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Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Strasse. 25, D-14476 Golm, Germany. M. G. Peter. Interdisciplinary Center for Mass Spectrometry of ...

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Structural Characterization of Mesquite (Prosopis velutina) Gum and its Fractions Yolanda L. Lo´pez-Franco, Ana M. Caldero´n de la Barca, Miguel A. Valdez, Martin G. Peter, Marguerite Rinaudo, Ge´rard Chambat, Francisco M. Goycoolea*

Structural and physicochemical characteristics of mesquite gum (from Prosopis velutina) were investigated using FT-IR spectroscopic, mass spectrometric and chromatographic methods. Four fractions (F-I, F-IIa, F-IIb and F-III) were isolated by hydrophobic interaction chromatography. The samples were characterized and analyzed for their monosaccharide and oligomers composition by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). L-Arabinose (L-Ara) and D-galactose (D-Gal) were found as the main carbohydrate constituent residues in the polysaccharides from mesquite gum and their ratio (L-Ara/D-Gal) varied within the range 2.54 to 3.06 among the various fractions. Small amounts of D-glucose (D-Glc), D-mannose (D-Man) and D-xylose (D-Xyl) were also detected, particularly in Fractions IIa, IIb and III. Infrared spectroscopy identified polysaccharides and protein in all the samples. Data from mass spectrometry (MALDI-TOF MS) was consistent with the idea that the structure corresponding to the periphereal chains of Fraction I is predominantly a chain of pentoses attached to uronic acid.

Y. L. Lo´pez-Franco, F. M. Goycoolea Laboratory of Biopolymers, Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C. (C.I.A.D., A.C.) P.O. Box 1735 Hermosillo, Sonora. 83000 Mexico E-mail: [email protected] A. M. Caldero´n de la Barca Laboratory of Proteins, Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C. (C.I.A.D., A.C.) P.O. Box 1735 Hermosillo, Sonora. 83000 Mexico M. A. Valdez Departamento de Investigacio´n en Polı´meros y Materiales, Departamento de Fı´sica, Universidad de Sonora. Blvd. Transversal y Rosales, 83000, Hermosillo, Sonora, Mexico Macromol. Biosci. 2008, 8, 749–757 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

M. G. Peter Institut fu ¨t Potsdam, Karl-Liebknecht-Strasse ¨r Chemie, Universita 25, D-14476 Golm, Germany M. G. Peter Interdisciplinary Center for Mass Spectrometry of Biopolymers, Universita ¨t Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 20, 14476 Golm, Germany M. Rinaudo, G. Chambat Centre de Recherches sur les Macromole´cules Ve´ge´tales, C.N.R.S. affiliated with University Joseph Fourier –B.P. 53, 38041, Grenoble, Cedex 9, France

DOI: 10.1002/mabi.200700285

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Introduction Plant gums are polysaccharides composed of several kinds of sugar units, some of which may be present as pyranose and others as furanose forms. Most plant gums contain the oxidized form of a hexose such as glucuronic or galacturonic acid, and some contain the deoxy form of hexose such as rhamnose (gum arabic) or fucose (gum tragacanth). Another characteristic of plant gums is that the units are joined by several types of glycosidic linkages.[1] The plant gums are classified into type I arabino-b-(1 ! 4)-galactans (e.g. arbinogalactans (AG) from potato fiber, onion and citrus pomace), type II arabino-b-(1 ! 3, 1 ! 6)-galactans (e.g. in gum arabic and larchwood arabinogalactan), substituted glucuronomannans (e.g. gum ghatti), or substituted rhamnogalacturonan (e.g. gum karaya). Gum tragacanth is a mixture of type II AG and glycanogalacturonan regions.[2,3] Gum arabic and related exudate gums are proteoglycans (arabinogalactan-protein, AGP), with 2–10% of protein[4] to which highly branched polysaccharide chains are attached. The polysaccharide component is predominantly formed by D-Gal and L-Ara (furanose and pyranose ring forms), and minor proportions of rhamnose (L-Rha), 4-O-methyl-D-glucuronic acid and D-glucuronic acid.[5–7] Mesquite gum is a polysaccharide extracted from the bark of Prosopis spp. trees, indigenous to arid and semi-arid regions in the world. In the rural areas of the state of Sonora, Mexico, mesquite gum (locally known as ‘‘chu´cata’’) is collected mostly from P. velutina wild trees and it is used in small amounts in various domestic applications, folk medicine and it is chewed as a sweet,[8,9] while in the arid highlands of central Mexico mesquite gum has been used at industrial scale for the formulation of soft drinks.[10,11] The first documented studies on the chemical composition of mesquite gum appeared about fifty years ago.[12–18] According with such studies, mesquite gum (from Prosopis juliflora), is a highly branched polysaccharide with residues of L-Ara, D-Gal and 4-O-methyl-D-glucuronic acid. Later,[19,20] it was demonstrated that after a partial acid hydrolysis, the inner backbone chains of the Prosopis juliflora gum were formed by D-Gal residues linked by 1 ! 3 and/or 1 ! 6 linkages with b-D-configuration and residues of glucuronic acid and 4-O-methyl-D-glucuronic acid bound to D-Gal by a-D-(1 ! 4) and b-D-(1 ! 6) linkages as the end groups. In addition, they found that the acid labile peripheral chains are very complex oligosaccharides containing L-Ara. Besides the carbohydrate component, mesquite gum contains about ca. 4% of protein.[21] In addition, other substances such as polyphenols (tannins) have been detected in mesquite gum. However, the detailed chemical nature and biological significance of these compounds remain unknown.[21] In previous studies, treatment of gum arabic with coffee bean a-galactosidase (EC 3.2.1.22) resulted in the release of

