Pectin quantity, composition and physicochemical behaviour as influenced by the purification process

Food Research International 42 (2009) 1197–1202

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Pectin quantity, composition and physicochemical behaviour as influenced by the purification process Beda M. Yapo * UFR-Sciences et Technologie des Aliments, Abobo-Adjamé Université, 02 BP 801 Abidjan 02, Cote d’Ivoire

a r t i c l e

i n f o

Article history: Received 10 December 2008 Accepted 7 June 2009

Keywords: Agro-residues Pectin extraction Purification method Molecular-weight distribution Gelling properties

a b s t r a c t To examine the purification procedure effect on the pectin amount, purity, macromolecular characteristics, and gelling ability, three pectin isolates, namely, alcohol (APP)-, dialysis (DPP)-, and metal ion (MPP)purified pectins are obtained from acid extracts of yellow passion fruit rind using alcohol-precipitation, dialysis, or ion-binding precipitation. The results show that the amount of MPP (4.1 g/100 g) is significantly (p < 0.05) lower than the amount of the two other pectins (6.8–7.5 g/100 g). In contrast, MPP has a higher galacturonic acid (78.9 g/100 g) and lower neutral sugar (9.7 g/100 g), ash (0.9 g/100 g), and protein (1.4 g/100 g) contents than the remainder (62.4–70.1, 16.0–17.8, 2.7–5.8, 3.1–3.2 g/100 g, respectively). Molecular-weight distribution patterns suggest that MPP is free of neutral sugar oligosaccharide contaminants unlike the others, especially APP. Therefore, the term of ‘the galacturonic acid yield’ is introduced to complement the conventional term of ‘the pectin yield’. Furthermore, MPP gel preparation is likely to set more rapidly, with the gel formed achieving a much higher strength. It is concluded that the pectin amount, composition, and physicochemical properties can be considerably affected by the purification mode. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Pectins are structural polysaccharides present within all higher plant cell walls. Their widely believed overall structure encompasses homogalacturonan blocks covalently linked to type one rhamnogalacturonan blocks bearing neutral sugar side-chains, with sometimes a few type two rhamnogalacturonan blocks (Yapo, Lerouge, Thibault, & Ralet, 2007) even though the way these blocks are assembled to form pectin macromolecules is still to be elucidated. Pectins can have diverse functional properties, but the one clearly known and common to various sources since the earliest studies (Braconnot, 1825; Vauquelin, 1790) is gel formation under specified conditions. Studies of extracted ‘non-modified’ pectins showed that not only intrinsic factors, such as the galacturonic acid (GalA) and neutral sugar (NS) contents, degrees of methyl-esterification (DM) and acetyl-esterification (DA), and average-molecular weight (O’Donoghue & Somerfield, 2008; Yapo, 2009a), but also extrinsic factors such as the ash content and ionic strength of pectin solutions can influence their gelling properties. Contrary to the pectin GalA content, DM, and average-molecular weight, an increase in the other factors cited above was reported to negatively affect the gel formation (Miyamoto & Chang, 1992; Pippen,

* Tel.: +225 015 511 50. E-mail address: [email protected] 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.06.002

McCready, & Owens, 1950; Sosulski, Lin, & Humbert, 1978). For instance, pectins which are naturally rich in acetyl-esterification and neutral sugars and/or have low average-molecular weights such as those extracted from chicory, pumpkin, and sugar beet by-products (Robert, Devillers, Wathelet, Van Herck, & Paquot, 2006; Shkodina, Zeltser, Selivanov, & Ignatov, 1998; Yapo, Robert, Etienne, Wathelet, & Paquot, 2007) exhibit rather poor gelling properties unless they are structurally-modified beforehand. On an industrial scale, pectins are usually extracted using hot water acidified with a strong mineral acid (the so-called conventional (acid) extraction) under pH, temperature, and duration conditions generally in the range of 1.3–3, 60–100 °C, and 20–360 min, respectively. Hot dilute solutions of strong mineral acids are indeed capable of releasing from pectin-rich cell wall materials (CWMs) not only water-, buffer-, and chelating agent-extractable pectin fractions but also pectins which are resistant to these agents, the so-called ‘protopectins’. The acid-solubilisation of the latter occurs through the cleavage of glycosidic bonds, primarily the NS linkages. In an acid medium (pH < 2), glycosidic linkage between two NS units is indeed more sensitive to acid than glycosidic linkages implicating one rhamnose and one GalA (pseudo-aldobiuronic or aldobiuronic linkages); the linkage between two GalA units being the most stable (BeMiller, 1967; Chambat & Joseleau, 1980; Yapo, Robert, et al., 2007; Yapo, Lerouge, et al., 2007). Consequently, acid-extraction generally enriches in GalA (homogalacturonic regions), pectins solubilised from CWMs, following

