Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins

Accepted Manuscript Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins Xiaoming Guo, Hecheng Meng, Qiang Tang, Runquan Pan, Siming Zhu, Prof. Shujuan Yu PII:

S0268-005X(15)30023-0

DOI:

10.1016/j.foodhyd.2015.07.013

Reference:

FOOHYD 3069

To appear in:

Food Hydrocolloids

Received Date: 13 September 2014 Revised Date:

5 July 2015

Accepted Date: 16 July 2015

Please cite this article as: Guo, X., Meng, H., Tang, Q., Pan, R., Zhu, S., Yu, S., Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.07.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins Xiaoming Guo a, Hecheng Meng a, Qiang Tang a, Runquan Pan a, Siming Zhu a,

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Shujuan Yu a, b, c, * College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China

Guangdong Province Key Laboratory for Green Processing of Natural Products and

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State Key Laboratory of Pulp and paper Engineering, Guangzhou 510640, China

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Product Safety, Guangzhou 510640, China

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*Corresponding author: Prof. Shujuan Yu

Address: 381 Wushan, Guangzhou, China E-mail: [email protected]

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Tel.: +86 20 87113668

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Fax: +86 20 87113668

This manuscript has been thoroughly edited by a native English speaker from an editing company. Editing Certificate will be provided upon request.

*

Corresponding author: e-mail: [email protected]; tel.: +86 20 87113668; fax:

+86 20 87113668. 1

ACCEPTED MANUSCRIPT Abstract: Sugar beet pectins (SBP) were precipitated from a purified extract in a 75%

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v/v ethanol solution at an initial extract pH (I-pH) ranging from 2.0 to 4.5. The effects

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of I-pH on pectin yield and pectin-cation interactions were studied. No simple

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correlation between pectin yield and I-pH was observed. The lowest pectin yield was

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obtained by precipitating the acidic pectin extract with ethanol at I-pH 4.5. Pectin

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yield increased to a maximum value as I-pH decreased from 4.5 to 3.0. These results

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indicated that decreased electrostatic repulsion between pectin chain segments

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enhanced pectin chain-chain interactions, thereby improving the precipitation effect. A

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decrease in pectin yield was observed as I-pH decreased below 3.0. The cation content

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of

samples

was

measured

by

high-performance

cation-exchange

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chromatography in order to determine the content of SBP-bound cations during

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precipitation. Cation content and degree of cation binding were measured for pectins

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precipitated at I-pH 3.0, 2.5, and 2.0, and the results revealed that a decrease in pectin

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yield accompanied a decrease in cation-pectin interactions. These results suggest that

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the precipitation of SBP from an aqueous extract involves complex interactions

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between cations, solvent molecules, and pectin chain segments. The larger

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precipitation effect observed with divalent ions compared to monovalent ions may be

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due to enhanced inter-chain interactions between pectin molecules, probably via the

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formation of intermolecular bonds.

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Keywords: Cation-pectin interactions; Ethanol precipitation; Sugar beet pectins;

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Precipitation pH

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1. Introduction Pectins, a family of polysaccharides located in the plant cell wall, have complex

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structures. Generally, pectins are characterized by an extended backbone of

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α-D-galacturonic acid units with 1-4 linkages (Buchholt, Christensen, Fallesen, Ralet,

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& Thibault, 2004). The galacturonic acid (GalA) units of the backbone can be

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substituted with 2-O-linked-α-L-rhamnose residues, which can bear neutral sugar

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side-chains, with arabinose and galactose as the major sugar constituents. The

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carboxyl groups of GalA residues within the backbone can be partially esterified by

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methanol. Depending on their degree of methylation (DM), pectins can be classified

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as high methoxyl pectins, with DM > 50%, or low methoxyl pectins, with DM < 50%

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(Ralet, Crépeau, Buchholt, & Thibault, 2003). In addition, the free hydroxyl groups of

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GalA residues can be partially acetyl-esterified at the O-2 and/or O-3 positions (Ralet,

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et al., 2005). The degree of acetylation (DA) of the extracted pectins depends greatly

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on the plant source used. Industrial pectins from either citrus peel or apple pomace

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have a low DA, while those from sugar beet pulp are likely to have a high DA (Ralet,

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et al., 2003).

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Pectins are usually purified and separated from the acidic plant extract, which

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contains pectins as well as a considerable amount of non-pectic compounds, notably

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salts and neutral polysaccharides (Yapo, 2009; Hwang, Roshdy, Kontominas, &

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Kokini, 1992). To investigate the structural and physical properties of pectins,

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precipitation methods are used to separate and purify pectins from the extract. Various

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methods have been developed for the separation of pectins, e.g., metal precipitation,

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ACCEPTED MANUSCRIPT protein-pectin complexation, alcohol precipitation, and ultrafiltration (Hwang, et al.,

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1992; Yapo, Wathelet, & Paquot, 2007; Garna, Emaga, Robert, & Paquot, 2011).

