Food Chemistry 123 (2010) 669678

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Isolation and characterisation of pectic substances from murta (Ugni molinae Turcz) fruits
Edelio Taboada a, Patricio Fisher b, Rory Jara c, Elisa Ziga d, Manuel Gidekel e, Juan Carlos Cabrera f, Eduardo Pereira g, Ana Gutirrez-Moraga h, Reynaldo Villalonga i, Gustavo Cabrera e,*
a

Escuela de Ingeniera Ambiental, Universidad Catlica de Temuco, Temuco, Chile Escuela de Agronoma, Facultad de Recursos Naturales, Universidad Catlica de Temuco, Temuco, Chile University of Maine, Orono, ME, USA d Centro de Investigacin y Desarrollo de Alimentos Funcionales (CIDAF), Facultad de Farmacia, Universidad de Valparaso, Valparaso, Chile e VentureLab, Escuela de Negocios, Universidad Adolfo Ibez, Santiago de Chile, Chile f Unit de Recherche en Biologie Cellulaire Vgtale, Facults Universitaires Notre-Dame de la Paix, Namur, Belgium g Analytical and Inorganic Chemistry Department, Faculty of Chemistry, University of Concepcin, Casilla 160-C, Concepcin, Chile h Facultad de Ingeniera y Ciencias, Universidad Adolfo Ibez, Santiago de Chile, Chile i Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Madrid, Spain
b c

a r t i c l e

i n f o

a b s t r a c t
Cell walls polysaccharides from murta fruit (Ugni molinae Turcz), an endemic Chilean species with relevant food uses, were fractionated by water, ammonium oxalate, hot diluted HCl and cold diluted NaOH extractions. The polysaccharide fractions were analysed for monosaccharide composition and physicochemical properties. Pectic substances were found in all extracts, but mainly in the oxalate and acid soluble fractions, in which they appear as homogalacturonan polymers. Murta pectin was further extracted by hot diluted acid treatment using industrial conditions, yielding 30% by weight of dry fruit. The polymer showed similar composition and physicochemical properties to those of commercial citrus pectin, presenting a galacturonic acid content of 70.9% (w/w), a molecular weight of 597 kDa, and a methoxylation degree of 57%. The FT-IR spectrum of murta pectin suggests the presence of ferulic acid residues on its structure and the NMR analysis conrmed the structure of this polysaccharide. It is concluded that murta fruit can be considered as a valuable source of high quality pectin. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 6 November 2009 Received in revised form 26 March 2010 Accepted 4 May 2010

Keywords: Pectin Murta Ugni molinae Polysaccharide extraction Pectin characterisation

1. Introduction Pectins are complex heteropolysaccharides ubiquitously presented in the cell wall of land plants, providing consistency and mechanical resistance to vegetable tissues. The overall structure of these macromolecules encompasses homogalacturonan blocks (1,4-linked a-D-GalA units, which can be partially methylesteried) covalently linked to type I rhamnogalacturonan blocks (repeating disaccharide [ ? 4)-a-D-GalA-(1 ? 2)-a-L-Rha-(1 ? ] units) bearing neutral sugar side chains (Mohnen, 2008; Ridley, ONeill, & Mohnen, 2001). Pectin is a valuable functional food ingredient widely employed as gelling, emulsifying and stabilizing agent. It is used worldwide in jams and jellies, fruit juices, fruit drink concentrates, desserts, baking fruit preparations, dairy and delicatessen products (Koubala, Mbome, et al., 2008; Tsoga, Richardson, & Morris, 2004). Consumption of this polysaccharide, which constitutes a soluble bre component of fruits and vegetables, has important positive effects on
* Corresponding author. Tel.: +56 2 3311298; fax: +56 2 3311906. E-mail address: gustavo.cabrera@uai.cl (G. Cabrera). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.030

