Structural Characterization, Degree of Esterification and Some Gelling Properties of Krueo Ma Noy (Cissampelos Pareira) Pectin
Structural Characterization, Degree of Esterification and Some Gelling Properties of Krueo Ma Noy (Cissampelos Pareira) Pectin
Structural Characterization, Degree of Esterification and Some Gelling Properties of Krueo Ma Noy (Cissampelos Pareira) Pectin
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Abstract
Pectins extracted from Krueo Ma Noy (Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral
sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked a-D-galacturonan by a combination of carboxyl
reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7
and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration
of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 8C.
Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy
of Krueo Ma Noy pectin increased with polysaccharide concentration.
Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved.
Keywords: Krueo Ma Noy pectin; Cissampelos pareira; FT-IR spectroscopy; Degree of esterification; HPSEC; DSC
Enzymatic hydrolysis, NMR, and FT-IR are proven to be The pectate was then incubated with pectate lyase, which
an effective method for determining structures of poly- cleaves the polygalacturonic acid and releases unsaturated
saccharides, including pectins (Voragen, Pilnik, Thibault, (4,5-ene) oligosaccharides that gave an absorbance at
Axelos, & Renard, 1995). The degree of esterification (DE) 235 nm.
and the percent of the total number of carboxyl groups The amount of unsaturated product produced was
esterified has a significant effect on the strength of the gel calculated as:
and the gelling mechanisms of pectins (Walter, 1991).
Unsaturated product Z DAbs !1=L !1=3
Several methods for determining the DE have been
reported, e.g. a titrimetric method adopted by Food where DAbs is the change of reaction absorbance minus
Chemical Codex (FCC, 1981) and USP 26 NF 21 (2003). blank absorbance measured after 30 min, L is the cuvette
The DE of pectins can also be determined by HPLC path length (Z1 cm) and 3 is the molar extinction
(Levigne, Thomas, Ralet, Quemener, & Thibault, 2002; coefficient of the reaction product (4600 MK1cmK1). A
Plöger, 1992) and 1H-NMR spectroscopy (Grasdalen, value of more than 0.5!10K5 of the unsaturated product
BakØy, & Larsen, 1988). Recently, FT-IR becomes a indicates the presence of pectin and conversely an
preferred technique for determining the DE due to its ease of unsaturated product concentration less than 0.5!10K5
use (Filippov, 1992; Manrique & Lajolo, 2002). In previous indicates the absence of pectin (Hansen, Thuesen, &
paper, we reported the chemical composition and solution Soderberg, 2001).
properties of the pectic polysaccharide extracted from
Krueo Ma Noy (Singthong et al., 2004). The objectives of
2.4. Methylation and GC–MS of partially methylated
the present paper were to determine the structural features of
alditol acetate (PMAA)
Krueo Ma Noy pectin, its DE, molecular characteristics and
gelling properties.
Methylation analysis of pectic substances has been a
difficult task due to the presence of large quantities of uronic
acids. In this study, we first reduced the uronic acid into
2. Materials and methods
neutral sugars then carried out the normal methylation
analysis for neutral sugars. The two steps are described in
2.1. Preparation of standard samples
the following sections.
Pectin standards with known DE, 26, 59 and 94%, were
obtained from SIGMA (Steinheim, Germany). Standards 2.4.1. Reduction of uronic acids
with known DE, such as 42.5 and 76.5%, were prepared by The reduction of the uronic acid was conducted
mixing appropriate amounts of the three commercial following a procedure described by Taylor and Conrad
standards. (1972) and York, Darvill, McNeil, Stevenson, and Alber-
sheim (1986) with slight modification (Fig. 1). Sample
2.2. Preparation of pectin from Krueo Ma Noy (5 mg) was dissolved in deuterium oxide (2 ml) and 50 mg
of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide
Krueo Ma Noy leaves, procured from the farm market in methyl-p-toluenesulfonate (CMC, Sigma) was added. The
the Northeast of Thailand, were cleaned with water then pH was adjusted and maintained at 4.75, using 0.1 M HCl in
dried at 60 8C for 3 h. The dried leaves were ground and deuterium oxide. After 1 h, 800 mg of sodium borodeuteride
stored at room temperature (25 8C) in vacuum. Krueo Ma dissolved in 5 ml of deuterium oxide was added over a
Noy pectins were extracteded according to a method period of 0.5 h, and the pH of the reaction mixture was
described previously (Singthong et al., 2004). To extract maintained at 7.0, using 2.0 M HCl in deuterium oxide
pectin, the dry powder (2% solids) was stirred in distilled during the reduction reaction.