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ca. 8% of D-Gal, corresponding to sugars occurring as non-reducing end groups, but not in a noticeable change in the overall sugar composition of the polymer,[22] indicating that most of the D-Gal residues were not accessible for cleavage by the enzyme. The same approach had not so far been tested to the analysis of the chemical structure of mesquite gum and was therefore considered as a strategy worth testing to glean further knowledge on the nature of this material. In previous studies, we have investigated the macromolecular and biophysical properties of mesquite gum and its comprising fractions by light scattering and Langmuir balance techniques,[23] concluding that the macromolecular structure of mesquite gum is consistent with the ‘‘twisted hairy rope’’ model, originally proposed by Qi et al.[24] to account for the AGP structure of gum arabic. The present work aimed to deepen the knowledge of the primary structure of mesquite gum from Sonora, Mexico, employing modern FT-IR spectroscopy, HPAEC-PAD chromatography and MALDI-TOF mass spectrometry techniques in order to address the characterization of the whole gum and its comprising fractions along with the products afforded after selective enzymatic hydrolysis of sugar residues at the peripheral branches.

Experimental Part A batch of crude mesquite gum collected was obtained from various local suppliers in Hermosillo, Sonora, Mexico. The gum was hand sorted so as to select only large and clear amber nodules to be used for these studies. The sample was dissolved in water at a concentration of ca. 4.5 vol.-%, the solution was filtered through a cellulose filter (Zeta Plus1, Cuno Inc. USA) and the filtrates were freeze-dried, as reported previously.[23] All chemicals were analytical grade, purchased from Sigma-Aldrich Chemicals (St Louis, MO, USA). The carbohydrate standards were all from Aldrich Chemical Co., Inc. (Milwaukee, WI).

Hydrophobic Interaction Chromatography A 100 cm3 volume of a 10 wt.-% solution of mesquite gum in 4.2 mol  dm3 NaCl was fractionated on a phenyl-Sepharose CL–4B column as described previously.[23] Mesquite gum solution was loaded and eluted successively by 4.2 mol  dm3 NaCl (Fraction I), 2 mol  dm3 NaCl (Fraction II), and finally with water (Fraction III) at a flow rate 40 cm3  h1. The obtained fractions were dialyzed against water and freeze dried.

Elemental Analysis Samples of freeze-dried mesquite gum and its fractions (5 mg) were burned at 1000 8C in flowing oxygen for C, H, N, and O analysis in the analyzer CHNS-932 (LECO Corp. St. Joseph, USA). The CO2, H2O, NOx and SO2 combustion gases were passed through a reduction tube with helium as the carrier gas for converting

DOI: 10.1002/mabi.200700285

Structural Characterization of Mesquite (Prosopis velutina) Gum . . .

the NOx nitrogen oxides into N2 and binding the free oxygen. Selective IR detectors measured the CO2, H2O, and SO2 combustion gases. After corresponding absorption of these gases, the content of the remaining nitrogen was determined by thermal conductivity detection.

