,(3)
X~
X,
x,(2)
iH - N M R
F50 F40 F30 F20 FI0 F0
3'48 1"78 0"90 0"77 1-29 1'25
49"7 60-0 65"2 66'8 64'8 63'6
I 1-2 14"4 18"3 18"8 15"4 16"2
3"05 1"53 0-75 0"66 1"06 1' 14
54"7 60"3 65'3 66'4 63'9 64"2
9" 1 13"9 18"0 18"4 15-5 14'5
39"1 25"6 16-5 14"4 19"8 20"2
Methylation
F50 F40 F30 F20 FI0 F0
l'0 0"7 0'5 0"6 0"8 0"9
31"1 22"3 13-8 12'5 17"2 17"5
4-1 2"8 2"4 2"1 2"6 2'9
" Abbreviations: Di/mono, disubstituted/monosubstituted xylose; X0, unsubstituted xylose; X~, monosubstituted xylose; Xl(2), monosubstituted xylose on position 2; XI(3), monosubstituted xylose on position 3; X2, disubstituted xylose; X,, terminal xylose.
was quite similar for both isolation methods; only small differences were found. Extraction of the flour with aqueous ethanol solutions yielded an arabinoxylan fraction most enriched in molecules with disubstituted Xyl (F50, lowest monosubstitution), while the fraction with lowest substitution degree (F515_20,highest mono- and lowest disubstitution) was obtained by the ethanol precipitation technique. In contrast to earlier results for rye f l o u r 1~'23, a virtually pure arabinoxylan with
only un- and monosubstitution Xyl residues was not obtained from wheat with either technique.
1H-NMR spectroscopy versus methylation
analysis
The use of methylation analysis showed that the proportion of 0-2 substituted Xyl residues in total water-extractable arabinoxylans from wheat flour were negligible. This implies that the ~H-NMR
82
G. Cleemputet al.
Table VI Glycosidiclinkage of compositions of arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions Amount (mol %)4 Partially methylated acetates 2,3,5-Me3-Arab 3,5-Me2-Ara 2,3-Me2-Ara 2,5-Me2-Ara 2,3,4-Me3-Xyl 2,3-Me2-Xyl 2-Me-Xyl 3-Me-Xyl Xyl 2,3,4,6-Me.rGlc 2,3,6-Me3-Glc 2,4,6-Me3-Glc 2,4-Me2-Gal
F50
F40
F30
F20
FI 0
F0
45-3 1"3 0-3
36"9 0'9 0"2
34"0 0-7
30-1 0"7 0-2
29'9 0-7
2"1 28' 1 4"7 0"5 16'0 0'3 0"6
1-7 36'9 8"5 0'4 13"7
1'6 43-9 12"1 0-3 9"3 0-6
1"4 44"4 12-3 0'4 8"4 0" 1 1' 1
30"8 0"8 0'3 0" 1 1-6 38'7 9"4 0-5 10'4 0" 1 0"7
0" 1
0' 1
0-1
0"8
6"5
9"8
0-8
2,3,6-Me:~-Man
0"8
1"7 37" 1 8"4 0"5 10-1 0" 1 1"6
"Expressed as proportion (mol/100 mol) of all partially methylated alditol acetates present. ~'2,3,5-Me3-Ara, 2,3,5-tri-O-methyl-l,4-di-O-acetyl-arabinitol, etc. technique described by Westerlund et al. ~5 and used previously by Cleemput et al.~4, can be used, with confidence, for comparison of the 'average' arabinoxylan structures of different wheat cultivars. When the arabinoxylans from one particular cultivar were fractionated further, however, fractions were recovered that were enriched in such moieties. Because the ~H-NMR signal of the anomeric protons of the Ara residues attached at 0 - 2 of Xyl overlaps with the signal originating from the anomeric protons of the Ara residues linked to 0 - 2 and 0 - 3 of the same Xyl residue 23, the I H - N M R data need to be supplemented by methylation analysis results to facilitate reliable interpretation. Methylation analysis illustrated that the more highly substituted structures contained greater levels of both substituted Ara residues of terminal Xyl units. This observation, which was also made by Gruppen et al. 21 and Izydorczyk and Biliaderis 22, has not been investigated in detail. The greater levels of terminal Xyl units can be ascribed only partly to the lower molecular weight of the highly substituted fractions. The proportion of terminal Xyl units cannot result only from terminal residues in the xylan backbone, however, since the observed molecular weight is much higher than that calculated from the data for terminal Xyl units. This may indicate the presence of Xyl units in side chains.
Variation in the range of arabinoxylan structures amongst wheat cultivars Cleemput et al. 14 showed that the 'average' arabinoxylan structures varied for different European wheat cultivars. Fractionation of the water-extractable arabinoxylan of one cultivar, using either European 2Lor Canadian 22 wheat cultivars, showed the same relationship between the A / X ratio and proportion of mono- and disubstituted xyloses of the fractions obtained. The increase in monosubstitution at the 0 - 2 position of Xyl with increasing degree of substitution was also reported for a European biscuit wheat cultivar 2~ and Canadian cultivars 22. Since the structures observed were quite comparable for different wheat cultivars, it seems that the reported variation in the 'average' arabinoxylan of different wheat cultivars ~4 results from the presence of varying proportions of the different polymers rather than a difference in the structures of the polymers present. Recently, Andersson et al. 3~ suggested that the pattern of variation in the structure of extractable wheat flour arabinoxylans may be explained on the basis of the presence of two differently composed arabinoxylan fractions in varying amount, both having the same amount of unsubstituted Xyl units but with different degree of mono- and disubstitution. The present study shows clearly, however, that more than two classes are present,
o-Xylose substitution in arabinoxylans
83
un- and disubstituted Xyl residues and the second with virtually all branched residues monosubstituted. In our fractionation experiments with wheat, we did not recoveF any such polymers. It seems, therefore, that they are either absent or that their separation is very difficult in the case of wheat. The variation in wheat arabinoxylan structures might therefore represent a continuum varying from one extreme to the other. Fso
Acknowledgments F4o
G. Cleemput gratefully acknowledges receipt of an EC Directorate-General X I I mobility grant. We thank Luc Van den Ende, K U Leuven, for technical assistance.
REFERENCES Fso F2o
Fzo
Fo
5.4
5.3
5.2
Figure 3 ~H-nuclear magnetic resonance spectra of the protons on the anomeric carbons of arabinose residues in arabinoxylan fractions of Camp Remy flour obtained by extraction of the flour with aqueous ethanol solutions.
not only varying in the levels of mono- and disubstitution but also in the level of unsubstituted Xyl units.