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substantial degradations of NS side chain-containing rhamnogalacturonic regions. However, the degree of purity of the final pectin isolates may be dependent on the used purification procedure. In general, the extracted cell wall fractions contain pectins as well as a considerable amount of contaminating free neutral components, which may include monomeric sugars, oligosaccharides and high molecular weight polysaccharides (Hwang, Roshdy, Kontominas, & Kokini, 1992). Commercial (apple or citrus) pectins are purified from filtered (and concentrated) aqueous acid extracts by means of an alcohol (ethanol, isopropanol, or methanol) precipitation process, which may involve subsequent alcohol or acid-alcohol washings for removing salts, free sugars, and other (acid-) alcohol soluble compounds and converting pectins to their free acid form in the latter case. On a laboratory scale, alcohol-precipitation or dialysis is frequently used for the purification of pectins from aqueous medium-soluble cell wall extracts. Even if either process is believed to effectually remove small molecular weight sugar components, notably, monosaccharides and disaccharides (Pagán, Ibarz, Llorca, & Coll, 1999; Panouillé, Thibault, & Bonnin, 2006), because of their high alcohol-solubility or ability to pass through dialysing membrane pores, it could not be the case for non-pectin-incorporated longer neutral oligosaccharides and polysaccharides. The final pectin samples are most likely to contain notable amounts of free neutral polysaccharides with high molecular weight, which are distinguished from neutral polysaccharides covalently incorporated into the side chains of pectins (Hwang et al., 1992). Furthermore, neutral sugars such as glucose, mannose, and fructose, which do not typically belong to pectin chains in situ have been reported in acid-extracted alcohol-precipitated apple pomace pectins (Garna et al., 2007), suggesting that the obtained final pectin samples are contaminated with sugar components from neighbouring polysaccharides. Up to now, comparative studies of more than two different pectin purification methods (e.g., alcohol-precipitation, dialysis, ultrafiltration–diafiltration, and ion-binding precipitation) were hardly reported in literature. Moreover, in previous comparative study, commercial (apple) pectins have been used as the starting pectin extracts to be purified (Hwang et al., 1992), which does not permit to clearly appraise the effective influence of compared procedures (dialysis versus metal precipitation). By using citric, nitric, or sulphuric acid extractant, it has been recently shown that acid type strongly influences the macromolecular and gelling properties of isolated pectins (Yapo, 2009a); with citric acid being the least pectin degrading (depolymerising and deesterifying) extracting agent, and therefore leading to pectin isolates with the best gelling properties. However, possible differences in quality characteristics of extracted pectins that the use of different purification methods could highlight haven’t been hitherto investigated. Keeping in mind the environmentfriendly aspect and consumers increasing attractiveness for natural (acid) agent-extracted pectins (Yapo, 2009b), pectins are therefore extracted from pectin-rich CWM from yellow passion fruit (YPF) rind, an unexploited industrial by-product available locally in large quantities (Yapo & Koffi, 2006, 2008), with a unique extractant type (citric acid) and then conventional alcohol-precipitation, dialysis, or ion-binding precipitation is used to purify pectin samples directly from the same crude aqueous acid extracts. This study aims at evaluating the actual effect of each kind of purification procedure on the chemical composition, macromolecular characteristics, and gelling properties of the final pectin products, thereby completely complementing the first study (Yapo, 2009a), a consequence of which would be the proposal of an improved environmentally friendly procedure for producing gelling pectins of high purity level on a larger (e.g., industrial) scale.