Methylation Analysis A sample of freeze-dried Fraction I (F-I) was subjected to methylation analysis.[25] The sample (5 mg) was dissolved in dry DMSO (1 cm3). A 0.5 cm3 solution of sodium methylsulfinylmethanide (2 mol  dm3) in 0.5 cm3 of dry DMSO was added and the mixture was stirred under N2 for 16 h at 20 8C. Methyl iodide (1 cm3) was added over a period of 2 h. The excess of methyl iodide was evaporated, and the mixture was dialyzed against distilled water and the purified solution containing the methylated derivative was freeze-dried. On the whole material a second methylation was performed in the same conditions. The procedure was repeated once, in order to ensure complete methylation. The permethylated polysaccharides were then hydrolyzed with formic acid (1 cm3, 90 vol.-% at 100 8C, 1 mg of samples) for 1 h. After drying, further hydrolysis with trifluoroacetic acid (1 cm3, 2 mol  dm3, 100 8C) was performed for 3 h. Samples were then reduced with a sodium borohydride solution for 16 h at room temperature. The reduced samples were acetylated with acetic anhydride/pyridine (vol.-/vol.-) at 100 8C for 1 h.[26] Alditol acetate derivatives were analyzed by gas chromatography equipped with a flame-ionization detector (CPG/FID) using a SP2380 macrobore column (0.53 mm  30 m) in a Hewlett Packard 5890A system using various methylated alditol carbohydrate as control. The carrier gas was high-purity nitrogen. For sample separation the following conditions were applied: 3 min at an initial temperature of 165 8C followed by an incremental increase (2.5 8C  min1) to a final of 225 8C during 3 min. Complementary insight identification were obtained by GC-MS (Delsi GC coupled to a Nermag R10-10C mass analyser) using the same chromatographic protocol.

Enzymatic Hydrolysis

FT-IR Spectroscopy FT-IR spectra were obtained using a Nicolet Prote´ge´ 460 E.S.P. spectrometer (Nicolet Instrument Corp. Madison, WI). The solid sample (1 mg) was mixed with KBr in a ratio of 1:100. Pellets were formed at 6000-psi pressure in a manually operated hydraulic press (International Crystal Laboratories, 12 Ton E-Z Press). The spectra were recorded in the transmission mode from 4 000 to 400 cm1 with a resolution of 2 cm1 and a total of 32 scans were accumulated. Three replicate spectra were collected for each sample.

Carbohydrate Analysis by Ion Chromatography (HPAEC-PAD) For acid hydrolysis of samples to be analyzed by HPAEC-PAD, 1 mg of sample was hydrolyzed with 1 cm3 of 2 mol  dm3 trifluoroacetic acid (TFA) for 3 h at 100 8C and concentrated to dryness under an air stream at 40 8C, followed by addition of water (1 cm3) and evaporated as described above. The vial contents were re-dissolved with 1 cm3 of water and the column was loaded with 100 mL of sample.

Ion Chromatography Ion exchange chromatography was carried out on a HPAEC-PAD DX 600 system equipped with a CarboPacTM-PA 1 column (Dionex, Idstein, Germany) coupled to a Dionex ED50 Electrochemical Detector in pulsed amperometric detection mode. For separations of monosaccharides, the column was flushed with 0.2 mol  dm3 NaOH for 15 min and then equilibrated with water for 20 min. Analytes were eluted isocratically with water at 1 cm3  min1. Sensitivity of the pulsed amperometric detection was enhanced by post-column addition of 0.3 mol  dm3 NaOH (flow rate 0.5 cm3  min1).