Variation in the range of arabinoxylan structures
amongst rye and wheat In the case of rye, Bengtsson et al. ~7J8 and Vinkx et al. ~9'2s reported that two extreme populations of arabinoxylan structures exist, one containing only
I. Hoseney, R.C. Functional pentosans in baked foods. Food Technol. 38 (1984) 114-147. 2. Delcour, J.A., Vanhamel, S. and Hoseney, R.C. Physicochemical and functional properties of rye nonstarch polysaccharides. II. Impact of a fraction containing watersoluble pentosans and proteins on gluten-starCh loaf volumes. Cereal Chemistry 68 (1991) 72-76. 3. Kulp, K. Pentosans of wheat endosperm. Cereal Science Today 13 (1968) 414--417, 426. 4. Jelaca, S.L. and Hlynka, I. Waterbinding capacity of wheat flour crude pentosans and their relation to mixing characteristics of dough. Cereal ChemistTy 48 (1971) 211222. 5. Neukom, H. and Markwalder, H.U. Oxidative gelation of wheat flour pentosans: a new way of cross-linking polymers. Cereal Foods World 23 (1978) 374-376. 6. Weegels, P.L., Marseille, J.P. and Voorpostel, A.M.B. Enzymes as processing aid in the separation of wheat into starch and gluten. In 'Gluten Proteins 1990' (W. Bushuk and R. Tkachuk, eds) AACC, St Paul, MN (1990) pp. 199-203. 7. Roels, S.P., Cleemput, G., Vandewalle, X. and Delcour, J.A. Bread volume potential determined by mixing time and baking absorption levels of variable-quality fours with constant protein level. Cereal Chemistry 70 (1993) 318-323. 8. D'Appolonia, B.L., Gilles, K.A. and Medcalf, D.G. Effect of water-soluble pentosans on gluten-starch loaves. Cereal Chemistry 47 (1970) 194-204. 9. D'Appolonia, B.L. Role ofpentosans in bread and dough. A review. Baker's D~,est 45 (1971) 20-23, 63. 10. D'Appolonia, B.L. Comparison of pentosans extracted from conventional and continuous bread. Cereal Chemistry 50 (1973) 27-36. 11. Casier,J.PJ., De Paepe, G.M.J. and Brummer,J. Einfluss der wasserunloslichen Weizen- und Roggen-Pentosane auf die Backeigenschaften yon Weizenmehlen und anderen Rohstoffen. GetreideMehl Brot 27 (1973) 36-44. 12. Casier, J.PJ., De Paepe, G.MO., Willems, H.E.J., Goffings, GJ.G., Hermans,J.L. and Noppen, H.E. Bread production from pure flours of tropical starchy crops: III. From pure and mixed flours of cassava, millet, sop
84
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ghum, corn, rice and the starches. Trop. Foods Chem. and aVutr. 1 (1979) 279-340. 13. Meuser, F. and Suckow, P. Non-starch polysaccharides. In 'Chemistry and Physics of Baking' (J.M.V. Blanshard, P.J. Frazier and T. Galliard, eds) Royal Society of Chemistry, London (1985) pp. 42-61. 14. Cleemput, G., Roels, S.P., van Oort, M., Grobet, P.J. and Delcour,J.A. Heterogeneity in the structure of watersoluble arabinoxylans in European wheat flours of variable bread-making quality. Cereal Chemist!y 70 (1993) 324-329. 15. Westerlund, E., Andersson, R., ,~man, P. and Theander, O. Effects of baking on water-soluble polysaccharides in white bread fractions. Journal of Cereal Science 12 (1990) 33-42. 16. Andersson, R. 'Wheat Flour Polysaccharides and Breadmaking', Ph.D. thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden (1993). 17. Bengtsson, S., /~nan, P. and Andersson, R. Structural studies on water-soluble arabinoxylans in rye grain using enzymatic hydrolysis. Carbohydr. Polym 17 (1992) 277-284. 18. Bengtsson, S., Andersson, R., Westerlund, E., .~anan, P. Content, structure and viscosity of soluble arabinoxylans in rye grain from several countries, aT. Sci. Food Agric. 58 (1992) 331-337. 19. Vinkx, CJ.A., Reynaert, H.R., Grobet, PJ. and Delcour, J.A. Physiochemical and functional properties of rye nonstarch polysaccharides. V. Variability in the structure and gelling capacity ofwater-soluble arabinoxylans. Cereal ChemistO, 70 (1993) 311-317. 20. Hoffmann, R.A., Roza, M., Maat, J., Kamerling, J.P. and Vliegenthart, J.F.G. Structural characteristics of the cold-water-soluble arabinoxylans from the white flour of the soft wheat variety Kadet. Carbohydr. Polym. 15 (1991) 415-430. 21. Gruppen, H., Hamer, RJ. and Voragen, A.GJ. Waterunextractable cell wall material from wheat flour. 2. Fractionation of alkali-extractable polymers and c o r n -
22. 23.
24. 25.
26.
27.
28.
29.
30.
31.
parison with water-extractable arabinoxylans. Journal of Cereal Science 16 (1992) 53-67. Izydorczyk, M.S. and Biliaderis, C.G. Structural heterogeneity of wheat endosperm arabinoxylans. Cereal CkemistO, 70 (1993) 641-646. Vinkx, C.J.A., Delcour, J.A., Verbruggen, M.A. and Gruppen, H. Rye water-soluble arabinoxylans also vary in their contents of 2-monosubstituted xylose. Cereal Chemistry (accepted for publication). Hakomori, S. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide..7. Biochem. 55 (1964) 205-208. Englyst, H.N. and Cummings, J.H. Simplified method for the measurement of total non-starch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Ana~sl 109 (1984) 937-942. Sandford, P.A. and Conrad, H.E. The structure of the Aerobacter aerogenes A3(S1) polysaccharide. I. A reexamination using improved procedures for methylation analysis. Biochemistry 5 (1966) 1508-1517. Gruppen, H., Hamer, R.J. and Voragen, A.G.J. Waterunextractable cell wall material from wheat flour. 1. Extraction of polymers with alkali. Journal of Cereal Science 16 (1992) 41-51. Sweet, D.P., Shapiro, R.H. and Albersheim, P. Quantitative analysis by various G.L.C. response-factor theories for partially methylated and partially ethylated alditol acetates. Carbohydr. Res. 40 (1975) 217-225. Jansson, P.E., Kenne, L., Liedgren, H., Lindberg, H. and LOnngren. A practical guide to the methylation analysis ofcarbohydrates.aT. Chem. Commun. Univ. Stockholm 8 (1976) 1-76. Hoffmann, R.A., Kamerling, J.P. and Vliegenthart, J.F.G. Structural features of a water-soluble arabinoxylan from the endosperm of wheat. Carbohydr. Res. 226 (1992) 303-311. Andersson, R., Westerlund, E. and ,~man, P. Natural variation in the contents of structural elements of waterextractable non-starch polysaccharides in white flour. Journal of Cereal Science 16 (1994) 77-82.
D-Xylose Substitution in Arabinoxylans Extracted from a European Wheat Flour
Variation in the Degree of
G. Cleemput*, M. van Oort*, M. Hessing*, M. E. F. BergmanslH. GruppenT, P.J. Grobet§ and J.A. Delcour§ *TNO Nutrition and Food ResearchInstitute, Division of Biochemistryand Gene Technology, P.O. Box 360, 3700 AJ Zeist, The Netherlands; t Wageningen Agricultural Universily, Departmentof Food Science, P.O. Box 8129, 6700 EV Wageningen, The Netherlands and §Katholieke Universiteit Leuven, ResearchUnit Food Chemistry, KardinaalMercierlaan 92, B-3001 Heverlee, Belgium Received4 July, 1994 ABSTRACT Total water-extractable arabinoxylan from flour of the European bread making wheat cultivar Camp Remy was fractionated by ethanol precipitation. Both LH-NMR spectroscopy and methylation analysis of the isolated arabinoxylan fractions showed wide variation in the degree of xylose substitution. More highly substituted arabinoxylan fractions were precipitated at higher ethanol concentrations. At ethanol concentrations of 10-30% (v/v) (1 ~ 3),(1 --*4)-13-o-glucanco-precipitated with arabinoxylan. Re-precipitation of the polymer mixture resulted in a partial separation of the arabinoxylan from the mixture. Arabinoxylans with wide structural variation were also isolated by extraction of the flour with aqueous ethanol solutions of decreasing concentations. By this isolation technique, fractions with the highest degree of substitution were extracted with the most concentrated (50%) ethanolic solutions. The range of structural variation in the fractions was quite similar for both isolation methods. A decrease in the proportion ofunsubstituted xylose residues occurred concomitantly with a decrease in the proportion of monosubstituted xylose units and with an increase in the proportion of disubstituted xylose units. An increase in the proportion of paired disubstituted xylose units as the arabinose to xylose ratio of the arabinoxylan fractions increased, and the presence of monosubstituted xylose residues next to disubstituted xylose residues in the highly substituted fractions, were illustrated by ~HN-MR spectroscopy. Methylation analysis indicated an increase in traces of side chains ofarabinose residues and in the levels of O-2 substituted xylose residues as the arabinose to xylose ratio increased. © 1995AcademicPress Limited
Ifevwords: arabinoxylans; wheat; flour; methylation; tH-NMR.
INTRODUCTION
v a r i o u s aspects o f the q u a l i t y o f w h e a t as a r a w m a t e r i a l for b r e a d m a k i n g . D e s p i t e n u m e r o u s earlier efforts, their i m p a c t has m o s t l y b e e n des c r i b e d in g e n e r a l terms, such as a n increase in viscosity ~'2, w a t e r b i n d i n g 3'4, oxidative g e l a t i o n 5 a n d steric h i n d r a n c e to g l u t e n n e t w o r k f o r m a t i o n 6' 7. T h e findings are r a t h e r c o n t r a d i c t o r y with r e g a r d to their c o n t r i b u t i o n to b r e a d v o l u m e 8-~3. R e c e n t l y , C l e e m p u t et.al. ~4 m e a s u r e d the p r o p o r t i o n o f un-, m o n o - a n d disubstituted xylose residues in the w a t e r - e x t r a c t a b l e a r a b i n o x y l a n s o f six E u r o p e a n w h e a t flours o f e q u a l p r o t e i n c o n t e n t
Nonstarch polysaccharides (NSP) have received c o n s i d e r a b l e a t t e n t i o n in literature d e a l i n g with
USED: A / X = a r a b i n o s e / x y l o s e ; A r a = arabinofuranosyl; FID = flame-ionisation detector; GC = g a s chromatography; m / e = m o l e c u l a r mass/ charge; MS = mass spectrometry; N M R = nuclear magnetic resonance; N S P = n o n s t a r c h polysaccharides; T W E A X = total water-extractable arabinoxylan; Xyl = xylopyranosyl. ABBREVIATIONS
0733-5210/95/010073 + 12 $12.00/0
73
© 1995 Academic Press Limited
74
G. Cleemput et al.
with well established and greatly varying baking performances7 using the Westerlund et al. ~5 and Andersson 16 approach. Correlation coefficients found between the NSP contents and structures for these flours and their baking absorption and mixing time characteristics increased when the structural information was taken into account. This indicates that an increased knowledge of the arabinoxylan structure may lead to enhanced understanding of the role of NSP in bread making. Lack of such information in the past, has probably been one of the main reasons for the above cited conflicting views on the precise role of arabinoxylans in bread making. In the case of both rye ~7-~9and wheat 2°-22, evidence exists for variation in the structure of waterextractable arabinoxylan within a cultivar. Hoffmann et al. 2° fractionated arabinoxylans by ethanol precipitation of water extracts from the European puff pastry wheat cultivar Kadet. The arabinose to xylose (A/X) ratios of the resulting fractions ranged from 0"41 to 0"57. Further fractionation by gel filtration chromatography resulted in fractions with A / X ratios ranging from 0"39 to 0"89. Gruppen et al. ~l observed a wide variation in the proportion of un-, mono- and disubstituted xylose residues in the fractionated water-extractable and water-unextractable arabinoxylans with the European biscuit wheat variety Arminda. Izydorczyk and Biliaderis ~2 prepared different subfractions of water-extractable arabinoxylans from Canadian wheat flours by ammonium sulphate precipitation. They demonstrated that wheat endosperm arabinoxylans constitute a group of polysaccharides that are heterogeneous with respect to molecular mass, A / X ratio, ferulic acid content and arabinose substitution along the xylan backbone. In the case of rye, however, a virtually pure arabinoxylan with only un- and monosubstituted xylose residues was described ~9'23, whereas in the above-mentioned studies on wheat no such arabinoxylans were found. The present work was carried out to investigate the structural variation in the extractable arabinoxylans isolated from a European wheat cultivar of good bread making performance (cv. Camp Remy). In this work, arabinoxylans were fractionated by precipitation with increasing levels of ethanol and also by extraction with aqueous ethanol solutions of decreasing concentrations in order to investigate whether the distinct arabinoxylan structures reported for rye are also present in wheat flour.
