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Structural features of pectic polysaccharides of red beet (Beta vulgaris conditiva)
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All fights reserved.
631
Structural features of pectic polysaccharides of red beet (Beta
vulgaris conditiva) Georg R. Strasser, Daniel E. Wechsler, Renato Amadb Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zurich, Switzerland
Abstract Cell-wall material from ripe red beet was extracted as alcohol-insoluble residue (AIR). The CDTA-soluble extract from AIR was fractionated by anion-exchange chromatography. Four fractions were isolated by a step-wise increase in the ionic strength of the elution buffer. The main fraction was further fractionated by gel filtration chromatography. This chromatogram showed one regular broad peak, which was divided into three parts and pooled. All fractions isolated from both chromatographic systems were freeze-dried and their neutral sugar compositions as well as uronic acid contents were determined. Furthermore methylation analysis of these fractions were performed prior and after reduction of the pectic polysacchaddes with NaBD4.
1. INTRODUCTION Pectins are a group of polysaccharides from the primary cell wall and the intercellular regions of higher plants [1]. They have been investigated for their structural features and their functions within the plant cell wall for many years, because changes in the texture of fruits and vegetables and in the properties of their products are related to changes in the pectic components [2]. From literature it is known, that the pectic backbone consists of ~t-(1---~)linked D-galacturonic acid units, interrupted by the insertion of o~-(1---)2)-linked L-rhamnosyl residues in adjacent or alternate positions [3]. Side chains consisting essentially of arabinans, galactans, arabinogalactans and single xylose residues are attached to this backbone. Other sugars have been found less frequently. In addition some non-sugar substituents, mainly methanol, acetic acid and phenolic acids are known to be present in pectins. Although the main structural elements of pectins are known, the complexity of these polymers has prevented a complete understanding of their fine structure so far. The aim of this project is to get additional information about the fine structure of pectic polysaccharides. Therefore pectins from red beet were isolated and fractionated by chromatographic methods. Some results obtained by methylation analysis of these pectin-rich fractions are presented.
632 2. MATERIALS AND METHODS
Extraction and fractionation of pectins (Figure 1): Red beets of the variety Red Ace F~ were purchased from a local store. Preparation of the AIR was done according to Selvendran and O'Neil [4]. Extraction with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) was performed according to Selvendran et al. [5]. The CDTA-soluble extract was dialysed first against running tap water and subsequently against distilled water. Then it was fractionated by anion-exchange chromatography on DEAE Sepharose CL-6B using 0.04M phosphate buffer (pH 6.5) as eluent with a step-wise increase in the ionic strength (0, 0.1, 0.2, 1.0M NaC1). Related fractions were pooled. The main fraction (IE 0.2M) was further fractionated by gel filtration chromatography on two coupled columns, one packed with Sephacryl S-500 and the other one with Sephacryl S-200 using 0.04M phosphate buffer (pH 6.0) as eluent. The peak on the chromatogram was divided into three parts, which were separately pooled. All samples were dialysed and freeze-dried before further analysis. Analytical techniques: Fractions from anion-exchange chromatography were assayed for uronic acid and neutral sugar contents using an automated segmented flow analyser [6]. Neutral sugars (NS) of all samples were analysed by GC as alditol-acetates [7]. Uronic acids (UA) were determined by the m-hydroxy-diphenyl method [8]. Methylation analysis [9,10] was performed with polysaccharides prior and after carbodiimide-activated reduction with NaBD4. Peaks were identified by GC-MS, whereas the quantification was done by GC (FID) using calculated relative response factors on an effective carbon response (e.c.r.) basis [ 11]. 3. RESULTS AND DISCUSSION The results are summarised in Table 1. As expected, for the IE samples higher amounts of uronic acid are found with increasing ionic strength of the elution buffer. For the GF samples larger molecules (GF1) contain half as much NS compared to smaller molecules (GF3). The sum of differently linked NS residues determined by methylation analysis is calculated to 100%. 1,4-Galp is difficult to determine because of the high amount of 1,4GalAp present in most samples. Therefore 1,4-Galp was determined by methylation analysis without prior reduction with NaBD4. Whereas the percentage of some NS residues remain nearly unchanged in the different samples (e.g. T-Araf, 1,5-Araf and 1,6-Galp), others differ considerably. The relative amounts of 1,3-Araf, 1,3,5-Araf, 1,2,3,5-Araf, 1,4-Galp, 1,2-Rhap and 1,2,4-Rhap increase at higher ionic strength elution on anion exchange chromatography and decrease for smaller molecules on gel filtration chromatography. 1,3-Galp and 1,3,6-Galp show the opposite behaviour. This indicates that pectins eluted at high ionic strength and larger pectic molecules of the IE 0.2M fraction contain on average smaller side chains because of the relatively high amount of 1,2,4-Rhap compared to the total amount of NS residues (1,2,4-Rhap is the main branching residue in the pectic backbone). Furthermore it can be assumed that the side chains of these pectins mainly consist of structures similar to arabinans and arabinogalactans type I, because of the differently linked arabinose and galactose residues present. Pectins eluted at low ionic strength and smaller pectin molecules of the IE 0.2M fraction contain more galactose, which is mainly 1,3- and 1,3,6-1inked. These residues are believed to be part of structures, which are known to be present in arabinogalactans type II.