MALDI-TOF Mass Spectrometry

Mass spectrometry was performed on a Reflex II MALDI-TOF A 2 cm3 volume of 2 wt.-% solution of mesquite gum fractions in instrument (Bruker Daltonik, Bremen, Germany) in the positive 0.1 mol  dm3 sodium acetate buffer (0.2 vol.-%, pH ¼ 4.5 adjusted with acetic acid) were incubated with 0.25 U of a-galactosidase from Aspergillus niger (EC 3.2.1.22 Sigma, St. Lous, MO, USA) for 1 h at 40 8C. The solutions were cooled to ca. 4 8C. After hydrolysis, the samples were dialyzed through a membrane of 6–8 kDa molecular weight cut-off (MWCO) during 24 h against several volumes of distilled water. The retentates (R) were freeze dried and the permeates (P) concentrated by ultrafiltration through a 1 kDa MWCO membrane. The concentrate (C) and filtrate (F) were freeze dried. The experimental protocol for enzymatic hydrolysis and separation of the various Scheme 1. Experimental protocol for enzymatic hydrolysis and separations using dialysis products is shown in Scheme 1. with 6 kDa membrane and ultrafiltration through a 1 kDa membrane. Macromol. Biosci. 2008, 8, 749–757 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Table 1. Elemental composition of mesquite gum (MG) and the fractions isolated by hydrophobic interaction chromatography (na ¼ data not available)

ion mode. For the ionization, a nitrogen laser (337 nm, 3 ns pulse width, 3 Hz) was used. All spectra were measured in the reflector mode using external calibration. 2,5-dihydroxybenzoic acid was used as matrix. The matrix solution was prepared by dissolving 20 mg dihydroxybenzoic acid in 1 cm3 of 20 vol.-% aqueous methanol. Concentrates and filtrates from Fraction I (F-I), Fraction IIa (F-IIa), Fraction IIb (F-IIb) and Fraction III (F-III) from mesquite gum were redissolved in 15–50 mL methanol/water (vol.-% 50:50). 0.5 mL of the sample was applied to the target followed by the addition of 1 mL of matrix solution, and dried under a gentle stream of air.

Component

MG

Gum recovery

F-Id)

F-IIad) F-IIbd)

F-IIId)

94.77

2.49

1.17

1.56

96.27

97.21

88.90

71.08

76.54

3.73

2.79

11.10

28.92

23.46

2.60

na

na

na

na

32.14

41.34

wt.-% Total sugara) % Proteinb) % Ashc)

Results and Discussions

% Fractionation by Hydrophobic Chromatography Hydrophobic interaction chromatography (HIC) of mesquite gum on Phenyl-Sepharose CL-4B yielded three fractions which, according to increasing hydrophobicity, eluted at 4.2 mol  dm3 NaCl (F-I), 2.0 mol  dm3 NaCl (F-II), and water (F-III; see Figure 1), respectively. The total recovery was 67.45 wt.-% of the material originally applied to the column. Fraction F-I represented 94.77 wt.-% of the totally recovered material. According to elemental analysis, performed on a CHNS-932 apparatus, the C/N ratio was 93.51. Assuming that the nitrogen value represents protein, it was estimated that F-I consists of ca. 97.2 wt.-% carbohydrates and 2.8 wt.-% protein (Table 1). The N value (0.43 wt.-%) was different from that obtained earlier with an LECO Model FP-528 analyzer (1.09 wt.-%)[23] but close to that obtained by Micro-Kjeldahl (0.61 wt.-%).[21]

Elemental composition % Carbon Hydrogen

41.71

40.21

40.36

6.09

6.15

6.41

4.96

6.16

Oxygen

51.48

53.12

51.46

57.85

48.54

Nitrogen

0.57

0.43

1.70

4.43

3.59

Sulfur

0.15

0.10

0.07

0.63

0.37

Carbon/Sulfur 278.07 402.10 576.57 51.02 111.73 a)

Calculated by difference (values in dry weight basis).; b)Calculated from total N contents using a conversion factor of 6.53 (Anderson and Weiping 1989)[37] (values in dry weight basis).; c) As determined by gravimetric method after burning of a sample in a furnace at 600 -C during 4 h (value in dry weight basis).; d)F-I, F-IIa and F-IIb, and FIII are fractions of mesquite gum obtained by hydrophobic interaction chromatography after elution with 4.2 mol  dmS3 NaCl, 2 mol  dmS3 NaCl and water, respectively.