EXPERIMENTAL
Flour Grain of the wheat cultivar Camp Remy (1992 harvest) was milled into white flour on a Miag Multomat KOMW0810 mill (MIAG, Germany). The extraction rate was 78% (0"49% ash, on a dry matter basis).
Fractionation of total water-extractable arabinoxylan
Isolation and purification of water-extractable arabinoxylan Water-extractable arabinoxylan was isolated from cv. Camp Remy flour by scaling up the method of Cleemput et al. 14. The isolation was carried out at room temperature unless indicated otherwise. Thus, flour (1"0 kg) that had been inactivated by heating at 130°C for 90 min was extracted with water (5:1 v/w; 15 min; 30°C) and centrifuged (6000g;, 15min). The supernatant (4"21) was heated to 90°C and residual starch was hydrolysed by addition o f alpha-amylase solution (2"0 ml, Type XII-A, from Bacillus licheniformis, A 3403, Sigma Chemical Co, St Louis, MO). The mixture was incubated at 90°C for 120 min. It was then allowed to cool to room temperature and centrifuged as above yielding 3"81 1ofarabinoxylan solution. The arabinoxylan was precipitated by stepwise addition of absolute ethanol to a final concentration of 65% (v/v). The mixture was stirred for 30 min, kept at 4°C overnight and recovered by centrifugation (3000g; 30min; 4°C). The precipitate was then dissolved in water (1"0 1), and ethanol was added to a final concentration of 60% (v/v). The mixture was then stirred as above and kept overnight at 4°C. The precipitate was recovered by centrifugation (6000g; 30 min; 4°C) and washed twice with 96% (v/v) ethanol (500 ml) and once with acetone (500ml) with intermediate stirring (120 min) and centrifugation (6000 g;, 30 min; 4°C). The final precipitate was dried for 24 h at 45°C in a drying oven. This fraction is referred to as total water-extractable arabinoxylan (TWEAX).
Fractionation of water-extractable arabinoxylan by ethanol precipitation TWEAX was separated into 3 fractions by graded ethanol precipitation. A sample (2"5 g) was dissolved in water (1"01). Aliquots of ethanol were added over a 30rain period under continuous
o-Xylosesubstitutionin arabinoxylans stirring to a final concentration of 30% (v/v) ethanol. The mixture was stirred for an additional 30min and kept overnight at 4°C. The precipitated material was recovered by centrifugation (10 000 g;, 30 min; 4°C), and dried by solvent washing as described above. This material is referred to as F10-3o. The ethanol concentration of the supernatant was increased to 50% in a similar manner and an arabinoxylan fraction, referred to as F23o-50, was recovered. The second supernatant was brought to an ethanol concentration of 65% and resulted in the recovery of an arabinoxylan fraction termed F350_65. The fractions Flo_30 and F230-50 were further fractionated as follows. To a solution (1 "0 g of the fraction/500 ml water) ethanol was added to raise the concentration in a stepwise manner. For F10-30, the ethanol concentration was raised first to 20% and then to 30% (v/v) yielding fractions Flo-20 and F120-30, respectively. These fractions were solvent washed and dried as described above. In a similar way, fraction F230-s0 was sub-fractionated into two subfractions F23o-40 and F240-50 (i.e. precipitates were obtained at alcohol concentrations of 40% and 50% (v/v) subsequently). A separate fractionation of T W E A X was also performed to obtain a better fractionation in the low ethanol concentration range. The ethanol concentration of a solution of T W E A X (1-5 g) in water (1"0 1) was increased to 15% (v/v) and then to 20% (v/v). The fractions obtained are referred to as F40_~5 and F5~s-20, respectively.
Extraction of flour with aqueous ethanol solutions Inactivated f o u r (l'0kg) was mixed with 70% (v/v) ethanol (3"01), stirred for 30 min and centrifuged (10 000g; 30min; 10°C). The supernatant was removed and the residue was air-dried for 16 h. The residue was then extracted with 60% (v/v) ethanol (3:1 v/w), centrifuged and air-dried as above. The 60% and 70% ethanol supernatants were discarded since they contained gluten proteins. The resulting air dried residue was fractionated sequentially in a similar way with 50, 40, 30, 20, 10, and 0% (v/v) aqueous ethanol yielding six extracts. The ethanol in the supernatants was evaporated under reduced pressure by rotary evaporation (40°C) to reduce the extract volumes. The extracts were dialysed against distilled water (48 h; 4°C), treated with alpha-amylase (0"05 ml/
75
100 ml extract) as described above and centrifuged (6000g; 15 min). The arabinoxylans in the solutions were precipitated by stepwise addition of absolute ethanol to a final' concentration of 65% and further purified and dried as described for the isolation and purification of TWEAX, yielding six arabinoxylan fractions (F50, F40, F30, F20, F10, F0). The fraction F50 was further fractionated as follows. An aliquot (0"5g) was dissolved in water (500 ml) and ethanol was added to obtain a final concentration of 60 % (v/v). The mixture was stirred for 30 min and kept overnight at 4°C. The precipitated arabinoxylan was covered by centrifugation (10 000g; 30 min; 4°C) and dried with ethanol and acetone, yielding fraction F50,60.
Sugar analysis Samples (14"0-16"0mg) were hydrolysed with 2"0ra trifluoroacetic acid (5"0ml) for 60min at 110°C. Myo-inositol solution (1"0 ml, 2"0 mg/ml) was added. Derivatisation was performed by the method of Englyst and Cummings 34. The resulting alditol acetates were injected (2 gl) onto a Supelco SP-2330 column (30 m x 0"75 mm) fitted in a Hewlett Packard 5890 Series II gas chromatograph (GC). Injection and detection (flame-ionisation detector, FID) temperatures were 250°C. The separation was with two isothermal steps (190°C; 5 min and 210°C; 11 min) with an intermediate temperature increase (20°C/min), and a final linear heating step (15°C over 36 s) to a temperature of 225°C. After two min at 225°C, the run was terminated.
Methylation analysis Methylation analysis was performed by the method of Hakomori 24 as modified by Sandford and Conrad ~6. After methylation, the samples were hydrolysed with 2"0 M trifluoroacetic acid, reduced with NaBD4 and converted to alditol acetates2k The samples were analysed by GC and GC-mass spectrometry (GC-MS) as described by Gruppen et al. 27. The amounts of the different methylated alditol acetates were calculated from the FID detector response according to their effective carbon responses 28, while identification of the derivates was confirmed by mass spectrometry. Since 2methylated xylitol (2-Me-Xyl) and 3-methylated xylitol (3-Me-Xyl) acetates were co-eluted, their
76
G.