2. Materials and methods 2.1. Pectin extraction Yellow passion fruit (Passiflora edulis var. flavicarpa) rind CWM utilised as the pectin source was prepared as previously described (Yapo, 2009a). Pectins were extracted from CWM with hot dilute citric acid (a natural extractant) under the following optimising conditions: dried CWM to extractant ratio (1 g/25 mL), pH 1.8, temperature 75 °C, duration 60 min, and an extraction number of two (Yapo, 2009a). At the end of each extraction, the slurry was centrifuged and filtered on G-3 sintered glass and the obtained pectin-containing solution was rapidly brought to pH 3, for the sake of its stability, using a 0.2 mol L1 sodium citrate solution. Crude aqueous extracts from the first and second extractions were combined and the resulting solution was concentrated, at 40 °C, to 10 g L1 pectin concentration using a rotary evaporation technique. The extraction of pectins was carried out in three independent runs. 2.2. Pectin purification 2.2.1. Alcohol-precipitation procedure Pectin-containing aqueous extract was divided into three equal parts and one-third of it was mixed with 95% (v/v) ethanol (1:3, v/ v) at 4 °C. The precipitate (ethanol-insoluble fraction) formed was recovered through centrifugation and filtration, washed with 55% (v/v), and then with 70% (v/v) ethanol until the filtrate gave a negative response with the colorimetric phenol–sulphuric acid assay (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The remaining fraction was dispersed in distilled water, freeze-dried and referred to as alcohol-purified pectin (APP). 2.2.2. Dialysis procedure The second one-third of pectin-containing solution was extensively dialysed in tubing having a pore size of 12,000 nominal molecular weight cut-off (MWCO), against distilled water at 4 °C for 72 h, with a changing of dialysing water three times a day. The material remaining within the dialysis tubing was freeze-dried and referred to as dialysis-purified pectin (DPP). 2.2.3. Metal ion-precipitation procedure A 70 g L1 solution of CuSO45H2O was mixed drop-wise with the last one-third of pectin-containing solution (1:10, v/v) and the mixture was gently stirred at 4 °C. The precipitate (insoluble Cu2+–pectin complexes) formed was recovered after centrifugation and filtration, washed three times with a mixture of 1% (v/v) HCl60% (v/v) ethanol to eliminate Cu2+ ions, which was tested for completeness with the colorimetric cuprizon test (Keijbets & Pilnik, 1974), followed by 60% (v/v) ethanol washings until the filtrate was chloride-free as demonstrated by a negative response with silver nitrate. The remaining fraction was dispersed in distilled water, freeze-dried, and referred to as metal ion-purified pectin (MPP). For each kind of purification method, experiment was performed in three independent runs. 2.3. Characterisation of pectin isolates Proximate compounds (ash, moisture, and protein) were analysed with conventional methods, uronic acid content was colorimetrically assessed using a 3,5-dimethylphenol chromogenic reagent and galacturonic acid (GalA) as external standard, and individual neutral sugars (NSs) were quantified as alditol acetate derivatives by gas chromatography using myo-inositol as internal standard (Yapo & Koffi, 2006). Molecular-weight distribution

B.M. Yapo / Food Research International 42 (2009) 1197–1202

(MWD) and weight-average-molecular weight (Mw) were determined as previously published (Yapo & Koffi, 2008) by high pressure size exclusion chromatography (HPSEC) in combination with on-line light scattering (LS) detector (photometer) and differential refractive index (RI) detector (refractometer), and viscosity-average-molecular weight (Mv) was evaluated from viscometric measurements (intrinsic viscosity ([g])) with the aid of an on-line differential pressure viscometer and the use of Mark–Houwink– Kuhn–Sakurada equation (1). To assess more accurately the average-molecular weights, the so-called universal calibration line (Grubisic, Rempp, & Benoit, 1967), i.e., the plot of log ([g]M) against the elution volume (Ve), was constructed with ‘monodisperse’ linear pullulans and relatively polydisperse branched dextran standards of a broad average-molecular weight range.