human health including lowering serum cholesterol and glucose levels (Behall & Reiser, 1986; Brown, Rosner, Willett, & Sacks, 1999; Yamada, 1996), reducing ulcer and cancer development (Jackson et al., 2007; Kiyohara et al., 1994; Nangia-Makker et al., 2002), and stimulating the immune response (Inngjerdingen et al., 2007). Despite its relevant uses in the food area, this polymer is also employed in the formulation of cosmetic and pharmaceutical products (Ahrabi, Madsen, Dyrstad, Sande, & Graffner, 2000; Willats, Knox, & Mikkelsen, 2006), as well as in enzyme immobilization (Gmez, Ramrez, Neira-Carrillo, & Villalonga, 2006; Gmez & Villalonga, 2000). Industry, traditionally uses citrus wastes (pulp and peel), apple pomace and sugar-beet pulp as raw material for pectin production (May, 1990). However, other sources can be potentially explored for producing this hydrocolloid, such as local industrial wastes and endogenous species, especially in developing countries. The Chilean guava, also known as murta or murtilla (Ugni molinae Turcz, Myrthaceae) is a wild native plant occurring in the downhill of the southern mountains of Chile. This plant has been historically appreciated because of the pleasant avour of its edible fruits, and has been also introduced in the UK and Australia for

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Fig. 1. Sequential extraction of pectic substances from murta fruits.

ornamental purposes. Due to its perennial shrub properties and applications in food manufacturing, it has been gaining attention from Chilean farmers, who have already domesticated this plant at large scale (Pastenes, Santa-Mara, Infante, & Franck, 2003). At present, murtilla fruits are industrially used for jam, jellies, tea and liquor production. In addition, the benecial effects of this plant on human health have been described previously (Aguirre et al., 2006). However, until now, the study of pectic polysaccharide fractions from this species, as well as their extraction conditions, has not been reported. The present work deals with the isolation and characterisation of cell wall polysaccharides from murta fruits. In a rst step, a sequential approach based on the extraction of pectic polysaccharides with different solvents, such as water, ammonium oxalate, HCl and NaOH (Fig. 1), was carried out. These polysaccharide fractions were characterised according to the extraction yield, pectin extractability, sugar composition, protein content, degree of methoxylation and average molecular weight. In a second step, the extraction of murta pectin using industrial conditions was evaluated, and its sugar composition and physicochemical properties was compared with commercial citrus and apple pectins. 2. Materials and methods 2.1. Materials Ripe light red murta (U. molinae Turcz) fruits were harvested in an orchard near Temuco City, La Araucania Region, south of Chile.

Citrus pectin was provided by Citrus Co. (USA) and apple pectin was obtained from Sigma (USA). Three samples of fresh murta fruit (100 g) were dried in a conventional air oven at 60 C for 24 h and the mean value of dry matter reported. 2.2. Preparation of alcohol insoluble solids (AIS) The fruits were grinded using a stainless steel knife mill obtaining a mixture of murta skin, pulp and seed. The skin and pulp were separated from seeds using a 1 mm sieve. The AIS was obtained by treating the skin and pulp mixture with ethanol (85%, v/v). The hydroalcoholic mixture was heated at 80 C for 15 min and the extraction was repeated three times using a solid/liquid ratio 1:3 (w/v). Finally, the mixture was ltered and the solid phase was dried at 40 C in a conventional oven during 12 h. The dry matter of AIS was obtained by drying 1 g of sample in a conventional air oven at 105 C for 24 h. Three replicates were used in this analysis and the mean value was reported. The moisture value was calculated from the AIS dry matter. 2.3. Sequential extraction of cell wall polysaccharides (Fig. 1) Different experimental conditions were employed for the sequential extraction of pectic substances from murta AIS: (1) distilled water at 20 C for 30 min; (2) 80 mM (NH4)2C2O4, pH 4.5 at 20 C for 30 min; (3) 50 mM HCl at 85 C for 30 min; and (4) 50 mM NaOH at 4 C for 30 min. Each sample was extracted three