Cleemputet al.
relative amounts were calculated from the relative abundance o f m / e (ratio molecular mass to charge) 118 and m / e 130 of the 2-Me-Xyl and 3-Me-Xyl peaks in GC-MS, respectively27'29. The relative amounts of 2,3- and 3,4-dimethylated xylitols (2, 3-Me2-Xyl and 3,4-Me2-Xyl), which were also coeluted, were estimated from the intensities of the peaks at m / e 118 and 117, respectively.
1H-NMR spectroscopy LH-NMR spectra were recorded on a Bruker 300MHz Fourier Transform-spectrometer at 85°C. The samples were dissolved in D20 (99%), stirred for 120 min and lyophilised. This step was repeated once and the resulting deuterium exchanged dry material was finally dissolved in D20 (1 "0 mg/ml). Pulse repetition was 2 s and number of scans varied from 1000 to 17 000. The proportions of unsubstituted, mono- and disubstituted xylose residues were calculated by combining the IH-NMR spectral data with the gas chromatography results as outlined before 15.
Gel permeation chromatography Aliquots of the isolated arabinoxylan fractions (5"0 mg) were solubilised in 1"0 ml of water and centrifuged (10 000 g; 15 min). Samples of the solutions (20 gl) were separated on a Shodex B-806 column (50 x 0"8 cm) (Showa Denko K.K., Tokyo, Japan) equipped with a pre-column (5 x 0"6 cm) by elution with water ( l ' 0 m l / h r at 30°C). The eluate was monitored by using a ECR-7510 (Erma Inc, Tokyo, Japan) refractive index detector. Molecular weight markers were Shodex standard P82 pullulan (1 "0 mg/ml) with molecular weights of 8"53x105 , 3"80x105 , 1"86x105 , 1"0x105 , 4"80 x 104, 2"37 x 104, 1"22 x 104 and 0"58 x 10~.
RESULTS Fractionation of total water-extractable arabinoxylan The isolation procedure yielded 0"42% (w/w on total flour) of TWEAX. Analysis of the monosaccharide composition showed (Table I) that apart from arabinose and xylose, a small amount of galactose but a substantial amount (10%) of glucose were present in this fraction. The glucose can be ascribed in part to the presence of [k)-glucan 14. The arabinose to xylose ratio (A/X) of 0"53 was
similar to that found in an earlier study t4. Graded ethanol precipitation of T W E A X yielded three arabinoxylan fractions (F10_30, F230-50 and F350-65). The yield and monosaccharide data of these fractions, representing 85% of TWEAX, are listed in Table I. These data show that fractions with varying A / X ratios, ranging from 0"43 to 0"82, were obtained and that the ratios increased with increasing ethanol concentration. F lo_3o and F230-5owere each further fractionated in two subfractions (F10-20, F120-30, F23o-40 and F24o_50). Fraction Flo_20 contained a large proportion of glucose, while fraction F12o_30appeared to consist of pure arabinoxylan. Both fractions had the same A / X ratio of 0"40, which was somewhat lower than the A / X ratio found for F10_30. The material in these two fractions corresponded to 47% of the F10_3o fraction. Fraction F10_20 contained only 33% arabinoxylan and was not characterised further. Fraction F230-50 could be fractionated in two fractions with obviously different arabinoxylan structures. Fraction F230~o was contaminated with [3-r)-glucan, as was demonstrated clearly by a broad doublet between 5 4"70 and 8 4"80 ppm in the tH-NMR spectrum (data not shown). The fraction of T W E A X that precipitated at 15% ethanol (F40_15) contained less than 5% arabinoxylan, and was not analysed further. Fraction F5L5-20 had the lowest A / X ratio of all fractions. Using the fractionation procedure described, fractions were obtained with varying A / X ratios, ranging from 0"36 to 0"82, indicating that there was a large variation in arabinoxylan structure between the different fractions.
1H-NMRanalysis The tH-NMR spectral data (protons on the anomeric carbon of the arabinose residues) of the fractions with largest variation in A / X ratios are shown in Figure 1. The relative intensity of the first peak (5 5-40 ppm), representing H-1 of a-Larabinofuranosyl (Ara) linked to 0 - 3 of xylopyranosyl (Xyl) residues, decreased with increasing A / X ratio, while the two peaks at 5 5"30 and 8 5"23 ppm, representing the anomeric protons of Ara linked to 0-2 and 0 - 3 of the same Xyl residue, became more pronounced. Table II lists the ratios of di- to monosubstituted Xyl residues obtained by the quantitative integration of the corresponding signals. Values range from 0"29 to 2"66. An increase in the amount of paired di-
D-Xylose substitution in arabinoxylans Table I
77
Yield, monosaccharide contents and compositions of arabinoxylan fractions obtained by ethanol precipitation Weight proportion (%)"
Fraction
ara
xyl
100
30"0
56"3
Flw30 F2~0-5o F35o-65
49 25 11
23"6 38"8 44"3
54"9 62"9 53"9
Flo-20 F12o-30
14 9
10'5 32"6
26'0 81"0
F23o-4o F24o_5o
3 13
22" 1 40'8
42"6 67-1
F4o-j5 F515_~0
7 2
2"8 28"2
2"6 78"4
TWEAX
Yield ~
man
gal
glc
"P~ (%)
A/X
0"7
9"7
75"9
0"53
1"4
14"0 6'9 4"3
69' 1 89'5 86"4
0'43 0'62 0"82
0"3
25'6
32-1 100-0
0"40 0"40
25"0 1"6
56'9 95'0
0'52 0'61
24'6 7'1
4'8 93"8
1"08 0"36
1" 1
"Abbreviations: ara, arabinose; xyl, xylose; man, mannose; gal, galactose; glc, glucose; AX, 0'88 (% a r a + % xyl); A / X , arabinose to xylose ratio. b Yield is expressed as weight percentage (as is basis) of TWEAX.
substituted Xyl units as the A / X ratio increased was shown by the intensity of the unresolved signals slightly downfield from the peaks at ~ 5"30 and 5 5" 23 ppm. L9.30The presence of a signal downfield from the 8 5"40 ppm peak in the spectra of fraction F350_65 results from a monosubstituted Xyl residue next to a disubstituted Xyl residue 3°. The proportions ofunsubstituted, mono- and disubstituted Xyl are given in Table II. A decrease in proportion of unsubstituted Xyl residues occurred concomitantly with a decrease in the proportion of monosubstituted Xyl units and an increase of disubstituted Xyl units.
