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Extraction and characterisation of pomace pectin from gold kiwifruit (Actinidia chinensis)
Food Chemistry 187 (2015) 290–296
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Extraction and characterisation of pomace pectin from gold kiwifruit (Actinidia chinensis) Oni Yuliarti a,b, Kelvin K.T. Goh a,⇑, Lara Matia-Merino a, John Mawson a,c, Charles Brennan a,d a
Massey Institute of Food Science and Technology, School of Food & Nutrition, Massey University, Palmerston North, New Zealand School of Chemical and Life Sciences, Singapore Polytechnic, Singapore c School of Agricultural and Wine Sciences, Charles Sturt University, Australia d Faculty of Agriculture and Life Sciences, Lincoln University, New Zealand b
a r t i c l e
i n f o
Article history: Received 11 January 2015 Received in revised form 24 March 2015 Accepted 26 March 2015 Available online 18 April 2015 Keywords: Pectin Pomace Gold kiwifruit Extraction Galacturonic acid, molar mass and viscosity
a b s t r a c t Gold kiwifruit pomace extracted using citric acid, water and enzyme (Celluclast 1.5L) were studied in terms of pectin yield, protein, ash, non-starch polysaccharide, galacturonic acid (GalA), neutral sugar composition, molar mass (Mw), viscosity and degree of branching. Water-extracted pectin was considered closest to its native form. Enzyme extracted pectin showed the highest yield (4.5% w/w) as compared with the acid and water extraction methods (3.6–3.8% w/w). Pectin obtained from different extraction methods showed different degree of branching. The Mw and root mean square (RMS) radius varied with the extraction methods with values of 8.4 105 g/mol and 92 nm, 8.5 105 g/mol and 102 nm, 6.7 105 g/mol and 52 nm for acid, water and enzymatic extraction methods, respectively. Similar trend was observed for pectin viscosity, with water-extracted pectin giving a slightly higher viscosity followed by acid and enzyme-extracted pectin. This study showed that gold kiwifruit pomace pectin has potential application in food products. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Pectin is a structural polysaccharide found in the cell walls of growing plants. Pectin is regarded as a complex heteropolysaccharide consisting mainly of a-(1,4)-linked D-galacturonic acid backbone with different degrees of esterification (Mohnen, 2008). Pectin can be further classified based on their occurrences as three main structures namely, homogalacturonan (HG), rhamnogalacturonan type I (RG-I) and substituted galacturonan (Ridley, O’Neill, & Mohnen, 2000). The HG group has a backbone with a linear chain of a-(1,4)-linked D-galacturonosyl acid. The RG-I group has a backbone of a-(1,2)-linked L-rhamnosyl and a-(1,4)-linked D-galacturonosyl acid residues, with neutral sugars such as arabinose and galactose as the side chains. The substituted galacturonan group consists of a-(1,4)-linked D-galacturonosyl acid residues as the backbone such as rhamnogalacturonan II and xylogalacturonan (O’Neill, Albersheim, & Darvil, 1990). Pectin is widely used as a thickener and stabiliser in food products such as jams and yoghurt drinks. Apple pomace and citrus peels are currently the two main sources of commercial pectin
⇑ Corresponding author. Tel.: +64 06 3569099; fax: +64 06 3505657. E-mail address: [email protected] (K.K.T. Goh). http://dx.doi.org/10.1016/j.foodchem.2015.03.148 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
(May, 1990). They are usually obtained from the by-products of fruit juice manufacture. Pectin polymers from other plant sources have been isolated and studied. They include sugar beet pulp (Wang & Chang, 1994), peach (Pagan, Ibarz, Llorca, Pagan, & Barbosa-Canovas, 2001), mango (Ketsa, Chidtragool, Klein, & Lurie, 1999), banana peel (Emaga, Ronkart, Robert, Wathelet, & Paquot, 2008) and chicory roots (Panouille, Thibault, & Bonnin, 2006). The extraction and isolation of pectin can be carried out using different methods including chemical, physical or enzymatic treatments (Panouille et al., 2006). Chemical extraction method using dilute acid solution is commonly employed in pectin manufacture. However, the use of chemical method to extract pectin could give a negative connotation as consumers increasingly prefer chemical-free ingredients to be used in food products. Instead, the use of specific enzymes could be advantageous due to the low enzyme concentrations required and the low amount of waste generated (Yuliarti, Matia-Merino, Goh, Mawson, & Brennan, 2012). New Zealand kiwifruit growers produced approximately 400,000 tonnes of kiwifruit from 13,000 hectares split between green (80%) and gold (20%) kiwifruit (MPI, 2013). Kiwifruit is either consumed as a fruit or processed into kiwifruit juice. In the processing of kiwifruit juice, a large proportion of the fruit goes into waste which includes the fruit peels, pomace and seeds. Kiwifruit
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waste has been reported by Wabnitz (2008) to be approximately 18% of the total kiwifruit crop. Gold kiwifruit pomace obtained after juice extraction constitutes about 40–50% of the weight of the fresh fruit (data not published). Despite the smaller percentage of gold kiwifruit crop based on the current data, the demand for gold kiwifruit is envisaged to grow as consumer demand increases. The physico-chemical properties of pectin from the whole gold kiwifruit have recently been reported (Yuliarti et al., 2015). However, the physico-chemical properties of pectin solely from the pomace of gold kiwifruit have not been well-understood. The chemical composition of pomace pectin and its functional properties could differ from pectin extracted from whole kiwifruit. It is useful for growers and manufacturers to understand the potential functional benefits of pomace where the whole kiwifruit is utilised for juice processing. The data from this study could provide useful knowledge to maximise the utilisation of gold kiwifruit that will not only value-add but also reduce kiwifruit waste generated. It was therefore deemed necessary to characterise and compare the properties of pomace pectin with the pectin obtained from the whole kiwifruit. Knowledge of the physical functionality of pomace pectin could provide useful information for manufacturers to consider utilising the kiwifruit pomace as another source of pectin that is capable of providing certain unique functional properties. The objectives of this study were to examine the yield, composition, molar mass and the rheological properties of gold kiwifruit pomace pectin extracted using different extraction methods (acid, water and enzyme). Comparisons were made with pectin isolated from the whole kiwifruit (main-harvested fruit; MHF), as previously reported by Yuliarti et al. (2015).
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Fig. 1. Schematic flow chart of the preparation of gold kiwifruit pomace.
2.2. Pectin extraction
cloths to remove the pulp and seeds that were not separated during centrifugation. Warm water at 50 °C was added to the pellet, in a 1:1 (w/v) ratio, and the mixture was stirred for 30 min (to solubilise the remaining soluble polysaccharides) and centrifuged again as described above. All supernatants and filtrates were combined and precipitated with ethanol as commonly carried out in pectin production (May, 1990). The polysaccharide in the supernatant was precipitated by ethanol to give a final concentration of 80% (v/v). This concentration was chosen because low molecular weight saccharides (e.g. monosaccharides and oligosaccharides) would remain soluble but polysaccharides would be precipitated out (Englyst, Quigley, Hudson, & Cummings, 1992). The mixture of ethanol and extract was stirred (10 min, ±25 °C) to obtain a uniform mixture and was then kept at 4 °C for 4 h to allow the polysaccharides to precipitate. To separate the polysaccharide precipitate from the solvent, the mixture was centrifuged (3300g, 10 min, 4 °C). The pellet was washed twice with 95% ethanol (1:1, w/v) and then centrifuged again as before. The pellet was vacuum dried (Eyela, Vacuum Oven, Voc-300 SD, Science Technique Ltd, New Zealand) at 58 ± 3 °C, 65 cm Hg, for approximately 7–10 h until a constant weight was achieved. For further purification of pectin, the vacuum-dried sample was dispersed in RO water (1.0% w/w) and stirred overnight (15 h) in a 4 °C chiller. The dispersion was then centrifuged at 30,000g for 60 min (4 °C) to separate out the remaining insoluble particles. The soluble pectin in the supernatant was recovered by ethanol precipitation (80%), centrifuged, ethanol washed and vacuum oven dried as described earlier. The pectin yield was calculated using the equation below (Ptichkina, Markina, & Rumyantseva, 2008) and was denoted as the acid extracted pomace pectin:
The pomace recovered from MHF was subjected to three different extraction methods using acid, water or enzyme respectively.
mpectin D ¼ 100 mkp
2. Materials and methods 2.1. Materials The main-harvested gold kiwifruit (MHF) obtained from Zespri International Ltd (Hastings, New Zealand) was the raw material used in this study. The fruit was harvested approximately 20 weeks after pollination, with a firmness of 32 Newton (N). The pomace of MHF used in this study was obtained by squashing whole kiwifruit in a fruit juicer (Avanti, Model 2000 Juicer, FED Australia and New Zealand, 0.5 mm mesh). As the juice contained a mixture of very coarse fruit pulp fractions, skin and seeds, centrifugation (3300g, 20 min at 4 °C) was carried out to isolate the insoluble fraction from the juice. The fruit pulp from the juicer was hydraulically pressed to separate the remaining juice. The pellet, which was considered to be part of the pomace, was pooled and mixed with the fruit pulp that was retained in the juicer. The skin and seeds of the fruit were included in the pomace preparation. A flow diagram for the preparation of the pomace is shown in Fig. 1.