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times with the same solvent under magnetic stirring; a solid/liquid ratio of 1:40 (w/v) was employed in all cases. The extracts were ltered from the solid residues and dialysed against distilled water during 3 days using Millipore dialysis membranes (MWCO 1000 Da). The extracts were then ltered through a 0.45 lm membrane (Sartorious, Germany) and further lyophilised. All samples were prepared in triplicate. 2.4. Murta pectin preparation using industrial conditions The murta AIS (100 g) was stirred with 4 l of 50 mM HCl solution at 85 C during 1 h. The mixture was ltered through a nylon cloth, and pectin was precipitated from the solution by adding 12 l of ethanol (96%, v/v). The precipitated polysaccharide was sequentially washed with 70%, 80%, 90% and 100% ethanol (v/v) and nally lyophilised. 2.5. Chemical analysis 2.5.1. Neutral sugars All samples were analysed by high performance anion exchange chromatography with pulsed amperometric detection (HPAECPAD). Total monosaccharide composition was determined after one step hydrolysis using 4% (v/v) H2SO4 at 120 C for AIS and 1 M H2SO4 at 100 C for pectin samples. Dried samples (20.0 1.0 mg) were mixed with the acidic solution (8.7 ml) and then heated in a hot plate for 1 h with magnetic stirring. Samples were diluted 10 times before injection in the HPAEC-PAD system. Chromatography of the samples was performed using a Dionex LC system coupled to an AS 50 autosampler. The HPAEC system was equipped with a CarboPac PA10 column (4 250 mm) in combination with a CarboPac guard column and run at 30 C. Separation was performed with a ow rate of 0.7 ml/min in the column and a total ow rate of 1 ml/min in the post-column using a combined gradient of two eluents prepared using degassed distilled HPLC grade water (Fisher Chemical): eluent A, 0.17 M sodium acetate in 0.2 M NaOH solution; eluent B, distilled water. Gradients of A and B were used in sequence to elute monosaccharides. This resulted in the following gradient: A 05 min, 100%; B 631.5 min. Samples (5 ll) were injected at 15.5 min. NaOH (0.3 M) was used post-column at a ow rate of 0.3 ml/min. The efuent was monitored using a pulsed-electrochemical detector in the pulse-amperometric mode with a gold working electrode and a Ag/AgCl reference electrode to which potentials of E1 = 0.1 V, E2 = 2.0 V, E3 = 0.6 V and E4 = 0.1 V were applied for duration times t1 = 0.4 s, t2 = 0.02 s, t3 = 0.01 s and t4 = 0.07 s, respectively. Quantication of the samples was performed using the response factors calculated from the peak areas of the mixed standard solutions for six sugars (glucose, mannose, rhamnose, galactose, arabinose and xylose). Fucose was used as internal standard. 2.5.2. Galacturonic acid, lignin and protein content The galacturonic acid content of the samples was determined colorimetrically by the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). Pure galacturonic acid monohydrate (purity >99%, Fluka Chemie AG, Buchs, Switzerland) was used as standard. Lignin was quantied by detergent analysis of the samples according to the Van Soest method (Van Soest, Robinson, & Lewis, 1991). Protein was estimated by Bradford method using bovine serum albumin as standard protein (Bradford, 1976). 2.6. Structural characterisation of pectin samples The degree of methoxylation of pectic substances was determined by acidbase titration according to the method described by Schultz (1965).