Methylation analysis The above results were confirmed by methylation analysis of fractions TWEAX, F10-30, F23o_~oand F350-65 (Table III). The main residues in all fractions were terminal Ara (2,3,5-Me3-Ara) and unsubstituted Xyl units (2,3-Me2-Xyl). The relative amount of 2,3-Me2-Xyl to 3,4-Me2-Xyl was larger than 9/1 for all samples (data not shown) and unsubstituted Xyl was therefore further referred to as 2,3-Me2-Xyl. With higher ethanol concentration, more 2,3,5-trimethylated Ara (2,3,5Me3-Ara) was found, but also larger traces of substituted Ara (3,5-, 2,3-, and 2,5-Me~-Ara). In fraction F35o-65up to 5% of the Ara residues were substituted. Variation was also found in the degree ofxylose substitution (Table II). Apart from 2-MeXyl, small quantities of 3-Me-Xyl were detected, indicating the presence of Xyl residues mono-
substituted at the 2-position with Ara residues. Such Xyl units made up 0"5% of total Xyl units in TWEAX, but the level increased to 2"0% for the fraction with the highest level of substitution. As pointed out by Vinkx et al. 23, under our experimental conditions O-2 substitution of Xyl by Ara units cannot be detected by ~H-NMR. The values found by methylation analysis for the proportions ofdisubstituted Xyl were lower than those calculated from the combination of ~H-NMR and monosaccharide composition data. This difference was reflected in the di- to monosubstituted Xyl ratios, which were much lower than those found by ~H-NMR analysis (Table II). The overall trend of substitution, however, was the same for both methods. Terminal Xyl residues (2,3,4-Me3-Xyl, X,) increased from 1"9 to 4"0% with increasing degree of substitution. The detection of 2,3,6-, and 2,4,6-Me3-Glc in the fractions confirmed the presence of (1~3), (l~4)-[3-o-glucan as a contaminant. Small amounts of 2,4-Me2-Gal were detected in F23o_50and F350-65, which indicated the presence of arabinogalactan in these fractions.
Gel permeation analysis The gel permeation profiles of TWEAX, Fl0_30, F230-50 and F350-65 are shown in Figure 2. All four fractions contained components of the same molecular weight but the relative proportion of these components differed among the isolated fractions. The fraction precipitated at the highest ethanol concentration (65%) had a somewhat
78
G. Cleemput et al.
F515-2o
Fl2o-zo
A ~
_J
30--40
240-50
I
I
I
5.4
5.3 5
5.2
F i g u r e 1 kH-nuclear magnetic resonance spectra of the protons on the anomeric carbons of arabinose residues in arabinoxylan fractions of Camp Remy flour obtained by ethanol precipitation.
higher proportion of low molecular weight components compared with the fraction precipitated at lower ethanol concentrations. These results also imply that an increase in the A / X ratio of arabinoxylan occurs concomitant with a decrease of their molecular weight. These results are in agreement with the results of Izydorczyk and Biliaderis 22 for arabinoxylan fractions isolated by graded ammonium sulphate precipitation.
Extraction of flour with aqueous ethanol solutions The yields and monosaccharide compositions of the fractions are listed in Table IV. Extraction
of the resulting residue with 50% (v/v) ethanol, resulted in an arabinoxylan fraction containing small amounts of galactose. This galactose, originating from the presence of arabinogalactan, could be removed by precipitation of the material with 60% (v/v) ethanol. A pure arabinoxylan fraction with A / X ratio of 0"89 (F50,60) was then obtained. The fractions extracted with 40, 30, 20% (v/v) ethanol were almost free of galactose and the A / X ratio decreased with decreasing ethanol concentration. Fractions F10 and F0 had similar A / X ratios and both fractions contained mannose and glucose. These monosaccharides may have resulted from the presence of glucomannans L3.
1H-NMR analysis Figure 3 shows the tH-NMR spectral data for the six arabinoxylan fractions. The spectra confirmed the large variation in arabinoxylan structures present in the different fractions. The relative intensity of the peak at 5 5"40 ppm to that of the two major peaks at 5 5-30 and 5 5-23 ppm decreased with decreasing ethanol concentration (from 50 to 20% v/v). The variation in di- to monosubstituted Xyl residues is listed in Table V. Ratios ranging from 3"48 to 0"77 were found. All the spectra showed unresolved signals downfield from the signals at 5"30 and 5 5"23ppm. This indicates 3° that the arabinoxylan fractions had both isolated and paired disubstituted Xyl residues. Fraction F50 clearly contained higher levels of paired disubstituted Xyl residues than the other fractions. The presence of a monosubstituted Xyl residue next to a disubstituted Xyl residue was also clear 3° for this fraction. The signal at 5 5"26 ppm in F50 could be ascribed to terminal Ara residues in arabinogalactan. Table V lists the proportions of un-, mono- and disubstituted Xyl residues in the arabinoxylan fractions. The levels ofunsubstituted Xyl ranged from 50 to 67%. A higher degree of substitution (decrease in unsubstituted Xyl) was accompanied by a decrease in the proportion of monosubstituted Xyl and an increase in proportion of disubstituted Xyl residues.
Methylation analysis Apart from terminal Ara units, 2 to 4% of the Ara units were present in longer side chains (3,5-Me2Ara, 2,3-Me2-Ara and 2,5-Me2-Ara; Table VI). While the proportion of 2-Me-Xyl increased in the series F50 to F30, the proportion of Xyl units methylated at position 3 was more constant for all
D-Xylose substitution in arabinoxylans
79
Table II Ratio of di- to monosubstituted xyloses and proportions (%) of unsubstituted, mono- and disubstituted xylosea in arabinoxylan fractions obtained by ethanol precipitation, calculated from ~H-NMR spectral data and methylation analysis data Fraction
Di/mono
X0
Xj X,(3)
X2
X,
X,(2)
H-NMR TWEAX
0"89
63"8
19-2
17"0
F 10-so F230-50 F350-~5
0"40 1' 19 2"66
66"5 60"0 52"4
24"0 18"3 13"0
9"5 21 '7 34-6
F120-3o
0"34
68'6
23-5
7-9
F2~0-40 F24o-s0
0"93 1"33
64"9 61 "4
18"2 16-6
16"9 22"0
F515-20
0"29
70"6
22"8
6"6
Methylation TWEAX
0"60
64"9
19"5
0"5
12"6
2"5
Flo-3o F230-50 F3s0-ts
0"37 0"48 1"99
72"5 66"3 50"0
18"4 19"6 13"5
0-3 1"3 1"9
6-9 10"0 30"6
1"9 2"8 4"0
Abbreviations: Di/mono, disubstituted/monosubstituted xylose; Xo, unsubstituted xylose; X~, monosubstituted xylose; X~(2), mono-substituted xylose on position 2; X~(3) monosubstituted xylose on position 3; X2, disubstituted xylose; X,, terminal xylose.