2.2.1. Acid extraction Gold kiwifruit pomace (200 g) was mixed with 600 mL of 1.0% (w/v) citric acid (CA) solution of pH 2.20 ± 0.01 (1:3 w/v pomace to acid solution ratio) in a container and the mixture (pH 3.1 ± 0.05) was heated in a 50 °C water bath for 60 min under continuous stirring. The container was covered with aluminium foil to reduce moisture loss due to evaporation. Purified water obtained by reverse osmosis (RO water) was used throughout the study. After extraction, the mixture was cooled to approximately 20 °C in a bath of crushed ice and then centrifuged at 3300g for 20 min at 4 °C. The supernatant was filtered through four layers of cheese
where D is the percentage yield of purified pectin (%), mpectin is the mass of recovered pectin (g) and mkp is the mass of kiwifruit pomace (g) used in the extraction. 2.2.2. Water extraction The water extraction method was similar to the acid extraction method except that RO water was used instead of the citric acid solution. The extraction was carried out in a 25 °C water bath for 30 min with a pomace to water ratio of 1:3 (w/v) (i.e. 200 g of pomace mixed with 600 mL of RO water). The pH value of the mixture was 3.70 ± 0.05. Recovery of the soluble fraction from the
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mixture was carried out as described for the acid extraction method. Isolation and purification of pectin were carried out based on the procedures described for the acid extraction method. The pectin yield was calculated and was denoted as the water extracted pomace pectin.
values obtained in this study were expressed as gram of polysaccharide per 100 g of sample.
2.2.3. Enzymatic extraction For enzymatic extraction method, the steps for pectin isolation, ethanol precipitation and purification were similar to those described for the acid extraction method. The extraction was conducted at 25 °C for 30 min with Celluclast 1.5L (Novozymes, Copenhagen, Denmark) at a concentration of 1.05 mL/kg. Prior to this extraction, the enzymatic activities of Celluclast 1.5L were pre-determined at 25 °C (pH 3.50 ± 0.01). The results showed that this enzyme contained a mixture of cellulase, polygalacturonase and arabinase with activities of 50.81, 40.82 and 42.42 nkatals/ mL respectively (Yuliarti et al., 2008). In addition, Celluclast 1.5L has been reported to contain xyloglucanase (1120 nkatals/mL) and rhamnogalacturonase (10 nkatals/mL) at 40 °C and pH 5.5 (Panouille et al., 2006). The extraction method of gold kiwifruit pectin by enzymatic treatment was based on a previous study reported by Yuliarti, Matia-Merino, Goh, Mawson, and Brennan (2011). To facilitate a good pomace and enzyme (Celluclast 1.5L) dispersion, RO water was added to the pomace at a ratio of 1:3 (w/v) and the pH value of the mixture was pH 3.70 ± 0.05. This pH value was within the optimum range of Celluclast activity. Recovery of the soluble fraction from the mixture and purification of the pectin were carried out based on the procedures described for the acid extraction method. The pectin obtained was denoted as the enzyme extracted pomace pectin.
The Mw, polydispersity index (Mw/Mn) and the root mean square (RMS) radius of purified pectin were determined by size exclusion chromatography (SEC) coupled to a multi-angle laser light scattering (MALLS) system (Mini Dawn, Wyatt Technology Corp., Santa Barbara, CA, USA) as described by Yuliarti et al. (2015). This system consisted of a high performance liquid chromatography (HPLC) system (GBC Scientific Equipment Ltd, Victoria, Australia), which comprised an HPLC pump (model LC 1150), an ultraviolet (UV) detector (model LC 1200), a system organiser (model LC 1440) and a differential refractive index (DRI) detector (Waters, model R401, Milford, MA, USA). For the molar mass calculation, the specific refractive index increment (dn/dc) of the pectin from the pomace was 0.189 mL/g which is very similar to the value reported earlier for pectin from MHF (Yuliarti et al., 2011). The data were analysed using Astra software (version 4.50, Wyatt Technology Corp., Santa Barbara, CA, USA) and the Zimm plot method to determine the Mw, Mw/Mn and RMS radius of the pectin fraction. A commercial citrus pectin (Sigma, P9135, unknown Mw) was also analysed for comparative purposes.
2.3. Analyses of pomace pectin The fresh pomace and the isolated pomace pectin (obtained from the three extraction methods) were analysed for dry matter which was measured by calculating the weight difference after drying the sample. The ash and protein contents were determined using gravimetric method and Kjeldahl method respectively (AOAC, 1990). Neutral sugar composition was determined by gas liquid chromatography (GLC, BPX-70 column), in the form of alditol acetate derivatives as described by Englyst, Quigley, and Hudson (1994) using an Englyst Kit for non-starch polysaccharide (NSP) determination (Englyst Carbohydrate Ltd, UK). The GLC column temperature was 220 °C, and the injector and detector were maintained at 180 °C. The carrier gas (hydrogen) flow rate was 8 mL/min. In this method, starch was removed from the NSP fraction by enzymatic hydrolysis. The recovered NSP fraction (0.5 mL) was acid hydrolysed to its sugar constituents by the addition of 2.5 mL of sulphuric acid (2.4 M), followed by heating in a boiling water bath for 1 h. An internal standard (which contained 1 mg/ mL of allose) was added to 1 mL of each sample hydrolysate and standard (consisting of known concentrations of sugars). The released constituent sugars were then derivatised by the reduction of monosaccharides with sodium borohydride to alditols and the acetylation of alditols to alditol acetate derivatives. The GalA content was determined based on the colorimetric method described by Scott (1979) using an UV-160A spectrophotometer (Shimadzu, Douglas Scientific, Singapore). The difference in absorbance of a sample at 400 nm and at 450 nm was measured against a blank consisting of 2 M H2SO4. A 0.3 mL aliquot sample was mixed with 0.3 mL of sodium chloride–boric acid solution and this was followed by the fast addition of 5 mL of concentrated H2SO4. The sample was then heated in a 70 °C water bath for 40 min. Approximately 0.2 mL of 3–5-dimethylphenol solution was added before the sample absorbance was taken. The GalA
2.4. The weight-average molecular weight (Mw) determination
2.5. Viscosity determination Vacuum-dried pectin samples isolated from kiwifruit pomace (based on 1% w/w GalA concentration) were re-dispersed in Milli-Q water under continuous stirring at room temperature for 15 min, followed by stirring for another 15 min at 60 °C. The samples were de-gassed in an ultrasonic water bath (for a few seconds only) and the pH values were adjusted to 3.50 ± 0.01 by the slow addition of 0.5 M HCl or 0.5 NaOH prior to the viscosity measurements. As a reference, a commercial citrus pectin sample (P9135, Sigma–Aldrich, Cheme GmbH, Germany) was analysed at a similar concentration and pH (based on 1% w/w GalA concentration, pH = 3.5). Viscosity curves were obtained using a controlled-stress rheometer (Paar Physica MCR 301; Anton-Paar, GmbH, Germany) with a cone and plate measuring system (CP 4/40) at 20 ± 0.1 °C and at shear rates between 1 and 1000 s1.
3. Results and discussion 3.1. Compositions of pomace The compositions of gold kiwifruit pomace as compared with the whole kiwifruit are presented in Table 1. Note that the data from MHF were published in Yuliarti et al. (2015) but are included in this manuscript for ease of comparison with data from the pomace pectin. The dried gold kiwifruit pomace used in this experiment contained approximately 4.5% (w/w) protein (%N 5.18) and 6.3% w/ w ash. The pomace had a relatively higher total NSP (23% w/w) as compared with MHF (11% w/w). The NSP of the pomace consisted of a large fraction of glucose (45% w/w), GalA (23% w/ w), xylose (15% w/w), galactose (7% w/w), rhamnose (5% w/ w), arabinose (4% w/w), mannose (4%) and fucose (1%). When compared with the MHF, the percentages of all the sugars are very similar i.e. glucose 47% w/w, GalA 26%, xylose 16%, galactose 8% w/w, arabinose 3% w/w, mannose 3% with the exception for rhamnose and fucose, where both sugars were not detected in the MHF. The absence of rhamnose could be due to
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O. Yuliarti et al. / Food Chemistry 187 (2015) 290–296 Table 1 Composition of fresh gold kiwifruit pomace and whole MHF (Yuliarti et al., 2015) based on dry matter.
Component Protein (% w/w) Ash (% w/w) Total-NSP (% w/w)
Pomace
MHFa
4.51 ± 0.34 6.33 ± 0.02 22.97 ± 0.21
4.24 ± 0.07 3.94 ± 0.09 11.07 ± 0.01
Neutral sugar composition based on total NSP (% w/w) Glucose 44.84 ± 0.18 Xylose 15.41 ± 0.30 Galactose 6.62 ± 0.14 Rhamnose 4.74 ± 0.00 Arabinose 4.35 ± 0.00 Mannose 4.16 ± 0.13 Fucose 0.61 ± 0.13 GalA (% w/w) 22.85 ± 0.74 Soluble 8.92 ± 0.47 Insoluble 13.93 ± 0.60
46.97 ± 0.27 15.63 ± 0.27 7.86 ± 0.27 ND 2.62 ± 0.27 2.62 ± 0.27 ND 25.93 ± 0.27 23.2 ± 0.27 2.71 ± 0.09
ND – Not Detected. a Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
the very low level of rhamnose present when analysed as a whole fruit. The considerable amount of glucose present is likely to originate from the cell wall of the kiwifruit which is made up of mainly cellulose. The pomace also contained a higher level of insoluble GalA fraction (61% w/w) than the soluble GalA fraction (12% w/w) based on the total GalA content. The higher insoluble GalA fraction could suggest that a larger proportion of the GalA remained trapped within the cell wall materials in pomace. 3.2. Effect of extraction methods on the pectin yield and composition Table 2 shows the yield, ash and protein contents of the purified pectin from the pomace based on the three extraction methods (acid, water and enzyme). The pomace pectin yields varied approximately between 3.6% and 4.5% w/w on a dry matter basis across the three extraction methods. These results were relatively similar to the whole MHF pectin yields (3.3–4.4% w/w) which were previously reported in Yuliarti et al. (2015). However, pomace pectin had a lower ash content (2.6–2.7% w/w) and a lower protein content (9.4–10.1% w/w) as compared with pectin from MHF (see Table 2). The highest pomace pectin yield was obtained using enzymatic extraction method (4.5% w/w) and the lower yields were obtained using acid (3.8% w/w) and water (3.6% w/w) extraction methods. The extraction of pomace pectin by enzyme (Celluclast 1.5L) which gave the highest pectin yield was due to enzymatic hydrolysis of cellulose which probably led to the release of pectin trapped within the cellulose matrix of the plant cell wall. Recent research on the extraction of pectin from apple pomace by Wikiera, Mika, and Grabacka (2015) revealed similar findings where extraction by Celluclast gave the highest pectin yield (18.95% w/w) as compared with the conventional acid based extraction method.