The average molar mass for different samples was measured by dynamic light scattering technique using a 90Plus Particle Size Analyser (Brookhaven Corp) equipped with a high power (35 mW) unit. For this, dilute solutions of each sample (0.1 g/ml) were prepared using sodium nitrate (0.1 mol/l) as solvent. Moreover, the samples were put in an ultrasonic bath and ltered through a syringe membrane lter with pore diameter of 0.45 lm. The time variation of the scattered intensity was measured at 90, and 5 runs were performed for each sample. All experiments were conducted at 25 C. In order to get the molar mass of the samples, a linear polysaccharide model of the Mark HouwinkSakurada equation was employed. Infrared spectra were measured on a Nicolet Magna 5PC Fourier transform infrared (FT-IR) spectrophotometer (USA) coupled to a personal computer with OMNIC analysis software. Pellets were prepared by blending pectin samples with KBr at 2% (w/w) concentration. Spectra were recorded at 4 cm1 of resolution and 64 scans were accumulated. Second derivative FT-IR spectrum was obtained using the OMNI analysis software. 2.7. NMR analysis 2.7.1. Partial hydrolysis of murta pectin (HP) Murta pectin (110 mg) was stirred in concentrated HCl (2 ml) at 20 C. After 120 min the solution was poured into acetone (10 ml) and the precipitate was washed three times with acetone (2 ml), centrifuged, and then dried in a non-vacuum glass desiccator over-night. The powder of the partially hydrolysed pectin (HP) (79.5 mg) was dissolved in HOD (1 ml) and then lyophilised. This operation was repeated three times. 2.7.2. NMR spectroscopy The 1H NMR (400.13 MHz) and 13C (100.62 MHz) spectra of the polysaccharides were recorded in D2O, after isotopic exchange (3 0.75 ml) at 80 C for HP derivative on a Bruker Avance DRX 400 spectrometer. All two-dimensional spectra were acquired using a pulse eld gradient incorporated into the NMR pulse sequence. The two-dimensional homonuclear 1H1H (COSY) spectra were acquired with 128 2040 data points, having a spectral width of 1085 Hz and were processed in a 1024 1024 matrix to give a nal resolution close to 2.3 Hz/point in the two dimensions. The number of scans in each experiment was dependent on the sample concentration. The chemical shifts were assigned relative to the HOD resonance.

3. Results and discussion 3.1. Alcohol insoluble solid Table 1 reports the AIS content and composition of the entire murta fruit. The fruit showed high moisture content (21%), and AIS represented 36.3% of the dry fruit. The total sugar content in AIS was estimated as 58.2%, representing 12.3 g/100 g dry matter in the whole fruit. Considering the high concentration of uronic acids in the AIS (24.3%), pectin seems to be the main macromolecular component in murta fruits. Neutral sugars represented 33.9% of the AIS, which also contains high amount of lignin (21.6%) and protein (15.8%). The acid/neutral sugars ratio in murta AIS was higher than those reported for apple (18.7/58.9) and Japanese quince (22/54) fruit (Bittner, Burritt, Moser, & Street, 1982; Thomas, Guillemin, Guillon, & Thibault, 2003). Glucose was the most abundant neutral sugar in murta AIS (20.5%), being probably associated with the presence of hemicelluloses, cellulose and different glucans in the fruit. Other neutral sugars were also found in minor

672 Table 1 AIS content and composition of murta fruits.a

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AIS Moisture (g/100 g fresh fruit) Dry matter (g/100 g fresh fruit) Yield (g AIS/100 g dry fruit) Lignin content (g/100 g AIS) Protein content (g/100 g AIS) Sugar composition (g/100 g AIS) GalA Ara Gal Rha Glc Xyl Man
a

21 1 79 1 36.3 0.2 21.6 0.4 15.8 0.7 24.3 0.6 4.8 0.1 4.5 0.2 0.8 0.1 20.5 0.7 2.1 0.1 1.2 0.2

tion conditions used. In this sense, higher content of uronic acids were found in the hot diluted acid and oxalate-soluble fractions. In order to quantify the effectiveness of these extractions, the pectin extractability (expressed as the amount of galacturonic acids extracted from the AIR) was estimated according to the following equation (Koubala, Mbome, et al., 2008):

Pectin extractability %

mGalAextract 100 mGalAAIS

Values are means SD of triplicate measurements.