½g ¼ KMav

ð1Þ

where [g] and Mv are the intrinsic viscosity and viscosity-averagemolecular weight of the polymer, respectively, and K and a, the Mark–Houwink constants for a given polymeric solute–solvent system. Degree of (methyl)-esterification (DE) was assessed by a direct titrimetric method (Schultz, 1965) as slightly modified previously (Yapo, 2009a). In this connection, samples of APP and DPP were first submitted to acid-alcohol washings as described above for MPP in order to convert these pectins to their free acid form before titration. Briefly, 20 mL of 10 g L1 pectin solutions were titrated with a 0.2 mol L1 NaOH solution in the presence of two drops of phenolphthalein indicator (Titration A) and then, 20 mL of 0.5 mol L1 NaOH were added under stirring for 30 min to deesterify pectin, after which 20 mL of 0.5 mol L1 HCl were added to exactly neutralize the NaOH. This mixture was titrated with a 0.2 mol L1 NaOH solution in the presence of two drops of phenolphthalein indicator (Titration B). Acetyl-esterification being negligible (Yapo & Koffi, 2006), the degree of esterification (DE), corresponding exclusively to DM, was calculated using Eq. (2) and the methoxy content was assessed according to Eq. (3).

DE ðmol%Þ ¼ ½Titration B=ðTitration A þ Titration BÞ  100

ð2Þ

Methoxy content ðg=100gÞ ¼ ½DE  31  anhydrogalacturonic acid content ðg=100gÞ=ð176  100Þ

ð3Þ 1

where 176 and 31 are the molar masses (g mol ) of anhydrogalacturonic acid and methoxy (–OCH3) groups, respectively. The gelling properties were analysed as the gelling velocity of pectin gel preparations at 30 °C (Joseph & Baier, 1949) and as the strength of demoulded gels after 24 h of ageing using conventional SAG method (IFT, 1959) as previously described (Yapo, 2009a). The gelling velocity was determined as the setting time, i.e., the duration from the end point of the jelly preparation to the first sign of gelation upon cooling at 30 °C in a water bath. Briefly, a jelly tumbler was prepared with citric acid solution containing coarsely ground pepper and placed in an incubator at 30 °C. The jelly preparation, at the end of boiling, was carefully poured into it to a desired height at time zero. At suitable intervals, the gelling process was controlled by twisting slightly the test glass and as the gel was setting the pepper was seen to swing back slightly before coming to rest. The setting time was recorded when this set reached 6 mm from the gel surface. Gel strength was measured by the Ridgelimeter method. Briefly, at the end of boiling, the gel preparation was completely filled in a Ridgelimeter glass and the surface was covered with a waxed paper disc (to minimize evaporation) and left undisturbed at room temperature for 2 h before aging for a further 22 h in an incubator at 30 °C. The gel was then carefully demoulded undamaged onto a Ridgelimeter glass plate. After ex-