633 Red Beet Alcohol soluble extract
Extraction with ethanol ,,~ Alcohol insoluble residue (AIR)
CDTA insoluble residue
Extraction with CDTA =~ CDTA soluble material (CDTAS)
Anion exchange chromatography on DEAE-Sepharose CL-6B 1200 T --o- Uronic acids ~ --.- Neutral sugars ~ ~~ 800 + .... 0.04M phosphate buffer ~o~.= containing NaC1 a~ ~=
1 I/ .ll
400
............
q- 1.0 ] ] "=~=~ 0.5
,
0 i ,
I
20 ~ IEOM
i I '
I
40 i 60 I 1 ' 1 IE0.1M IE0.2M
i
=o
80 (I, IE1.0M
Fraction number
Gel filtration chromatography on Sephacryl S-500 and on Sephacryl S-200
~
Mw
(Pullulan)
6
]
i
L60
80
rlE 0.2M GF1 [IE 0.2M GF2] IE 0.2M GF3"[ Figure 1. Extraction and fractionation of pectins from ripe red beets
Fraction number
634
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635
636 The ratios between linear and branched residues are presented as well. All values obtained without prior reduction (NS residues only), are in the range between one and two. From this it can be assumed, that side chains contain only slightly more linear than branched residues. After prior reduction (NS and UA residues) much more linear than branched residues are found. This surplus of linear residues originates from the 1,4-1inked galacturonic acid backbone, which is also known as smooth region. The results presented allow the following assumptions to be made: CDTA-soluble material eluted at low ionic strength (IE 0M) contains hardly any uronic acids and consists mainly of neutral sugar residues, which indicate the presence of arabinans and arabinogalactans type II. Pectins eluted at high ionic strength (IE 1.0M) contain large amounts of uronic acids. The side chains consist mainly of arabinans, galactans and mixtures of them. High molecular weight pectins eluted at intermediate ionic strength (IE 0.2M GF1) contain large smooth regions of 1,4-1inked galacturonic acid and relatively small side chains. These side chains consist of sugar residues, which probably belong to arabinans, arabinogalactans type I and mixtures of them. Low molecular weight pectins of the same ionic strength fraction (IE 0.2M GF3) contain larger side chains, consisting of sugar residues, which indicate the presence of arabinogalactan type II similar structures. Additionally other sugar residues (e.g. T-Galp, T-Xylp, T-GlcAp) and non-sugar residues (e.g. methanol and acetic acid; results are not shown) are attached to these pectic polysaccharides, but further investigations are needed to clarify the fine structure in detail.
4. REFERENCES
8
9 10 11
Voragen, A.G.J., Pilnik, W., Thibault, J-F., Axelos, M.A.V., Renard, C.M.G.C. (1995). Food polysaccharides and their applications (Stephen A.M., ed.), Marcel Dekker, Inc., 287-339. Carpita, N.C., Gibeaut D.M. (1993). Plant J. 3, 1-30. McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Ann. Rev. Biochem. 53, 625-663. Selvendran, R.R., O'Neil, M.A. (1987). Meth. Biochem. Anal. 32, 25-153. Selvendran, R.R., Stevens, B.J.H., O'Neil M.A. (1985). Biochemistry of plant cell walls (Brett, C.T., Hillman, J.R., eds.) Cambridge University Press, Cambridge, 39-75. Thibault, J.F. (1979). Lebensnt-Wiss. u. Technol. 12, 247-251. Blakeney, A.B., Harris, P.J., Henry, R.J., Stone, B.A. (1983). Carbohydr. Res. 113, 291-299. Blumenkrantz, N., Asboe-Hansen, G. (1973). Anal Biochent 54, 484-489. Kvemheim, A.L. (1987). Acta Chent Scand. Ser. B41, 150-152. Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). Carbohydr. Res. 127, 5973. Sweet, D.P., Shapiro, R.H., Albersheim, P. (1975). Carbohydr. Res. 40, 217-225.