quantities, and other constituents, such as minerals may also be present in the AIS prepared. 3.2. Sequential extract of AIS In order to determine the proportion and structural properties of the different pectic substances in the fruit, the AIS was sequentially extracted with water, ammonium oxalate, hot diluted HCl and cold diluted NaOH. It is well known that high methoxylated pectins, generally located in the middle lamella of the cell walls, can be easily extracted with water. On the other hand, chelating agents, such as oxalate ions solubilises low methoxylated pectin fractions by chelating calcium ions. Disruptive treatment with hot diluted acids yields pectin fractions with lower molecular weight due to cleavage of glycosidic bonds, and dilute alkali treatment release pectins from its ester derivatives with phenolic acids (man & Westerlund, 1996). Table 2 reports the yields, sugar composition and physicochemical properties of the pectin fractions extracted. The acid soluble fraction showed the highest extraction yield, which was 1.6-, 2.7and 13.5-fold higher than those achieved through water, oxalate and dilute alkali extraction, respectively. The extraction yield of the acid soluble pectin fraction was higher than that reported for tomato pectin (Sharma, Liptay, & Le Magner, 1998), but similar to that described for ambarella and mango pectins (Koubala, Kansci, et al., 2008; Koubala, Mbome, et al., 2008) under the same extraction conditions. Uronic acids are present as galacturonic acid in all extracted fractions, but concentration varies with the extrac-

where m(GalA)extract and m(GalA)AIS are the amount of galacturonic acid in the extracts and AIS, respectively. Pectin extractability was also signicantly higher for the diluted HCl treatment, and these facts suggest that acid extraction is the best approach for large scale production of murta pectin. The neutral sugar composition also varies with the extraction conditions. Water soluble polysaccharide fraction is enriched in neutral sugars (Fig. 2A), representing 59.2% of the total molar content of monosaccharides. Arabinose (25.6%), glucose (12.1%), galactose (9.0%) and rhamnose (8.4%) were found to be the predominant neutral sugars in the water-soluble fraction. A high Gal/Rha molar ratio of 4.8 (Fig. 2F) was found for this fraction, suggesting a high proportion of rhamnogalacturonic regions in this pectic polysaccharide. On the other hand, a relatively high (Ara + Gal)/GalA molar ratio of 0.85 was found in this fraction, which characterise highly branched pectins. According to the monosaccharide composition, it seems that glucans, arabinogalactans and other water-soluble polysaccharides were extracted along with this pectin. Additionally, the water-soluble fraction contained about 3.1% (w/w) of protein. As expected, the highest degree of methoxylation was found for this water-soluble fraction, which also showed a high average molecular weight. The oxalate-soluble fraction showed the highest molar concentration of GalA, as can be observed in Fig. 2B. Arabinose was the most abundant neutral sugar in this fraction, which showed very low (Ara + Gal)/GalA and high Gal/Rha molar ratios of 0.17 and 20.8, respectively. According to these ratios, pectin extracted with (NH4)2C2O4 should have a low proportion of rhamnogalacturonic regions with few neutral sugar side chains. It is known that the oxalate-soluble pectin fraction is the homogalacturonan fraction associated with calcium ions in the cell wall (man & Westerlund, 1996). In murta AIS, the yield of this fraction was lower than was in the water and acid fractions, which indicates the low proportion of this type of association in the murta cell wall. It is worth noting that the murta fruit is a highly brous fruit with a large postharvest life. Thus, even when fruits are harvested after ripening, it is ex-

Table 2 Yield, chemical composition, average molecular weight, degree of methoxylation and degree of acetylation of pectins extracted from murta fruits.a Extraction conditions H2O Yield (g/100 g AIS) Pectin extractability (%) Sugar compositionb GalA Ara Gal Rha Glc Xyl Man Protein (g/100 g AIS) Mw (kDa) DM (%, mol)
a b

(NH4)2C2O4 10 1 10.6 258 2 22.4 0.9 14.0 0.8 10.5 0.2 12.7 0.8 1.5 0.1 0.9 0.2 3.2 0.1 970 52

HCl 27 4 78.6 707 1 136.6 0.4 94.6 0.6 9.9 0.2 21.1 0.8 4.7 0.2 2.1 0.3 2.9 0.1 334 48

NaOH 21 0.4 39 1 10.6 0.1 146.4 0.3 12.2 0.1 87.0 0.6 87.6 0.4 35.1 0.2 3.9 0.1 132 3

AISR 44 2 4.1 23 1 2.9 0.7 28.1 0.5 3.4 0.4 436.0 3.0 39.2 0.2 21.9 0.1 2.4 0.3

17 2 6.1 87 1 42.2 0.7 17.8 0.5 15.2 0.7 23.9 0.5 4.6 0.6 2.7 0.4 3.1 0.2 429 62

Values are means SD of triplicate measurements. mg/g extract.