3.3. Sugar compositions The sugar compositions of the pectin isolated from gold kiwifruit pomace and MHF obtained by the three extraction methods are presented in Table 3. The pomace pectin had a total NSP composition in the range 78–80% w/w which can be regarded as fairly high level of polysaccharide isolated. The total NSP of the pomace (79% w/w) was higher than the MHF (64% w/w) (Yuliarti et al., 2015) across the three extraction methods. This was expected since the MHF solids contained small sugar molecules found in the juice which caused the percentage of total NSP in MHF to be lower. The proportions of GalA based on total NSP from the pomace by water, acid and enzymatic extraction methods were 83% w/w, 82% w/w and 85% w/w respectively. GalA concentrations from pomace were slightly lower compared with the amounts of GalA obtained from the MHF pectin (i.e. 86% w/w, 81% w/w and 91% w/w, respectively). It is worth noting that the amount of GalA recovered from pectin was slightly increased by enzymatic extraction method for both pomace and MHF. The enzyme Celluclast 1.5L appeared to have facilitated the release of trapped pectin without causing hydrolysis of the GalA. This finding was similar to a study conducted on chicory roots by Panouille et al. (2006). The authors showed that pectin extracted by enzyme had the highest GalA concentration compared with the acid extraction method. The cellulolytic activity of Celluclast appeared to be effective in solubilising most of cellulosic materials of gold kiwifruit pectin. Both enzymatic-extracted pectin of pomace and MHF (Yuliarti et al., 2015) showed the lowest amount of glucose (2.16% and 0.01% w/w) compared with acid (4.43% and 2.92% w/w) and water-extracted pectins (2.27% and 6.19% w/w) for pomace and MHF, respectively (see Table 3). The occurrence of other sugars such as rhamnose, galactose and arabinose in all pectin extracts could indicate that the pectin fraction of gold kiwifruit pomace was in the form of arabinans, galactans and arabinogalactans. The presence of arabinogalactans as part of pectin molecule was also observed by Redgwell, Fischer, Kendal, and MacRae (1997) in the case of green kiwifruit (Actinidia deliciosa, cv Hayward) cell wall materials. From the sugar composition of the pomace pectin, the extraction methods showed some variations in the sugar ratios across the different extraction methods. For instance, a lower amounts of arabinose were found in pectin obtained by acid-extraction method (3.1% w/w) and water-extraction method (3.6% w/w) than in the enzyme-extraction method (3.9% w/w). The decrease in arabinose in acid could be due to the hydrolysis of arabinan chains. The arabinofuranosyl linkages appeared to be more easily degraded under the acidic extraction conditions. Similar findings were reported by Levigne, Ralet, and Thibault (2002) and Garna et al. (2007) for acid extraction from fresh sugar beet pulp and apple pomace. The rhamnose contents were fairly similar across the three extraction methods (1.71–1.77% w/w). However, a different trend was observed for MHF pectin as reported in Yuliarti et al. (2015), where water and acid extraction methods showed higher rhamnose levels (1.41–1.52% w/w) than the enzymatic method
Table 2 Purified pectin yield (% w/w dry matter) of gold kiwifruit pomace and whole gold kiwifruit (Yuliarti et al., 2015). Extraction methods
Acid Water Enzyme a
Whole MHF pectina
Pomace pectin Purified yield
Ash
Protein
Purified yield
Ash
Protein
3.83 ± 0.07 3.62 ± 0.03 4.48 ± 0.21
2.59 ± 0.01 2.65 ± 0.00 2.70 ± 0.10
10.09 ± 0.04 9.38 ± 0.02 9.82 ± 0.02
3.27 ± 0.03 3.27 ± 0.37 4.39 ± 0.64
2.42 ± 0.05 5.11 ± 0.01 7.09 ± 0.01
17.98 ± 0.05 9.63 ± 0.05 13.85 ± 0.05
Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
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Table 3 Total-NSP and monosaccharide compositions of purified gold kiwifruit pectin extracted from pomace and whole fruit (Yuliarti et al., 2015) using different extraction methods. Whole MHF pectina
Pomace pectin Extraction methods
a
Extraction methods
Acid
Water
Enzyme
Acid
Water
Enzyme
% w/w total NSP (based on dried pectin)
77.59 ± 0.99
79.16 ± 0.27
80.12 ± 0.57
64.49 ± 0.93
63.77 ± 1.60
64.02 ± 0.14
% w/w sugar composition (based on total NSP) Galactose Glucose Arabinose Rhamnose Mannose Xylose Fucose GalA
6.39 ± 0.05 4.43 ± 0.22 3.09 ± 0.13 1.74 ± 0.45 1.38 ± 0.09 1.12 ± 0.09 0.50 ± 0.01 82.36 ± 2.18
8.12 ± 0.30 2.27 ± 0.10 3.55 ± 0.18 1.77 ± 0.10 0.91 ± 0.19 1.09 ± 0.18 0.21 ± 0.03 82.67 ± 1.41
6.86 ± 0.12 2.16 ± 0.01 3.87 ± 0.12 1.71 ± 0.34 0.67 ± 0.07 0.25 ± 0.00 0.27 ± 0.02 84.62 ± 0.19
5.69 ± 0.19 2.92 ± 0.02 2.68 ± 0.17 1.41 ± 0.09 0.31 ± 0.00 0.76 ± 0.12 0.33 ± 0.05 86.96 ± 0.91
8.73 ± 0.22 6.19 ± 0.41 2.79 ± 0.19 1.52 ± 0.03 0.28 ± 0.00 1.07 ± 0.00 0.38 ± 0.08 81.34 ± 2.56
4.33 ± 0.14 0.01 ± 0.37 1.97 ± 0.03 0.89 ± 0.30 0.25 ± 0.15 0.12 ± 0.05 0.30 ± 0.02 91.49 ± 0.39
Molecular parameters Mw (106/mol) Mw/Mn RMS radius (nm)
0.84 ± 0.03 9.61 ± 1.15 91.80 ± 2.65
0.85 ± 0.02 11.53 ± 2.24 101.5 ± 0.80
0.67 ± 0.03 10.93 ± 1.80 51.50 ± 1.20
2.20 ± 0.05 1.77 ± 0.06 112.6 ± 1.00
3.75 ± 0.11 2.43 ± 0.09 182.7 ± 1.10
1.65 ± 0.04 2.49 ± 0.05 162.0 ± 0.60
Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
(0.89% w/w). There is no clear explanation for this observation. It could be possible that rhamnose in pomace pectin could occur at different position in the pectin molecular chain where it could be less susceptible to enzymatic hydrolysis as compared with pectin from the MHF. In the case of galactose, water extraction method yielded the highest level (8.1% w/w) as compared with acid (6.4% w/w) and enzymatic (6.9% w/w) extraction methods. Similar trend of galactose was observed for pectin in MHF (Yuliarti et al., 2015). This could suggest that both acid and enzymatic methods could cause some hydrolysis of side chains where galactose sugar moieties reside. The difference in sugar composition suggested that pomace pectin could result in different degrees of branching depending on the extraction method used. Xylose, mannose and glucose were also detected in all extracted samples. The occurrence of these sugars could indicate that other cell wall polymers such as hemicelluloses and cellulose were also isolated during the extraction of gold kiwifruit pomace. Redgwell, Melton, and Brasch (1988) reported that the bulk of hemicellulose from green kiwifruit (A. deliciosa, cv. Hayward) at harvest consists mainly of xyloglucan. This could imply that the presence of xylose and glucose in the NSP extract of gold kiwifruit pomace may also occur as xyloglucan.
3.4. Linearity and branching Fig. 2a–c shows the possible polymeric degree of branching, linearity and extent of RG-1 branching of pomace pectin derived based on the GalA and neutral sugar ratios. Firstly, the degree of branching was calculated by dividing the amount of rhamnose by GalA concentration (Parkar et al., 2010). Secondly, according to Houben, Jolie, Fraeye, Van Loey, and Hendrickx (2011), the sugar ratio of GalA to neutral sugars reflects the linearity of pectin backbone structure, which is defined as the molar amount of GalA relative to the molar amount of pectin neutral sugars (fucose, rhamnose, arabinose, galactose and xylose) of the different pectin fractions. Thirdly, the ratio of the amount of arabinose and galactose to the amount of rhamnose indicates the extent of branching of RG-I. The composition of rhamnose, galactose and arabinose should therefore be interpreted with reference to the amount of GalA since rhamnose, galactose and arabinose usually occur as the branched portions along the GalA backbone of the pectin structure.