631
Structural features of pectic polysaccharides of red beet (Beta
vulgaris conditiva) Georg R. Strasser, Daniel E. Wechsler, Renato Amadb Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zurich, Switzerland
Abstract Cell-wall material from ripe red beet was extracted as alcohol-insoluble residue (AIR). The CDTA-soluble extract from AIR was fractionated by anion-exchange chromatography. Four fractions were isolated by a step-wise increase in the ionic strength of the elution buffer. The main fraction was further fractionated by gel filtration chromatography. This chromatogram showed one regular broad peak, which was divided into three parts and pooled. All fractions isolated from both chromatographic systems were freeze-dried and their neutral sugar compositions as well as uronic acid contents were determined. Furthermore methylation analysis of these fractions were performed prior and after reduction of the pectic polysacchaddes with NaBD4.
1. INTRODUCTION Pectins are a group of polysaccharides from the primary cell wall and the intercellular regions of higher plants [1]. They have been investigated for their structural features and their functions within the plant cell wall for many years, because changes in the texture of fruits and vegetables and in the properties of their products are related to changes in the pectic components [2]. From literature it is known, that the pectic backbone consists of ~t-(1---~)linked D-galacturonic acid units, interrupted by the insertion of o~-(1---)2)-linked L-rhamnosyl residues in adjacent or alternate positions [3]. Side chains consisting essentially of arabinans, galactans, arabinogalactans and single xylose residues are attached to this backbone. Other sugars have been found less frequently. In addition some non-sugar substituents, mainly methanol, acetic acid and phenolic acids are known to be present in pectins. Although the main structural elements of pectins are known, the complexity of these polymers has prevented a complete understanding of their fine structure so far. The aim of this project is to get additional information about the fine structure of pectic polysaccharides. Therefore pectins from red beet were isolated and fractionated by chromatographic methods. Some results obtained by methylation analysis of these pectin-rich fractions are presented.
632 2. MATERIALS AND METHODS
Extraction and fractionation of pectins (Figure 1): Red beets of the variety Red Ace F~ were purchased from a local store. Preparation of the AIR was done according to Selvendran and O'Neil [4]. Extraction with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) was performed according to Selvendran et al. [5]. The CDTA-soluble extract was dialysed first against running tap water and subsequently against distilled water. Then it was fractionated by anion-exchange chromatography on DEAE Sepharose CL-6B using 0.04M phosphate buffer (pH 6.5) as eluent with a step-wise increase in the ionic strength (0, 0.1, 0.2, 1.0M NaC1). Related fractions were pooled. The main fraction (IE 0.2M) was further fractionated by gel filtration chromatography on two coupled columns, one packed with Sephacryl S-500 and the other one with Sephacryl S-200 using 0.04M phosphate buffer (pH 6.0) as eluent. The peak on the chromatogram was divided into three parts, which were separately pooled. All samples were dialysed and freeze-dried before further analysis. Analytical techniques: Fractions from anion-exchange chromatography were assayed for uronic acid and neutral sugar contents using an automated segmented flow analyser [6]. Neutral sugars (NS) of all samples were analysed by GC as alditol-acetates [7]. Uronic acids (UA) were determined by the m-hydroxy-diphenyl method [8]. Methylation analysis [9,10] was performed with polysaccharides prior and after carbodiimide-activated reduction with NaBD4. Peaks were identified by GC-MS, whereas the quantification was done by GC (FID) using calculated relative response factors on an effective carbon response (e.c.r.) basis [ 11]. 3. RESULTS AND DISCUSSION The results are summarised in Table 1. As expected, for the IE samples higher amounts of uronic acid are found with increasing ionic strength of the elution buffer. For the GF samples larger molecules (GF1) contain half as much NS compared to smaller molecules (GF3). The sum of differently linked NS residues determined by methylation analysis is calculated to 100%. 1,4-Galp is difficult to determine because of the high amount of 1,4GalAp present in most samples. Therefore 1,4-Galp was determined by methylation analysis without prior reduction with NaBD4. Whereas the percentage of some NS residues remain nearly unchanged in the different samples (e.g. T-Araf, 1,5-Araf and 1,6-Galp), others differ considerably. The relative amounts of 1,3-Araf, 1,3,5-Araf, 1,2,3,5-Araf, 1,4-Galp, 1,2-Rhap and 1,2,4-Rhap increase at higher ionic strength elution on anion exchange chromatography and decrease for smaller molecules on gel filtration chromatography. 1,3-Galp and 1,3,6-Galp show the opposite behaviour. This indicates that pectins eluted at high ionic strength and larger pectic molecules of the IE 0.2M fraction contain on average smaller side chains because of the relatively high amount of 1,2,4-Rhap compared to the total amount of NS residues (1,2,4-Rhap is the main branching residue in the pectic backbone). Furthermore it can be assumed that the side chains of these pectins mainly consist of structures similar to arabinans and arabinogalactans type I, because of the differently linked arabinose and galactose residues present. Pectins eluted at low ionic strength and smaller pectin molecules of the IE 0.2M fraction contain more galactose, which is mainly 1,3- and 1,3,6-1inked. These residues are believed to be part of structures, which are known to be present in arabinogalactans type II.