water at 25–28 8C and natural pH (3.8–4.0). Alcohol The reaction was allowed to continue with constant
precipitation and drying produced a crude extract; further stirring for 0.5 h at pH 7.0, after the addition of sodium
dialysis against distilled water and lyophilization produced borodeuteride. After titration of the solution to pH 4.0, the
a purified extract. reduced polysaccharide was separated from salts by dialysis
against distilled water overnight at 22 8C (3500 molecular
2.3. Enzyme assay for identification of pectin weight cut off), and the solution was lyophilized. The
polysaccharide was dissolved in distilled water and 10%
The identification and confirmation of pectins from acetic acid in methanol was added. The mixture was dried
Krueo Ma Noy leaf extract was carried out using an assay with a stream of nitrogen to remove boric acid. This process
available in a kit from Megazyme (Megazyme International was repeated 3–4 times to ensure that most of the boric acid
Ireland Ltd, Ireland). The sample was dissolved in was removed. Finally, a few drops of methanol were added
de-ionized water, and the pH adjusted to 12 to catalyze and the solution evaporated (two times) to remove any boric
the deesterification of the 6-methyl galacturonic acid. acid remained.
J. Singthong et al. / Carbohydrate Polymers 58 (2004) 391–400 393
Fig. 1. Flow chart of the carboxyl reduction procedure of uronic acids. Fig. 2. Flow chart of methylation analysis procedure.
Samples of reduced uronic acid (2–3 mg) were dried at 2.5 h after adding 0.3 ml methyl iodide. The methylated
80 8C for 4–5 h and then stored over night under vacuum polysaccharide was then extracted with 1 ml methylene
over phosphorus pentoxide (P2O5) in desiccators. chloride. The methylene chloride extract was passed
through a sodium sulphate column (0.5!15 cm) to remove
2.4.2. Methylation analysis water, and then evaporated by a stream of nitrogen. The
The methylation analysis of the pectin samples after dried methylated polysaccharide was hydrolyzed in 0.5 ml
reduction of the uronic acid was carried out according to the of 4.0 M trifluoroacetic acid (TFA) in a sealed test tube at
method of Ciucanu and Kerek (1984) with slight modifi- 100 8C for 6 h and the TFA was removed by evaporation
cation (Fig. 2). The dried samples were dissolved in under a stream of nitrogen and dissolved in 0.3 ml distilled
anhydrous DMSO at 85 8C for 2 h with constant stirring water. The hydrolysate was reduced, using sodium borodeu-
and then sonicated for 4 h to ensure that the samples were teride (1–5 mg) and acetylated with acetic anhydride
completely dissolved. Dry sodium hydroxide (20 mg) was (0.5 ml). Aliquots of the resultant partially methylated
added, and the mixture was stirred for 3 h at room alditol acetates (PMAA) were injected on to GC–MS system
temperature (22 8C). The mixture was stirred for additional (ThermoQuest Finnigan, San Diego, CA) fitted with
394 J. Singthong et al. / Carbohydrate Polymers 58 (2004) 391–400
a SP-2330 (Supelco, Bellefonte, Pa) column (30 m! Shimadzu Scientific Instruments Inc., Columbia, MA,
0.25 mm, 0.2 mm film thickness, 160–210 8C at 2 8C /min, USA) according to a method described by Wang, Wood,
and then 210–240 8C at 5 8C/min) equipped with an ion trap Huang, and Cui (2003). The column set consisted of two