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actly 2 min of standing, the pointer of the apparatus was carefully lowered until it touched the gel surface and the percentage of sagging under its specific gravity was measured, from which the gel strength (°sag) was calculated using an appropriate correction factor from established standard tables. Gel preparations contained 65.0% soluble solids (sucrose) and 0.70 wt% pectin at pH 2.3 (fine-tuned with a citric acid solution). 2.4. Statistical analysis The obtained data were statistically evaluated by the global test of a single-factor analysis of variance (ANOVA), followed by the Bonferroni’s posthoc test for multiple comparisons (Motulsky, 1999), whenever applicable, using a GraphPad Prism V.3 software (GraphPad software Inc., San Diego, CA). Means of different treatments were considered significantly different at p-values < 0.05. 3. Results 3.1. Pectin recovery The total recovery of pectin isolates varies from 4.1 to 7.5 g/ 100 g according to the mode of purification (Table 1). The amount of MPP is very significantly (p-values < 0.01) lower than the amount of APP or DPP; the amounts of the two latter pectins being similar. By using successively dialysis and pure (acid-free) alcoholprecipitation or the latter and acid-alcohol washing, the amount of isolated pectin appeared inferior to the amount of APP or DPP (results not shown). 3.2. Pectin compositional, molecular weight and gelling characteristics The chemical composition of pectins is shown in Table 1. The GalA contents of APP, DPP, and MPP are 62.4, 70.1, and 78.9 g/ 100 g, respectively, which are extremely significantly (p-values < 0.001) different from one another. Hence, the GalA content of MPP is the highest, followed by that of DPP. The total NS contents of APP, DPP, and MPP are 17.8, 16.0, and 9.7 g/100 g, respectively. In terms of total NS content, the three pectin samples are significantly (p-values < 0.001) different, with a more marked effect from MPP. The latter indeed contains much less total NS than the former. This large difference in total NS content of MPP and the two other pectins chiefly stems from significant (p-values < 0.001) differences in the amounts of two neutral glycosyl residue constituents, namely, arabinose and galactose. The ash contents of the three pectin isolates are different (p-values < 0.001) and the protein content of MPP is different (p-values < 0.01) from the protein content of the remainder. MPP has the fewest ash and protein contents, and DPP contains less ash than APP. On the other hand, when DPP samples were further treated by pure (acid-free) alcohol-precipitation or when APP samples were further washed with acidified alcohol, the NS and ash contents of resulting pectin samples were considerably lower than the NS and ash contents of DPP and APP, but got closer to those of MPP (results not shown). This indicated that dialysis followed by pure alcohol-precipitation as well as pure alcohol-precipitation followed by acidified alcohol washing was more effective than dialysis or pure alcohol-precipitation-andwashing alone for removing pectin extract contaminants, notably, ash and free NS components. Hence, dialysis appears efficacious against small components such as ash and short neutral oligosaccharides, probably due to the rather high membrane pore size (12,000 nominal MWCO). However, the removal of free sugar components could be affected by their degrees of polymerisation and interaction with the membrane. Pure alcohol-precipitation-andwashing is likely to be effectual against free and rather short NS

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Table 1 Yield, biochemical composition, viscosity-average-molecular weight (Mv), gel setting time and gel strength of APP, DPP, and MPP.a Pectin samples APP Yield (g/100 g of dry weight) Composition (g/100 g of dried pectin) Uronic acid (as GalA) Rhamnose Galactose Arabinose Mannose Xylose Glucose Neutral sugar Ash Protein DE (mol%) Mv (kDa) Gel setting time (s) Gel strength (°sag)

DPP

7.5 ± 0.6b 62.4 ± 2.3b 3.2 ± 0.1b 4.8 ± 0.3b 5.9 ± 0.5b 0.7 ± 0.1 2.3 ± 0.1b 0.9 ± 0.1 17.8 ± 0.9b 5.8 ± 0.3b 3.2 ± 0.1b 58 ± 3 83.4 ± 3.2bc 1082 ± 6b 143 ± 0.7b

MPP

6.8 ± 0.9b

4.1 ± 0.2c

70.1 ± 1.6c 2.8 ± 0.2c 4.6 ± 0.4b 5.2 ± 0.4c 0.5 ± 0.2 2.1 ± 0.2b 0.8 ± 0.1 16.0 ± 1.2c 2.7 ± 0.2c 3.1 ± 0.2b 59 ± 4 85.2 ± 1.8b 1038 ± 3c 152 ± 0.5c

78.9 ± 1.8d 2.4 ± 0.1d 2.9 ± 0.2c 2.7 ± 0.2d – 1.7 ± 0.1c – 9.7 ± 0.5d 0.9 ± 0.1d 1.4 ± 0.1c 54 ± 2 79.6 ± 1.2c 853 ± 3d 168 ± 0.3d

a Data are means ± standard deviations; n = 5, with the exception of the yield, where n = 3. Mean values in the same line with different letters are significantly different (p < 0.05) using the Bonferroni’s posthoc test.