Fraction F-II was separated further into F-IIa and F-IIb. Fraction F-IIa accounted for 2.49 wt.-% of the totally recovered material and had 11.1 wt.-% of protein, while the F-IIb (1.17 wt.-% recovered) had 28.9 wt.-% of protein. F-III represented 1.56 wt.-% of recovery and had 23.5 wt.-% of protein (Table 1). Sulfur values were higher in F-IIb, and F-III. This is in general agreement with the relatively higher amounts of protein in those fractions. However, F-IIa, despite having protein contents higher than those of F-I and whole mesquite gum, its sulfur value is the lowest. Varying contents of Met and Cys aminoacids among the different fractions can account for this apparent discrepancy. In gum arabic from Acacia senegal, different aminoacid profiles have been found for the different HIC molecular fractions.[27] Methylation Analysis Figure 1. Separation of mesquite gum on Phenyl-Sepharose CL4B. [23] Molecular fractions (as labeled) were eluted using 4.2 mol  dm3 NaCl, 2 mol  dm3 NaCl and water.

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Methylation of Fraction I (F-I), comprised predominantly of polysaccharides, followed by total hydrolysis, reduction

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Structural Characterization of Mesquite (Prosopis velutina) Gum . . .

with NaBH4, acetylation, and gas chromatography resulted in identification of the following monosaccharide derivatives: 2,3,5-tri-O-methyl-Ara (14 mol-%), 3,5-di-O-methyl-Ara (45 mol-%), 2,5-di-O-methyl-Ara (9.5mol-%), 2,3-di-O-methylAra (2.5 mol-%), 2,3,4,6-tetra-O-methyl-Gal (0.4 mol-%), 2,4,6-tri-O-methyl-Gal (3 mol-%), 2,3,6-tri-O-methyl-Gal (4 mol-%), 2,3,4-tri-O-methyl-Gal (3 mol-%), 2,4-di-O-methylGal (11 mol-%), 2-O-methyl-Gal (5 mol-%), confirming previously reported data.[16,17,19] Identification of 2,3,5-tri-O-methyl-Ara indicates the presence of terminal L-Ara residues. While 2,4-di-O-methylGal arises from !3,6)-linked D-Gal residues located at branching points of the central galactan backbone and 2-O-methyl-Gal corresponds with !3,4,6)-linked D-Gal located presumably at branching points where arabinan oligosaccharides are attached. The presence of 2,3,4-triO-methyl-Gal and 2,3,6-tri-O-methyl-Gal is consistent with the occurrence of the aldobiuronic acids of (1 ! 6) and (1 ! 4) D-Gal residues linked to GlcUA, respectively, as has been reported earlier.[16,17] The rather high relative amounts of 2,3,5-tri-O-methyl-Ara, 2,4-di-O-methyl-Gal and of uronic acid (4 mol-%) confirm the highly branched structure of mesquite gum in agreement with that proposed Cunnen and Smith.[17] The remaining L-Ara units appear as 3,5-di-O-methyl derivatives, meaning that they are located between the terminal L-Ara units and the uronic acids.

Enzymatic Hydrolysis The macromolecular structure of mesquite gum has been proposed to be consistent with the ‘‘twisted hairy rope’’ model originally proposed to describe the AGP molecules in gum Arabic.[23] The action of a-galactosidase on the whole gum and its HIC fractions along with the characterization of the products formed was thought to provide valuable insight into the fine structure of the carbohydrate moieties comprising the branches of these proteoglycans. The rational behind this approach, was to selectively hydrolyze the a-D-Gal residues present at branching chains, as previously documented for the study of gum arabic.[22] Scheme 1 summarizes the experimental protocol for the analysis of mesquite gum (MG) and its separated fractions. Treatment with a-galactosidase (a-D-galactoside galactohydrolase EC 3.2.1.22), was followed by dialysis of the reaction mixture to give retentates (R) which contained arabinogalacto-oligomers of Mw > 6 kDa, and permeates (P) containing principally hexoses derivatives of Mw < 6 kDa. The P were fractionated by ultrafiltration through a 1 kDa membrane filter to afford a filtrate (F) (Mw < 1 kDa) and a concentrate (C) (1 kDa < Mw < 6 kDa) (Table 2–4). These results indicated that the a-galactosidase was able Macromol. Biosci. 2008, 8, 749–757 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 2. Carbohydrate composition of mesquite gum (MG) and fractions F-I to F-III before enzymatic hydrolysis (nd ¼ not detected).