Fig. 2a shows the degree of branching based on the ratio of Rha/GalA. A higher ratio indicates a higher degree of branching. The branching of pomace pectin obtained by three extraction methods occurred at 45–50 of GalA residues (calculated based on the ratio of GalA/Rha). Pectin extracted by enzymatic method showed the lowest degree of branching (carried side chains every 50 GalA residues) as compared with pectin extracted by the acid and water extraction methods (carried side chains every 48 and 45 GalA residues respectively). Based on the water extraction method, pomace pectin appeared to have a higher degree of branching (i.e. carried side chains for every 45 GalA residues) than the whole MHF pectin which carried side chains for every 53 GalA residues (Yuliarti et al., 2015) (Fig. 2a). However, when compared with the cell wall materials content of green kiwifruit (A. deliciosa, cv. Hayward) at harvest stage, the degree of branching appeared to be even higher (carry side chains approximately every 23 GalA residues) as reported by Redgwell, Melton, and Brasch (1992). The difference in the degree of branching could suggest a difference in pectin functionality between the gold and green kiwifruits. For the linearity parameter, pectin extracted by acid and enzymatic extraction methods indicated more linear structures than pectin from the water extraction method (Fig. 2b). This could imply that both acid and enzymatic extraction methods could cause some degree of hydrolysis of the polysaccharide at the branched portion of the pectin molecules. In the case of pectin from MHF, the extent of hydrolysis of the polysaccharides containing the neutral sugars was very obvious for pectin isolated by the enzyme methods (see Fig. 2a and b). Pectin obtained by water extraction method (Fig. 2c) also showed the highest extent of RG-I branching indicating that the water extraction method was able to retain the highest amount of RG-I side chains. Pectin obtained by the water extraction method could be regarded as the native form of pomace pectin since the conditions used in the water extraction method were considered very mild. Similar trends to the pomace pectin were observed for pectin isolated from the whole MHF. 3.5. Weight-average molecular weight (Mw) Table 3 shows the molar masses (Mw), polydispersity indices (Mw/Mn) and RMS radii of pectin from pomace and MHF obtained by different extraction methods. For the enzyme-extracted pomace pectin, the Mw (6.7 105 g/mol) was lower than the waterextracted pomace pectin (8.5 105 g/mol) and acid-extracted
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It is important to note that the pomace pectin showed markedly lower Mw (6.7–8.5 105 g/mol) and RMS radius (52–102 nm) but a higher polydispersity index (9.6–11.5) than the whole MHF pectin (Mw 1.7–3.8 106 g/mol; RMS radius 113– 183 nm; Mw/Mn 1.8–2.5) for all the extraction methods. The results clearly indicated that the pectin fraction obtained from the MHF (which included the kiwifruit juice) was different from pectin isolated from the pomace. Studies on the pectin extracted from sugar beet and sugar beet pomace showed similar results where the pectin from sugar beet pomace had a lower Mw (1.3 105 g/mol) than whole sugar beet pectin (3.5 105 g/mol) (Levigne et al., 2002; Wang & Chang, 1994). 3.6. Viscosity Fig. 3 shows the viscosity curves of pomace and MHF pectin solutions prepared in Milli-Q water. The samples were prepared based on the same GalA content (1.0% w/w). The pomace pectin obtained using the three extraction methods exhibited almost similar viscosities (30–34 mPa s) at shear rates above 50 s1. Slight differences in the viscosity was observed at the lower shear rate range (<10 s1) in the order consistent with the molar mass and RMS radius (i.e. the viscosity of water-extracted pectin > acid-extracted pectinenzyme-extracted pectin). As shown in Fig. 3, the viscosity of the pomace pectin solution was higher than a commercial citrus-derived pectin (1.66 105 g/mol, Sigma pectin P9135, 13 mPa s) suggesting pomace pectin’s potential to act as a thickener. 4. Conclusions This study showed that pomace pectin was different from pectin obtained from MHF based on the molecular parameters. Pomace NSP accounted for approximately 23% w/w of the gold kiwifruit pomace (on dry weight basis). The extraction methods (acid, water or enzyme) were observed to have some effects on the yield, degree of branching of the pectin molecules, Mw and viscosity of pomace pectin solutions. In terms of yield, the enzymatic method showed highest pectin recovery. On the other hand, pectin obtained from the water extraction method had a lower yield but was considered closest to the native native pectin present in the pomace. All three extraction methods gave slightly different
Fig. 2. Sugar ratios for the different pectin fractions in pomace and whole gold kiwifruit pectin (MHF): (a) degree of branching (GalA/Rha); (b) the linearity pectin backbone (GalA/Fuc, Rha, Ara, Gal, Xyl) and (c) the extent of branching of RG-I (Ara + GalA/Rha).
pomace pectin (8.4 105 g/mol). The trend is consistent with the RMS radius data which showed that the largest polymer molecules were obtained using the water extraction method (102 nm), followed by acid extraction method (92 nm). The smallest size pectin molecules were obtained by the enzymatic extraction method (52 nm). This finding showed a consistent trend to the degree of branching, linearity and extent of RG1 branching as well as other studies on pectin extracted by enzymatic extraction methods reported by Panouille et al. (2006) and Ptichkina et al. (2008).
Fig. 3. Viscosity of purified gold kiwifruit pomace and whole gold kiwifruit (MHF) pectin extracted using different extraction methods; and commercial citrus pectin (sigma pectin P9135) at 1.0% w/w GalA concentration, pH 3.50 ± 0.01.
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degrees of branching and sizes based RMS radii. The significance of the degree of branching, linearity and extent of RG1 branching of the pectin is currently not well understood. All these parameters will ultimately affect the physical functionality of the pectin. Further work is required to relate degree of branching to specific functional advantages in different applications such as thickening, stabilising, gelling properties and in the formation of novel structures to deliver different technical functionality and sensorial properties, etc. Acknowledgments The authors gratefully acknowledge Ian Andrew for his help in the analysis for sugar compositions and Zespri International Ltd for funding this research. References AOAC. (1990). Official methods of analysis (15th ed.). Washington, DC: Association of Official Analytical Chemists. Emaga, T. H., Ronkart, S. N., Robert, C., Wathelet, B., & Paquot, M. (2008). Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design. Food Chemistry, 108(2), 463–471. Englyst, H. N., Quigley, M. E., Hudson, G. J., & Cummings, J. H. (1992). Determination of dietary fiber as non-starch polysaccharides by gas-liquid-chromatography. Analyst, 117(11), 1707–1714. Englyst, H. N., Quigley, M. E., & Hudson, G. J. (1994). Determination of dietary fiber as non-starch polysaccharides with gas-liquid-chromatographic, highperformance liquid-chromatographic or spectrophotometric measurement of constituent sugars. Analyst, 119(7), 1497–1509. Garna, H., Mabon, N., Robert, C., Cornet, C., Nott, K., & Legros, H. (2007). Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol. Journal of Food Science, 72(1), C1–C9. Houben, K., Jolie, R. P., Fraeye, I., Van Loey, A. M., & Hendrickx, M. E. (2011). Comparative study of the cell wall composition of broccoli, carrot, and tomato: Structural characterization of the extractable pectins and hemicelluloses. Carbohydrate Research, 346(9), 1105–1111. Ketsa, S., Chidtragool, S., Klein, J. D., & Lurie, S. (1999). Firmness, pectin components and cell wall hydrolases of mango fruit following low-temperature stress. Journal of Horticultural Science & Biotechnology, 74(6), 685–689. Levigne, S., Ralet, M. C., & Thibault, J. F. (2002). Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49(2), 145–153. May, C. D. (1990). Industrial pectins – Sources, production and applications. Carbohydrate Polymers, 12(1), 79–99.
Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant Biology, 11(3), 266–277. MPI (Ministry for Primary Industry) (2013). Kiwifruit. Accessed 02.12.14. O’Neill, M., Albersheim, P., & Darvil, A. (1990). The pectic polysaccharides of primary cell walls. In P. M. Dey (Ed.). Methods in plant biochemistry (Vol. 2, pp. 415–441). London: Academic Press. Pagan, J., Ibarz, A., Llorca, M., Pagan, A., & Barbosa-Canovas, G. V. (2001). Extraction and characterization of pectin from stored peach pomace. Food Research International, 34(7), 605–612. Panouille, M., Thibault, J. F., & Bonnin, E. (2006). Cellulase and protease preparations can extract pectins from various plant byproducts. Journal of Agricultural and Food Chemistry, 54(23), 8926–8935. Parkar, S. G., Redgate, E. L., Wibisono, R., Luo, X., Koh, E. T. H., & Schröder, R. (2010). Gut health benefits of kiwifruit pectins: Comparison with commercial functional polysaccharides. Journal of Functional Foods, 2(3), 210–218. Ptichkina, N. M., Markina, O. A., & Rumyantseva, G. N. (2008). Pectin extraction from pumpkin with the aid of microbial enzymes. Food Hydrocolloids, 22(1), 192–195. Redgwell, R. J., Fischer, M., Kendal, E., & MacRae, E. A. (1997). Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls. Planta, 203(2), 174–181. Redgwell, R. J., Melton, L. D., & Brasch, D. J. (1988). Cell-wall polysaccharides of kiwifruit (Actinidia deliciosa) – Chemical-features in different tissue zones of the fruit at harvest. Carbohydrate Research, 182(2), 241–258. Redgwell, R. J., Melton, L. D., & Brasch, D. J. (1992). Cell-wall dissolution in ripening kiwifruit (Actinidia deliciosa) – Solubilisation of the pectic polymers. Plant Physiology, 98(1), 71–81. Ridley, B. L., O’Neill, M. A., & Mohnen, D. A. (2000). Pectins: Structure, Biosynthesis, and Oligogalacturonide-Related Signaling. In Paper presented at the Symposium of the American-Chemical-Society, San Francisco. Scott, R. W. (1979). Colorimetric determination of hexuronic acids in plant materials. Analytical Chemistry, 51(7), 936–941. Wabnitz, G. (2008). Biogas from kiwifruit waste. Wellington: Maunsell Ltd. Wang, C. C. H., & Chang, K. C. (1994). Beet pulp and isolated pectin physicochemical properties as related to freezing. Journal of Food Science, 59(6), 1153. Wikiera, A., Mika, M., & Grabacka, M. (2015). Multicatalytic enzyme preparations as effective alternative to acid in pectin extraction. Food Hydrocolloids, 44, 156–161. Yuliarti, O., Goh, K., Matia-Merino, L., Mawson, J., Drummond, L., & Brennan, C. S. (2008). Effect of extraction techniques and conditions on the physicochemical properties of the water soluble polysaccharides from gold kiwifruit (Actinidia chinensis). International Journal of Food Science & Technology, 43, 2268–2277. Yuliarti, O., Matia-Merino, L., Goh, K. T., Mawson, J. A., & Brennan, C. (2011). Effect of Celluclast 1.5L on the physicochemical characterization of gold kiwifruit pectin. International Journal of Molecular Sciences, 12, 6407–6417. Yuliarti, O., Matia-Merino, L., Goh, K. T., Mawson, J. A., & Brennan, C. (2012). Characterization of gold kiwifruit pectin isolated by enzymatic treatment. International Journal of Food and Science & Technology, 47, 633–639. Yuliarti, O., Matia-Merino, L., Goh, K. K. T., Mawson, J. A., Williams, M. A. K., & Brennan, C. (2015). Characterization of gold kiwifruit pectin from fruit of different maturities and extraction methods. Food Chemistry, 166(0), 479–485.
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Extraction and characterisation of pomace pectin from gold kiwifruit (Actinidia chinensis) Oni Yuliarti a,b, Kelvin K.T. Goh a,⇑, Lara Matia-Merino a, John Mawson a,c, Charles Brennan a,d a
Massey Institute of Food Science and Technology, School of Food & Nutrition, Massey University, Palmerston North, New Zealand School of Chemical and Life Sciences, Singapore Polytechnic, Singapore c School of Agricultural and Wine Sciences, Charles Sturt University, Australia d Faculty of Agriculture and Life Sciences, Lincoln University, New Zealand b
a r t i c l e
i n f o
Article history: Received 11 January 2015 Received in revised form 24 March 2015 Accepted 26 March 2015 Available online 18 April 2015 Keywords: Pectin Pomace Gold kiwifruit Extraction Galacturonic acid, molar mass and viscosity
a b s t r a c t Gold kiwifruit pomace extracted using citric acid, water and enzyme (Celluclast 1.5L) were studied in terms of pectin yield, protein, ash, non-starch polysaccharide, galacturonic acid (GalA), neutral sugar composition, molar mass (Mw), viscosity and degree of branching. Water-extracted pectin was considered closest to its native form. Enzyme extracted pectin showed the highest yield (4.5% w/w) as compared with the acid and water extraction methods (3.6–3.8% w/w). Pectin obtained from different extraction methods showed different degree of branching. The Mw and root mean square (RMS) radius varied with the extraction methods with values of 8.4 105 g/mol and 92 nm, 8.5 105 g/mol and 102 nm, 6.7 105 g/mol and 52 nm for acid, water and enzymatic extraction methods, respectively. Similar trend was observed for pectin viscosity, with water-extracted pectin giving a slightly higher viscosity followed by acid and enzyme-extracted pectin. This study showed that gold kiwifruit pomace pectin has potential application in food products. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Pectin is a structural polysaccharide found in the cell walls of growing plants. Pectin is regarded as a complex heteropolysaccharide consisting mainly of a-(1,4)-linked D-galacturonic acid backbone with different degrees of esterification (Mohnen, 2008). Pectin can be further classified based on their occurrences as three main structures namely, homogalacturonan (HG), rhamnogalacturonan type I (RG-I) and substituted galacturonan (Ridley, O’Neill, & Mohnen, 2000). The HG group has a backbone with a linear chain of a-(1,4)-linked D-galacturonosyl acid. The RG-I group has a backbone of a-(1,2)-linked L-rhamnosyl and a-(1,4)-linked D-galacturonosyl acid residues, with neutral sugars such as arabinose and galactose as the side chains. The substituted galacturonan group consists of a-(1,4)-linked D-galacturonosyl acid residues as the backbone such as rhamnogalacturonan II and xylogalacturonan (O’Neill, Albersheim, & Darvil, 1990). Pectin is widely used as a thickener and stabiliser in food products such as jams and yoghurt drinks. Apple pomace and citrus peels are currently the two main sources of commercial pectin
⇑ Corresponding author. Tel.: +64 06 3569099; fax: +64 06 3505657. E-mail address: [email protected] (K.K.T. Goh). http://dx.doi.org/10.1016/j.foodchem.2015.03.148 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
(May, 1990). They are usually obtained from the by-products of fruit juice manufacture. Pectin polymers from other plant sources have been isolated and studied. They include sugar beet pulp (Wang & Chang, 1994), peach (Pagan, Ibarz, Llorca, Pagan, & Barbosa-Canovas, 2001), mango (Ketsa, Chidtragool, Klein, & Lurie, 1999), banana peel (Emaga, Ronkart, Robert, Wathelet, & Paquot, 2008) and chicory roots (Panouille, Thibault, & Bonnin, 2006). The extraction and isolation of pectin can be carried out using different methods including chemical, physical or enzymatic treatments (Panouille et al., 2006). Chemical extraction method using dilute acid solution is commonly employed in pectin manufacture. However, the use of chemical method to extract pectin could give a negative connotation as consumers increasingly prefer chemical-free ingredients to be used in food products. Instead, the use of specific enzymes could be advantageous due to the low enzyme concentrations required and the low amount of waste generated (Yuliarti, Matia-Merino, Goh, Mawson, & Brennan, 2012). New Zealand kiwifruit growers produced approximately 400,000 tonnes of kiwifruit from 13,000 hectares split between green (80%) and gold (20%) kiwifruit (MPI, 2013). Kiwifruit is either consumed as a fruit or processed into kiwifruit juice. In the processing of kiwifruit juice, a large proportion of the fruit goes into waste which includes the fruit peels, pomace and seeds. Kiwifruit
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waste has been reported by Wabnitz (2008) to be approximately 18% of the total kiwifruit crop. Gold kiwifruit pomace obtained after juice extraction constitutes about 40–50% of the weight of the fresh fruit (data not published). Despite the smaller percentage of gold kiwifruit crop based on the current data, the demand for gold kiwifruit is envisaged to grow as consumer demand increases. The physico-chemical properties of pectin from the whole gold kiwifruit have recently been reported (Yuliarti et al., 2015). However, the physico-chemical properties of pectin solely from the pomace of gold kiwifruit have not been well-understood. The chemical composition of pomace pectin and its functional properties could differ from pectin extracted from whole kiwifruit. It is useful for growers and manufacturers to understand the potential functional benefits of pomace where the whole kiwifruit is utilised for juice processing. The data from this study could provide useful knowledge to maximise the utilisation of gold kiwifruit that will not only value-add but also reduce kiwifruit waste generated. It was therefore deemed necessary to characterise and compare the properties of pomace pectin with the pectin obtained from the whole kiwifruit. Knowledge of the physical functionality of pomace pectin could provide useful information for manufacturers to consider utilising the kiwifruit pomace as another source of pectin that is capable of providing certain unique functional properties. The objectives of this study were to examine the yield, composition, molar mass and the rheological properties of gold kiwifruit pomace pectin extracted using different extraction methods (acid, water and enzyme). Comparisons were made with pectin isolated from the whole kiwifruit (main-harvested fruit; MHF), as previously reported by Yuliarti et al. (2015).
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Fig. 1. Schematic flow chart of the preparation of gold kiwifruit pomace.