MS detector. columns in series, a Shodex OhPak KB-806M (Showa
Denko K.K., Tokyo, Japan) and an Ultrahydrogel linear
2.5. Determination of the degree of esterification (Waters, Milford, CT, USA) maintained at 40 8C during
measurements. The mobile phase was 50 mM NaNO3 (pH
2.5.1. Titrimetric method 5.8) with 0.03% (w/w) NaN3 with a flow rate of 0.6 ml/min.
The DE of pectin from Krueo Ma Noy pectin was Triple detectors, a right angle laser light detector, a
determined by the titrimetric method of Food Chemical refractive index detector and a viscosity detector, were
Codex (FCC, 1981) and USP 26 NF 21 (2003) with slight used for characterizing the molecular weight and molecular
modification. Dried sample (500 mg) was transferred to a weight distribution.
250 ml flask, moistened with 2 ml of ethanol and dissolved
in 100 ml of carbon dioxide-free water. After the sample 2.7. Rheological properties
was completely dissolved, five drops of phenolphthalein
were added, the sample was titrated with 0.5 M sodium All rheological properties were determined on a Bohlin
hydroxide and the result was recorded as the initial titer. CVO Rheometer (Bohlin Instruments, East Brunswick, NJ).
Then, 10 ml of 0.5 M sodium hydroxide were added, the A parallel plate geometry (40 mm diameter, 1.0 mm gap)
sample was shaken vigorously, and allowed to stand for was used for oscillatory measurements. The viscoelastic
15 min; 10 ml of 0.5 M hydrochloric acid were added and properties, storage modulus (G 0 ) and loss modulus (G 00 ),
the sample was shaken until the pink color disappeared. were determined through small amplitude oscillatory test at
Phenolphthalein (five drops) were added and the solution frequencies from 0.1 to 10 Hz. Prior to any dynamic
was titrated with 0.5 M sodium hydroxide to a faint pink experiments, a strain sweep test at a constant frequency of
color that persisted after vigorous shaking (end-point). This 0.1 Hz determined the linear viscoelastic region. All
volume of titration was recorded as the saponification titer oscillatory tests were performed at a strain value of 0.02
(the final titer). (2%) (within the linear viscoelastic region). A thin layer of
The DE was calculated from the following formula: low viscosity mineral oil was used to cover the sample in
%DEZthefinaltiter=ðtheinitialtiterCthefinaltiterÞ!100 order to prevent solvent evaporation during measurements.
Temperature sweeps were performed between 5 and 80 8C.
Samples were loaded onto the rheometer in a gel state at
2.5.2. FT-IR spectroscopic method
5 8C and the heating rate was 1 8C/min.
Pectin standards and Krueo Ma Noy pectin were dried
and desiccated in a vacuum jar prior to FT-IR analysis. FT-
IR spectra of pectins were obtained using a Golden-gate 2.8. Differential scanning calorimetry (DSC)
Diamond single reflectance ATR in a FTS 7000 FT-IR
spectrometer equipped with a DTGS detector (DIGILAB, Thermal analyses were performed using a differential
Randolph, MA). The spectra were recorded at the scanning calorimeter (2920 modulated DSC; TA
absorbance mode from 4000 to 400 cmK1(mid infrared Instruments, New Castle, DE, USA). Sample size was
region) at a resolution of 4 cmK1 with 128 co-added scans. about 80–90 mg, and the scanning rate was 5 8C /min. The
At least triplicate spectra were recorded for each sample. reported values are means of duplicate measurements.
Because the DE is defined as (number of esterified
carboxylic groups/number of total carboxylic groups)!