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Unlike other methods, alcohol precipitation changes the physical properties of the

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solvent in order to precipitate pectins. The addition of alcohol decreases the polarity

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of the solvent because alcohol is much less polar than water. According to the “like

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dissolves like” principle, the pectic polysaccharides that are initially soluble will

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eventually precipitate if enough alcohol is added to decrease the polarity of the

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solvent. Hence, the precipitation of polymers with simple and specific structures is

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hypothesized to be due to the decreased interactions between polymer and solvent

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molecules. The strength of such interactions during alcohol precipitation may be

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affected by both intrinsic factors (molecular size and hydrophobic and hydrophilic

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character) and extrinsic factors (precipitation pH, ethanol volume, and the presence of

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inorganic salts) (Xu, et al., 2014; Kalapathy & Proctor, 2001; Faravash & Ashtiani,

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2007; Smidsrød & Haug, 1967). Among them, precipitation pH is an important factor

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that affects the precipitation of pectins during alcohol precipitation (Kalapathy, et al.,

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2001; Faravash, et al., 2007). From an electrostatic point of view, pectins differ from

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neutral polysaccharides, such as starch and hemicellulose, because pectin chain

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segments can be either charged or uncharged, depending on the protonation state of

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their carboxylic groups (Capel, Nicolai, Durand, Boulenguer, & Langendorff, 2006;

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Li, Al-Assaf, Fang, & Phillips, 2013). Therefore, the alcoholic precipitation of pectin

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is a complex process that may involve multiple interactions between ionized pectins,

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solvent molecules, and positively charged cations.

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ACCEPTED MANUSCRIPT Numerous studies have examined how precipitation conditions affect the

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precipitation process. Most of these studies focused on the yield and chemical features

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of the pectin precipitates. A few studies investigated the effect of pH on pectin

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precipitation. These studies demonstrated that the precipitation pH affected the pectin

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yield, with relatively high yield observed when alcohol precipitation was performed at

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a pH of ~3.5 (Faravash, et al., 2007; Kalapathy, et al., 2001). The pH sensitivity of

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pectin precipitation was attributed to changes in the hydrophobic-hydrophilic balance

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of the pectin at different pHs. However, it is still unclear how the structure of the

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pectin polymer contributes to its different precipitation behaviors.

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In the present report, we studied the precipitation behaviors of sugar beet pectins

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(SBP) under different precipitation pHs, and we correlated these behaviors to the state

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of the pectin chains and to the interactions between the pectins and cations. We

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summarize our findings in a schematic illustration of the proposed precipitation

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behaviors of SBP during alcohol precipitation.

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2. Materials and methods

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2.1. Materials

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Fresh sugar beet pulp with sucrose removed was provided by Lvxiang beet sugar

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company (Xinjiang, China). Fresh sugar beet pulp was sliced into thin chips and

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immersed in water at 75°C for 70 min to remove sucrose. The pulp was pressed by

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screw presses to squeeze the remaining juice out of the pulp, and then air-dried at

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45°C for 48 h. The processed dry pulp was subsequently ground until able to pass

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through a 20-mesh screen, and then stored at –20°C until used for extraction.

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2.2. Acidic extraction Dried sugar beet pulp was dispersed in water (solid-liquid ratio 1:20, w/v), and the

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suspension was adjusted to a pH of 1.5 with HCl. Extractions were performed by

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heating the suspension to 80°C with stirring (250 rpm) for 1 h. Then, the resulting

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slurries were filtered through nylon cloth and centrifuged (10,000 g × 20 min) to

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remove the residue. After centrifugation, the supernatants were pooled. In order to

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minimize the effect of excess free salt on precipitation, the extract was dialyzed in a

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dialysis membrane bag (14,000 g/mol nominal molecular weight cut-off) against

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distilled de-ionized water for 48 h at room temperature, with the dialysis water being

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changed every 6 h. After dialysis, the dialyzed extract was centrifuged at 10,000 g for

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5 min to remove insoluble non-pectic substances formed during dialysis. The dialyzed

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extract was then used for alcohol precipitation.

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2.3. Ethanol precipitation

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2.3.1. Effect of I-pH

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Prior to precipitation, the pH of pectin-containing aqueous extracts was precisely

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adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 using 1 M NaOH or 1 M HCl. A volume of

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100 mL of pH-adjusted extract was used for ethanol precipitation. Three volumes of

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ethanol were gradually added to the acidic extract through a fluoroelastomer tube

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using a peristaltic pump (Longerpump Ltd., Hebei, China) at a flow rate of 10 mL/min

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with stirring (350 rpm). The pH of the resulting precipitation mixtures changed to 2.7,

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3.1, 3.7, 4.5, 5.5, and 6.1, respectively, just after the addition of ethanol. The mixtures

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were allowed to stand for 10 h at 25 °C to ensure complete precipitation of SBP. The

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ACCEPTED MANUSCRIPT pectin precipitates and supernatants were separated by centrifugation (12,000 g × 20

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min). Pectins were subsequently washed with 75% and 95% ethanol (twice each), and

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air-dried at 45°C until a constant weight was measured. These samples were named

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alcohol-precipitated pectins (APP). APP2.0/2.7, APP2.5/3.1, APP3.0/3.7, APP3.5/4.5,

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APP4.0/5.5, and APP4.5/6.1 refer to pectins precipitated at I-pH 2.0, 2.5, 3.0, 3.5, 4.0,

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and 4.5, respectively. The supernatant of the ethanol solution at precipitation pH 2.7

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was recovered independently and prepared for secondary precipitation of the

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remaining pectins. In detail, pectins were precipitated in situ by adjusting the

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supernatant pH to 3.7 using 1 M NaOH. Then, the resulting precipitation solution was

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allowed to stand for 10 h at 25°C. Pectin precipitates were centrifuged, washed, and

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dried as described above. These pectins were referred to as secondary precipitated

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pectins (SPP) and named SPP2.7/3.7.