2.2. Pectin extraction
cloths to remove the pulp and seeds that were not separated during centrifugation. Warm water at 50 °C was added to the pellet, in a 1:1 (w/v) ratio, and the mixture was stirred for 30 min (to solubilise the remaining soluble polysaccharides) and centrifuged again as described above. All supernatants and filtrates were combined and precipitated with ethanol as commonly carried out in pectin production (May, 1990). The polysaccharide in the supernatant was precipitated by ethanol to give a final concentration of 80% (v/v). This concentration was chosen because low molecular weight saccharides (e.g. monosaccharides and oligosaccharides) would remain soluble but polysaccharides would be precipitated out (Englyst, Quigley, Hudson, & Cummings, 1992). The mixture of ethanol and extract was stirred (10 min, ±25 °C) to obtain a uniform mixture and was then kept at 4 °C for 4 h to allow the polysaccharides to precipitate. To separate the polysaccharide precipitate from the solvent, the mixture was centrifuged (3300g, 10 min, 4 °C). The pellet was washed twice with 95% ethanol (1:1, w/v) and then centrifuged again as before. The pellet was vacuum dried (Eyela, Vacuum Oven, Voc-300 SD, Science Technique Ltd, New Zealand) at 58 ± 3 °C, 65 cm Hg, for approximately 7–10 h until a constant weight was achieved. For further purification of pectin, the vacuum-dried sample was dispersed in RO water (1.0% w/w) and stirred overnight (15 h) in a 4 °C chiller. The dispersion was then centrifuged at 30,000g for 60 min (4 °C) to separate out the remaining insoluble particles. The soluble pectin in the supernatant was recovered by ethanol precipitation (80%), centrifuged, ethanol washed and vacuum oven dried as described earlier. The pectin yield was calculated using the equation below (Ptichkina, Markina, & Rumyantseva, 2008) and was denoted as the acid extracted pomace pectin:
The pomace recovered from MHF was subjected to three different extraction methods using acid, water or enzyme respectively.
mpectin D ¼ 100 mkp
2. Materials and methods 2.1. Materials The main-harvested gold kiwifruit (MHF) obtained from Zespri International Ltd (Hastings, New Zealand) was the raw material used in this study. The fruit was harvested approximately 20 weeks after pollination, with a firmness of 32 Newton (N). The pomace of MHF used in this study was obtained by squashing whole kiwifruit in a fruit juicer (Avanti, Model 2000 Juicer, FED Australia and New Zealand, 0.5 mm mesh). As the juice contained a mixture of very coarse fruit pulp fractions, skin and seeds, centrifugation (3300g, 20 min at 4 °C) was carried out to isolate the insoluble fraction from the juice. The fruit pulp from the juicer was hydraulically pressed to separate the remaining juice. The pellet, which was considered to be part of the pomace, was pooled and mixed with the fruit pulp that was retained in the juicer. The skin and seeds of the fruit were included in the pomace preparation. A flow diagram for the preparation of the pomace is shown in Fig. 1.
2.2.1. Acid extraction Gold kiwifruit pomace (200 g) was mixed with 600 mL of 1.0% (w/v) citric acid (CA) solution of pH 2.20 ± 0.01 (1:3 w/v pomace to acid solution ratio) in a container and the mixture (pH 3.1 ± 0.05) was heated in a 50 °C water bath for 60 min under continuous stirring. The container was covered with aluminium foil to reduce moisture loss due to evaporation. Purified water obtained by reverse osmosis (RO water) was used throughout the study. After extraction, the mixture was cooled to approximately 20 °C in a bath of crushed ice and then centrifuged at 3300g for 20 min at 4 °C. The supernatant was filtered through four layers of cheese
where D is the percentage yield of purified pectin (%), mpectin is the mass of recovered pectin (g) and mkp is the mass of kiwifruit pomace (g) used in the extraction. 2.2.2. Water extraction The water extraction method was similar to the acid extraction method except that RO water was used instead of the citric acid solution. The extraction was carried out in a 25 °C water bath for 30 min with a pomace to water ratio of 1:3 (w/v) (i.e. 200 g of pomace mixed with 600 mL of RO water). The pH value of the mixture was 3.70 ± 0.05. Recovery of the soluble fraction from the
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mixture was carried out as described for the acid extraction method. Isolation and purification of pectin were carried out based on the procedures described for the acid extraction method. The pectin yield was calculated and was denoted as the water extracted pomace pectin.
values obtained in this study were expressed as gram of polysaccharide per 100 g of sample.
2.2.3. Enzymatic extraction For enzymatic extraction method, the steps for pectin isolation, ethanol precipitation and purification were similar to those described for the acid extraction method. The extraction was conducted at 25 °C for 30 min with Celluclast 1.5L (Novozymes, Copenhagen, Denmark) at a concentration of 1.05 mL/kg. Prior to this extraction, the enzymatic activities of Celluclast 1.5L were pre-determined at 25 °C (pH 3.50 ± 0.01). The results showed that this enzyme contained a mixture of cellulase, polygalacturonase and arabinase with activities of 50.81, 40.82 and 42.42 nkatals/ mL respectively (Yuliarti et al., 2008). In addition, Celluclast 1.5L has been reported to contain xyloglucanase (1120 nkatals/mL) and rhamnogalacturonase (10 nkatals/mL) at 40 °C and pH 5.5 (Panouille et al., 2006). The extraction method of gold kiwifruit pectin by enzymatic treatment was based on a previous study reported by Yuliarti, Matia-Merino, Goh, Mawson, and Brennan (2011). To facilitate a good pomace and enzyme (Celluclast 1.5L) dispersion, RO water was added to the pomace at a ratio of 1:3 (w/v) and the pH value of the mixture was pH 3.70 ± 0.05. This pH value was within the optimum range of Celluclast activity. Recovery of the soluble fraction from the mixture and purification of the pectin were carried out based on the procedures described for the acid extraction method. The pectin obtained was denoted as the enzyme extracted pomace pectin.
The Mw, polydispersity index (Mw/Mn) and the root mean square (RMS) radius of purified pectin were determined by size exclusion chromatography (SEC) coupled to a multi-angle laser light scattering (MALLS) system (Mini Dawn, Wyatt Technology Corp., Santa Barbara, CA, USA) as described by Yuliarti et al. (2015). This system consisted of a high performance liquid chromatography (HPLC) system (GBC Scientific Equipment Ltd, Victoria, Australia), which comprised an HPLC pump (model LC 1150), an ultraviolet (UV) detector (model LC 1200), a system organiser (model LC 1440) and a differential refractive index (DRI) detector (Waters, model R401, Milford, MA, USA). For the molar mass calculation, the specific refractive index increment (dn/dc) of the pectin from the pomace was 0.189 mL/g which is very similar to the value reported earlier for pectin from MHF (Yuliarti et al., 2011). The data were analysed using Astra software (version 4.50, Wyatt Technology Corp., Santa Barbara, CA, USA) and the Zimm plot method to determine the Mw, Mw/Mn and RMS radius of the pectin fraction. A commercial citrus pectin (Sigma, P9135, unknown Mw) was also analysed for comparative purposes.
2.3. Analyses of pomace pectin The fresh pomace and the isolated pomace pectin (obtained from the three extraction methods) were analysed for dry matter which was measured by calculating the weight difference after drying the sample. The ash and protein contents were determined using gravimetric method and Kjeldahl method respectively (AOAC, 1990). Neutral sugar composition was determined by gas liquid chromatography (GLC, BPX-70 column), in the form of alditol acetate derivatives as described by Englyst, Quigley, and Hudson (1994) using an Englyst Kit for non-starch polysaccharide (NSP) determination (Englyst Carbohydrate Ltd, UK). The GLC column temperature was 220 °C, and the injector and detector were maintained at 180 °C. The carrier gas (hydrogen) flow rate was 8 mL/min. In this method, starch was removed from the NSP fraction by enzymatic hydrolysis. The recovered NSP fraction (0.5 mL) was acid hydrolysed to its sugar constituents by the addition of 2.5 mL of sulphuric acid (2.4 M), followed by heating in a boiling water bath for 1 h. An internal standard (which contained 1 mg/ mL of allose) was added to 1 mL of each sample hydrolysate and standard (consisting of known concentrations of sugars). The released constituent sugars were then derivatised by the reduction of monosaccharides with sodium borohydride to alditols and the acetylation of alditols to alditol acetate derivatives. The GalA content was determined based on the colorimetric method described by Scott (1979) using an UV-160A spectrophotometer (Shimadzu, Douglas Scientific, Singapore). The difference in absorbance of a sample at 400 nm and at 450 nm was measured against a blank consisting of 2 M H2SO4. A 0.3 mL aliquot sample was mixed with 0.3 mL of sodium chloride–boric acid solution and this was followed by the fast addition of 5 mL of concentrated H2SO4. The sample was then heated in a 70 °C water bath for 40 min. Approximately 0.2 mL of 3–5-dimethylphenol solution was added before the sample absorbance was taken. The GalA
2.4. The weight-average molecular weight (Mw) determination
2.5. Viscosity determination Vacuum-dried pectin samples isolated from kiwifruit pomace (based on 1% w/w GalA concentration) were re-dispersed in Milli-Q water under continuous stirring at room temperature for 15 min, followed by stirring for another 15 min at 60 °C. The samples were de-gassed in an ultrasonic water bath (for a few seconds only) and the pH values were adjusted to 3.50 ± 0.01 by the slow addition of 0.5 M HCl or 0.5 NaOH prior to the viscosity measurements. As a reference, a commercial citrus pectin sample (P9135, Sigma–Aldrich, Cheme GmbH, Germany) was analysed at a similar concentration and pH (based on 1% w/w GalA concentration, pH = 3.5). Viscosity curves were obtained using a controlled-stress rheometer (Paar Physica MCR 301; Anton-Paar, GmbH, Germany) with a cone and plate measuring system (CP 4/40) at 20 ± 0.1 °C and at shear rates between 1 and 1000 s1.