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Fig. 2. Molar composition of murta polysaccharides in H2O (A), (NH4)2C2O4 (B), HCl (C) and NaOH (D) extracts and AISR (E). Molar GalA/Rha ratios in these polymers (F).

pected that most pectin will be covalently linked to other cell wall polysaccharides, and drastic conditions must be employed in order to remove them. The oxalate-soluble pectin fraction also showed the highest average molecular weight, which was 2.2-, 2.9- and 7.4-fold higher than that corresponding to the water, hot diluted acid and cold diluted alkali fractions, respectively. The oxalate-soluble fraction showed a degree of methoxylation of 52% and a protein content of 3.2%. Arabinose and galactose were the predominant neutral sugars found in the acid soluble fraction, as can be observed in Fig. 2C. The GalA/Rha molar ratio of the acid soluble pectin fraction was signicantly higher than was that of the other treatments, as shown in Fig. 2F. This fact indicates a very low proportion of rhamnogalacturonic regions in this polymer, compared to the other

ones. In fact, the oxalate and diluted acid soluble pectin fractions can be classied as homogalacturonan, while rhamnogalacturonans-I are predominant in the other extracts (Schols & Voragen, 1996). This fraction also showed a low (Ara + Gal)/GalA molar ratio of 0.39, suggesting that the polymeric backbones are not extensively branched with neutral sugar chains. This fact is related to the drastic extraction conditions employed, which cause polymer debranches. Moreover, the disruptive nature of this extraction approach yielded pectin molecules with low average molecular weight, in comparison with those extracted with water and ammonium oxalate (Table 2). It should also be noted that the acid soluble fraction showed a degree of methoxylation of 48%, and a protein content of 2.9%. As can be observed in Table 2 and Fig. 2D, the alkali soluble fraction was enriched in neutral sugars, representing 91.7% of the total

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monosaccharide content. Galactose (33.6%), xylose (24.1%) and glucose (19.9%) were the predominant sugars found in this extract. Only a small amount of pectic substances were extracted in this fraction (pectin extractability about 0.4%), which should be mainly composed by hemicelluloses-related polysaccharides. Table 2 and Fig. 2E show that about 56% of the AIS were extracted through the sequential approach employed, leaving a residue mainly composed of neutral sugars, such as glucose (77.6%), xylose (8.4%) and mannose (3.9%). This composition suggests that the AISR fraction is enriched in cellulose and alkali-insoluble hemicelluloses. On the other hand, analysis of the uronic acids content in the AISR fraction revealed that about 98% of all pectic substances were extracted with the solvents used. 3.3. Characterisation of murta pectin extracted using industrial conditions Despite the relatively low average molecular weight showed by pectin extracted by the hot diluted acid method, this approach seems to be the most effective in terms of yield, extractability, and structural composition, as described above. For this reason, murta pectin was extracted under similar conditions to other industrial pectins (HCl pH 1.5 at 85 C) (May, 1990) and its structural composition was compared with known commercial samples. The yield of pectin extraction from murta fruit was 30% on a dry matter basis, while pectin extractability was estimated at 87.6%. This extraction yield was higher than those previously reported for apple (16%) (Rascn-Chu et al., 2009). Fig. 3A shows the FT-IR spectra of murta pectin along with commercial apple and citrus pectins. All spectra were similar, showing the characteristic bands at 3440 cm1 (t(OH)), 2930 cm1 (t(CH)), 1740 cm1 tC@OCOOMe;COO , 1640 cm1 tasCOO , 1435 cm1 tsCOO , 1240 cm1 (d(CH)), 1145 cm1 (t(COC) glycosidic bond,

ring), and 1100 cm1 (t(CC)) (Barros et al., 2002; Manrique & Lajolo, 2002). In Fig. 3B, the second derivative FT-IR spectra of murta pectin is shown. Several bands between 1700 and 1500 cm1 assigned to amide function of protein (Aguirre, Isaacs, Matsuhiro, Mendoza, & Ziga, 2009; Boulet, Williams, & Doco, 2007) can be observed. This nding agrees with the results from Table 2, showing the presence of small amount of protein in the pectin fractions. These amide I and II bands usually overlap with the vibration of uronic acid carboxylate groups and also with the aromatic ring urkov et al., 1999). vibrational bands from lignin residues (Kac In this spectrum, the bands at 1519.1 cm1 (t (CC) aromatic ring) and 1049.1 cm1 (t (OCH3)) suggest the presence of small amounts of ferulic acid linked to murta pectin (Sebastiana, Sundaraganesana, & Manoharanb, 2009). It has been demonstrated that

Table 3 Chemical composition, average molecular weigh and degree of methoxylation of pectins from murta, apple and citrus fruits.a Pectin Murta Sugar compositionb GalA Ara Gal Rha Glc Xyl Man Mw (kDa) DM (%, mol)
a b

Apple 695.3 0.8 14.1 0.3 28.5 0.8 8.7 0.1 7.9 0.9 2.9 0.4 1.2 0.2 2030 69

Citrus 743.2 0.5 23.1 0.8 66.1 0.5 8.5 0.1 17.8 0.5 1.1 0.1 0.7 0.1 615 63

709.4 0.6 22.0 0.2 54.9 0.7 9.1 0.1 14.0 0.2 3.3 0.2 1.0 0.4 597 57

Values are means SD of triplicate measurements. mg/g extract.

Fig. 3. (A) FT-IR spectra of pectin samples from different sources: murta (A), apple (B) and citrus (C) pectins and (B) second derivative FT-IR spectra of murta pectin.

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Fig. 4. Molar composition of murta (A), apple (B) and citrus (C) pectins, and molar Gal/Rha ratios in these polysaccharides (D).

sugar-beet pectin also contains signicant amount of ferulates on its structure (Oosterveld, Grabber, Beldman, Ralph, & Voragen, 1997). These analyses conrm the pectic nature of the extracted polysaccharide. Table 3 reports the monosaccharide composition and structural properties of murta pectin, as well as the pectin derived from apple and citrus fruits. The galacturonic acid content in murta pectin was similar to those in the commercial samples. This uronic acid content was also higher than that reported for yellow passion fruit pectins isolated by alcohol precipitation or dialysis (Yapo, 2009). Galactose, arabinose and glucose are the predominant neutral sugars in these polymers; similar molar pattern for monosaccharide distribution were found for murta compared to the commercial samples (Fig. 4). The GalA/Rha molar ratio of murta pectin was high and similar to those for commercial apple and citrus polysaccharides (Fig. 4D), suggesting that these pectins contained low proportion of rhamnogalacturonic regions. Murta, apple and citrus pectins showed (Ara + Gal)/GalA molar ratios of 0.12, 0.07 and 0.14, respectively. For this reason, a low degree of neutral sugar branching for these polymers is expected. Pectin extracted from murta fruits showed an average molecular weight of 597 kDa, which was higher than the pectin in the acid soluble fraction, previously described. This fact conrms that some of the pectic substances that were previously extracted in the water and oxalate-soluble fractions were now co-extracted under the hot diluted acid treatment. The average molecular weight of murta pectin was similar to that of the citrus polymer, but significantly lower than apple pectin. The molecular weight of this polysaccharide was also higher than that reported for ambarella, mango and lime pectins (Koubala, Kansci, et al., 2008). The degree of methoxylation of the extracted pectin was estimated to be 57%, indicating that the polysaccharide was of the

high-methoxyl type (Thakur, Singh, & Handa, 1997). Similar degree of methyl esterication was observed for citrus pectin.

