HAL Id: hal-02278861
https://hal.archives-ouvertes.fr/hal-02278861
Submitted on 4 Sep 2019
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Distributed under a Creative Commons Attribution| 4.0 International License
Characterisation by SEC and High Field NMR of Limit-Dextrins from Enzymatically Degraded Cationic
Starches
Ali Ayoub, Christophe Bliard
To cite this version:
Ali Ayoub, Christophe Bliard. Characterisation by SEC and High Field NMR of Limit-Dextrins from Enzymatically Degraded Cationic Starches. 12th European Carbohydrate Symposium, Jul 2003, Grenoble, France. 258, pp.115 - 121, 2003. �hal-02278861�
Characterisation by SEC and High Field NMR of Limit-Dextrins from Enzymatically Degraded Cationic Starches
A.Ayoub * & C. Bliard
Laboratoire de Pharmacognosie UMR C.N.R.S. 6013 Bat 18 Moulin de la Housse Université de Reims Champagne Ardenne 51687 REIMS cedex 2 France
[email protected]
Chemically modified starches are widely used derivatives in fields such as paper-making and textile industries. Based on the results of enzymatic degradation Kavitha and Bemiller [3] showed that a commercial hydroxy-
The physicochemical macroscopic properties of these products are closely linked to their structural organisation propyl starch seemed to more modified in areas near to the branching zones. Commercial cationic at the molecular level. There is a wide range of possible modification pattern on macromolecules such as starches are synthesised either in solution or more likely by dry process, grafting ammonium groups amylopectins. Different reactions may lead to a variety of structures. In a earlier work we observed that high ether bonds [4]. In a recent work we describe an alternative synthetic route to cationic starches using
Acetylated starches of the same DS, but synthesised via two different processes, had different structures as via a glycerol-water plasticised molten medium to achieve the reaction [5]. The products of the b- seen from their NMR spectra [1]. Woortman and Steeneken [2] reported that granular starch was methylated amylolytic degradation are separated by SEC and analysed by NMR Spectroscopy. The results of In a more block-wise manner whereas when reacted in solution it was modified in a random fashion. structural characterisation are presented.
[1] B. Laignel, C. Bliard, G. Massiot and J.M. Nuzillard, Carbohyd. Res., 298 (1997) 251-260 [2] P.A.M Steeneken, A.J.JL Wortman, Carbohyd. Res., 258 (1994) 207-221
[3] R. Kavitha, J.N. BeMiller, Carbohyd. Polym., 37 (1998) 115-121
[4] D.B. Solarek, O.D. Wurzburg ed. Modified Starches : properties and use. CRC Press, Boca Raton, FL,USA, 1986.
[5] A. Ayoub, C. Bliard, Starch/Stärke, (2003) (accepted)
Introduction
H ydroxpropyltrimethylammonium starches used in paper industries are synthesised by reaction of either 3-chloro-2-hydroxypropyltrimetylammonium chloride (1) or 2-epoxypropyltrimethylammoniumchloride (2) catalysed with sodium hydroxide.
The cationisation was performed in a glycerol-water molten plasticised starch mixture, using a reactive extrusion system (Fig.1). b-amylolysis, size exclusion chromatography (SEC)and NMR spectroscopy was used in this study to examine the molecular architecture
of these modified polysaccharides.
Native starch (semi-crystalline)
Fig 1 : Starch Cationisation in molten medium
Melting and reaction
Thermo-Mechanical Energy
[T=120°C ; speed of twin screw = 120 rpm]
OH
N + CH 3 CH 3
Cl - H 3 C HPTMA =
O
O O
O O
O
O OH
OH HO
HO
HO OH
OH
OH
OH HPTMA
NaOH,H 2 O
O N
Cl - CH 3
CH 3 CH 3 OH +
N + CH 3 CH 3
Cl - H 3 C
Cl
Purification, 10 KD
NaOH
Results and discussion
A) Size-exclusion Chromatography (SEC)
Amylose and amylopectin fractions of unmodified and HPTMA-modified starches were analysed using
Size-Exclusion Chromatography
(SEC) on a Sepharose® CL-2B Column (Pharmacia). The void and total volume of the column were determined usingBlue dextran
(MW 2,000,000) andglucose
respectively. An aliquot of native, molten and chemically chemically wheat starches (10mg / 1ml, dry starch basis) was taken and 200µl of NaOH 1N was added. The sample was dissolved by heating at 60°C and stirring for 20 min. The solution was then filtered at 5µm. The filtered sample was placed on the Sepharose® column (25 x 680 mm) and eluted with a 50mM NaCl / 50mM NaOH aqueous solution stabilised with 0.01% NaN3, at a flow rate of 1.68 ml/min.A.1) Fractionation of wheat starches
A decrease of the excluded fraction [MW >2 000 000 between (40) and (85) ml] corresponding to the amylopectin fraction was observed for both melted samples (Fig 2). This chain length reduction was attributed to the thermo- mechanical energy action. A shift towards the shorter chains was observed with the melted samples, but the cationisation treatment did not cause further macromolecular reduction.