components but clearly not against ash and longer neutral oligosaccharides, whereas acidified alcohol washing proves to be effective against ash, monosaccharides, disaccharides, and longer oligosaccharides, probably as a result of further acid-degradation of the latter, to have been removed via solubilisation in acid-alcohol mixture. The DEs of isolated pectins are similar, ranging from 54 to 59 (Table 1). Hence, the DE of solubilised pectins has not been significantly affected by the purification procedure. The DEs of the three pectins are more than 50%, and hence they can be conventionally taken as high ester-methyl pectins. The Mv of isolated pectins ranges from 79 to 86 kDa, with MPP having a significantly (p-values < 0.01) lower Mv than DPP. It is worth underlining that the Mv of a given pectin, determined from viscometric measurements and application of the Mark–Houwink equation was notably different from (lower than) corresponding Mw, assessed from LS measurements; this discrepancy being more pronounced with APP (data not shown). This might be caused by the presence of high molecular weight (HMW) ‘impurities’ (aggregates and/or microgels) in pectin solutions, disturbing LS measurements and therefore leading to overestimated Mw and/or by the polydispersity of pectins as indicated by their MWD patterns covering a wide elution volume range (Fig. 1). Berth (1988) observed a dominant influence of these HMW impurities on LS measurements

550

Detector response (mV)

500 450

V0

400

Vt

350 300 250 200 150 15

20

25

30

Retention time (min) Fig. 1. High pressure size exclusion chromatography elution patterns of APP (upper), DPP (intermediate), and MPP (lower) from yellow passion fruit rind aqueous acid extracts.

of citrus pectin Mw in that partial removal of the latter from original commercial high methoxy citrus pectin, by ion exchange chromatography or ultracentrifugation purification, led to a considerable decrease in Mw (from 3000 to 165–154 kDa) while the effect on intrinsic viscosities was only small (from 345 to 326–318 mL/g). On the other hand, Mv was reported to differ from Mw in a polydisperse polymer sample (Kasaai, Arul, & Charlet, 2000). Unlike APP and DPP, MPP exhibits a polydisperse but ‘monomodal-like’ MWD pattern, with the absence of distinguishable lower molecular size material peaks (Fig. 1). Hence, MPP sample is the least heterogeneous in terms of the size of contained polymeric materials. The setting time of gel preparations and the strength of gels formed vary from 853 to 1082 s and from 143 to 168 °sag, respectively (Table 1). MPP gel preparation displays the lowest setting time and the gel formed achieves the highest firmness (p-values < 0.001). DPP seems better than APP with respect to gelling properties. 4. Discussion The well-known gel-forming ability of pectins has been long since unambiguously established that they are indeed the genuine gelling agents in diverse food formulations, notably, preserves, jellies, and marmalades. However, various extrinsic factors such as ash and NS contaminants can negatively impact on the gelling properties of pectins. By using three different purification methods, namely, dialysis, pure alcohol-precipitation, and metal ion-binding precipitation, three pectin samples (DPP, APP, and MPP) of significantly different degrees of chemical composition (purity), macromolecular characteristics, and gelling abilities have been isolated from acid extracts of CWM from YPF rind. Indeed, MPP gel strength appears higher than APP or DPP gel strength, which can be ascribed to the significantly lower ash, NS, and possibly protein contents along with higher GalA content of the former. It has been reported that pectin with a low ash content is more favourable to gel formation (Sosulski et al., 1978) and pectin with a high NS content has a low gelling power (Miyamoto & Chang, 1992). Unlike APP or DPP, MPP contains much less total NS and exhibits a monomodal-like MWD pattern, which strongly suggests that it does not comprise free neutral oligosaccharides and/or polysaccharides of relatively low molecular weights. The presence of quantifiable free neutral components within DPP and APP is in accord with the reports of considerable amounts of free neutral polysaccharides, of high