3. Results and discussion 3.1. Compositions of pomace The compositions of gold kiwifruit pomace as compared with the whole kiwifruit are presented in Table 1. Note that the data from MHF were published in Yuliarti et al. (2015) but are included in this manuscript for ease of comparison with data from the pomace pectin. The dried gold kiwifruit pomace used in this experiment contained approximately 4.5% (w/w) protein (%N 5.18) and 6.3% w/ w ash. The pomace had a relatively higher total NSP (23% w/w) as compared with MHF (11% w/w). The NSP of the pomace consisted of a large fraction of glucose (45% w/w), GalA (23% w/ w), xylose (15% w/w), galactose (7% w/w), rhamnose (5% w/ w), arabinose (4% w/w), mannose (4%) and fucose (1%). When compared with the MHF, the percentages of all the sugars are very similar i.e. glucose 47% w/w, GalA 26%, xylose 16%, galactose 8% w/w, arabinose 3% w/w, mannose 3% with the exception for rhamnose and fucose, where both sugars were not detected in the MHF. The absence of rhamnose could be due to
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Component Protein (% w/w) Ash (% w/w) Total-NSP (% w/w)
Pomace
MHFa
4.51 ± 0.34 6.33 ± 0.02 22.97 ± 0.21
4.24 ± 0.07 3.94 ± 0.09 11.07 ± 0.01
Neutral sugar composition based on total NSP (% w/w) Glucose 44.84 ± 0.18 Xylose 15.41 ± 0.30 Galactose 6.62 ± 0.14 Rhamnose 4.74 ± 0.00 Arabinose 4.35 ± 0.00 Mannose 4.16 ± 0.13 Fucose 0.61 ± 0.13 GalA (% w/w) 22.85 ± 0.74 Soluble 8.92 ± 0.47 Insoluble 13.93 ± 0.60
46.97 ± 0.27 15.63 ± 0.27 7.86 ± 0.27 ND 2.62 ± 0.27 2.62 ± 0.27 ND 25.93 ± 0.27 23.2 ± 0.27 2.71 ± 0.09
ND – Not Detected. a Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
the very low level of rhamnose present when analysed as a whole fruit. The considerable amount of glucose present is likely to originate from the cell wall of the kiwifruit which is made up of mainly cellulose. The pomace also contained a higher level of insoluble GalA fraction (61% w/w) than the soluble GalA fraction (12% w/w) based on the total GalA content. The higher insoluble GalA fraction could suggest that a larger proportion of the GalA remained trapped within the cell wall materials in pomace. 3.2. Effect of extraction methods on the pectin yield and composition Table 2 shows the yield, ash and protein contents of the purified pectin from the pomace based on the three extraction methods (acid, water and enzyme). The pomace pectin yields varied approximately between 3.6% and 4.5% w/w on a dry matter basis across the three extraction methods. These results were relatively similar to the whole MHF pectin yields (3.3–4.4% w/w) which were previously reported in Yuliarti et al. (2015). However, pomace pectin had a lower ash content (2.6–2.7% w/w) and a lower protein content (9.4–10.1% w/w) as compared with pectin from MHF (see Table 2). The highest pomace pectin yield was obtained using enzymatic extraction method (4.5% w/w) and the lower yields were obtained using acid (3.8% w/w) and water (3.6% w/w) extraction methods. The extraction of pomace pectin by enzyme (Celluclast 1.5L) which gave the highest pectin yield was due to enzymatic hydrolysis of cellulose which probably led to the release of pectin trapped within the cellulose matrix of the plant cell wall. Recent research on the extraction of pectin from apple pomace by Wikiera, Mika, and Grabacka (2015) revealed similar findings where extraction by Celluclast gave the highest pectin yield (18.95% w/w) as compared with the conventional acid based extraction method.
3.3. Sugar compositions The sugar compositions of the pectin isolated from gold kiwifruit pomace and MHF obtained by the three extraction methods are presented in Table 3. The pomace pectin had a total NSP composition in the range 78–80% w/w which can be regarded as fairly high level of polysaccharide isolated. The total NSP of the pomace (79% w/w) was higher than the MHF (64% w/w) (Yuliarti et al., 2015) across the three extraction methods. This was expected since the MHF solids contained small sugar molecules found in the juice which caused the percentage of total NSP in MHF to be lower. The proportions of GalA based on total NSP from the pomace by water, acid and enzymatic extraction methods were 83% w/w, 82% w/w and 85% w/w respectively. GalA concentrations from pomace were slightly lower compared with the amounts of GalA obtained from the MHF pectin (i.e. 86% w/w, 81% w/w and 91% w/w, respectively). It is worth noting that the amount of GalA recovered from pectin was slightly increased by enzymatic extraction method for both pomace and MHF. The enzyme Celluclast 1.5L appeared to have facilitated the release of trapped pectin without causing hydrolysis of the GalA. This finding was similar to a study conducted on chicory roots by Panouille et al. (2006). The authors showed that pectin extracted by enzyme had the highest GalA concentration compared with the acid extraction method. The cellulolytic activity of Celluclast appeared to be effective in solubilising most of cellulosic materials of gold kiwifruit pectin. Both enzymatic-extracted pectin of pomace and MHF (Yuliarti et al., 2015) showed the lowest amount of glucose (2.16% and 0.01% w/w) compared with acid (4.43% and 2.92% w/w) and water-extracted pectins (2.27% and 6.19% w/w) for pomace and MHF, respectively (see Table 3). The occurrence of other sugars such as rhamnose, galactose and arabinose in all pectin extracts could indicate that the pectin fraction of gold kiwifruit pomace was in the form of arabinans, galactans and arabinogalactans. The presence of arabinogalactans as part of pectin molecule was also observed by Redgwell, Fischer, Kendal, and MacRae (1997) in the case of green kiwifruit (Actinidia deliciosa, cv Hayward) cell wall materials. From the sugar composition of the pomace pectin, the extraction methods showed some variations in the sugar ratios across the different extraction methods. For instance, a lower amounts of arabinose were found in pectin obtained by acid-extraction method (3.1% w/w) and water-extraction method (3.6% w/w) than in the enzyme-extraction method (3.9% w/w). The decrease in arabinose in acid could be due to the hydrolysis of arabinan chains. The arabinofuranosyl linkages appeared to be more easily degraded under the acidic extraction conditions. Similar findings were reported by Levigne, Ralet, and Thibault (2002) and Garna et al. (2007) for acid extraction from fresh sugar beet pulp and apple pomace. The rhamnose contents were fairly similar across the three extraction methods (1.71–1.77% w/w). However, a different trend was observed for MHF pectin as reported in Yuliarti et al. (2015), where water and acid extraction methods showed higher rhamnose levels (1.41–1.52% w/w) than the enzymatic method
Table 2 Purified pectin yield (% w/w dry matter) of gold kiwifruit pomace and whole gold kiwifruit (Yuliarti et al., 2015). Extraction methods
Acid Water Enzyme a
Whole MHF pectina
Pomace pectin Purified yield
Ash
Protein
Purified yield
Ash
Protein
3.83 ± 0.07 3.62 ± 0.03 4.48 ± 0.21
2.59 ± 0.01 2.65 ± 0.00 2.70 ± 0.10
10.09 ± 0.04 9.38 ± 0.02 9.82 ± 0.02
3.27 ± 0.03 3.27 ± 0.37 4.39 ± 0.64
2.42 ± 0.05 5.11 ± 0.01 7.09 ± 0.01
17.98 ± 0.05 9.63 ± 0.05 13.85 ± 0.05
Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
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Table 3 Total-NSP and monosaccharide compositions of purified gold kiwifruit pectin extracted from pomace and whole fruit (Yuliarti et al., 2015) using different extraction methods. Whole MHF pectina
Pomace pectin Extraction methods
a
Extraction methods
Acid
Water
Enzyme
Acid
Water
Enzyme
% w/w total NSP (based on dried pectin)
77.59 ± 0.99
79.16 ± 0.27
80.12 ± 0.57
64.49 ± 0.93
63.77 ± 1.60
64.02 ± 0.14
% w/w sugar composition (based on total NSP) Galactose Glucose Arabinose Rhamnose Mannose Xylose Fucose GalA
6.39 ± 0.05 4.43 ± 0.22 3.09 ± 0.13 1.74 ± 0.45 1.38 ± 0.09 1.12 ± 0.09 0.50 ± 0.01 82.36 ± 2.18
8.12 ± 0.30 2.27 ± 0.10 3.55 ± 0.18 1.77 ± 0.10 0.91 ± 0.19 1.09 ± 0.18 0.21 ± 0.03 82.67 ± 1.41
6.86 ± 0.12 2.16 ± 0.01 3.87 ± 0.12 1.71 ± 0.34 0.67 ± 0.07 0.25 ± 0.00 0.27 ± 0.02 84.62 ± 0.19
5.69 ± 0.19 2.92 ± 0.02 2.68 ± 0.17 1.41 ± 0.09 0.31 ± 0.00 0.76 ± 0.12 0.33 ± 0.05 86.96 ± 0.91
8.73 ± 0.22 6.19 ± 0.41 2.79 ± 0.19 1.52 ± 0.03 0.28 ± 0.00 1.07 ± 0.00 0.38 ± 0.08 81.34 ± 2.56
4.33 ± 0.14 0.01 ± 0.37 1.97 ± 0.03 0.89 ± 0.30 0.25 ± 0.15 0.12 ± 0.05 0.30 ± 0.02 91.49 ± 0.39
Molecular parameters Mw (106/mol) Mw/Mn RMS radius (nm)
0.84 ± 0.03 9.61 ± 1.15 91.80 ± 2.65
0.85 ± 0.02 11.53 ± 2.24 101.5 ± 0.80
0.67 ± 0.03 10.93 ± 1.80 51.50 ± 1.20
2.20 ± 0.05 1.77 ± 0.06 112.6 ± 1.00
3.75 ± 0.11 2.43 ± 0.09 182.7 ± 1.10
1.65 ± 0.04 2.49 ± 0.05 162.0 ± 0.60
Data extracted from Yuliarti et al. (2015), mean ± standard error (n = 3).