633 Red Beet Alcohol soluble extract
Extraction with ethanol ,,~ Alcohol insoluble residue (AIR)
CDTA insoluble residue
Extraction with CDTA =~ CDTA soluble material (CDTAS)
Anion exchange chromatography on DEAE-Sepharose CL-6B 1200 T --o- Uronic acids ~ --.- Neutral sugars ~ ~~ 800 + .... 0.04M phosphate buffer ~o~.= containing NaC1 a~ ~=
1 I/ .ll
400
............
q- 1.0 ] ] "=~=~ 0.5
,
0 i ,
I
20 ~ IEOM
i I '
I
40 i 60 I 1 ' 1 IE0.1M IE0.2M
i
=o
80 (I, IE1.0M
Fraction number
Gel filtration chromatography on Sephacryl S-500 and on Sephacryl S-200
~
Mw
(Pullulan)
6
]
i
L60
80
rlE 0.2M GF1 [IE 0.2M GF2] IE 0.2M GF3"[ Figure 1. Extraction and fractionation of pectins from ripe red beets
Fraction number
634
o
0
0
~ l,,-i
0
N
9 v,,,~
~ ~,,,i
0 ~ i...i
0
Z
~
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9
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o
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9
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~
~
V'~ .-4
9
o
o
9
Z
0
0
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l ~.~
.v,,q
0.) 0
4..a
635
636 The ratios between linear and branched residues are presented as well. All values obtained without prior reduction (NS residues only), are in the range between one and two. From this it can be assumed, that side chains contain only slightly more linear than branched residues. After prior reduction (NS and UA residues) much more linear than branched residues are found. This surplus of linear residues originates from the 1,4-1inked galacturonic acid backbone, which is also known as smooth region. The results presented allow the following assumptions to be made: CDTA-soluble material eluted at low ionic strength (IE 0M) contains hardly any uronic acids and consists mainly of neutral sugar residues, which indicate the presence of arabinans and arabinogalactans type II. Pectins eluted at high ionic strength (IE 1.0M) contain large amounts of uronic acids. The side chains consist mainly of arabinans, galactans and mixtures of them. High molecular weight pectins eluted at intermediate ionic strength (IE 0.2M GF1) contain large smooth regions of 1,4-1inked galacturonic acid and relatively small side chains. These side chains consist of sugar residues, which probably belong to arabinans, arabinogalactans type I and mixtures of them. Low molecular weight pectins of the same ionic strength fraction (IE 0.2M GF3) contain larger side chains, consisting of sugar residues, which indicate the presence of arabinogalactan type II similar structures. Additionally other sugar residues (e.g. T-Galp, T-Xylp, T-GlcAp) and non-sugar residues (e.g. methanol and acetic acid; results are not shown) are attached to these pectic polysaccharides, but further investigations are needed to clarify the fine structure in detail.
4. REFERENCES
8
9 10 11
Voragen, A.G.J., Pilnik, W., Thibault, J-F., Axelos, M.A.V., Renard, C.M.G.C. (1995). Food polysaccharides and their applications (Stephen A.M., ed.), Marcel Dekker, Inc., 287-339. Carpita, N.C., Gibeaut D.M. (1993). Plant J. 3, 1-30. McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Ann. Rev. Biochem. 53, 625-663. Selvendran, R.R., O'Neil, M.A. (1987). Meth. Biochem. Anal. 32, 25-153. Selvendran, R.R., Stevens, B.J.H., O'Neil M.A. (1985). Biochemistry of plant cell walls (Brett, C.T., Hillman, J.R., eds.) Cambridge University Press, Cambridge, 39-75. Thibault, J.F. (1979). Lebensnt-Wiss. u. Technol. 12, 247-251. Blakeney, A.B., Harris, P.J., Henry, R.J., Stone, B.A. (1983). Carbohydr. Res. 113, 291-299. Blumenkrantz, N., Asboe-Hansen, G. (1973). Anal Biochent 54, 484-489. Kvemheim, A.L. (1987). Acta Chent Scand. Ser. B41, 150-152. Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). Carbohydr. Res. 127, 5973. Sweet, D.P., Shapiro, R.H., Albersheim, P. (1975). Carbohydr. Res. 40, 217-225.