B.M. Yapo / Food Research International 42 (2009) 1197–1202

and/or low molecular weights, within dialysed commercial apple pectins (Hwang et al., 1992), alcohol-precipitated but not washed acid-extracted apple pomace pectin fractions (Garna et al., 2007), and alcohol-precipitated and washed acid-solubilised pectin fractions from buttercup squash flesh (O’Donoghue & Somerfield, 2008). However, a stepwise use of dialysis and alcohol-precipitation-and-washing (Yapo, 2009a; Yapo & Koffi, 2006) significantly lowered free NS components in the final pectin samples compared to either procedure alone (unpublished results). On the basis of its MWD pattern, it is very likely that NSs other than rhamnose within MPP are all incorporated into the side chains of pectin polymers. Indeed, ion-purified YPF pectins have been recently shown to consist of at least three types of copolymer building blocks of different relative proportions, namely, predominantly homogalacturonans, followed by type I rhamnogalacturonans bearing arabinogalactan side-chains and finally a few xylogalacturonans (Yapo, 2009c). Metal ion-binding precipitation method proves to be the most effective for removing free (NS) components based on the data obtained from chemical composition and MWD analyses of the three pectin isolates. This supports the allegation that some multivalent metal ions such as Cu2+ and Al3+ ions can form complexes exclusively with pectins primarily through the anionic carboxyl groups, but not with free neutral components (Hwang et al., 1992). However, it cannot be ruled out the fact that the included subsequent acid-alcohol washing, in that purification procedure, might have released some neutral glycosyl residues initially incorporated within pectin chains, thus enhancing the lowering of the NS level of MPP. The ash content of MPP is the lowest, followed by that of DPP, whereas the ash content of APP is by far the highest. Also recently, another membrane procedure (ultrafiltration–diafiltration) with a 10 kDa nominal MWCO membrane has allowed to lower by 1.6-, 1.9-, and 4.6-fold, the NS, ash, and protein contents, respectively, and to increase by 1.3-fold the GalA content of acid-extracted sugar beet pectins, compared to a pure alcoholprecipitation-and-washing method (Yapo, Wathelet, & Paquot, 2007). These results clearly indicate that metal ion purification process is the most efficacious for removing ash and proteins, whereas pure alcohol-precipitation is the least effective, which could be attributable to the poor solubility of these components, including longer neutral oligosaccharides and polysaccharides, in alcohols. On the other hand, the yield of MPP is significantly lower than the yields of the other pectin isolates, showing that the total recovery of pectins from the same crude aqueous acid extracts can be dependent on the employed purification process. These substantial differences in the amount of MPP compared with the remainder could be explained by the nearly total absence in MPP of contaminating components, particularly, free neutral oligosaccharides and/or polysaccharides of relatively high molecular weights. A probable loss of neutral sugar-containing highly methyl-esterified pectin chains, following a failure to form insoluble complexes with Cu2+ ions, as has been reported with Al3+ ions (Joslyn & de Luca, 1957), might also participate in the obtained lower yield of MPP. Indeed, calculations of the total GalA recovered from the same initial extracts showed that APP and DPP led to similar amounts of GalA, whereas MPP led to less than 70% of the GalA amounts of the former. A pure alcohol-precipitation-and-washing process is far from yielding a final pectin product devoid of free neutral oligomeric and/or polymeric components that are released, during extraction, not only from pectin side-chains but also from neighbouring polysaccharide materials such as cellulose, hemicelluloses, and possibly starch. Therefore, to the conventional ‘yield of pectin’, which is defined as the weight ratio of the quantity of dried alcohol precipitates of aqueous medium (especially dilute acid)soluble cell wall extracts to the dried starting pectin-containing material multiplied by 100, it could be proposed a complementary term, namely, ‘the yield of galacturonic acid’. The main compound