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2.3.2. Effect of inorganic salts

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The effect of salts on pectin precipitation was studied at I-pH 4.5. Varying amounts

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of NaCl were added to the extracts to achieve desired salt concentrations (0 to 80

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mM). The addition of salt was conducted under stirring conditions to ensure complete

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dissolution of the salt. Pectins were then precipitated and purified from the extracts

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using the same procedures as described above (Section 2.3.1).

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2.4. Determination of electrophoretic mobility

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The electrophoretic mobility of the acidic extracts and the pectin-ethanol solutions

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was measured with a Nano-Zetasizer (Malvern Ltd., UK). Solutions (1 mL) were

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carefully pipetted into a DTS 1060 cuvette (Malvern Ltd., UK), and measurements

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were carried out in duplicate at 25°C. Data are presented as mean ± SD of the

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duplicate measurements.

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2.5. Analysis GalA content was quantified colorimetrically by the automated m-phenylphenol

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technique (Blumenkrantz & Asboe-Hansen, 1973). The degree of methylation and

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degree of acetylation were determined by HPLC (Levigne, Thomas, Ralet, Quemener,

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& Thibault, 2002).

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The molecular mass of the samples was determined by size exclusion

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chromatography (SEC) using Ultrahydrogel Linear and Ultrahydrogel 1000 columns

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(300 mm × 7.5 mm, Waters, USA). After the sample solution (0.1%) was filtered

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through a 0.45 µm filter, an aliquot (100 µL) was injected into the columns at a flow

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rate of 0.6 mL/min with 100 mM NaNO3 as the eluent. The eluent from the column

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was then passed through a multi-angle light scattering apparatus (DAWN HELEOS,

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Wyatt Corp., USA) and an interferometric refractometer (Optilab, Wyatt Corp., USA).

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A dn/dc of 0.135 mL/g was used. Runs were carried out at 35°C.

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Neutral Sugar (NS) content was determined by high-performance anion-exchange

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chromatography (HPAEC) with a pulsed amperometric detector, following the method

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reported by Garna, Mabon, Wathelet, and Paquot (2004) with minor modifications.

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Pectin (20 mg) was decomposed by 0.2 M trifluoroacetic acid, followed by an

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enzymatic hydrolysis by viscozyme L (Novozymes, Tianjin, China). The reaction

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mixture was denatured at 100°C for 3 min to stop the reaction. After being filtered

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through a membrane filter of 0.22 µm pore size, the hydrolysate (25 µL) was injected

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ACCEPTED MANUSCRIPT into the HPAEC system (ICS-5000, Dionex Corp., USA) equipped with a CarboPac

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PA1 column (4 × 250 mm) and a CarboPac PA1 guard column (4 × 50 mm). The

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neutral monosaccharides were eluted using 10 mM NaOH for 25 min. Uronic acids

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were eluted with a gradient of 170 mM CH3COONa and 100 mM NaOH for 10 min.

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All samples were analyzed at 30°C. The amount of total neutral sugars was calculated

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by summing the amounts of rhamnose, arabinose, galactose, glucose, and xylose. Data

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are presented as means ± SD of duplicates.

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Quantification of cation content was performed using a high-performance

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cation-exchange chromatography system (HPCEC) (ICS-5000, Dionex Inc., USA)

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equipped with a conductivity detector and a cation suppressor CSRS (4 mm). Pectins

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(100 mg) were decomposed at 500°C in a muffle furnace (STUART, UK) for 16 h

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according to the dry ashing procedure (Van paemel, De Rycke, Millet, Hesta, &

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Janssens, 2005). After the ashing, the sample was washed into a 100 mL volumetric

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flask with 10 mL methanesulfonic acid (100 mM). The solution was treated with

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ultrasound for 15 min to ensure complete dissolution of the material, and was diluted

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to 100 mL with distilled de-ionized water. The solution (25 µL) was then filtered

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through a 0.22 µm filter and injected into the HPCEC system. All steps were carried

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out at 30°C. Cation separation was performed using an IonPac-CS12A column (4 ×

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250 mm) in combination with an IonPac-CG12A guard column (4 × 50 mm). Mineral

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ions were eluted isocratically with 20 mM methanesulfonic acid at a flow rate of 1

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mL/min.

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2.6. Degree of cation binding

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Degree of cation binding (DC) was defined as the molar equivalent ratio of

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carboxyl groups to cations present in the pectins. DC was calculated according to

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equation 1: % DC = B ⁄ [A × (100 – DM)] × 100

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where A is the molar equivalent of GalA, DM is the degree of pectin methylation, B

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is the sum of the molar equivalents of sodium, potassium, magnesium, and calcium

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ions.

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3. Results

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3.1. Electrophoretic mobility of pectin extracts

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Electrophoretic mobility was measured to determine the protonation state of

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carboxyl groups on the pectin chains. The higher the degree of protonation of

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carboxyl groups, the weaker the repulsion between pectin chain segments. As shown

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in Figure 1, at pH below 2.0, the electrophoretic mobility was almost zero due to the

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full protonation of the carboxyl groups in the pectin backbones. The suppressed

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ionization of the carboxyl groups converted the pectins from anionic to neutral

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polymers. Thus, due to the lower degree of ionization of carboxyl groups, electrostatic

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interactions between pectins and oppositely-charged compounds could not occur. The

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electrophoretic mobility decreased as the pH increased from 2.0 to 4.5 and then

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remained fairly constant, indicating that the carboxyl groups of the pectins were fully

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charged. The observed changes in electrophoretic mobility as a function of pH were

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consistent with that obtained from sugar beet pectin solution as reported by Li, Fang,

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Phillips, and Al-Assaf (2013).