Carbohydrate

MG

F-Ia)

F-IIaa) F-IIba) F-IIIa)

wt.-% L-Ara

73.19 73.28

72.76

71.87

69.49

D-Gal

26.57 26.40

23.78

24.58

27.37

D-Glc

0.03

0.14

1.60

2.27

1.27

D-Xyl

nd

nd

0.50

nd

0.44

nd

nd

D-Man Total

1.11

0.90

1.05

99.79 99.82

99.75

99.62

99.62

2.75

3.06

2.92

2.54

wt.-% L-Ara/D-Gal ratio

2.78

a)

F-I, F-IIa, F-IIb and F FIII are hydrophobic chromatography fractions (as defined in Table 1).

to by-pass the branching points of a-D-Gal residues, bearing L-Ara containing oligosaccharides.

FT-IR Spectroscopy The FT-IR spectra of mesquite gum (MG) and its fractions in solid state showed broad absorptions of OH (nOH, hydrogen bonded) and CH (nCH) at 3 375 and 2 932 cm1 (Figure 2). An intense band centered at 1 650–1 600 cm1, observed in all spectra, is assigned to amide I, while a less intense band at 1 540–1 550 cm1, noticeable in fractions F-IIa, F-IIb and FIII is assigned to amide II. Amide I and II bands are characteristic of peptidic bonds and confirm the presence of protein, particularly in fractions FIIa, FIIb and FIII. In addition, COO asymmetric stretching bands located at 1 425 cm1 were also identified in all of the samples. The absorption bands at 1 153, 1 031, and 902 cm1 are from the glycosidic acetal groups of pyranoses. After enzymatic digestion with a-galactosidase, FT-IR spectra were recorded of the materials obtained after lyophylization from the concentrates of F-I, F-IIa and F-IIb (Figure 3), as well as those obtained from the filtrates prepared from MG and Fractions I–III (Figure 4). In most samples, the band of nOH was shifted to lower wave numbers, i.e. ca. 3 240 cm1, indicating a lower degree of hydrogen bonding in the smaller oligosaccharide fragments obtained after enzymatic hydrolysis. Close inspection of the spectra of MG-F, FIIa-F and FIIb-F (Figure 4), reveals a less intense band at 1 730 cm1 (nC –– O), which could be assigned to the ester functional groups in lipids.[28] With the exception of the concentrate of F-I which was very similar to the sample of F-I before

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Table 3. Carbohydrate composition in dialysis retentates after enzymatic hydrolysis of mesquite gum and fractions F-I to F-III (nd ¼ not detected).

MG-Ra)

F-I-Ra)

F-IIa-Ra)

F-IIb-Ra)

F-III-Ra)

L-Ara

53.28

71.22

71.78

67.25

60.52

D-Gal

41.78

28.40

25.45

27.40

23.94

D-Glc

0.68

0.26

0.97

3.98

7.42

D-Xyl

nd

nd

0.61

1.06

nd

D-Man

nd

nd

nd

nd

nd

95.74

99.88

98.81

99.69

91.88

1.27

2.51

2.82

2.45

2.53

Carbohydrate wt.-%

Total wt.-% L-Ara/ D-Gal ratio a)

Retentates of mesquite gum (MG-R) and of its hydrophobic interaction chromatography fractions (as defined in Table 1) obtained after digestion with a-galactosidase and dialysis against water (6 kDa molecular weight cut-off membrane).

enzymatic treatment, all spectra of the concentrates and the filtrates showed a strong increase of the carbohydrate absorptions at 1 420 (nC –OH), and 1 140 cm1 (nR –O –C –O –R). In addition, a band appeared at ca. 620 cm1, indicative for out-of-plane deformation vibration of C –OH groups. Carbohydrate analysis Table 2 shows the results of carbohydrate analysis of mesquite gum and fractions F-I to F-III before enzymatic hydrolysis. L-Ara and D-Gal were the most abundant sugars in both whole MG and all fractions, containing also

traces of D-Glc. Traces of D-Xyl occurred in F-IIa and F-III, only. In AG from other plants, D-Xyl was described in the gum exudates of Spondias dulcis (Anacardiaceae)[29] and Acacia gum[27] while Glc was found in green coffee.[30] In contrast to mesquite gum from P. juliflora [19,20], P. glandulosa [31], and P. laevigata, [32] L-Rha could not be detected in the gum of P. velutina. This has also been corroborated by 1H NMR analysis.[33] On the other hand, D-Man was detected in F-IIa, FII-b and F-III in nearly similar concentrations (0.90 to 1.11% wt.-%), which were in fairly good agreement with values previously reported in A. senegal (1.5 wt.-%).[27] The

Table 4. Carbohydrate composition in fractions obtained by ultrafiltration of dialysis permeate of mesquite gum and fractions F-I to F-III (nd ¼ not detected).