100, it is inferred that the ratio of the area of the band at Table 1
1730 cmK1 (corresponding to the number of esterified Determination of content of unsaturated oligosaccharides in pectin and non-
pectin polysaccharides
carboxylic groups) over the sum of the areas of the bands at
1730 and 1600 cmK1 (corresponding to the number of total Polysaccharide type Unsaturated
carboxylic groups) should be proportional to the DE, i.e. oligosaccharides !10K4
DEZA1730/(A1730CA1600) (Manrique & Lajolo, 2002; Carrageenan 0.009
Chatjigakis et al., 1998). Amidated low ester pectin 1.660
Low ester pectin 2.738
Sugar beet pectin 0.928
2.6. Molecular characterization High ester pectin 1.522
Dialyzed extract 2.362
Molecular weight, molecular weight distribution, radius
Average of duplicate results. Calculations: blank absorbanceZenzyme
of gyration and intrinsic viscosity of Krueo Ma Noy pectin blank Csample blank, DabsorbanceZreaction absorbanceKblank absor-
were determined by high performance size exclusion bance, unsaturated productZDAbs!1/L!1/3, where: L, cuvette path
chromatography (HPSEC, Shimadzu SCL-10Avp, length (Z1 cm); 3, molar extinction coefficient (4600 MK1 cmK1).
J. Singthong et al. / Carbohydrate Polymers 58 (2004) 391–400 395
Fig. 3. Chromatogram of GC and Mass spectrum of dialyzed extract. (a) GC, (b) MS.
396 J. Singthong et al. / Carbohydrate Polymers 58 (2004) 391–400
Fig. 4. Fourier transform infrared spectra of commercial pectin standards and Krueo Ma Noy pectins.
(Table 1), suggesting the presence of pectin (Hansen et al., 3.3. FT-IR spectrum and the degree of esterification
2001). For comparison purposes, commercial pectin stan- of pectins
dards and non-pectin samples were also examined (Table 1).
The FT-IR spectra of Krueo Ma Noy pectin
and commercial pectin standards are presented in Fig. 4.
3.2. Methylation analysis
The functional groups of pectins and their corresponding
Carbodiimide-activated reduction of the carboxyl frequencies and the nature of the bands are presented in
groups of glycosyluronic acids with sodium borodeuteride Table 2. The broad, strong areas of absorption between 3600
(NaBD4) resulted in an easily identified sugar (deuter- and 2500 cmK1 are caused by O–H stretching absorption
ized). There was only one major peak detected from the due to inter- and intra-molecular hydrogen bonds. The O–H
GC–MS analysis of the partially methylated alditol stretching vibrations occur within a broad range of
acetate (PMAA) derived from the carboxyl reduced
Krueo Ma Noy pectin (Fig. 3a), and its corresponding Table 2
mass spectrum is displayed in Fig. 3b. The combination FT-IR spectrum of pectin: wave numbers and intensities of functional
groups
of the fragmentation pattern and retention time of the
PMAA suggested that the reduced polysaccharide is made Wave number (cmK1) Functional groups Intensity
of 1,4-linked D-galactosyl residues. The diagnostic 3600–2500 O–H stretching Broad, strong
fragment m/z 235 is shifted two mass units higher than 3000–2800 C–H stretching, Sharp, occasionally
the m/z 233 expected from a 4-linked hexopyranosyl unit symmetric, double overlapping
(Biermann & McGinnis, 1989). Because there were no asymmetric with O–H
1760–1730 CZO, esterified Strong
GC peaks detected from the non-reduced Krueo Ma Noy 1630–1600 COO-asymmetric Strong
extract, there were no D-galactosyl residues in the stretching
polymer. The major peak in Fig. 3a represents 4-O- 1400 COO-symmetric Weak
substituted D-galacturonic acid. This result indicates that stretching
1380 C–H bending Weak
Krueo Ma Noy extract is a pectin that has a linear
1300–1000 CZO stretching Weak
backbone chain of 1/4-linked a-D-galacturonic acid
units (Walter, 1991). Adopted from Gnanasambandam and Proctor (2000) and Filippov (1992).
J. Singthong et al. / Carbohydrate Polymers 58 (2004) 391–400 397
4. Conclusion
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