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3.2. Electrophoretic mobility of the pectin-containing ethanol solution The electrophoretic mobility of the pectin-containing ethanol solution was

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measured to determine the change in pectin charge density after ethanol precipitation.

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It should be noted that I-pH increased when the pectins were mixed with ethanol

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(Faravash, et al., 2007). Therefore, electrophoretic mobility values for each

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precipitation medium were measured at the precipitation pH (P-pH), which was

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defined as the pH of the pectin-containing ethanol-water solution. The P-pH of the

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precipitation medium increased when mixed with ethanol (Figure 2). The increase in

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pH may be due to a decrease in dielectric constant caused by the addition of ethanol

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(Wang & Anderko, 2001).

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3.2.1. Effect of P-pH

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Figure 3a shows the electrophoretic mobility values of different pectin-ethanol

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solutions at different P-pHs. In the absence of added salt, the rank order of the

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absolute values of electrophoretic mobility was the following: P-pH 6.1 > P-pH 5.5 >

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P-pH 4.5 > P-pH 3.7 > P-pH 3.1 = P-pH 2.7. These results indicate that the

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electrophoretic mobility was pH-dependent. The values of electrophoretic mobility for

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P-pH 2.7 and P-pH 3.1 were almost zero, suggesting that the charge of the pectins was

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negligible, likely because the carboxyl groups of the pectins were fully protonated.

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However, electrophoretic mobility decreased as P-pH increased above 3.1, reflecting

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the increase in degree of dissociation of carboxyl groups with increasing P-pH.

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Soluble pectins in ethanol solution therefore exhibit different extents of ionization of

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their carboxyl groups.

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3.2.2. Effect of salts Figure 3b shows that electrophoretic mobility initially decreased with increasing

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NaCl content but leveled off at 40 mM NaCl. The decreased electrophoretic mobility

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with increasing salt concentration indicated that the sodium cation was capable of

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neutralizing the charge of the pectins, likely via the formation of ion pairs with the

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pectins. On the other hand, the initial amount of salt may have been insufficient for

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complete neutralization of the charged pectins because the excess free salt of the

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extract was removed during dialysis.

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3.3. Pectin yield

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3.3.1. Effect of I-pH

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Pectin yields obtained by precipitating various extracts under different pHs are

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shown in Figure 4a. The extracted pectins represented 4.1–9.2% w/w of the starting

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material, depending on the I- or P-pH. The lowest pectin yield (4.1%) was obtained at

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I-pH 4.5, which corresponded to a P-pH of 6.1. Pectin yield increased as I-pH

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decreased from 4.5 to 3.0 (P-pH 3.7), at which a maximum yield (9.2%) was obtained.

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Pectin yield decreased at I-pH values below 3.0. The highest pectin yield achieved

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with I-pH 3.0 indicated a low affinity between pectins and solvent molecules at that

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pH. Therefore, P-pH 3.7 could be the optimum pH for obtaining a satisfactory pectin

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yield. Lower pectin yields were obtained for pectins precipitated at higher I-pH values

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(3.5–4.5), where pectins were negatively charged, suggesting that negatively charged

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pectin molecules had a higher affinity than protonated ones for solvent molecules. On

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the other hand, a decrease in I-pH (from 3.0 to 2.0) resulted in a decreased, not

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increased, pectin yield. This implies that solubilization of pectins may also occur at

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low pHs.

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For comparison, the pectin yield from the secondary precipitated pectin group, SPP2.7/3.7 (section 2.4), was 0.9%.

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3.3.2. Effect of salt

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Figure 4b shows the pectin yield from precipitation reactions performed at an I-pH

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of 4.5 in the presence of different concentrations of NaCl. Pectin yield initially

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increased with increasing NaCl concentration, but leveled off at 20 mM NaCl. This

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indicates that the precipitation of charged pectins depends greatly on the concentration

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of salts. In this case, the effect of cations on pectin precipitation was ascribed to the

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formation of ion pairs, which is believed to disrupt the interactions between pectins

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and solvent molecules (Smidsrød, et al., 1967). From an electrostatic point of view,

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the formation of ion pairs also neutralizes the charge of the pectin chains, converting

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charged pectins in an aqueous solution to neutral pectins in the concentrated ethanol

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solution.

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3.4. Pectin composition and molecular weight

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The GalA content of the extracted SBP varied from 54.0% to 63.8% (Table 1).

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These values were higher than those previously obtained from beet pectins (Levigne,

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Ralet, & Thibault, 2002), probably due to the difference in raw materials

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(alcohol-insoluble materials was used in their study; sucrose-free pulp was used in the

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present work). APP2.0/2.7 had the highest GalA content (63.8%) among all samples,

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followed by SPP2.7/3.7 (58.8%). The higher GalA content observed with low I-pH or

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ACCEPTED MANUSCRIPT P-pH was likely due to the solubilization of some compounds, such as minerals and

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free sugars, by the strong acidic conditions during precipitation. This pH-dependent

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change in GalA content is in disagreement with a report by Kalapathy and Proctor

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(2001), who observed no marked difference in GalA content of soy hull pectins after

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alcohol precipitation at pH 3.5 and at 2.0. The P-pH effect on GalA that we observed

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in the present study may be due to differences in pectin source and/or in the quantities

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of co-precipitated non-pectic compounds.

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The neutral sugar content of SBP ranged from 16.9 to 19.3%, with arabinose

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(7.5–8.1%), galactose (5.7–7.6%), and rhamnose (2.8–3.5%) being the main

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constituent sugars (Table 1). Other neutral sugars, such as glucose (0.2–0.4%) and

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xylose (0.1–0.4%), were present in small quantities. APP4.5/6.1 had a relatively low

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NS content among the pectins studied due to an increased weight of non-pectic

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compounds, e.g. cations.

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No direct correlation was observed between P-pH and the degree of SBP

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methylation or the degree of SBP acetylation (Table 1). DM of the different pectins

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ranged from 40.7% to 47.8%, indicating the extraction of low-methylated pectins

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from the pulp. DA ranged from 21.5% to 28.0%. These DA values were consistent

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with those previously reported (3.1−29.2%) (Yapo, Robert, Etienne, Wathelet, &

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Paquot, 2007).

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The Mw of SBP varied from 184 to 106 kg/mol. APP2.0/2.7 had the highest Mw

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(184 kg/mol) among the pectins, followed by APP2.5/3.1 (173 kg/mol). The Mw of

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APP3.0/3.7 (148 kg/mol) was comparable to that of APP3.5/4.5 (142 kg/mol).

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relatively low Mw values. For comparison, the refractive index (RI) elution profiles of

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APP2.0/2.7, APP4.0/5.5, and APP4.5/6.1 are presented in Figure 5. All three pectins

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were found to exhibit similar broad molar mass distributions. As compared to

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APP2.0/2.7, APP4.0/5.5 and APP4.5/6.1 exhibited lower RI signal intensities at

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elution volumes between 11.0 and 14.5 mL, which corresponded to materials of high

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molecular mass. These results indicate that high I-pH conditions (≥ 4.0) may be more

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suitable than low I-pH conditions for ethanolic precipitation of relatively low molar

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mass fractions.

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3.5. Cation content and DC

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The cation content of SBP was measured to determine whether the cation-pectin

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interaction had an effect on precipitation. DC, defined as the mole equivalent ratio of

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free carboxylic groups to cations, was used to reflect the degree of binding of cations

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to carboxyl groups within the pectin backbone.

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Figure 6a shows the differences in cation content of pectins obtained under

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different precipitation conditions. The cation content increased with I-pH, indicating

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that the interaction between pectins and cations was sensitive to pH. These results

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emphasize the importance of P-pH on the composition of the resulting products. The

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bound content of each cation followed the order: potassium < sodium < magnesium <

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calcium, irrespective of P-pH. The content of sodium, magnesium, and calcium ions

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increased as I-pH increased from 2.0 to 4.5. The potassium content was the lowest and

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was almost independent of the precipitation conditions, indicating that this cation was

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ACCEPTED MANUSCRIPT not specifically bound by SBP. In contrast, the calcium content was always found to

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be the highest among the four cations, indicating that calcium had a higher binding

311

affinity for SBP. Moreover, for any sample, the content of divalent ions was higher

312

than the content of monovalent ions. These results suggest that the selectivity of SBP

313

for binding a specific cation is associated with the nature of the counter-cation, and

314

that divalent cations may have a larger effect on precipitation than monovalent ions.

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As shown in Figure 6b, the DC increased with increasing I-pH, which suggests that

316

the interaction between pectins and cations was dependent on the number of ionized

317

carboxylic groups of GalA residues that were available to interact with the

318

counterions. The DC of APP3.0/3.7 was 53%, which was 3.1-fold greater than that of

319

APP2.0/2.7 (17%). We observed that a decrease in DC accompanied a decrease in

320

pectin yield for APP2.0/2.7 and APP2.5/3.1 (Figure 4a), which suggests a correlation

321

between pectin solubility and cation-pectin interactions. To verify this assumption,

322

soluble pectins in the supernatant of the P-pH 2.7 medium were recovered and

323

subjected to cation analysis. Results showed that the cation content of SPP2.7/3.7 was

324

1.3% w/w, which was 2-fold higher than that of APP2.0/2.7. The sodium, potassium,

325

magnesium, and calcium contents of SPP2.7/3.7 were 0.17, 0.05, 0.09, and 1.03%

326

w/w, respectively. We noted that despite the addition of a considerable amount of

327

sodium ions to the ethanol solution during pH adjustment, the bound calcium content

328

of SPP2.7/3.7 was higher than its bound sodium content. This again confirmed that

329

calcium had a higher affinity than sodium for SBP.

330

3.6. Precipitation behaviors of SBP during ethanolic precipitation

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15

ACCEPTED MANUSCRIPT In aqueous solution, negatively-charged sugar beet pectin domains are surrounded

332

by a layer of counterions. When mixed with ethanol, the dielectric constant of the

333

solution decreases. Consequently, SBP forms ion-pairs with cations in the ethanol

334

solution, leading to enhanced cation-pectin interactions. Such interactions appear to

335

influence the degree of hydration of the pectin chains, and thus the precipitation of

336

SBP. We therefore propose that cation-pectin interactions influence pectin yields.

337

Figure 7 shows a schematic illustration of the proposed precipitation behaviors of SBP

338

in a 75% ethanol solution under different P-pHs.

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During ethanol precipitation at P-pH 6.1, one fraction of the SBP precipitated out of

340

solution, while another fraction remained soluble because of strong repulsive forces

341

that inhibited pectin aggregation (Figure 7a). Precipitation of the strongly anionic SBP

342

was characterized by an extensive cation-pectin interaction, as indicated by its high

343

bound cation content (Figure 6). In the present case, the binding of cations to SBP can

344

relieve the strong repulsion between pectin chain segments, thereby promoting the

345

aggregation of SBP.

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Ethanol precipitation at P-pH 3.7 resulted in almost complete SBP precipitation, as

347

indicated by the maximum yield obtained with I-pH 3.0 (Figure 7b). This sample

348

exhibited the lowest affinity between SBP and solvent molecules, which likely

349

facilitated the extensive precipitation of SBP. Suppressed ionization of the carboxyl

350

groups decreased the charge of the pectin chains, leading to enhanced pectin

351

segment-segment associations and pectin chain aggregation. Moderate cation-pectin

352

interactions also contributed to charge neutralization and strengthened intermolecular

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ACCEPTED MANUSCRIPT 353

interactions via the formation of intermolecular bonds. When precipitated with ethanol at I-pH 2.0, most of the highly protonated SBP

355

precipitated out of solution due to the interrupted interactions between SBP and

356

solvent molecules (Figure 7c). However, a small amount of SBP remained soluble,

357

even though the repulsive forces between pectin chain segments were negligible.

358

Unlike the highly-charged SBP, solubilization of these protonated SBP was chiefly

359

due to insufficient cation-pectin interactions, with a consequent decrease in

360

inter-chain interactions. When P-pH was increased to 3.7, SPP2.7/3.7 precipitated out

361

of solution, likely due to the re-establishment of the interactions between SBP and the

362

cations.

363

4. Discussion

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Precipitation of protonated pectic saccharides with alcohol is commonly thought to

365

be due to the disruption of the interactions between polymer and solvent molecules.

366

With charged pectins, this situation is complicated by the fact that the dissociated

367

anionic groups will induce complex interactions among the negatively-charged

368

pectins, solvent molecules, and positively-charged cations.

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Depending on the protonation state of its carboxyl groups, SBP exhibited different

370

precipitation behaviors. Our work demonstrated that precipitation pH significantly

371

influenced the precipitation of SBP. For the highly negatively-charged SBP, the

372

repulsions between chain segments allowed for strong interactions between SBP and

373

solvent molecules. Such interactions can not be easily broken by increasing the

374

ethanol concentration to 75%. Hence, in order to achieve a higher degree of SBP

17

ACCEPTED MANUSCRIPT precipitation, it was necessary to decrease the charge density and the extent of

376

repulsion between polymer segments. Maximum SBP precipitation was observed at

377

P-pH 3.7, where SBP were moderately charged. This optimum P-pH was consistent

378

with previous reports (P-pH ~3.5) (Kalapathy, et al., 2001; Faravash, et al., 2007).

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It is interesting to note that pectin yield did not necessarily increase with decreasing

380

I-pH or P-pH; rather, the yield started to decrease when I-pH was below 3.0. These

381

results indicate that the solubility of SBP in ethanol is highly pH-dependent. In

382

agreement with this observation, the fraction of SBP in the P-pH 2.7 ethanol solution

383

was successfully recovered by adjusting the P-pH to 3.7. Comparison of the DC and

384

cation content of APP2.0/2.7 and SPP2.7/3.7 revealed that cations, particularly

385

calcium ions, participated in the precipitation of SPP2.7/3.7. When cation-pectin

386

interactions are reduced to a certain level, some precipitated SBP may start to become

387

soluble again. These results indicate that specific interactions between cations and

388

SBP may also play a key role in the precipitation of SBP in ethanol.

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Determination of the cation composition of the different pectins allows us to

390

compare the affinity of SBP for different cations during ethanol precipitation. In the

391

present work, SBP was found to preferentially bind divalent ions over monovalent

392

ions, even though the acetyl groups (Table 1) may cause some steric hindrance for the

393

binding of divalent ions to the carboxyl groups of D-galacturonic acid residues (Kohn

394

& Furda, 1968; Dronnet, Renard, Axelos, & Thibault, 1996). A more detailed study of

395

the affinity of pectins for divalent and monovalent ions can be conducted using

396

pectins with a low degree of acetylation, such as those from citrus and apple pomace.

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ACCEPTED MANUSCRIPT Monovalent ions may primarily serve to neutralize the pectin chain during

398

precipitation, while divalent cations may promote intermolecular interactions, likely

399

by forming intermolecular bonds. This suggests that divalent ions induce the

400

formation of a ‘compact’ pectin lump that may be less hydrated by solvent molecules.

401

However, further studies are needed to provide evidence for this hypothesis.

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All ethanol solutions with a fixed concentration have an equal ability to solubilize a

403

polymer. Hence, it may be deduced that the polymer itself determines the nature of its

404

interaction with solvent molecules. Knowledge of the conformational features of SBP

405

in ethanol solution may be helpful to unveil the relationship between pectin structural

406

features and precipitation behaviors. For a more comprehensive understanding of the

407

precipitation behaviors of SBP, the effects of conformational characteristics on pectin

408

precipitation behaviors should be further investigated.

409

5. Conclusion

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Precipitation pH had a significant effect on the alcoholic precipitation of SBP from

411

aqueous extracts. The highest precipitation efficiency was observed at a moderate

412

I-pH (i.e., I-pH 3.0). In contrast, at other I-pH values, some SBP fractions interacted

413

with solvent molecules and remained soluble, either due to stronger repulsions

414

between SBP chain segments (I-pH > 3.0) or decreased strength of cation-pectin

415

interaction (I-pH < 3.0). Furthermore, the precipitation of SBP was associated with

416

different extents of cation-pectin interactions, depending on the I-pH used. Altogether,

417

the results obtained in this work indicate that ethanol precipitation of SBP involves

418

complex interactions among cations, solvent molecules, and pectin chain segments,

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ACCEPTED MANUSCRIPT 419

which lead to different precipitation behaviors of SBP under different precipitation

420

pHs.

421

Acknowledgements

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This work was supported by the Achievements Transformation Fund Project

424

(No.2013GB23600669) and the State Key Laboratory of Pulp and paper Engineering

425

(No.C712035z). Special thanks to Lvxiang beet sugar company for providing sugar

426

beet pulp.

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427 428

References

429

Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative

430

determination of uronic acids. Analytical biochemistry(54), 484-489. Buchholt, H. C., Christensen, T. M. I. E., Fallesen, B., Ralet, M.-C., & Thibault, J.-F.

432

(2004). Preparation and properties of enzymatically and chemically modified

433

sugar beet pectins. Carbohydrate Polymers, 58(2), 149-161.

TE D

431

Capel, F., Nicolai, T., Durand, D., Boulenguer, P., & Langendorff, V. (2006). Calcium

435

and acid induced gelation of (amidated) low methoxyl pectin. Food

436

Hydrocolloids, 20(6), 901-907.

EP

434

Dronnet, V. M., Renard, C. M. G. C., Axelos, M. A. V., & Thibault, J.-F. (1996).

438

Characterisation and selectivity of divalent metal ions binding by citrus and

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sugar-beet pectins. Carbohydrate Polymers, 30(4), 253-263.

440

Faravash, R. S., & Ashtiani, F. Z. (2007). The effect of pH, ethanol volume and acid

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washing time on the yield of pectin extraction from peach pomace.

442

International Journal of Food Science & Technology, 42(10), 1177-1187.

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Garna, H., Emaga, T. H., Robert, C., & Paquot, M. (2011). New method for the

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purification of electrically charged polysaccharides. Food Hydrocolloids,

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25(5), 1219-1226. 20

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Garna, H., Mabon, N., Wathelet, B., & Paquot, M. (2004). New method for a two-step

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hydrolysis and chromatographic analysis of pectin neutral sugar chains.

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Journal of agricultural and food chemistry, 52(15), 4652-4659. Hwang, J., Roshdy, T., Kontominas, M., & Kokini, J. (1992). Comparison of dialysis

450

and metal precipitation effects on apple pectins. Journal of food science, 57(5),

451

1180-1184.

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Kalapathy, U., & Proctor, A. (2001). Effect of acid extraction and alcohol

453

precipitation conditions on the yield and purity of soy hull pectin. Food

454

chemistry, 73(4), 393-396.

456

Kohn, R., & Furda, I. (1968). Binding of calcium ions to acetyl derivatives of pectin. Collection of Czechoslovak Chemical Communications, 33(7), 2217-2225.

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Levigne, S., Ralet, M.-C., & Thibault, J.-F. (2002). Characterisation of pectins

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extracted from fresh sugar beet under different conditions using an

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experimental design. Carbohydrate Polymers, 49(2), 145-153. Levigne, S., Thomas, M., Ralet, M.-C., Quemener, B., & Thibault, J.-F. (2002).

461

Determination of the degrees of methylation and acetylation of pectins using a

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C18 column and internal standards. Food Hydrocolloids, 16(6), 547-550.

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Li, X., Al-Assaf, S., Fang, Y., & Phillips, G. O. (2013). Characterisation of

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commercial LM-pectin in aqueous solution. Carbohydrate Polymers, 92(2),

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1133-1142.

EP

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Li, X., Fang, Y., Phillips, G. O., & Al-Assaf, S. (2013). Improved sugar beet

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pectin-stabilized emulsions through complexation with sodium caseinate.

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Ralet, M.-C., Cabrera, J. C., Bonnin, E., Quéméner, B., Hellìn, P., & Thibault, J.-F.

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AC C

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(2005). Mapping sugar beet pectin acetylation pattern. Phytochemistry, 66(15),

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1832-1843.

468

Journal of agricultural and food chemistry, 61(6), 1388-1396.

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Ralet, M.-C., Crépeau, M.-J., Buchholt, H.-C., & Thibault, J.-F. (2003).

473

Polyelectrolyte behaviour and calcium binding properties of sugar beet pectins

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differing in their degrees of methylation and acetylation. Biochemical

475

Engineering Journal, 16(2), 191-201. 21

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Smidsrød, O., & Haug, A. (1967). Precipitation of acidic polysaccharides by salts in

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ethanol–water mixtures. In

Journal of Polymer Science Part C: Polymer

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Symposia (Vol. 16, pp. 1587-1598): Wiley Online Library. Van paemel, M. R., De Rycke, H., Millet, S., Hesta, M., & Janssens, G. P. (2005).

480

Evaluation of dry ashing in conjunction with ion chromatographic

481

determination of transition metal ions in pig feed samples. Journal of

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agricultural and food chemistry, 53(6), 1873-1877.

484

Wang, P., & Anderko, A. (2001). Computation of dielectric constants of solvent mixtures and electrolyte solutions. Fluid Phase Equilibria, 186(1), 103-122.

SC

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Xu, J., Yue, R.-Q., Liu, J., Ho, H.-M., Yi, T., Chen, H.-B., & Han, Q.-B. (2014).

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Structural diversity requires individual optimization of ethanol concentration

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in

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macromolecules, 67, 205-209.

polysaccharide

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precipitation.

International

journal

of

biological

Yapo, B. M. (2009). Biochemical characteristics and gelling capacity of pectin from

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yellow passion fruit rind as affected by acid extractant nature. Journal of

491

agricultural and food chemistry, 57(4), 1572-1578.

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Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of

493

extraction conditions on the yield, purity and surface properties of sugar beet

494

pulp pectin extracts. Food chemistry, 100(4), 1356-1364.

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Yapo, B. M., Wathelet, B., & Paquot, M. (2007). Comparison of alcohol precipitation

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and membrane filtration effects on sugar beet pulp pectin chemical features

497

and surface properties. Food Hydrocolloids, 21(2), 245-255.

498 499

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63.8±1 3.5±0.4 7.9±0.5 7±0.5 0.2±0 0.4±0 19 44.1±1.5 22.3±0.4 184±4

56.5±0.6 3.2±0.1 7.6±0.6 7.4±1 0.2±0.1 0.1±0 18.5 43.3±1.3 26.5±0.3 173±3

55.2±0.7 3.3±0.4 8.1±0 7.5±0.4 0.2±0.2 0.2±0.1 18 47.6±2.1 28.0±1.4 148±5

55.9±1.2 3±0.3 8±0.9 7±0.8 0.2±0.1 0.2±0 19.3 47.8±0.7 27.7±0.6 142±4

ND: not detected

APP4.0/5.5

APP4.5/6.1

SPP2.7/3.7

55.7±0.2 2.8±0.1 7.5±0.2 7.6±0.8 0.3±0.1 0.2±0 18.4 40.7±3.9 21.9±0.6 112±2

54.0±0.5 3.1±0.4 7.6±1 5.7±0.5 0.4±0 0.1±0 16.9 41.1±1.3 21.5±0.8 106±5

58.8±0.1 3.3±0 7.7±0.6 6.8±0.2 0.2±0.1 0.2±0 18.2 41.3±1 25.6±1.1 ND a

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APP3.5/4.5

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a

APP3.0/3.7

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501

APP2.5/3.1

AC C

GalA rhamnose arabinose galactose glucose xylose NS DM DA Mw

APP2.0/2.7

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Table 1

500

23

ACCEPTED MANUSCRIPT Figure captions:

503

Figure 1. Electrophoretic mobility as a function of pH for the dialyzed-extract.

504

Figure 2. P-pH of the resulting ethanol solution as a function of I-pH.

505

Figure 3. Electrophoretic mobility values for the pectin containing ethanol solutions

506

as a function of pH (a) and concentration of added salts (b). Effect of salt addition in

507

electrophoretic mobility was studied at I-pH 4.5.

508

Figure 4. Pectin yields as a function of I-pH (a) and concentration of added NaCl (b).

509

Effect of salt addition on pectin yield was studied at I-pH 4.5.

510

Figure 5. Molecular weight distribution profiles of sugar beet pectins using RI

511

detection.

512

Figure 6. Contents of cations (a) and degrees of cation-binding (b) of sugar beet

513

pectins precipitated under various conditions.

514

Figure 7. Schematic illustrations of precipitation behaviors of pectins.

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502

515

Table caption:

517

Table 1 Chemical composition (% w/w), degree of methylation (% mol) and degree of

518

acetylation (% mol), and weight-average molar mass (kg/mol) of the different pectins

519

precipitated under different conditions

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24

ACCEPTED MANUSCRIPT 1

pH

0 -0.5 1

2

3

4

5

6

7

-1 -2 -2.5 -3

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Figure 1.

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-1.5

SC

UE (µm·cm / V·s)

0.5

25

1.5

2.0

2.5

3.0

3.5

Initial pH

4.5

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Figure 2.

4.0

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7 6 5 4 3 2 1 0

26

5.0

SC

Corresponding P-pH

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

3

4

5

6

7

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b

Concentration of added NaCl (mM)

0.1 0 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8

20

40

60

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UE ( µm·cm / V·s )

P-pH

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Figure 3.

27

80

SC

UE ( µm·cm / V·s )

a 0.1 0.0 -0.1 2 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8

100

ACCEPTED MANUSCRIPT

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Pectin yield (% w/w)

a 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

SC

I-pH 2.0 I-pH 2.5 I-pH 3.0 I-pH 3.5 I-pH 4.0 I-pH 4.5

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Pectin yield (% w/w)

b 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

0

4

8

20

40

Concentration of added NaCl (mM)

AC C

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Figure 4.

28

80

ACCEPTED MANUSCRIPT

10

15

SC

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RI signal (a.u.)

APP2.0/2.7 APP4.0/5.5 APP4.5/6.1

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Elution volume (mL)

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Figure 5.

29

20

ACCEPTED MANUSCRIPT

Na K Mg Ca

2.0

1.5

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Cation content (% w/w)

a

1.0

0.5

SC

0.0

90

b

80

60 50 40

EP

30

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DC (%)

70

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2.7 3.1 3.7 4.5 5.5 6.1 3.7 .0/ .5/ .0/ .5/ .0/ .5/ .7/ P2 P2 P3 P3 P4 P4 P2 P P P P P P P S A A A A A A

20

AC C

10

3.7 6 .1 5.5 4.5 3.7 3.1 2.7 .7/ .5/ .0/ .5/ .0/ .5/ .0/ P2 P4 P4 P3 P3 P2 P2 P P P P P P P S A A A A A A

Figure 6.

30

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ACCEPTED MANUSCRIPT

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Figure 7.

31

ACCEPTED MANUSCRIPT Highlights
Solubility of pectins in the ethanolic solution depended on the pH at which pectins were precipitated.
No simple correlation between precipitation pH and pectin yield was observed.

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Effects of precipitation pH on the pectin-cation interactions were investigated.
Interactions between charged pectins and divalent cations promoted the

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precipitation of pectins.