Effect of enzyme de-esterified pectin on the electrostatic complexation with pea protein isolate under different mixing conditions

Journal Pre-proof Effect of enzyme de-esterified pectin on the electrostatic complexation with pea protein isolate under different mixing conditions

Prasanth K.S. Pillai, Blanca E. Morales-Contreras, Louise Wicker, Michael T. Nickerson PII:

S0308-8146(19)31548-1

DOI:

https://doi.org/10.1016/j.foodchem.2019.125433

Reference:

FOCH 125433

To appear in:

Food Chemistry

Received date:

19 April 2019

Revised date:

2 August 2019

Accepted date:

27 August 2019

Please cite this article as: P.K.S. Pillai, B.E. Morales-Contreras, L. Wicker, et al., Effect of enzyme de-esterified pectin on the electrostatic complexation with pea protein isolate under different mixing conditions, Food Chemistry(2019), https://doi.org/10.1016/ j.foodchem.2019.125433

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© 2019 Published by Elsevier.

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Effect of enzyme de-esterified pectin on the electrostatic complexation with pea protein isolate under different mixing conditions

Prasanth K. S. Pillai1, Blanca E. Morales-Contreras2,3, Louise Wicker2 and Michael T. Nickerson1* 1

Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive,

School of Nutrition & Food Sciences, Louisiana State University, Baton Rouge, LA, USA,

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2

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Saskatoon, SK, Canada, S7N 5A8

Institution: Tecnológico Nacional de México/I. T. Durango, Posgrado en Ingeniería Bioquímica,

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70803

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Felipe Pescador 1803, Nueva Vizcaya, 34080 Durango, Dgo., México.

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Running title: Coacervation of enzymatically modified pectin and pea protein

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*Corresponding author:

Michael T. Nickerson, PhD

Department of Food and Bioproduct Sciences, University of Saskatchewan 51 Campus Drive, Saskatoon, SK, S7N 5A8, Canada Tel: (306) 966-5030 Fax: (306) 966-8898 E-mail: [email protected]

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ABSTRACT Native high methoxy citrus pectin (NP) was de-esterified by pectin methyl esterase to produce modified pectins [MP (42, 37, and 33)] having different degrees of esterification. Complex coacervation between a pea protein isolate (PPI) and each pectin was investigated as a function of pH (8.0 – 1.5) and mixing ratio (1:1 - 30:1, PPI-pectin). Complex formation was found to be optimal for biopolymer-mixing ratios of 8:1, 8:1, 25:1 and 25:1 for PPI complexed

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with NP, MP42, MP37 and MP33, respectively, at pHs 3.6, 3.5, 3.9 and 3.9. And, the critical

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pHs associated with complex formation (accessed by turbidity) was found to shift significantly to

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higher pHs as the degree of esterification of the pectin decreased, whereas the shift in the pH

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corresponding to their initial interactions was minimal with degree of esterification.

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Complexation of PPI with NP and MP42 greatly improved the protein solubility.

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KEYWORDS:

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solubility

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Enzyme modification of pectin; complex coacervation; de-esterification; pea protein isolate;

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1. INTRODUCTION Protein-polysaccharide complexes formed through controlled electrostatic attraction (i.e., complex coacervation) play important roles in controlling the flow, stability, texture and mouthfeel of food products (Turgeon, Beaulieu, Schmitt, & Sanchez, 2003), in the development of edible packaging films or hydrogels (Hunt, Feldman, Lynd, Deek, Campos, Spruell, et al., 2011; Lalevée, David, Montembault, Blanchard, