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Emulsifying and structural properties of pectin enzymatically extracted from pumpkin
LWT - Food Science and Technology xxx (2014) 1e8
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Emulsifying and structural properties of pectin enzymatically extracted from pumpkin Steve W. Cui a, Yoon Hyuk Chang b, * a b
Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada Department of Food and Nutrition, Kyung Hee University, Seoul, 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 31 March 2014 Accepted 6 April 2014 Available online xxx
The present study investigated the emulsifying and structural properties of pectin enzymatically extracted from pumpkin. Pumpkin pectin fraction A was obtained from raw pumpkin with an enzymatic preparation of cellulase and a-amylase. Pumpkin pectin fraction B was achieved by treating the fraction A solution with pronase to reduce protein content. According to the findings (on protein content, galacturonic acid content, neutral sugar composition, and molecular weight distribution), the pronase treatment could remove protein from the fraction A without considerably influencing any other chemical and molecular properties. Moreover, the fraction A exhibited emulsifying properties in water and oil mixture, whereas the removal of protein in the fraction B resulted in the loss of emulsifying properties. The FT-IR and 1D NMR analysis revealed that the backbone of pumpkin pectin is mainly composed of a1,4-D-galacturonic acid in which a considerable portion of galacturonic acid residues is present as methyl esters, and some L-rhamnose are involved in the linear region of the backbone through a-1,2-linkages. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Keywords: Pumpkin pectin Emulsifying properties Structural characterization
1. Introduction Pectin, the most abundant polysaccharides in the middle lamella and cell wall of many higher plants, has been widely used in the food industry as a gelling agent, thickener, and stabilizer of jam, jelly, and acid milk products (Singthong, Cui, Ningsanond, & Goff, 2004; Tamaki, Konishi, Fukuta, & Tako, 2008). The identification of the molecular structure for pectin is of fundamental importance because the physicochemical and functional properties of pectin are highly dependent on its structure. However, pectin is very heterogeneous in its composition, which renders the structural study of the polysaccharides extremely challenging. Pectin is considered as an alternation of homogalacturonan regions, generally called “smooth regions”, and rhamnogalacturonan regions I, also called “hairy regions” (Waldron, Parker, & Smith, 2003). Homogalacturonan is a repetition of (1 / 4)-linked a-Dgalactopyranosyluronic acid (GalpA) residues. Rhamnogalacturonan region I is constituted by an alternating sequence of (1 / 4)linked a-D-GalpA and (1 / 2)-linked a-L-rhamnopyranose (Rhap) residues. Various side chains (arabinan, galactan, and arabinogalactan) can be linked to the rhamnose residues (Vincken et al., 2003; Voragen, Pilnik, Thibault, Axelos, & Renard, 1995). * Corresponding author. Tel.: þ82 2 960 0264; fax: þ82 2 961 0261. E-mail address: [email protected] (Y.H. Chang).
Citrus peel and apple pomace are the major raw materials used for the production of commercially acceptable pectin. Other raw materials, including pumpkin (Matora et al., 1995), sugar beet (Levigne, Ralet, & Thibault, 2002), sunflower (Iglesias & Lozano, 2004), honey pomelo (Guo et al., 2014), and papaya peels (Koubala, Christiaens, Kansci, Loey, & Hendrickx, 2014) have been considered as possible pectin sources and their potential has been discussed. Several studies on the extraction of pumpkin pectin have been performed using different extraction methods, such as an acidic extraction method and enzymatic extraction method (Fissore, Ponce, Stortz, Rojas, & Gerschenson, 2007; Matora et al., 1995; Ptichkina, Markina, & Rumyantseva, 2008; Shkodina, Zeltser, Selivanov, & Ignatov, 1998). Matora et al. (1995) observed that the enzyme extraction method (multi-enzyme culture supernatants from various strains of Bacillus polymyxa) gave much bigger yields of pumpkin pectin than did the acid extraction method (0.1 M HCl). Moreover, the current acid pectin extraction methods not only do not allow pectin to be extracted fully with no damage to its structure, but also lead to the environmental concerns due to acid usage (Ptichkina et al., 2008). Different enzymes, like cellulase, hemicellulase, protease, amylase, and so on, have been employed for the enzymatic extraction of pumpkin pectin (Fissore et al., 2007; Shkodina et al., 1998). Shkodina et al. (1998) employed cellulase (Trichoderma
http://dx.doi.org/10.1016/j.lwt.2014.04.012 0023-6438/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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S.W. Cui, Y.H. Chang / LWT - Food Science and Technology xxx (2014) 1e8
viride) or hemicellulase (Aspergillus niger)] for the extraction of pectin from pumpkin pulp (following juice extraction), and reported that the cellullase treatment led to an increase in pectin yields and in galacturonic acid contents, as compared to that of the hemicellulose treatment. The same result was also observed in the literature performed by Fissore et al. (2007), who found that the amounts of galacturonic acid in pumpkin pectin obtained from cellulase were greater than those obtained from hemicellulase. The emulsifying properties of pectin have been demonstrated in a few literatures (Dickinson, 2003; Funami et al., 2011; Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Siew, Williams, Cui, & Wang, 2008). A small percentage of hydrophobic protein (about 2%) in sugar beet pectin plays major role in stabilizing oil-inwater emulsions (Siew et al., 2008). Leroux et al. (2003) also noted that beet pectin and citrus pectin may efficiently decrease the interfacial tension between oil and water phase in the emulsions; however, the emulsion stabilizing features of pectin change due to the contents difference of calcium ions, acetyl groups, and proteins. As far as the authors are aware, no studies have apparently reported the effect of pronase treatment on the emulsifying properties of pumpkin pectin, nor have elucidated the structural features of pectin enzymatically extracted from pumpkin. In the present study, pectin fraction A was obtained from raw pumpkin using cellulase and a-amylase, and then pumpkin pectin fraction B was produced after pronase treatment with the fraction A. The main objectives of the present study are to (1) evaluate the potential of pumpkin for the enzymatic extraction of pectin and (2) investigate the emulsifying and structural features of pectin enzymatically extracted from pumpkin. 2. Materials and methods 2.1. Materials Raw pumpkin (Cucurbita mixta), harvested in ON, Canada, was used in the present study. Cellulase from Trichoderma viride (5 U/ mg) and a-amylase from Bacillus licheniformis (3500 U/mL) were obtained from Sigma (Oakville, ON, Canada). Pronase from Streptomyces griseus (7.0 U/mg) was provided by Roche Molecular Biochemicals (Indianapolis, USA). Standards for rhamnose, arabinose, galactose, glucose, xylose, mannose, and galacturonic acid were purchased from Sigma (Oakville, ON, Canada). All chemicals were of reagent grade unless otherwise specified. 2.2. Enzymatic extraction for the production of pumpkin pectin fractions A and B Pumpkin pectin fractions A and B were extracted from raw pumpkin using the procedure described by Fissore et al. (2007) with some modifications. Raw pumpkin was first cut, and the seeds and skin were removed. The remainder was reduced to fragments (about 4 cm 0.5 cm), freeze-dried, and ground to make dried pumpkin powder. Fifty g of pumpkin powder were suspended in 1.5 L of 50 mmol/L sodium citrate buffer (pH 5.2), and then cellulase (600 mg) and a-amylase (500 mg) were added. Our preliminary study indicated that starch contents of dried pumpkin powder were 7.93 g/100 g dry solids; therefore, a-amylase was employed to remove starch existed in pumpkin powder. The mixture was stirred at 30 C for 20 h and centrifuged at 15,000 g for 30 min. The supernatant was collected. The remaining pellet was resuspended in 100 mL of distilled water and subjected to a further centrifugation. The supernatants were combined and treated with two volumes of absolute ethanol (Sigma, Oakville, ON, Canada). The resulting precipitate was collected, solubilised in
distilled water, freeze-dried, and ground. This procedure produced pumpkin pectin fraction A. To produce pumpkin pectin fraction B, 0.5 g/100 mL solution of pumpkin pectin fraction A was stirred with pronase for 60 min at 50 C to reduce protein content. The solution was heated at 85 C for 30 min, cooled, and centrifuged at 15,000 g for 20 min. The supernatant was collected and then treated with two volumes of absolute ethanol (Sigma, Oakville, ON, Canada). The resulting precipitate was collected, solubilised in distilled water, freeze-dried, and ground. The resulting protein-reduced pectin fraction was termed as pumpkin pectin fraction B. The details of enzymatic extraction for pumpkin pectin fractions A and B are summarised in Fig. 1. 2.3. Moisture content, protein content, galacturonic acid content, degree of esterification, and degree of acetylation Pumpkin pectin fraction A and fraction B were analysed for their moisture contents using AACCI Approved Method 44-15A (AACCI, 2000). The protein contents of pumpkin fraction A and fraction B were analysed by NA2100 Nitrogen and Protein Analyser (Strada Rivoltana, Milan, Italy) using the factor of 5.7 to convert measured nitrogen to protein. The galacturonic acid contents of pumpkin pectin fraction A and fraction B were determined by the mhydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). Standard galacturonic acid solutions (10e100 mg/mL) were used to construct the standard curve for the determination. The degree of esterification (DE) and degree of acetylation for the fractions A and B were determined by the titrimetric method of Food Chemical Codex (FCC, 1981, pp. 283e286) and the method of Matora et al. (1995), respectively. 2.4. Neutral sugar composition The neutral sugar composition of pumpkin pectin fraction A and fraction B was determined by modifying the procedure of Wood, Weisz, and Blackwell (1994). Each sample was hydrolysed in 1 mol/L H2SO4 at 100 C for 4 h and diluted 20 times. The diluted samples were passed through a 0.45 mm filter and injected to a high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex-5500, Dionex Corporation, Canada). Separation of each neutral sugar was performed on a CarboPac PA1 column (250 4 mm I.D., Dionex Corporation, Canada) and a guard column (3 25 mm, Dionex Corporation, Canada). The solvents were A: 100 mmol/L NaOH, B: 300 mmol/L NaOH, and C: water (distilled water filtered through Nanopure Infinity, Model: D8971, Dubuque, IA, USA). Elution was with 8% A and 92% C for 7 min, then with a gradient to 100% eluent C for 28 min. The column system was cleaned after each analysis with 100% eluent B for 15 min. After cleaning, the initial conditions were maintained for 10 min between each injection of sample. The solvent flow rate was 1.0 mL/min and the injection volume was 50 mL. A post-column delivery system of 600 mmol/L NaOH with a flowrate of 0.5 mL/min was added to the HPAEC-PAD system. Standard solutions of the individual neutral sugar (rhamnose, arabinose, galactose, glucose, xylose, and mannose) were used at varying concentrations (10e50 mg/mL) for identification and quantification. The instrument was controlled and data were processed using Dionex AI 450 software (Dionex Corporation, Canada). 2.5. Molecular weight distribution The molecular weight distribution of pumpkin pectin fraction A and fraction B was evaluated by high performance size exclusion chromatography (HPSEC) equipped with a refractive index (RI)
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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Raw pumpkin Cutting freeze-drying grinding
Pumpkin powder Sodium citrate buffer (pH 5.2) cellulase α-amylase centrifuge
Residue
Supernatant Ethanol precipitation solubilization freeze-drying
Fraction A Solubilization pronase centrifuge
Residue
Supernatant Ethanol precipitation solubilization freeze-drying
Fraction B Fig. 1. Scheme of enzymatic extraction for pumpkin pectin fraction A and fraction B from raw pumpkin.
detector (Model Dual 250, Viscotek, Houston, TX, USA). The chromatographic system includes a Shimadzu SCL-10Avp pump, automatic injector (Shimadzu Scientific Instruments Inc., Columbia, MD, USA), and two columns in series: a Shodex OHpak KB-806M (Showa Denko K.K., Tokyo, Japan) and an Ultrahydrogel linear (Waters, Milford, CT, USA). The columns and RI detector were maintained at 40 C. The mobile phase, injection volume, and flow rate were 0.1 mol/L NaNO3, 100 mL, and 0.6 mL/min, respectively. Each sample was solubilized in distilled water at 90 C for 1 h, cooled, and filtered through a 0.45 mm filter prior to injection onto the column. The molecular weight distribution was estimated by the pullulan standards (molecular weights: 10, 20, 50, and 400 kDa) (Sigma, Oakville, ON, Canada). 2.6. Surface tension The surface tension of pumpkin pectin fraction A, fraction B, and pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) was determined using a semi-automatic surface tensionmeter model Surface Tensionmat 21 (Fisher Scientific, Toronto, Canada) at 21e22 C. This tensionmeter employed the Du Nouy ring method to measure static surface tension. Distilled water was used to calibrate the tensionmeter for surface tension measurements. Sample concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 g/100 mL were used for surface tension measurements.
2.7. Emulsion capacity and stability The emulsion capacity and emulsion stability of pumpkin pectin fraction A, fraction B, and pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) were investigated according to Yasumatsu et al. (1972) with some modifications. The sample (0.01 g) was suspended in 10 mL of distilled water before mixing with 10 mL of vegetable oil. The water/oil mixture was emulsified using a homogenizer at 10,000 rpm for 1 min, and then the mixture was centrifuged at 1300 g for 5 min. Emulsion capacity was calculated as follows:
ðHeight of emulsion layer=total height of fluidÞ 100%
(1)
Emulsion stability was determined by heating the emulsion at 80 C for 30 min, cooling with tap water for 15 min, and centrifuging at 1300 g for 5 min. Emulsion stability was calculated as follows:
ðHeight of remainingemulsion layer=total heightof fluidÞ 100%
(2)
2.8. Structural analysis Only pumpkin pectin fraction B was used to analyse the structural characteristics because pectin with little or no proteins is better for the structural analysis.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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2.8.1. Fourier transform-infrared spectroscopy The pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) and pumpkin pectin fraction B were dried in a vacuum oven for 5 h and stored in a desiccator prior to Fourier transforminfrared (FT-IR) analysis. FT-IR spectra of each sample were obtained according to the procedure of Singthong et al. (2004). 2.8.2. 1H and 13C NMR spectroscopy Pumpkin pectin fraction B was dissolved in deuterium oxide (D2O, 80 C, 1 h) and freeze-dried for three times to replace the exchangeable protons with deuterons before being finally redissolved in D2O (3 g/100 mL) for NMR analysis. High-resolution 1H and 13C NMR spectra were recorded in D2O at 500.13 and 125.78 MHz, respectively, on a Bruker AM500 NMR spectrometer operating at 25 C. A 5 mm inverse geometry 1H/13C/15N probe was used. The chemical shifts were reported relative to external standards, trimenthysily propionate (TSP in D2O, 4.76 ppm, for 1H) and 1,4-dioxane (in D2O, 66.5 ppm, for 13C). 2.9. Statistical analysis All statistical analyses were performed using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was performed using the general linear models (GLM) procedure to determine significant differences among the samples. Means were compared by using Fisher’s least significant difference (LSD) procedure. Significance was defined at the 5% level. 3. Results and discussion 3.1. Chemical composition The yields of pumpkin pectin fractions A and B were calculated as a percentage of the weight of dried pumpkin powder, which were 10.03 and 8.08 g/100 g, respectively. The protein content (derived from nitrogen) of pumpkin pectin fraction A was 4.55 g/ 100 g, while no nitrogen was detected in pumpkin pectin fraction B (Table 1). The DE values (about 47%) of pumpkin pectin fractions A and B were not significantly different. The galacturonic acid contents of pumpkin fractions A and B represent 75.02 and 78.22 g/ 100 g, respectively. This finding indicated that both of the fractions are mainly composed of homogalacturonan (Vincken et al., 2003; Waldron et al., 2003). Small amounts (about 10 g/100 g) of six different neutral sugars were found in both of the pectin fractions, including rhamnose, arabinose, galactose, glucose, xylose, and mannose (Table 1). The presence of rhamnose, galactose, and arabinose in the pumpkin pectin fractions could indicate the existence of rhamnogalacturonan I with side chains, such as arabinan, galactan, and arabinogalactan (Tamaki et al., 2008; Wu, Ai, Wu, & Cui, 2013). The presence of glucose and xylose in the pumpkin pectin fractions could explain the presence of xyloglucan as one of the side chains and the presence of xyloglacturonan (Fissore et al., 2007; Kost’álová, Hromádková, & Ebringerová, 2013; Vincken et al., 2003). More specific structural analysis for pumpkin pectin will be discussed in the next section.
Table 1 Chemical composition of pumpkin pectin fraction A and fraction B.a Pectin sample Fraction A Moisture (g/100 g) Protein (g/100 g) Degree of esterification (%) Degree of acetylation (%) Galacturonic acid (g/100 g) Neutral sugar (g/100 g) Rhamnose Arabinose Galactose Glucose Xylose Mannose
Fraction B a
9.61 4.58 47.30 6.87 75.02
0.27 0.32 0.12a 0.22a 0.93b
9.65 0.12a NDþ 47.10 0.15a 6.79 0.20a 78.22 0.45a
1.73 1.77 3.15 2.26 0.41 0.31
0.09a 0.09a 0.03a 0.01a 0.02a 0.00a
1.72 1.78 3.15 2.24 0.41 0.30
0.04a 0.04a 0.05a 0.03a 0.04a 0.05a
þ
ND: not detectable. a Results are the means of two determinations standard deviation. Values with different letters within the same row differ significantly (P < 0.05).
galactomannans. In the present study, the observations (on protein content, galacturonic acid content, neutral sugar composition, and molecular weight distribution) suggest that the treatment of pronase could selectively remove the residual proteinaceous components from pumpkin pectin without considerably influencing any other chemical and molecular properties. 3.3. Surface tension Changes in surface tension of water by pumpkin pectin fraction A and fraction B with different concentrations (0.05e1.00 g/100 g) at 21e22 C are shown in Fig. 3. The surface tension of water was 0.0723 N/m. Adding pumpkin pectin fraction A, pumpkin pectin fraction B, or pectin standard at all the concentrations studied into water led to a decrease in the surface tension of water, indicating that the pectin fractions A and B as well as pectin standard have the surface activity. Moreover, the surface tension of water decreased with increasing the concentration of pumpkin pectin fractions (fraction A from 0 to 0.2 g/100 g; fraction B from 0 to 0.1 g/100 g) and finally reached a plateau. Similar trend was also found in other polysaccharide samples, such as arabinoxylan and arabinogalactan (Izydorczyk, Biliaderis, & Bushuk, 1991), Portulaca oleracea gum (Garti, Slavin, & Aserin, 1999), and fenugreek gum (Brummer et al., 2003).
3.2. Molecular weight distribution The molecular weight distribution of pumpkin pectin fraction A and fraction B was very similar (Fig. 2). The finding was consistent with Brummer, Cui, and Wang (2003), who applied pronase for the purification of fenugreek gum. They found that treating fenugreek gum solution with pronase considerably decreased the protein content of fenugreek gum from 2.36 to 0.57 g/100 g without affecting the molecular weight and galactose/mannose ratio of the
Fig. 2. Molecular weight distribution of pumpkin pectin fraction A (–) and fraction B (d).
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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Table 2 Emulsion capacity and stability of pumpkin pectin fraction A, fraction B, and pectin standard.a Pectin sample
Emulsifying property Emulsion capacity (%)
Fraction A Fraction B Pectin standard
a
63.7 2.0 NDþ 62.5 0.4a
Emulsion stability (%) 58.3 0.2a ND 58.0 0.3a
þ
ND: not detectable. a Results are the means of two determinations standard deviation. Values with different letters within the same column differ significantly (P < 0.05).
Fig. 3. Changes in surface tension of water by pumpkin pectin fraction A (,), fraction B (>), and pectin standard (6) with different concentrations at 21e22 C.
In the present study, the surface tension values of water for pumpkin pectin fraction A at all the concentrations were lower than those for pectin standard, indicating that the effect of fraction A on the surface activity of water was superior to that of pectin standard. In particular, the surface tension values of water for pumpkin pectin fraction A at all the concentrations were remarkably lower than those for pumpkin pectin fraction B. This finding suggests that pumpkin pectin fraction A had better capability to decrease the surface tension of water than did pumpkin pectin fraction B. Until now, the effects of residual protein existing in polysaccharides on the surface activity of water are still controversial. For instance, Garti, Madar, Aserin, and Sternheim (1997) studied the surface activity of non-purified fenugreek gum and purified fenugreek gum which was obtained by physical separation of contaminating protein from the non-purified fenugreek gum. They reported that reducing the protein content did not influence the surface tension of water. On the other hand, Brummer et al. (2003) used a purification step to remove the contaminating protein from fenugreek gum with pronase, and they compared the surface activity of the non-purified fenugreek gum and purified fenugreek gum. According to them (Brummer et al., 2003), the surface tension of water for non-purified fenugreek gum was much lower than that for purified fenugreek gum, explaining that the proteins closely associated with the galactomannans can enhance the surface activity of fenugreek gum. Dickinson, Murray, Stainsby, and Anderson (1988) also showed positive correlation between the surface activity and protein content of Acacia gum (nitrogen contents in the range from 0.1 to 7.5 g/100 g). Thus, it is inferred in the present study that the existence of the residual proteinaceous matters in pumpkin pectin can play a major functional role in improving its surface activity.
polysaccharides can adsorb at oilewater interfaces to form stabilizing layers around oil droplets (Siew et al., 2008). Leroux et al. (2003) noted that the emulsifying ability of beet pectin and citrus pectin is due to a small amount of protein which is covalently bound to a highly branched polysaccharide structure. Therefore, in the present study, it is evident that the emulsifying properties of pumpkin pectin fraction A can be considerably related to the presence of the residual hydrophobic proteinaceous components in the pectin structure. 3.5. Structural characterization 3.5.1. Fourier transform-infrared spectroscopy The FT-IR spectrum of pumpkin pectin fraction B was similar to that of pectin standard (citrus pectin), as illustrated in Fig. 4. In particular, both of pumpkin pectin fraction and pectin standard
3.4. Emulsion capacity and stability The emulsion capacity and stability of pumpkin pectin fraction A were 63.7 and 58.3%, respectively, and were not significantly different from those of pectin standard while both of them were not detected in pumpkin pectin fraction B (Table 2). In general, it has been widely known that many commercial polysaccharide samples include a small amount of protein, either as a contaminant or as an intrinsic part of the polysaccharide structure. The protein components can provide the polysaccharides with good emulsifying ability, since those are usually strongly hydrophobic (Dickinson, 2003). That is, the hydrophobic protein components in the
Fig. 4. Fourier transform-infrared spectra of pumpkin pectin fraction B and pectin standard.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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Fig. 5. 1H NMR spectrum of pumpkin pectin fraction B.
exhibited strong absorbances between 950 and 1200 cm1 wave number regions. The regions between 950 and 1200 cm1, generally called as the “finger print” region for each polysaccharide, provide information regarding the identification of major chemical groups in polysaccharides since the position and intensity of the individual band in the regions are unique for each polysaccharide (Kalapathy & Proctor, 2001). According to Kost’álová et al. (2013), the fingerprint region of pumpkin pectin represented the characteristic bands at 1145, 1103, 1077, 1050, and 1017 cm1. Thus, it is confirmed in the present study that pectin was successfully extracted from pumpkin using our enzymatic extraction method (amylase, cellulase, and pronase). Chatjigakis et al. (1998) reported that the regions between 1500 and 1800 cm1 wave number are the most important regions for the examination of structural features of pectin because carboxylic acid and carboxylic ester groups of pectin are found in the regions. More specifically, according to the work conducted by Manrique and Lajolo (2002), the band at 1630 cm1 wave number regions indicated the ionized carboxylate groups (COO), whereas the band at 1740 cm1 wave number regions was assigned to carbonyl groups (C]O) from COOCH3 groups. Furthermore, the bands at 1730 and 1600 cm1 correspond to esterified carboxylic acid groups and nonesterified carboxylic acid groups, respectively (Singthong et al., 2004; Winning, Viereck, Salomonsen, Larsen, & Engelsen, 2009). Therefore, in the present study, the occurrence of two distinguished bands at 1730 and 1600 cm1 apparently indicated the nonesterifed and esterified carboxyl groups of galacturonic acid in pumpkin pectin. 3.5.2. 1H and 13C NMR analysis The 1H NMR spectrum of pumpkin pectin fraction B is shown in Fig. 5. From the data, it was possible to distinguish and assign the resonances originating from a- and b-configured residues. In the anomeric region, the strong signals at 5.08 and 5.11 ppm were assigned to H-1 of 4-linked a-D-GalpA and H-1 of 2elinked a-LRhap, respectively (Tamaki et al., 2008; Wu et al., 2013). The signals at 4.45 and 5.17 ppm were attributed to H-1 of b-galactose and aarabinose, respectively (Duan, Wang, Dong, Fang, & Li, 2003; Golovchenko, Ovodova, Shashkov, & Ovodov, 2002). The very large signal at 3.81 ppm was derived from methyl groups binding to carboxyl groups of 4-linked a-D-GalpA (Cozzolino
et al., 2006; Kost’álová et al., 2013; Tamaki et al., 2008). Two signals at around 2.15 ppm were attributed to acetyl groups binding at O-2 and O-3 of GalpA. The identification of the acetyl groups was achieved by comparison of the observed spectrum against literature values (Bédouet, Courtois, & Courtois, 2003; Cozzolino et al., 2006; Kost’álová et al., 2013; Perrone et al., 2002). The methyl rhamnose signals at 1.13 and 1.25 ppm were associated with the 1,2-linked and 1,2,4-linked Rhap (Perrone et al., 2002; Wu et al., 2013). The 13C NMR spectrum of pumpkin pectin fraction B explained the resonances from C-1 of 4-linked a-D-GalpA at 100.2 and 101.0 ppm which were the most intensive in the anomeric region (Fig. 6). Both of the signals can be related to C-1 of nonesterifed and esterified carboxyl groups of 4-linked a-D-GalpA, respectively, by comparison with literature values of the anomeric chemical shifts (Cozzolino et al., 2006; Westerlund, Åman, Andersson, & Andersson, 1991). The respective corresponding typical resonances of C-6 of the carboxyl groups of 4-linked a-D-GalpA were observed at 171.7 and 171.5 ppm, and the existence of two carboxyl signals confirmed the presence of nonesterifed and esterified carboxyl groups of 4-linked a-D-GalpA (Cozzolino et al., 2006; Wu et al., 2013). The carbon signals at 21.08 ppm were assigned to CH3 of the O-acetyl groups (Westerlund et al., 1991). Based on the findings obtained from the galacturonic acid content, neutral sugar composition, FT-IR spectra, and positions of signals in the 1H and 13C NMR spectra of pumpkin pectin, it is suggested in the present study that the backbone of pumpkin pectin proved to mainly represent linear a-1,4-D-galacturonic acid in which a considerable portion of galacturonic acid residues is present as methyl esters, and some L-rhamnose are involved in the linear region of the backbone through a-1,2-linkages. Furthermore, it is suggested that the side chains representing galactans and arabinans are attached to the 4-position of the rhamnose residues of the backbone. 4. Conclusion The present study focused on the potential of pumpkin as a source for the extraction of pectin using enzymatic methods. In particular, the effect of pronase treatment on emulsifying properties of pumpkin pectin and the structural properties of pumpkin
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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Fig. 6.
7
13
C NMR spectrum of pumpkin pectin fraction B.
pectin were investigated. The emulsifying properties of pumpkin pectin resulted from the presence of hydrophobic proteinaceous components in the pectin structure. Pumpkin pectin could be used as an emulsifier in oil-in-water emulsions for the beverage industry unless residual hydrophobic proteinaceous components are removed. The backbone of pumpkin pectin was considered as an alternation of homogalacturonan regions, repetition of (1 / 4)linked a-D-GalpA, and rhamnogalacturonan regions. The GalpA residues of the backbone were partially methyl esterified and partially acetylated on C-2 and/or C-3. References AACC International American Association of Cereal Chemists. (2000). Approved methods of the AACC. St. Paul, MN: The Association. Bédouet, L., Courtois, B., & Courtois, J. (2003). Rapid quantification of O-acetyl and O-methyl residues in pectin extracts. Carbohydrate Research, 338, 379e383. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484e489. Brummer, Y., Cui, W., & Wang, Q. (2003). Extraction, purification and physicochemical characterization of fenugreek gum. Food Hydrocolloids, 17, 229e236. Chatjigakis, A. K., Pappas, C., Proxenia, N., Kalantzi, O., Rodis, P., & Polissiou, M. (1998). FT-IR spectroscopic determination of the degree of esterification of cell wall pectins from stored peaches and correlation to textural changes. Carbohydrate Polymers, 37, 395e408. Cozzolino, R., Malvagna, P., Spina, E., Giori, A., Fuzzati, N., Anelli, A., et al. (2006). Structural analysis of the polysaccharides from Echinacea angustifolia radix. Carbohydrate Polymers, 65, 263e272. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25e39. Dickinson, E., Murray, B. S., Stainsby, G., & Anderson, D. M. W. (1988). Surface activity and emulsifying behaviour of some Acacia gums. Food Hydrocolloids, 2, 477e490. Duan, J., Wang, X., Dong, Q., Fang, J., & Li, X. (2003). Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki. Carbohydrate Research, 338, 1291e1297. FCC. (1981). Food chemical codex. Washington, DC: National Academy of Sciences. Fissore, E. N., Ponce, N. M., Stortz, C. A., Rojas, A. M., & Gerschenson, L. N. (2007). Characterisation of fiber obtained from pumpkin (cucumis moschata duch.) mesocarp through enzymatic treatment. Food Science and Technology International, 13, 141e151. Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011). Structural modifications of sugar beet pectin and the relationship of structure to functionality. Food Hydrocolloids, 25, 221e229. Garti, N., Madar, Z., Aserin, A., & Sternheim, B. (1997). Fenugreek galactomannans as food emulifiers. Lebensmittel-Wissenschaft und-Technologie, 30, 305e311. Garti, N., Slavin, Y., & Aserin, A. (1999). Surface and emulsification properties of a new gum extracted from Portulaca oleracea L. Food Hydrocolloids, 13, 145e155. Golovchenko, V. V., Ovodova, R. G., Shashkov, A. S., & Ovodov, Y. S. (2002). Structural studies of the pectic polysaccharide from duckweed Lemma minor L. Phytochemistry, 60, 89e97.
Guo, X., Zhao, W., Pang, X., Liao, X., Hu, X., & Wu, J. (2014). Emulsion stabilizing properties of pectins extracted by high hydrostatic pressure, high-speed shearing homogenization and traditional thermal methods: a comparative study. Food Hydrocolloids, 35, 217e225. Iglesias, M. T., & Lozano, J. E. (2004). Extraction and characterization of sunflower pectin. Journal of Food Engineering, 62, 215e223. Izydorczyk, M., Biliaderis, C. G., & Bushuk, W. (1991). Physical properties of watersoluble pentosans from different wheat varieties. Cereal Chemistry, 68, 145e150. Kalapathy, U., & Proctor, A. (2001). Effect of acid extraction and alcohol precipitation conditions on the yield and purity of soy hull pectin. Food Chemistry, 73, 393e 396. Kost’álová, Z., Hromádková, Z., & Ebringerová, A. (2013). Structural diversity of pectins isolated from the Styrian oilepumpkin (Cucurbitapepo var. styriaca) fruit. Carbohydrate Polymers, 93, 163e171. Koubala, B. B., Christiaens, S., Kansci, G., Loey, A. M. V., & Hendrickx, M. E. (2014). Isolation and structural characterisation of papaya peel pectin. Food Research International, 55, 215e221. Leroux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion stabilizing properties of pectin. Food Hydrocolloids, 17, 455e462. Levigne, S., Ralet, M., & Thibault, J. (2002). Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49, 145e153. Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest Biology and Technology, 25, 99e107. Matora, A. V., Korshunova, V. E., Shkodina, O. G., Zhemerichkin, D. A., Ptitchkina, N. M., & Morris, E. R. (1995). The application of bacterial enzymes for extraction of pectin from pumpkin and sugar beet. Food Hydrocolloids, 9, 43e46. Perrone, P., Hewage, C. M., Thomson, A. R., Bailey, K., Sadler, I. H., & Fry, S. C. (2002). Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity. Phytochemistry, 60, 67e77. Ptichkina, N. M., Markina, O. A., & Rumyantseva, G. N. (2008). Pectin extraction from pumpkin with the aid of microbial enzymes. Food Hydrocolloids, 22, 192e195. Shkodina, O. G., Zeltser, O. A., Selivanov, N. Y., & Ignatov, V. V. (1998). Enzymic extraction of pectin preparations from pumpkin. Food Hydrocolloids, 12, 313e 316. Siew, C. K., Williams, P. A., Cui, S. W., & Wang, Q. (2008). Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer. Journal of Agricultural and Food Chemistry, 56, 8111e8120. Singthong, J., Cui, S. W., Ningsanond, S., & Goff, H. D. (2004). Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin. Carbohydrate Polymers, 58, 391e400. Tamaki, Y., Konishi, T., Fukuta, M., & Tako, M. (2008). Isolation and structural characterisation of pectin from endocarp of Citrus depressa. Food Chemistry, 107, 352e361. Vincken, J., Schols, H. A., Oomen, R. J. F. J., McCann, M. C., Ulvskov, P., Voragen, A. G. J., et al. (2003). If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture. Plant Physiology, 132, 1781e1789. Voragen, A. G. J., Pilnik, W., Thibault, J.-F., Axelos, M. A. V., & Renard, C. M. G. C. (1995). Pectins. In A. M. Stephen (Ed.), Food polysaccharides and their applications (pp. 287e340). New-York: Marcel Dekker. Waldron, K. W., Parker, M. L., & Smith, A. C. (2003). Plant cell walls and food quality. Comprehensive Reviews in Food Science and Food Safety, 2, 128e146.
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Westerlund, E., Åman, P., Andersson, R., & Andersson, R. E. (1991). Chemical characterization of water-soluble pectin in papaya fruit. Carbohydrate Polymers, 15, 67e78. Winning, H., Viereck, N., Salomonsen, T., Larsen, J., & Engelsen, S. B. (2009). Quantification of blockiness in pectins-A comparative study using vibrational spectroscopy and chemometrics. Carbohydrate Research, 344, 1833e1841. Wood, P. J., Weisz, J., & Blackwell, B. A. (1994). Structural studies of (1 / 3),(1 / 4)b-D-glucans by 13C-nuclear magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using high-performance anion-exchange
chromatography of oligosaccharides released by lichenase. Cereal Chemistry, 71, 301e307. Wu, Y., Ai, L., Wu, J., & Cui, S. W. (2013). Structural analysis of a pectic polysaccharide from boat-fruited sterculia seeds. International Journal of Biological Macromolecules, 56, 76e82. Yasumatsu, K., Sawada, K., Moritaka, S., Misaki, M., Toda, J., Wada, T., et al. (1972). Whipping and emulsifying properties of soybean products. Agricultural and Biological Chemistry, 36, 719e727.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Emulsifying and structural properties of pectin enzymatically extracted from pumpkin Steve W. Cui a, Yoon Hyuk Chang b, * a b
Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada Department of Food and Nutrition, Kyung Hee University, Seoul, 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 31 March 2014 Accepted 6 April 2014 Available online xxx
The present study investigated the emulsifying and structural properties of pectin enzymatically extracted from pumpkin. Pumpkin pectin fraction A was obtained from raw pumpkin with an enzymatic preparation of cellulase and a-amylase. Pumpkin pectin fraction B was achieved by treating the fraction A solution with pronase to reduce protein content. According to the findings (on protein content, galacturonic acid content, neutral sugar composition, and molecular weight distribution), the pronase treatment could remove protein from the fraction A without considerably influencing any other chemical and molecular properties. Moreover, the fraction A exhibited emulsifying properties in water and oil mixture, whereas the removal of protein in the fraction B resulted in the loss of emulsifying properties. The FT-IR and 1D NMR analysis revealed that the backbone of pumpkin pectin is mainly composed of a1,4-D-galacturonic acid in which a considerable portion of galacturonic acid residues is present as methyl esters, and some L-rhamnose are involved in the linear region of the backbone through a-1,2-linkages. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Keywords: Pumpkin pectin Emulsifying properties Structural characterization
1. Introduction Pectin, the most abundant polysaccharides in the middle lamella and cell wall of many higher plants, has been widely used in the food industry as a gelling agent, thickener, and stabilizer of jam, jelly, and acid milk products (Singthong, Cui, Ningsanond, & Goff, 2004; Tamaki, Konishi, Fukuta, & Tako, 2008). The identification of the molecular structure for pectin is of fundamental importance because the physicochemical and functional properties of pectin are highly dependent on its structure. However, pectin is very heterogeneous in its composition, which renders the structural study of the polysaccharides extremely challenging. Pectin is considered as an alternation of homogalacturonan regions, generally called “smooth regions”, and rhamnogalacturonan regions I, also called “hairy regions” (Waldron, Parker, & Smith, 2003). Homogalacturonan is a repetition of (1 / 4)-linked a-Dgalactopyranosyluronic acid (GalpA) residues. Rhamnogalacturonan region I is constituted by an alternating sequence of (1 / 4)linked a-D-GalpA and (1 / 2)-linked a-L-rhamnopyranose (Rhap) residues. Various side chains (arabinan, galactan, and arabinogalactan) can be linked to the rhamnose residues (Vincken et al., 2003; Voragen, Pilnik, Thibault, Axelos, & Renard, 1995). * Corresponding author. Tel.: þ82 2 960 0264; fax: þ82 2 961 0261. E-mail address: [email protected] (Y.H. Chang).
Citrus peel and apple pomace are the major raw materials used for the production of commercially acceptable pectin. Other raw materials, including pumpkin (Matora et al., 1995), sugar beet (Levigne, Ralet, & Thibault, 2002), sunflower (Iglesias & Lozano, 2004), honey pomelo (Guo et al., 2014), and papaya peels (Koubala, Christiaens, Kansci, Loey, & Hendrickx, 2014) have been considered as possible pectin sources and their potential has been discussed. Several studies on the extraction of pumpkin pectin have been performed using different extraction methods, such as an acidic extraction method and enzymatic extraction method (Fissore, Ponce, Stortz, Rojas, & Gerschenson, 2007; Matora et al., 1995; Ptichkina, Markina, & Rumyantseva, 2008; Shkodina, Zeltser, Selivanov, & Ignatov, 1998). Matora et al. (1995) observed that the enzyme extraction method (multi-enzyme culture supernatants from various strains of Bacillus polymyxa) gave much bigger yields of pumpkin pectin than did the acid extraction method (0.1 M HCl). Moreover, the current acid pectin extraction methods not only do not allow pectin to be extracted fully with no damage to its structure, but also lead to the environmental concerns due to acid usage (Ptichkina et al., 2008). Different enzymes, like cellulase, hemicellulase, protease, amylase, and so on, have been employed for the enzymatic extraction of pumpkin pectin (Fissore et al., 2007; Shkodina et al., 1998). Shkodina et al. (1998) employed cellulase (Trichoderma
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viride) or hemicellulase (Aspergillus niger)] for the extraction of pectin from pumpkin pulp (following juice extraction), and reported that the cellullase treatment led to an increase in pectin yields and in galacturonic acid contents, as compared to that of the hemicellulose treatment. The same result was also observed in the literature performed by Fissore et al. (2007), who found that the amounts of galacturonic acid in pumpkin pectin obtained from cellulase were greater than those obtained from hemicellulase. The emulsifying properties of pectin have been demonstrated in a few literatures (Dickinson, 2003; Funami et al., 2011; Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Siew, Williams, Cui, & Wang, 2008). A small percentage of hydrophobic protein (about 2%) in sugar beet pectin plays major role in stabilizing oil-inwater emulsions (Siew et al., 2008). Leroux et al. (2003) also noted that beet pectin and citrus pectin may efficiently decrease the interfacial tension between oil and water phase in the emulsions; however, the emulsion stabilizing features of pectin change due to the contents difference of calcium ions, acetyl groups, and proteins. As far as the authors are aware, no studies have apparently reported the effect of pronase treatment on the emulsifying properties of pumpkin pectin, nor have elucidated the structural features of pectin enzymatically extracted from pumpkin. In the present study, pectin fraction A was obtained from raw pumpkin using cellulase and a-amylase, and then pumpkin pectin fraction B was produced after pronase treatment with the fraction A. The main objectives of the present study are to (1) evaluate the potential of pumpkin for the enzymatic extraction of pectin and (2) investigate the emulsifying and structural features of pectin enzymatically extracted from pumpkin. 2. Materials and methods 2.1. Materials Raw pumpkin (Cucurbita mixta), harvested in ON, Canada, was used in the present study. Cellulase from Trichoderma viride (5 U/ mg) and a-amylase from Bacillus licheniformis (3500 U/mL) were obtained from Sigma (Oakville, ON, Canada). Pronase from Streptomyces griseus (7.0 U/mg) was provided by Roche Molecular Biochemicals (Indianapolis, USA). Standards for rhamnose, arabinose, galactose, glucose, xylose, mannose, and galacturonic acid were purchased from Sigma (Oakville, ON, Canada). All chemicals were of reagent grade unless otherwise specified. 2.2. Enzymatic extraction for the production of pumpkin pectin fractions A and B Pumpkin pectin fractions A and B were extracted from raw pumpkin using the procedure described by Fissore et al. (2007) with some modifications. Raw pumpkin was first cut, and the seeds and skin were removed. The remainder was reduced to fragments (about 4 cm 0.5 cm), freeze-dried, and ground to make dried pumpkin powder. Fifty g of pumpkin powder were suspended in 1.5 L of 50 mmol/L sodium citrate buffer (pH 5.2), and then cellulase (600 mg) and a-amylase (500 mg) were added. Our preliminary study indicated that starch contents of dried pumpkin powder were 7.93 g/100 g dry solids; therefore, a-amylase was employed to remove starch existed in pumpkin powder. The mixture was stirred at 30 C for 20 h and centrifuged at 15,000 g for 30 min. The supernatant was collected. The remaining pellet was resuspended in 100 mL of distilled water and subjected to a further centrifugation. The supernatants were combined and treated with two volumes of absolute ethanol (Sigma, Oakville, ON, Canada). The resulting precipitate was collected, solubilised in
distilled water, freeze-dried, and ground. This procedure produced pumpkin pectin fraction A. To produce pumpkin pectin fraction B, 0.5 g/100 mL solution of pumpkin pectin fraction A was stirred with pronase for 60 min at 50 C to reduce protein content. The solution was heated at 85 C for 30 min, cooled, and centrifuged at 15,000 g for 20 min. The supernatant was collected and then treated with two volumes of absolute ethanol (Sigma, Oakville, ON, Canada). The resulting precipitate was collected, solubilised in distilled water, freeze-dried, and ground. The resulting protein-reduced pectin fraction was termed as pumpkin pectin fraction B. The details of enzymatic extraction for pumpkin pectin fractions A and B are summarised in Fig. 1. 2.3. Moisture content, protein content, galacturonic acid content, degree of esterification, and degree of acetylation Pumpkin pectin fraction A and fraction B were analysed for their moisture contents using AACCI Approved Method 44-15A (AACCI, 2000). The protein contents of pumpkin fraction A and fraction B were analysed by NA2100 Nitrogen and Protein Analyser (Strada Rivoltana, Milan, Italy) using the factor of 5.7 to convert measured nitrogen to protein. The galacturonic acid contents of pumpkin pectin fraction A and fraction B were determined by the mhydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). Standard galacturonic acid solutions (10e100 mg/mL) were used to construct the standard curve for the determination. The degree of esterification (DE) and degree of acetylation for the fractions A and B were determined by the titrimetric method of Food Chemical Codex (FCC, 1981, pp. 283e286) and the method of Matora et al. (1995), respectively. 2.4. Neutral sugar composition The neutral sugar composition of pumpkin pectin fraction A and fraction B was determined by modifying the procedure of Wood, Weisz, and Blackwell (1994). Each sample was hydrolysed in 1 mol/L H2SO4 at 100 C for 4 h and diluted 20 times. The diluted samples were passed through a 0.45 mm filter and injected to a high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex-5500, Dionex Corporation, Canada). Separation of each neutral sugar was performed on a CarboPac PA1 column (250 4 mm I.D., Dionex Corporation, Canada) and a guard column (3 25 mm, Dionex Corporation, Canada). The solvents were A: 100 mmol/L NaOH, B: 300 mmol/L NaOH, and C: water (distilled water filtered through Nanopure Infinity, Model: D8971, Dubuque, IA, USA). Elution was with 8% A and 92% C for 7 min, then with a gradient to 100% eluent C for 28 min. The column system was cleaned after each analysis with 100% eluent B for 15 min. After cleaning, the initial conditions were maintained for 10 min between each injection of sample. The solvent flow rate was 1.0 mL/min and the injection volume was 50 mL. A post-column delivery system of 600 mmol/L NaOH with a flowrate of 0.5 mL/min was added to the HPAEC-PAD system. Standard solutions of the individual neutral sugar (rhamnose, arabinose, galactose, glucose, xylose, and mannose) were used at varying concentrations (10e50 mg/mL) for identification and quantification. The instrument was controlled and data were processed using Dionex AI 450 software (Dionex Corporation, Canada). 2.5. Molecular weight distribution The molecular weight distribution of pumpkin pectin fraction A and fraction B was evaluated by high performance size exclusion chromatography (HPSEC) equipped with a refractive index (RI)
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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3
Raw pumpkin Cutting freeze-drying grinding
Pumpkin powder Sodium citrate buffer (pH 5.2) cellulase α-amylase centrifuge
Residue
Supernatant Ethanol precipitation solubilization freeze-drying
Fraction A Solubilization pronase centrifuge
Residue
Supernatant Ethanol precipitation solubilization freeze-drying
Fraction B Fig. 1. Scheme of enzymatic extraction for pumpkin pectin fraction A and fraction B from raw pumpkin.
detector (Model Dual 250, Viscotek, Houston, TX, USA). The chromatographic system includes a Shimadzu SCL-10Avp pump, automatic injector (Shimadzu Scientific Instruments Inc., Columbia, MD, USA), and two columns in series: a Shodex OHpak KB-806M (Showa Denko K.K., Tokyo, Japan) and an Ultrahydrogel linear (Waters, Milford, CT, USA). The columns and RI detector were maintained at 40 C. The mobile phase, injection volume, and flow rate were 0.1 mol/L NaNO3, 100 mL, and 0.6 mL/min, respectively. Each sample was solubilized in distilled water at 90 C for 1 h, cooled, and filtered through a 0.45 mm filter prior to injection onto the column. The molecular weight distribution was estimated by the pullulan standards (molecular weights: 10, 20, 50, and 400 kDa) (Sigma, Oakville, ON, Canada). 2.6. Surface tension The surface tension of pumpkin pectin fraction A, fraction B, and pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) was determined using a semi-automatic surface tensionmeter model Surface Tensionmat 21 (Fisher Scientific, Toronto, Canada) at 21e22 C. This tensionmeter employed the Du Nouy ring method to measure static surface tension. Distilled water was used to calibrate the tensionmeter for surface tension measurements. Sample concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 g/100 mL were used for surface tension measurements.
2.7. Emulsion capacity and stability The emulsion capacity and emulsion stability of pumpkin pectin fraction A, fraction B, and pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) were investigated according to Yasumatsu et al. (1972) with some modifications. The sample (0.01 g) was suspended in 10 mL of distilled water before mixing with 10 mL of vegetable oil. The water/oil mixture was emulsified using a homogenizer at 10,000 rpm for 1 min, and then the mixture was centrifuged at 1300 g for 5 min. Emulsion capacity was calculated as follows:
ðHeight of emulsion layer=total height of fluidÞ 100%
(1)
Emulsion stability was determined by heating the emulsion at 80 C for 30 min, cooling with tap water for 15 min, and centrifuging at 1300 g for 5 min. Emulsion stability was calculated as follows:
ðHeight of remainingemulsion layer=total heightof fluidÞ 100%
(2)
2.8. Structural analysis Only pumpkin pectin fraction B was used to analyse the structural characteristics because pectin with little or no proteins is better for the structural analysis.
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2.8.1. Fourier transform-infrared spectroscopy The pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Canada) and pumpkin pectin fraction B were dried in a vacuum oven for 5 h and stored in a desiccator prior to Fourier transforminfrared (FT-IR) analysis. FT-IR spectra of each sample were obtained according to the procedure of Singthong et al. (2004). 2.8.2. 1H and 13C NMR spectroscopy Pumpkin pectin fraction B was dissolved in deuterium oxide (D2O, 80 C, 1 h) and freeze-dried for three times to replace the exchangeable protons with deuterons before being finally redissolved in D2O (3 g/100 mL) for NMR analysis. High-resolution 1H and 13C NMR spectra were recorded in D2O at 500.13 and 125.78 MHz, respectively, on a Bruker AM500 NMR spectrometer operating at 25 C. A 5 mm inverse geometry 1H/13C/15N probe was used. The chemical shifts were reported relative to external standards, trimenthysily propionate (TSP in D2O, 4.76 ppm, for 1H) and 1,4-dioxane (in D2O, 66.5 ppm, for 13C). 2.9. Statistical analysis All statistical analyses were performed using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was performed using the general linear models (GLM) procedure to determine significant differences among the samples. Means were compared by using Fisher’s least significant difference (LSD) procedure. Significance was defined at the 5% level. 3. Results and discussion 3.1. Chemical composition The yields of pumpkin pectin fractions A and B were calculated as a percentage of the weight of dried pumpkin powder, which were 10.03 and 8.08 g/100 g, respectively. The protein content (derived from nitrogen) of pumpkin pectin fraction A was 4.55 g/ 100 g, while no nitrogen was detected in pumpkin pectin fraction B (Table 1). The DE values (about 47%) of pumpkin pectin fractions A and B were not significantly different. The galacturonic acid contents of pumpkin fractions A and B represent 75.02 and 78.22 g/ 100 g, respectively. This finding indicated that both of the fractions are mainly composed of homogalacturonan (Vincken et al., 2003; Waldron et al., 2003). Small amounts (about 10 g/100 g) of six different neutral sugars were found in both of the pectin fractions, including rhamnose, arabinose, galactose, glucose, xylose, and mannose (Table 1). The presence of rhamnose, galactose, and arabinose in the pumpkin pectin fractions could indicate the existence of rhamnogalacturonan I with side chains, such as arabinan, galactan, and arabinogalactan (Tamaki et al., 2008; Wu, Ai, Wu, & Cui, 2013). The presence of glucose and xylose in the pumpkin pectin fractions could explain the presence of xyloglucan as one of the side chains and the presence of xyloglacturonan (Fissore et al., 2007; Kost’álová, Hromádková, & Ebringerová, 2013; Vincken et al., 2003). More specific structural analysis for pumpkin pectin will be discussed in the next section.
Table 1 Chemical composition of pumpkin pectin fraction A and fraction B.a Pectin sample Fraction A Moisture (g/100 g) Protein (g/100 g) Degree of esterification (%) Degree of acetylation (%) Galacturonic acid (g/100 g) Neutral sugar (g/100 g) Rhamnose Arabinose Galactose Glucose Xylose Mannose
Fraction B a
9.61 4.58 47.30 6.87 75.02
0.27 0.32 0.12a 0.22a 0.93b
9.65 0.12a NDþ 47.10 0.15a 6.79 0.20a 78.22 0.45a
1.73 1.77 3.15 2.26 0.41 0.31
0.09a 0.09a 0.03a 0.01a 0.02a 0.00a
1.72 1.78 3.15 2.24 0.41 0.30
0.04a 0.04a 0.05a 0.03a 0.04a 0.05a
þ
ND: not detectable. a Results are the means of two determinations standard deviation. Values with different letters within the same row differ significantly (P < 0.05).
galactomannans. In the present study, the observations (on protein content, galacturonic acid content, neutral sugar composition, and molecular weight distribution) suggest that the treatment of pronase could selectively remove the residual proteinaceous components from pumpkin pectin without considerably influencing any other chemical and molecular properties. 3.3. Surface tension Changes in surface tension of water by pumpkin pectin fraction A and fraction B with different concentrations (0.05e1.00 g/100 g) at 21e22 C are shown in Fig. 3. The surface tension of water was 0.0723 N/m. Adding pumpkin pectin fraction A, pumpkin pectin fraction B, or pectin standard at all the concentrations studied into water led to a decrease in the surface tension of water, indicating that the pectin fractions A and B as well as pectin standard have the surface activity. Moreover, the surface tension of water decreased with increasing the concentration of pumpkin pectin fractions (fraction A from 0 to 0.2 g/100 g; fraction B from 0 to 0.1 g/100 g) and finally reached a plateau. Similar trend was also found in other polysaccharide samples, such as arabinoxylan and arabinogalactan (Izydorczyk, Biliaderis, & Bushuk, 1991), Portulaca oleracea gum (Garti, Slavin, & Aserin, 1999), and fenugreek gum (Brummer et al., 2003).
3.2. Molecular weight distribution The molecular weight distribution of pumpkin pectin fraction A and fraction B was very similar (Fig. 2). The finding was consistent with Brummer, Cui, and Wang (2003), who applied pronase for the purification of fenugreek gum. They found that treating fenugreek gum solution with pronase considerably decreased the protein content of fenugreek gum from 2.36 to 0.57 g/100 g without affecting the molecular weight and galactose/mannose ratio of the
Fig. 2. Molecular weight distribution of pumpkin pectin fraction A (–) and fraction B (d).
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Table 2 Emulsion capacity and stability of pumpkin pectin fraction A, fraction B, and pectin standard.a Pectin sample
Emulsifying property Emulsion capacity (%)
Fraction A Fraction B Pectin standard
a
63.7 2.0 NDþ 62.5 0.4a
Emulsion stability (%) 58.3 0.2a ND 58.0 0.3a
þ
ND: not detectable. a Results are the means of two determinations standard deviation. Values with different letters within the same column differ significantly (P < 0.05).
Fig. 3. Changes in surface tension of water by pumpkin pectin fraction A (,), fraction B (>), and pectin standard (6) with different concentrations at 21e22 C.
In the present study, the surface tension values of water for pumpkin pectin fraction A at all the concentrations were lower than those for pectin standard, indicating that the effect of fraction A on the surface activity of water was superior to that of pectin standard. In particular, the surface tension values of water for pumpkin pectin fraction A at all the concentrations were remarkably lower than those for pumpkin pectin fraction B. This finding suggests that pumpkin pectin fraction A had better capability to decrease the surface tension of water than did pumpkin pectin fraction B. Until now, the effects of residual protein existing in polysaccharides on the surface activity of water are still controversial. For instance, Garti, Madar, Aserin, and Sternheim (1997) studied the surface activity of non-purified fenugreek gum and purified fenugreek gum which was obtained by physical separation of contaminating protein from the non-purified fenugreek gum. They reported that reducing the protein content did not influence the surface tension of water. On the other hand, Brummer et al. (2003) used a purification step to remove the contaminating protein from fenugreek gum with pronase, and they compared the surface activity of the non-purified fenugreek gum and purified fenugreek gum. According to them (Brummer et al., 2003), the surface tension of water for non-purified fenugreek gum was much lower than that for purified fenugreek gum, explaining that the proteins closely associated with the galactomannans can enhance the surface activity of fenugreek gum. Dickinson, Murray, Stainsby, and Anderson (1988) also showed positive correlation between the surface activity and protein content of Acacia gum (nitrogen contents in the range from 0.1 to 7.5 g/100 g). Thus, it is inferred in the present study that the existence of the residual proteinaceous matters in pumpkin pectin can play a major functional role in improving its surface activity.
polysaccharides can adsorb at oilewater interfaces to form stabilizing layers around oil droplets (Siew et al., 2008). Leroux et al. (2003) noted that the emulsifying ability of beet pectin and citrus pectin is due to a small amount of protein which is covalently bound to a highly branched polysaccharide structure. Therefore, in the present study, it is evident that the emulsifying properties of pumpkin pectin fraction A can be considerably related to the presence of the residual hydrophobic proteinaceous components in the pectin structure. 3.5. Structural characterization 3.5.1. Fourier transform-infrared spectroscopy The FT-IR spectrum of pumpkin pectin fraction B was similar to that of pectin standard (citrus pectin), as illustrated in Fig. 4. In particular, both of pumpkin pectin fraction and pectin standard
3.4. Emulsion capacity and stability The emulsion capacity and stability of pumpkin pectin fraction A were 63.7 and 58.3%, respectively, and were not significantly different from those of pectin standard while both of them were not detected in pumpkin pectin fraction B (Table 2). In general, it has been widely known that many commercial polysaccharide samples include a small amount of protein, either as a contaminant or as an intrinsic part of the polysaccharide structure. The protein components can provide the polysaccharides with good emulsifying ability, since those are usually strongly hydrophobic (Dickinson, 2003). That is, the hydrophobic protein components in the
Fig. 4. Fourier transform-infrared spectra of pumpkin pectin fraction B and pectin standard.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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S.W. Cui, Y.H. Chang / LWT - Food Science and Technology xxx (2014) 1e8
Fig. 5. 1H NMR spectrum of pumpkin pectin fraction B.
exhibited strong absorbances between 950 and 1200 cm1 wave number regions. The regions between 950 and 1200 cm1, generally called as the “finger print” region for each polysaccharide, provide information regarding the identification of major chemical groups in polysaccharides since the position and intensity of the individual band in the regions are unique for each polysaccharide (Kalapathy & Proctor, 2001). According to Kost’álová et al. (2013), the fingerprint region of pumpkin pectin represented the characteristic bands at 1145, 1103, 1077, 1050, and 1017 cm1. Thus, it is confirmed in the present study that pectin was successfully extracted from pumpkin using our enzymatic extraction method (amylase, cellulase, and pronase). Chatjigakis et al. (1998) reported that the regions between 1500 and 1800 cm1 wave number are the most important regions for the examination of structural features of pectin because carboxylic acid and carboxylic ester groups of pectin are found in the regions. More specifically, according to the work conducted by Manrique and Lajolo (2002), the band at 1630 cm1 wave number regions indicated the ionized carboxylate groups (COO), whereas the band at 1740 cm1 wave number regions was assigned to carbonyl groups (C]O) from COOCH3 groups. Furthermore, the bands at 1730 and 1600 cm1 correspond to esterified carboxylic acid groups and nonesterified carboxylic acid groups, respectively (Singthong et al., 2004; Winning, Viereck, Salomonsen, Larsen, & Engelsen, 2009). Therefore, in the present study, the occurrence of two distinguished bands at 1730 and 1600 cm1 apparently indicated the nonesterifed and esterified carboxyl groups of galacturonic acid in pumpkin pectin. 3.5.2. 1H and 13C NMR analysis The 1H NMR spectrum of pumpkin pectin fraction B is shown in Fig. 5. From the data, it was possible to distinguish and assign the resonances originating from a- and b-configured residues. In the anomeric region, the strong signals at 5.08 and 5.11 ppm were assigned to H-1 of 4-linked a-D-GalpA and H-1 of 2elinked a-LRhap, respectively (Tamaki et al., 2008; Wu et al., 2013). The signals at 4.45 and 5.17 ppm were attributed to H-1 of b-galactose and aarabinose, respectively (Duan, Wang, Dong, Fang, & Li, 2003; Golovchenko, Ovodova, Shashkov, & Ovodov, 2002). The very large signal at 3.81 ppm was derived from methyl groups binding to carboxyl groups of 4-linked a-D-GalpA (Cozzolino
et al., 2006; Kost’álová et al., 2013; Tamaki et al., 2008). Two signals at around 2.15 ppm were attributed to acetyl groups binding at O-2 and O-3 of GalpA. The identification of the acetyl groups was achieved by comparison of the observed spectrum against literature values (Bédouet, Courtois, & Courtois, 2003; Cozzolino et al., 2006; Kost’álová et al., 2013; Perrone et al., 2002). The methyl rhamnose signals at 1.13 and 1.25 ppm were associated with the 1,2-linked and 1,2,4-linked Rhap (Perrone et al., 2002; Wu et al., 2013). The 13C NMR spectrum of pumpkin pectin fraction B explained the resonances from C-1 of 4-linked a-D-GalpA at 100.2 and 101.0 ppm which were the most intensive in the anomeric region (Fig. 6). Both of the signals can be related to C-1 of nonesterifed and esterified carboxyl groups of 4-linked a-D-GalpA, respectively, by comparison with literature values of the anomeric chemical shifts (Cozzolino et al., 2006; Westerlund, Åman, Andersson, & Andersson, 1991). The respective corresponding typical resonances of C-6 of the carboxyl groups of 4-linked a-D-GalpA were observed at 171.7 and 171.5 ppm, and the existence of two carboxyl signals confirmed the presence of nonesterifed and esterified carboxyl groups of 4-linked a-D-GalpA (Cozzolino et al., 2006; Wu et al., 2013). The carbon signals at 21.08 ppm were assigned to CH3 of the O-acetyl groups (Westerlund et al., 1991). Based on the findings obtained from the galacturonic acid content, neutral sugar composition, FT-IR spectra, and positions of signals in the 1H and 13C NMR spectra of pumpkin pectin, it is suggested in the present study that the backbone of pumpkin pectin proved to mainly represent linear a-1,4-D-galacturonic acid in which a considerable portion of galacturonic acid residues is present as methyl esters, and some L-rhamnose are involved in the linear region of the backbone through a-1,2-linkages. Furthermore, it is suggested that the side chains representing galactans and arabinans are attached to the 4-position of the rhamnose residues of the backbone. 4. Conclusion The present study focused on the potential of pumpkin as a source for the extraction of pectin using enzymatic methods. In particular, the effect of pronase treatment on emulsifying properties of pumpkin pectin and the structural properties of pumpkin
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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Fig. 6.
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13
C NMR spectrum of pumpkin pectin fraction B.
pectin were investigated. The emulsifying properties of pumpkin pectin resulted from the presence of hydrophobic proteinaceous components in the pectin structure. Pumpkin pectin could be used as an emulsifier in oil-in-water emulsions for the beverage industry unless residual hydrophobic proteinaceous components are removed. The backbone of pumpkin pectin was considered as an alternation of homogalacturonan regions, repetition of (1 / 4)linked a-D-GalpA, and rhamnogalacturonan regions. The GalpA residues of the backbone were partially methyl esterified and partially acetylated on C-2 and/or C-3. References AACC International American Association of Cereal Chemists. (2000). Approved methods of the AACC. St. Paul, MN: The Association. Bédouet, L., Courtois, B., & Courtois, J. (2003). Rapid quantification of O-acetyl and O-methyl residues in pectin extracts. Carbohydrate Research, 338, 379e383. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484e489. Brummer, Y., Cui, W., & Wang, Q. (2003). Extraction, purification and physicochemical characterization of fenugreek gum. Food Hydrocolloids, 17, 229e236. Chatjigakis, A. K., Pappas, C., Proxenia, N., Kalantzi, O., Rodis, P., & Polissiou, M. (1998). FT-IR spectroscopic determination of the degree of esterification of cell wall pectins from stored peaches and correlation to textural changes. Carbohydrate Polymers, 37, 395e408. Cozzolino, R., Malvagna, P., Spina, E., Giori, A., Fuzzati, N., Anelli, A., et al. (2006). Structural analysis of the polysaccharides from Echinacea angustifolia radix. Carbohydrate Polymers, 65, 263e272. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25e39. Dickinson, E., Murray, B. S., Stainsby, G., & Anderson, D. M. W. (1988). Surface activity and emulsifying behaviour of some Acacia gums. Food Hydrocolloids, 2, 477e490. Duan, J., Wang, X., Dong, Q., Fang, J., & Li, X. (2003). Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki. Carbohydrate Research, 338, 1291e1297. FCC. (1981). Food chemical codex. Washington, DC: National Academy of Sciences. Fissore, E. N., Ponce, N. M., Stortz, C. A., Rojas, A. M., & Gerschenson, L. N. (2007). Characterisation of fiber obtained from pumpkin (cucumis moschata duch.) mesocarp through enzymatic treatment. Food Science and Technology International, 13, 141e151. Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011). Structural modifications of sugar beet pectin and the relationship of structure to functionality. Food Hydrocolloids, 25, 221e229. Garti, N., Madar, Z., Aserin, A., & Sternheim, B. (1997). Fenugreek galactomannans as food emulifiers. Lebensmittel-Wissenschaft und-Technologie, 30, 305e311. Garti, N., Slavin, Y., & Aserin, A. (1999). Surface and emulsification properties of a new gum extracted from Portulaca oleracea L. Food Hydrocolloids, 13, 145e155. Golovchenko, V. V., Ovodova, R. G., Shashkov, A. S., & Ovodov, Y. S. (2002). Structural studies of the pectic polysaccharide from duckweed Lemma minor L. Phytochemistry, 60, 89e97.
Guo, X., Zhao, W., Pang, X., Liao, X., Hu, X., & Wu, J. (2014). Emulsion stabilizing properties of pectins extracted by high hydrostatic pressure, high-speed shearing homogenization and traditional thermal methods: a comparative study. Food Hydrocolloids, 35, 217e225. Iglesias, M. T., & Lozano, J. E. (2004). Extraction and characterization of sunflower pectin. Journal of Food Engineering, 62, 215e223. Izydorczyk, M., Biliaderis, C. G., & Bushuk, W. (1991). Physical properties of watersoluble pentosans from different wheat varieties. Cereal Chemistry, 68, 145e150. Kalapathy, U., & Proctor, A. (2001). Effect of acid extraction and alcohol precipitation conditions on the yield and purity of soy hull pectin. Food Chemistry, 73, 393e 396. Kost’álová, Z., Hromádková, Z., & Ebringerová, A. (2013). Structural diversity of pectins isolated from the Styrian oilepumpkin (Cucurbitapepo var. styriaca) fruit. Carbohydrate Polymers, 93, 163e171. Koubala, B. B., Christiaens, S., Kansci, G., Loey, A. M. V., & Hendrickx, M. E. (2014). Isolation and structural characterisation of papaya peel pectin. Food Research International, 55, 215e221. Leroux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion stabilizing properties of pectin. Food Hydrocolloids, 17, 455e462. Levigne, S., Ralet, M., & Thibault, J. (2002). Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49, 145e153. Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest Biology and Technology, 25, 99e107. Matora, A. V., Korshunova, V. E., Shkodina, O. G., Zhemerichkin, D. A., Ptitchkina, N. M., & Morris, E. R. (1995). The application of bacterial enzymes for extraction of pectin from pumpkin and sugar beet. Food Hydrocolloids, 9, 43e46. Perrone, P., Hewage, C. M., Thomson, A. R., Bailey, K., Sadler, I. H., & Fry, S. C. (2002). Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity. Phytochemistry, 60, 67e77. Ptichkina, N. M., Markina, O. A., & Rumyantseva, G. N. (2008). Pectin extraction from pumpkin with the aid of microbial enzymes. Food Hydrocolloids, 22, 192e195. Shkodina, O. G., Zeltser, O. A., Selivanov, N. Y., & Ignatov, V. V. (1998). Enzymic extraction of pectin preparations from pumpkin. Food Hydrocolloids, 12, 313e 316. Siew, C. K., Williams, P. A., Cui, S. W., & Wang, Q. (2008). Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer. Journal of Agricultural and Food Chemistry, 56, 8111e8120. Singthong, J., Cui, S. W., Ningsanond, S., & Goff, H. D. (2004). Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin. Carbohydrate Polymers, 58, 391e400. Tamaki, Y., Konishi, T., Fukuta, M., & Tako, M. (2008). Isolation and structural characterisation of pectin from endocarp of Citrus depressa. Food Chemistry, 107, 352e361. Vincken, J., Schols, H. A., Oomen, R. J. F. J., McCann, M. C., Ulvskov, P., Voragen, A. G. J., et al. (2003). If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture. Plant Physiology, 132, 1781e1789. Voragen, A. G. J., Pilnik, W., Thibault, J.-F., Axelos, M. A. V., & Renard, C. M. G. C. (1995). Pectins. In A. M. Stephen (Ed.), Food polysaccharides and their applications (pp. 287e340). New-York: Marcel Dekker. Waldron, K. W., Parker, M. L., & Smith, A. C. (2003). Plant cell walls and food quality. Comprehensive Reviews in Food Science and Food Safety, 2, 128e146.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012
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S.W. Cui, Y.H. Chang / LWT - Food Science and Technology xxx (2014) 1e8
Westerlund, E., Åman, P., Andersson, R., & Andersson, R. E. (1991). Chemical characterization of water-soluble pectin in papaya fruit. Carbohydrate Polymers, 15, 67e78. Winning, H., Viereck, N., Salomonsen, T., Larsen, J., & Engelsen, S. B. (2009). Quantification of blockiness in pectins-A comparative study using vibrational spectroscopy and chemometrics. Carbohydrate Research, 344, 1833e1841. Wood, P. J., Weisz, J., & Blackwell, B. A. (1994). Structural studies of (1 / 3),(1 / 4)b-D-glucans by 13C-nuclear magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using high-performance anion-exchange
chromatography of oligosaccharides released by lichenase. Cereal Chemistry, 71, 301e307. Wu, Y., Ai, L., Wu, J., & Cui, S. W. (2013). Structural analysis of a pectic polysaccharide from boat-fruited sterculia seeds. International Journal of Biological Macromolecules, 56, 76e82. Yasumatsu, K., Sawada, K., Moritaka, S., Misaki, M., Toda, J., Wada, T., et al. (1972). Whipping and emulsifying properties of soybean products. Agricultural and Biological Chemistry, 36, 719e727.
Please cite this article in press as: Cui, S. W., & Chang, Y. H., Emulsifying and structural properties of pectin enzymatically extracted from pumpkin, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.012