(0.89% w/w). There is no clear explanation for this observation. It could be possible that rhamnose in pomace pectin could occur at different position in the pectin molecular chain where it could be less susceptible to enzymatic hydrolysis as compared with pectin from the MHF. In the case of galactose, water extraction method yielded the highest level (8.1% w/w) as compared with acid (6.4% w/w) and enzymatic (6.9% w/w) extraction methods. Similar trend of galactose was observed for pectin in MHF (Yuliarti et al., 2015). This could suggest that both acid and enzymatic methods could cause some hydrolysis of side chains where galactose sugar moieties reside. The difference in sugar composition suggested that pomace pectin could result in different degrees of branching depending on the extraction method used. Xylose, mannose and glucose were also detected in all extracted samples. The occurrence of these sugars could indicate that other cell wall polymers such as hemicelluloses and cellulose were also isolated during the extraction of gold kiwifruit pomace. Redgwell, Melton, and Brasch (1988) reported that the bulk of hemicellulose from green kiwifruit (A. deliciosa, cv. Hayward) at harvest consists mainly of xyloglucan. This could imply that the presence of xylose and glucose in the NSP extract of gold kiwifruit pomace may also occur as xyloglucan.
3.4. Linearity and branching Fig. 2a–c shows the possible polymeric degree of branching, linearity and extent of RG-1 branching of pomace pectin derived based on the GalA and neutral sugar ratios. Firstly, the degree of branching was calculated by dividing the amount of rhamnose by GalA concentration (Parkar et al., 2010). Secondly, according to Houben, Jolie, Fraeye, Van Loey, and Hendrickx (2011), the sugar ratio of GalA to neutral sugars reflects the linearity of pectin backbone structure, which is defined as the molar amount of GalA relative to the molar amount of pectin neutral sugars (fucose, rhamnose, arabinose, galactose and xylose) of the different pectin fractions. Thirdly, the ratio of the amount of arabinose and galactose to the amount of rhamnose indicates the extent of branching of RG-I. The composition of rhamnose, galactose and arabinose should therefore be interpreted with reference to the amount of GalA since rhamnose, galactose and arabinose usually occur as the branched portions along the GalA backbone of the pectin structure.
Fig. 2a shows the degree of branching based on the ratio of Rha/GalA. A higher ratio indicates a higher degree of branching. The branching of pomace pectin obtained by three extraction methods occurred at 45–50 of GalA residues (calculated based on the ratio of GalA/Rha). Pectin extracted by enzymatic method showed the lowest degree of branching (carried side chains every 50 GalA residues) as compared with pectin extracted by the acid and water extraction methods (carried side chains every 48 and 45 GalA residues respectively). Based on the water extraction method, pomace pectin appeared to have a higher degree of branching (i.e. carried side chains for every 45 GalA residues) than the whole MHF pectin which carried side chains for every 53 GalA residues (Yuliarti et al., 2015) (Fig. 2a). However, when compared with the cell wall materials content of green kiwifruit (A. deliciosa, cv. Hayward) at harvest stage, the degree of branching appeared to be even higher (carry side chains approximately every 23 GalA residues) as reported by Redgwell, Melton, and Brasch (1992). The difference in the degree of branching could suggest a difference in pectin functionality between the gold and green kiwifruits. For the linearity parameter, pectin extracted by acid and enzymatic extraction methods indicated more linear structures than pectin from the water extraction method (Fig. 2b). This could imply that both acid and enzymatic extraction methods could cause some degree of hydrolysis of the polysaccharide at the branched portion of the pectin molecules. In the case of pectin from MHF, the extent of hydrolysis of the polysaccharides containing the neutral sugars was very obvious for pectin isolated by the enzyme methods (see Fig. 2a and b). Pectin obtained by water extraction method (Fig. 2c) also showed the highest extent of RG-I branching indicating that the water extraction method was able to retain the highest amount of RG-I side chains. Pectin obtained by the water extraction method could be regarded as the native form of pomace pectin since the conditions used in the water extraction method were considered very mild. Similar trends to the pomace pectin were observed for pectin isolated from the whole MHF. 3.5. Weight-average molecular weight (Mw) Table 3 shows the molar masses (Mw), polydispersity indices (Mw/Mn) and RMS radii of pectin from pomace and MHF obtained by different extraction methods. For the enzyme-extracted pomace pectin, the Mw (6.7 105 g/mol) was lower than the waterextracted pomace pectin (8.5 105 g/mol) and acid-extracted
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It is important to note that the pomace pectin showed markedly lower Mw (6.7–8.5 105 g/mol) and RMS radius (52–102 nm) but a higher polydispersity index (9.6–11.5) than the whole MHF pectin (Mw 1.7–3.8 106 g/mol; RMS radius 113– 183 nm; Mw/Mn 1.8–2.5) for all the extraction methods. The results clearly indicated that the pectin fraction obtained from the MHF (which included the kiwifruit juice) was different from pectin isolated from the pomace. Studies on the pectin extracted from sugar beet and sugar beet pomace showed similar results where the pectin from sugar beet pomace had a lower Mw (1.3 105 g/mol) than whole sugar beet pectin (3.5 105 g/mol) (Levigne et al., 2002; Wang & Chang, 1994). 3.6. Viscosity Fig. 3 shows the viscosity curves of pomace and MHF pectin solutions prepared in Milli-Q water. The samples were prepared based on the same GalA content (1.0% w/w). The pomace pectin obtained using the three extraction methods exhibited almost similar viscosities (30–34 mPa s) at shear rates above 50 s1. Slight differences in the viscosity was observed at the lower shear rate range (<10 s1) in the order consistent with the molar mass and RMS radius (i.e. the viscosity of water-extracted pectin > acid-extracted pectinenzyme-extracted pectin). As shown in Fig. 3, the viscosity of the pomace pectin solution was higher than a commercial citrus-derived pectin (1.66 105 g/mol, Sigma pectin P9135, 13 mPa s) suggesting pomace pectin’s potential to act as a thickener. 4. Conclusions This study showed that pomace pectin was different from pectin obtained from MHF based on the molecular parameters. Pomace NSP accounted for approximately 23% w/w of the gold kiwifruit pomace (on dry weight basis). The extraction methods (acid, water or enzyme) were observed to have some effects on the yield, degree of branching of the pectin molecules, Mw and viscosity of pomace pectin solutions. In terms of yield, the enzymatic method showed highest pectin recovery. On the other hand, pectin obtained from the water extraction method had a lower yield but was considered closest to the native native pectin present in the pomace. All three extraction methods gave slightly different
Fig. 2. Sugar ratios for the different pectin fractions in pomace and whole gold kiwifruit pectin (MHF): (a) degree of branching (GalA/Rha); (b) the linearity pectin backbone (GalA/Fuc, Rha, Ara, Gal, Xyl) and (c) the extent of branching of RG-I (Ara + GalA/Rha).
pomace pectin (8.4 105 g/mol). The trend is consistent with the RMS radius data which showed that the largest polymer molecules were obtained using the water extraction method (102 nm), followed by acid extraction method (92 nm). The smallest size pectin molecules were obtained by the enzymatic extraction method (52 nm). This finding showed a consistent trend to the degree of branching, linearity and extent of RG1 branching as well as other studies on pectin extracted by enzymatic extraction methods reported by Panouille et al. (2006) and Ptichkina et al. (2008).
Fig. 3. Viscosity of purified gold kiwifruit pomace and whole gold kiwifruit (MHF) pectin extracted using different extraction methods; and commercial citrus pectin (sigma pectin P9135) at 1.0% w/w GalA concentration, pH 3.50 ± 0.01.
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degrees of branching and sizes based RMS radii. The significance of the degree of branching, linearity and extent of RG1 branching of the pectin is currently not well understood. All these parameters will ultimately affect the physical functionality of the pectin. Further work is required to relate degree of branching to specific functional advantages in different applications such as thickening, stabilising, gelling properties and in the formation of novel structures to deliver different technical functionality and sensorial properties, etc. Acknowledgments The authors gratefully acknowledge Ian Andrew for his help in the analysis for sugar compositions and Zespri International Ltd for funding this research. References AOAC. (1990). Official methods of analysis (15th ed.). Washington, DC: Association of Official Analytical Chemists. Emaga, T. H., Ronkart, S. N., Robert, C., Wathelet, B., & Paquot, M. (2008). Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design. Food Chemistry, 108(2), 463–471. Englyst, H. N., Quigley, M. E., Hudson, G. J., & Cummings, J. H. (1992). Determination of dietary fiber as non-starch polysaccharides by gas-liquid-chromatography. Analyst, 117(11), 1707–1714. Englyst, H. N., Quigley, M. E., & Hudson, G. J. (1994). Determination of dietary fiber as non-starch polysaccharides with gas-liquid-chromatographic, highperformance liquid-chromatographic or spectrophotometric measurement of constituent sugars. Analyst, 119(7), 1497–1509. Garna, H., Mabon, N., Robert, C., Cornet, C., Nott, K., & Legros, H. (2007). Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol. Journal of Food Science, 72(1), C1–C9. Houben, K., Jolie, R. P., Fraeye, I., Van Loey, A. M., & Hendrickx, M. E. (2011). Comparative study of the cell wall composition of broccoli, carrot, and tomato: Structural characterization of the extractable pectins and hemicelluloses. Carbohydrate Research, 346(9), 1105–1111. Ketsa, S., Chidtragool, S., Klein, J. D., & Lurie, S. (1999). Firmness, pectin components and cell wall hydrolases of mango fruit following low-temperature stress. Journal of Horticultural Science & Biotechnology, 74(6), 685–689. Levigne, S., Ralet, M. C., & Thibault, J. F. (2002). Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49(2), 145–153. May, C. D. (1990). Industrial pectins – Sources, production and applications. Carbohydrate Polymers, 12(1), 79–99.
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