Table HI
Glycosidic linkage compositions of arabinoxylan fractions obtained by ethanol precipitation Amount (mol %)" TWEAX
Flo-3o
F2~o-so
F35~5
2,3,5-Me3-Ara b 3,5-Me2-Ara 2,3-Me2-Ara 2,5-Me2-Ara
36" 1 0"6
25"3 0"2 0"2
37"2 1"0
41 "7 1"5 0"3 0"3
2,3,4-Me3-Xyl 2,3-Me~-Xyl 2-Me-Xyl 3-Me-Xyl Xyl
1'4 36'2 10'9 0"3 7'0
1"2 46"9 11"9 0"2 4"5
1"6 37 '5 11" 1 0"7 5"7
2" 1 26"0 7"0 1"0 15"9
6"5 1"0
0" 1 8" 1 1"4
0" 1 5'0
0" 1 3"6
0' 1
0"5
Partial methylated acetates
2,3,4,6-Me4-Glc 2,3,6-Me3-Glc 2,4,6-Mea-Glc 2,4-Me2-Gal
Expressed as a proportion (mol/100 mol) of all partially methylated alditol acetates present. b 2,3,5-Me3-Ara,2,3,5-tri-O-methyl- 1,4-di-O-acetyl-arabinitol, etc.
fractions in c o m p a r i s o n w i t h the f r a c t i o n s o b t a i n e d by ethanol precipitation. More variation was found in the p r o p o r t i o n o f d i s u b s t i t u t e d X y l units, resulting in di- to m o n o s u b s t i t u t e d X y l ratios r a n g i n g
f r o m (3"05 to 0"66; T a b l e ' V ) . T h e v a r i a t i o n in the c o n c e n t r a t i o n o f t e r m i n a l X y l units w a s c o m p a r a b l e with t h a t o b t a i n e d for the fractions obtained by ethanol precipitation.
80
G. Cleemputet al.
The presence of arabinogalactan in fraction F50 and glucomannan in fraction F20, F10 and F0 was indicated by the occurrence of 2,4Me~-Gal and 2,3,6-Me3-Glc and 2,3,6-Me3-Man respectively.
DISCUSSION Graded ethanol precipitation versus extraction with aqueous ethanol solutions
A
~]~
TWEAX
Flo-3o
F23o-5o
F35o-65
I
I
I
10
15 ml
20
F i g u r e 2 Gel permeation profiles ofarabinoxylan fractions obtained by ethanol precipitation. Elution volumes of the puUulan standards of molecular weight 8"53 x 105, 3'80 x 105, 1"86x105 , l ' 0 x l 0 ~, 4'80x104 . 2-37x104 , 1'22x104 , 0"55 x 104 (1 through 8, respectively) are indicated.
In this work two techniques, graded ethanol precipitation and extraction with ethanol solutions of different concentration, were used to fractionate the water-extractable arabinoxylans from the European bread making wheat cultivar Camp Remy. Both fractionation methods yielded arabinoxylan fractions with variation in their chemical structures as demonstrated by ~H-NMR spectroscopy and methylation analysis. With gradual ethanol precipitation, more highly substituted arabinoxylan fractions were precipitated at higher ethanol concentrations. The same observation was made by Gruppen et al. 2', who also used ethanol as the precipitant to fractionate the arabinoxylan from the biscuit wheat cultivar Arminda, as well as by Izydorczyk and . . . . ~2 Bahadens", who used gradual ammonium sulphate precipitation as the fractionation technique for Canadian wheat flours. The solubility of the extracted arabinoxylans from wheat is therefore very dependent on the degree of substitution of the polymer. At low ethanol concentrations (1~3), (l~4)-~-D-glucan was co-precipitated with arabinoxylan. Reprecipitation of the polymer mixture resulted in a partial separation of the arabinoxylan from the (1 ~3),(1--*4)-[3-D-glucan, however. This observation may be of interest in the purification of cereal cell wall polysaccharides in general. Structural analysis of the fractions obtained by aqueous ethanol extraction of the flour indicated that this fractionation technique is equally effective in yielding arabinoxylan fractions with varying degree of D-xylose substitution. The fraction with the highest degree of disubstitution was extracted from the flour with 50% (v/v) ethanol. Fractions extracted with decreasing ethanol concentrations (down to 20% v/v) had lower A / X ratios and lower levels of disubstitution. This indicates not only that the solubility of extracted arabinoxylans but also that the extractability itself depends on the level of substitution. The range of structural variation in the fractions
o-Xylose substilution in arabinoxylans Table IV
81
Yield, monosaccharide contents and compositions of arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions Weight proportion (%)°
Fraction F50 F40 F30 F20 FI0 F0
Yield b
ara
xyl
0"07 0" 18 0" 14 0"07 0"03 0-02
43'3 45-0 35"6 29"2 29"8 28'2
48-4 68"7 69-6 61"5 54-1 49"8
51"5
58"0
F50,6o
man
0"5 7-5 12'8
gal
glc
AX (%)
A/X
3"0 0" I
l'0 2-2 1"0 1-8 1-7 10"2
80'7 100"0 92'6 79'8 73'8 68"9
0"89 0"66 0'51 0-47 0"55 0-56
I'0
96"4
0"89
0"2 0" I
"Abbreviations: ara, arabinose; xyl, xylose; man, mannose; gal, galactose; glc, glucose; AX, 0"88 (% ara + % xyl); A/X, arabinose to xylose ratio. b Yield is expressed as weight percentage (as is basis) of flour.