F-I-Ca)

F-IIa-Ca)

F-IIb-Ca)

MG-Fb)

F-I-Fb)

L-Ara

32.72

23.46

39.41

10.34

D-Gal

31.61

27.58

21.38

10.29

D-Glc

35.67

40.88

33.44

D-Xyl

nd

8.07

5.77

Carbohydrate

F-IIa-Fb)

F-IIb-Fb)

F-III-Fb)

32.53

1.57

9.50

45.93

16.73

55.21

9.40

32.52

53.45

26.37

32.70

65.06

21.55

10.19

13.90

10.52

0.55

nd

wt.-%

D-Man

nd

nd

nd

15.02

1.17

nd

15.16

nd

Total

100

99.99

100

99.29

90.70

100

99.67

100

1.03

0.85

1.84

1.00

1.94

0.03

1.01

1.41

wt.-% L-Ara/D-Gal ratio a)

Concentrates of hydrophobic interaction chromatography fractions of mesquite gum (as defined in Table 1) obtained after ultrafiltration through a membrane of 1 kDa molecular weigh cut-off of the permeates of the dialysis treatment; b)Filtrates of hydrophobic interaction chromatography fractions of mesquite gum (as defined in Table 1) obtained after ultrafiltration through a membrane 1 kDa molecular weigh cut-off of the permeates of the dialysis treatment.

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DOI: 10.1002/mabi.200700285

Structural Characterization of Mesquite (Prosopis velutina) Gum . . .

Figure 2. FT-IR spectra of (a) Mesquite gum and (b) F-I, (c) F-IIa, (d) FIIb and (e) F-III fractions (as described in Figure 1) obtained by hydrophobic interaction chromatography.

presence of D-Man in F-IIa, F-IIb, and F-III, probably located on the side chains, indicates a more complex structure of mesquite gum, however, more studies are underway to confirm this suggestion. The L-Ara/D-Gal ratio values of mesquite gum and F-I were similar (2.75 vs. 2.78; Table 2), but they were lower than those of F-IIa (3.06) and F-IIb (2.92), indicating a higher degree of branching in these fractions. On the other hand, F-III had a lower ratio of L-Ara/D-Gal (2.54). The L-Ara/D-Gal ratios found here for gum extracts from P. velutina and its fractions were similar to the one that has previously been reported in gum from P. juliflora by Aspinall and Whitehead,[19,20] who reported an L-Ara/ D-Gal ratio of 2.7.

Figure 3. FT-IR spectra of (a) F-I-C (b) F-IIa-C, (c) F-IIb-C fractions (as described in Figure 1) obtained after ultrafiltration through a membrane of 1 kDa molecular weigh cut-off of the permeates of the dialysis treatment. The concentrates (C) contain components of 1 kDa < Mw < 6 kDa. Macromol. Biosci. 2008, 8, 749–757 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 4. FT-IR spectra of (a) mesquite gum and (b) F-I-F, (c) F-IIa-F, (d) F-IIb-F, (e) F-III-F fractions (as described in Figure 1) obtained after ultrafiltration through a membrane of 1 kDa molecular weigh cut-off of the permeates of the dialysis treatment. The filtrates (F) contain low Mw components of < 1 kDa.

Digestion of MG with a-galactosidase and subsequent dialysis gave a retentate (R) which showed relative amounts of sugars lower in fractions F-I-R to F-III-R, but the L-Ara/D-Gal ratio was rather similar to the values measured in such fractions before enzyme treatment (cf. Table 2 and 3). According to our results, enzymatic hydrolysis has not cleaved all the a-D-Gal ! L-Ara linkages in the gum and its fractions, because not all of the oligosaccharides comprising these were accessible to the enzyme. However, an increase in the relative amount of D-Glc was observed in all enzyme-treated material (Table 3) with respect the untreated one (Table 2). Ultrafiltration of the permeate from dialysis of the fractions treated with galactosidase gave a concentrate (C) containing components of 1 kDa < Mw < 6 kDa and filtrates (F) containing low Mw components of

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