3.4. NMR analysis of murta pectin Ambiguous information about the murta pectin structure was obtained from 1H and 13C NMR spectra (gure not shown); for this reason, partially hydrolysed pectin (HP) was obtained and analysed. The yield of the partially hydrolysed pectin (72.3%) may be related to the removal of neutral sugars and protein residues present in the original sample of pectin. In fact, it is known that acid hydrolysis of pectin could cause polymer debranching, which in turn would improve the NMR spectra quality. The 1H NMR spectrum of the HP sample (Fig. 5A) shows the anomeric H-1 (d 5.06 ppm) and H-5 (d 4.82 ppm) proton signals from non-esteried galacturonic acid residues, which are in agreement with previous reports, where the H-5 protons in pectin samples having free carboxylic acid groups were found around d 4.84.9 ppm (Marcon, Carneiro, Wosiacki, Beleski-Carneiro, & Petkowicz, 2005; Rosenbohm, Lundt, Christensen, & Young, 2003). By other side, the H-1 and H-5 protons from esteried carboxyl groups in the galacturonic acid residues appear as two overlapped signals around d 4.9 ppm. Signals due to the H-2, H-3 and H-4 protons of the HP sample were assigned with the aid of a COSY spectrum (Fig. 5B). The H-4 protons signal overlapped with the HOD signal (d 4.3 ppm), whereas the H-3 and H-2 appeared at d 3.87 and 3.62 ppm, respectively. The spectra of this sample contained a sharp signal at d 3.67 ppm, which could be assigned to the protons of the methoxyl group from the esteried units of galacturonic acid (Bdouet, Courtois, & Courtois, 2003; Rosenbohm et al., 2003; Winning, Viereck, Nrgaard, Larsen, & Engelsena, 2007).

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Fig. 5. (A) 1H NMR spectrum of partially hydrolysed pectin and (B) 1H1H COSY RMN spectrum.

Fig. 6.

13

C NMR spectrum of partially hydrolysed pectin.

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The 13C NMR spectrum of HP (Fig. 6) did not show the signals assigned to arabinofuranosyl residues, which may indicate that this monosaccharide is part of the pectin hairy region (Aguirre et al., 2009; Marcon et al., 2005). In the low eld region, it showed two major signals assignable to the C-6 of the carboxyl group of the galacturonic acid units at d 174.9 ppm (esteried) and d 173.6 ppm (non-esteried). The anomeric region signals showed signals at d 103.7 and 102.0 ppm which were assigned to the esteried and non-esteried carboxyl groups of the galacturonic acid units. The signals at d 80.5 (C-4), 73.9 (C-5), 71.6 (C-3) and 70.2 (C-2), were assigned to galacturonic acid units. The signal at d 55.1 ppm corresponds to the O-methyl ester groups (O-Me) at C-6 (Marcon et al., 2005). Overall, NMR spectroscopy was a useful technique for structural characterisation of the murta pectin. 4. Conclusions The present work described the sequential extraction of cell wall polysaccharides from murta fruits, demonstrating the presence of pectic substances in all extracts. The oxalate and acid soluble fractions were mainly enriched in homogalacturonan-type pectins, which were recovered in high yield. Murta pectin, prepared by hot diluted acid extraction, showed high molecular weight, low neutral sugars content, and high degree of methoxylation. The presence of ferulic acid on murta pectin was suggested by second derivative FT-IR analysis; however, further analysis will have to be conducted in order to conrm this nding. 13C and 1H NMR spectroscopy conrmed the presence of uronic acids in the free and methyl ester forms in murta pectin. Finally, the chemical composition of pectin extracted from murta fruit was similar to commercial citrus pectin. Additional experiments testing different extraction conditions of murta pectin, as well as their jellifying properties, are currently in progress. Acknowledgements G. Cabrera and E. Taboada would like to acknowledge the Innova Chile Grant number 08CT11PUT-23 for their nancial support for this work. R. Villalonga acknowledges the Ramn & Cajal contract from the Spanish Ministry of Science and Innovation. E. Pereira would like to thank Centro de Investigacin de Polmeros Avanzados (CIPA-Chile). References
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