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of pectins being indeed GalA, the pectin yield as defined above can be accompanied with ‘the GalA yield’, which can be defined as the amount of extracted GalA within alcohol precipitates relative to the total GalA present in the starting pectin-containing plant material. This second term can be very useful for the evaluation of the degree of extractability of cell wall pectins and consequently the extent of efficacy of the extraction process. 5. Conclusions The chemical composition, molecular weight distribution, average-molecular weight, and gelling ability of extracted pectins can be affected by the purification method. Amongst the purification procedures here examined, the metal ion-binding precipitation one proves to be the most effective. However, the latter is likely to generate a large amount of effluents on an industrial scale with a lot of demanding treatments to avoid causing environmental damages upon getting rid of them. Alcohol waste, in contrast, can be more easily managed with by recycling it for instance. Therefore, it would be more advantageous to precede alcohol-precipitation with an industrially-practical membrane procedure such as ultrafiltration–diafiltration using membranes of sufficiently great nominal molecular weight cut-offs for an effective removal of pectin contaminants (ash, free neutral and protein components, etc.), thereby improving the compositional quality and possibly gelling properties of the final pectin products. Acknowledgement Special thanks to V. Besson for invaluable assistance. References BeMiller, J. N. (1967). Acid-catalysed hydrolysis of glycosides. Advanced in Carbohydrate Chemistry, 22, 25–91. Berth, G. (1988). Studies on the heterogeneity of citrus pectin by gel permeation chromatography on Sepharose 2 B/Sepharose 4 B. Carbohydrate Polymers, 8, 105–117. Braconnot, H. (1825). Recherches sur un nouvel acide universellement répandu dans tous les végétaux. Annales de Chimie et de Physiques, 28(2), 173–178. Chambat, G., & Joseleau, J.-P. (1980). Isolation and characterization of a homogalacturonan in the primary walls of Rosa cells cultures in vitro. Carbohydrate Research, 85, C10–C12. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. Garna, H., Mabon, N., Robert, C., Cornet, C., Nott, K., Legros, H., et al. (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. Grubisic, Z., Rempp, P., & Benoit, H. (1967). A universal calibration for gel permeation chromatography. Journal of Polymer Science Part B, 5(7), 753–759. Hwang, J., Roshdy, T. H., Kontominas, M., & Kokini, J. L. (1992). Comparison of dialysis and metal precipitation effects on apple pectins. Journal of Food Science, 57, 1180–1184. IFT (1959). Pectin standardisation, final report of the IFT committee. Food Technology, 13, 496–501. Joseph, G. H., & Baier, W. E. (1949). Methods of determining the firmness and setting time of pectin test jellies. Food Technology, 3, 18–22. Joslyn, M. A., & De Luca, G. (1957). The formation and properties of aluminium pectinates. Journal of Colloid Science, 12, 108–130. Kasaai, M. R., Arul, J., & Charlet, G. (2000). Intrinsic viscosity-molecular weight relationship for chitosan. Journal of Polymer Science Part B: Polymer Physics, 38, 2591–2598. Keijbets, M. J. H., & Pilnik, W. (1974). Some problems in the analysis of pectin in potato tuber tissue. Potato Research, 17, 169–177. Miyamoto, A., & Chang, K. C. (1992). Extraction and physicochemical characterization of pectin from sunflower head residues. Journal of Food Science, 57, 1439–1443. Motulsky, H. J. (1999). Analyzing data with GraphPad Prism. San Diego, CA: GraphPad Software Inc.. O’Donoghue, E. M., & Somerfield, S. D. (2008). Biochemical and rheological properties of gelling pectic isolates from buttercup squash fruit. Food Hydrocolloids, 22, 1326–1336. Pagán, J., Ibarz, A., Llorca, M., & Coll, L. (1999). Quality of industrial pectin extracted from peach pomace at different pH and temperatures. Journal of the Science of Food and Agriculture, 79, 1038–1042.

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Yapo, B. M. (2009b). Lemon juice improves the extractability and quality characteristics of pectin from yellow passion fruit by-product as compared with commercial citric acid extractant. Bioresource Technology, 100, 3147–3151. Yapo, B. M. (2009c). Pineapple and banana pectins comprise fewer homogalacturonan building blocks with a smaller degree of polymerization as compared with yellow passion fruit and lemon pectins: Implication for gelling properties. Biomacromolecules, 10, 717–721. Yapo, B. M., & Koffi, K. L. (2006). Yellow passion fruit rind—A potential source of low-methoxyl pectin. Journal of Agriculture and Food Chemistry, 54, 2738–2744. Yapo, B. M., & Koffi, K. L. (2008). The polysaccharide composition of yellow passion fruit rind cell wall: Chemical and macromolecular features of extracted pectins and hemicellulosic polysaccharides. Journal of the Science of Food and Agriculture, 88, 2125–2133. Yapo, B. M., Lerouge, P., Thibault, J.-F., & Ralet, M.-C. (2007). Pectins from citrus peel cell walls contain homogalacturonans homogenous with respect to molar mass, rhamnogalacturonan I and rhamnogalacturonan II. Carbohydrate Polymers, 69, 426–435. Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chemistry, 100, 1356–1364. Yapo, B. M., Wathelet, B., & Paquot, M. (2007). Comparison of alcohol precipitation and membrane filtration effects on sugar beet pulp pectin chemical features and surface properties. Food Hydrocolloids, 21, 245–255.