Table V Ratio of di- to monosubstituted xyloses and proportions (%) of unsubstituted, mono- and disubstituted xylose" in arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions, calculated from ~H-NMR spectral data and methylation analysis data Fraction
Di/mono
Xo
X~
x,(3)
X~
X,
x,(2)
iH - N M R
F50 F40 F30 F20 FI0 F0
3'48 1"78 0"90 0"77 1-29 1'25
49"7 60-0 65"2 66'8 64'8 63'6
I 1-2 14"4 18"3 18"8 15"4 16"2
3"05 1"53 0-75 0"66 1"06 1' 14
54"7 60"3 65'3 66'4 63'9 64"2
9" 1 13"9 18"0 18"4 15-5 14'5
39"1 25"6 16-5 14"4 19"8 20"2
Methylation
F50 F40 F30 F20 FI0 F0
l'0 0"7 0'5 0"6 0"8 0"9
31"1 22"3 13-8 12'5 17"2 17"5
4-1 2"8 2"4 2"1 2"6 2'9
" Abbreviations: Di/mono, disubstituted/monosubstituted xylose; X0, unsubstituted xylose; X~, monosubstituted xylose; Xl(2), monosubstituted xylose on position 2; XI(3), monosubstituted xylose on position 3; X2, disubstituted xylose; X,, terminal xylose.
was quite similar for both isolation methods; only small differences were found. Extraction of the flour with aqueous ethanol solutions yielded an arabinoxylan fraction most enriched in molecules with disubstituted Xyl (F50, lowest monosubstitution), while the fraction with lowest substitution degree (F515_20,highest mono- and lowest disubstitution) was obtained by the ethanol precipitation technique. In contrast to earlier results for rye f l o u r 1~'23, a virtually pure arabinoxylan with
only un- and monosubstitution Xyl residues was not obtained from wheat with either technique.
1H-NMR spectroscopy versus methylation
analysis
The use of methylation analysis showed that the proportion of 0-2 substituted Xyl residues in total water-extractable arabinoxylans from wheat flour were negligible. This implies that the ~H-NMR
82
G. Cleemputet al.
Table VI Glycosidiclinkage of compositions of arabinoxylan fractions obtained by extraction of flour with aqueous ethanol solutions Amount (mol %)4 Partially methylated acetates 2,3,5-Me3-Arab 3,5-Me2-Ara 2,3-Me2-Ara 2,5-Me2-Ara 2,3,4-Me3-Xyl 2,3-Me2-Xyl 2-Me-Xyl 3-Me-Xyl Xyl 2,3,4,6-Me.rGlc 2,3,6-Me3-Glc 2,4,6-Me3-Glc 2,4-Me2-Gal
F50
F40
F30
F20
FI 0
F0
45-3 1"3 0-3
36"9 0'9 0"2
34"0 0-7
30-1 0"7 0-2
29'9 0-7
2"1 28' 1 4"7 0"5 16'0 0'3 0"6
1-7 36'9 8"5 0'4 13"7
1'6 43-9 12"1 0-3 9"3 0-6
1"4 44"4 12-3 0'4 8"4 0" 1 1' 1
30"8 0"8 0'3 0" 1 1-6 38'7 9"4 0-5 10'4 0" 1 0"7
0" 1
0' 1
0-1
0"8
6"5
9"8
0-8
2,3,6-Me:~-Man
0"8
1"7 37" 1 8"4 0"5 10-1 0" 1 1"6
"Expressed as proportion (mol/100 mol) of all partially methylated alditol acetates present. ~'2,3,5-Me3-Ara, 2,3,5-tri-O-methyl-l,4-di-O-acetyl-arabinitol, etc. technique described by Westerlund et al. ~5 and used previously by Cleemput et al.~4, can be used, with confidence, for comparison of the 'average' arabinoxylan structures of different wheat cultivars. When the arabinoxylans from one particular cultivar were fractionated further, however, fractions were recovered that were enriched in such moieties. Because the ~H-NMR signal of the anomeric protons of the Ara residues attached at 0 - 2 of Xyl overlaps with the signal originating from the anomeric protons of the Ara residues linked to 0 - 2 and 0 - 3 of the same Xyl residue 23, the I H - N M R data need to be supplemented by methylation analysis results to facilitate reliable interpretation. Methylation analysis illustrated that the more highly substituted structures contained greater levels of both substituted Ara residues of terminal Xyl units. This observation, which was also made by Gruppen et al. 21 and Izydorczyk and Biliaderis 22, has not been investigated in detail. The greater levels of terminal Xyl units can be ascribed only partly to the lower molecular weight of the highly substituted fractions. The proportion of terminal Xyl units cannot result only from terminal residues in the xylan backbone, however, since the observed molecular weight is much higher than that calculated from the data for terminal Xyl units. This may indicate the presence of Xyl units in side chains.
Variation in the range of arabinoxylan structures amongst wheat cultivars Cleemput et al. 14 showed that the 'average' arabinoxylan structures varied for different European wheat cultivars. Fractionation of the water-extractable arabinoxylan of one cultivar, using either European 2Lor Canadian 22 wheat cultivars, showed the same relationship between the A / X ratio and proportion of mono- and disubstituted xyloses of the fractions obtained. The increase in monosubstitution at the 0 - 2 position of Xyl with increasing degree of substitution was also reported for a European biscuit wheat cultivar 2~ and Canadian cultivars 22. Since the structures observed were quite comparable for different wheat cultivars, it seems that the reported variation in the 'average' arabinoxylan of different wheat cultivars ~4 results from the presence of varying proportions of the different polymers rather than a difference in the structures of the polymers present. Recently, Andersson et al. 3~ suggested that the pattern of variation in the structure of extractable wheat flour arabinoxylans may be explained on the basis of the presence of two differently composed arabinoxylan fractions in varying amount, both having the same amount of unsubstituted Xyl units but with different degree of mono- and disubstitution. The present study shows clearly, however, that more than two classes are present,
o-Xylose substitution in arabinoxylans
83
un- and disubstituted Xyl residues and the second with virtually all branched residues monosubstituted. In our fractionation experiments with wheat, we did not recoveF any such polymers. It seems, therefore, that they are either absent or that their separation is very difficult in the case of wheat. The variation in wheat arabinoxylan structures might therefore represent a continuum varying from one extreme to the other. Fso
Acknowledgments F4o
G. Cleemput gratefully acknowledges receipt of an EC Directorate-General X I I mobility grant. We thank Luc Van den Ende, K U Leuven, for technical assistance.
REFERENCES Fso F2o
Fzo
Fo
5.4
5.3
5.2
Figure 3 ~H-nuclear magnetic resonance spectra of the protons on the anomeric carbons of arabinose residues in arabinoxylan fractions of Camp Remy flour obtained by extraction of the flour with aqueous ethanol solutions.
not only varying in the levels of mono- and disubstitution but also in the level of unsubstituted Xyl units.
Variation in the range of arabinoxylan structures
amongst rye and wheat In the case of rye, Bengtsson et al. ~7J8 and Vinkx et al. ~9'2s reported that two extreme populations of arabinoxylan structures exist, one containing only
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