A.2) Fractionation of wheat starch b-limit dextrins
Wheat starches (unmodified and modified, 5 mg) were dispersed in 2 ml of 50mM sodium acetate buffer, pH 4.8; 2 units of b-amylase (Sigma, A 7005) were added, and the mixture was incubated for 24 h at 20°C. The enzyme was inactivated by placing the tube in a boiling water bath for 10 minutes. The digest was centrifuged to remove the precipitated enzyme. The supernatant was fractionated on Biogel® P- 10 to separate two fractions - b-dextrins and
maltose
. The carbohydrate contents of each fraction was estimated by the phenol-sulphuric acid method (Dubois et al., 1956). Appropriate fractions were pooled to obtain b-dextrins and the solutions were dialyzed and freeze-dried. The resulting fractions were placed on a Sepharose column CL-2B.A.3) Fractionation of b-dextrins separated after b-amylase enzyme on modified and non modified wheat starches
The quantity of liberated maltose depends on the chemical modification of starches
The results of the enzymatic degradation suggests that some of the chemical grafting happens on external a(1-4) chains. However the enzymatic action was not affected when the number of cationic groups were increased up to DS 0.13, suggesting that the subsequent modifications occur almost exclusively on internal chains. (Fig 3)
Degree of Substitution 36
48
0.02 0.06 0.1 0.14 0.18 24
12
O
OH O O
H O H
OH
O
OH OH O
H
+
OHb-amylase
Maltose (DP=2) b-dextrins
B.2 NMR 2D COSY – Sample B
(ppm)
5.2 4.4 3.6
4.8 4.0 3.2
(pp m)
B.1 NMR 1D – Samples A & B
(ppm) 4.2 3.4 2.6
5.0 5.8
H
1a(1-4)Sample (A)
Sample (B) H
1a(1-6)H
8H
3H
5,6,6*H
2,4H
9H
10(ppm)
45 55
65 75
85 95
105
C
1C
6C
10C
7C
9C
8C
4C
3C
2C
5Unmodified and HPTMA-modifiedb-dextrins were partitioned using SEC on the
Sepharose® CL-2B column
. An aliquot of each dextrin ( from native, melted or chemically modified starch in molten medium at DS 0.13) was dissolved in 1 ml water, 200µl of 1M NaOH was added. The solution was filtered at 5µm. The solution was placed on theSepharose® column
and eluted in a 50mM (NaCl)/50mM (NaOH).Native starch b-limit dextrin Native Starch
30 85 140 195 245
Elution volume (ml) 50
150 250 350 450
OR de te ct ion (m V) (non norm al ised)
B) NMR Spectroscopy
Two samples: A) cationic starch synthesised in molten medium with DS 0.13 and B) the corresponding b-limit-dextrin (DS 0.175), were studied by NMR multidimensional spectroscopy
A relative increase of the amylopectin peak between 55-85 ml, and the disappearance of the amylose peak(linear chains), was observed in native starch b-dextrins (Fig 4). But with modified and non-modified melted starch a different behaviour was observed (Fig 5). After the action of thermo-mechanical energy and b-amylolysis, the excluded amylopectin peak almost disappears in melted starch limit dextrin. Whereas after the cationisation treatment, this excluded peak was partly retained, showing an inhibiting effect of the grafted groups on the enzymatic action.
[This work was funded by Europol’Agro through the AMIVAL research program, devoted to the development of agricultural resources]
Proton spectra [500MHz, T =298K, Solvent : D
2O (400ml+ 100ml NaOD)
13
C spectrum
[500MHz, T =298K, Solvent : D
2O (400ml+ 100ml NaOD)]
Université de Reims - France (UE) Faculté de Pharmacie
Laboratoire de Pharmacognosie UMR 6013 – CNRS
Eurocarb XII – Grenoble (France-EU) July 2003
O O
O O
(HMPTA)
H H H
H O H
OH
3 possible positions of grafting (HPTMA) groups via ether bonds on the anydroglucose macromolecules
Modification on position 2 :
Modification on position 3 :
Modification on position 6 :
O O
O O H H
H
H O
H
OH
O O
OH O H H
H
H O
HMPTA-
OH
2 1 3
4 6
5
O O
OH O H H
H
H O
H
O-HPTMA
2 1 3
4 5
6
Plasticising
(destructured starch)
Sample (B)
H
(2)-1 H
(2)-1/ H
(2)-2
O O
O O
(HMPTA)
H H H
H O H
OH
H
12(melted modified starch)
Maltose and b-dextrins separated by SEC ( Biogel ® P-10 column )
Elution volume (ml)
0 40 80 120 160
DP = 2
b-dextrins from native starch
RI detection
2 3
4 5
6
. . . . .
+
+ +
+
+ +
+ + +
(1) (2)
Elution volume (ml)
Native Starch
OR detection (mV) (non normalised)
30 85 140 195 245
50 100 150 200
V t
Amylopectin fraction (MW > 2 000 000)
Amylose and reduced size chains fractions
Melted Starch
Molten medium modified Starch (DS 0.02)
Fig 2 : Native, extruded unmodified and modified starches profiles by SEC (Sepharose CL-2B)
Fig 3 : % of liberated maltose vs. DS
Melted starch b-limit dextrin
Modified starch b-limit dextrin (DS 0.13)
30 85 140 195 245
Elution volume (ml) 50
150 250 350 450
OR de te ct ion (m V) (non norm al ised)
Fig 4 : Native starch and the corresponding b-limit dextrins profiles by SEC (Sepharose® CL-2B) Fig 5 : The corresponding b-limit dextrins of « Melted unmodified » and « modified (DS0.13) » starches profiles by SEC (Sepharose® CL-2B)
H(2)-1
H
(2)-2/ H
(2)-3 H
(2)-3/ H
(2)-4
H
(2)-1 = 5.560 (ppm) H
(2)-2 = 3.301 (ppm) H
(2)-3 = 3.837 (ppm) H
(2)-4 = 3.480 (ppm)
OH
N + CH 3 CH 3
Cl - H 3 C HPTMA =
10
8 9 7
+ ++ ++
+++
+++ (Plasticiser)
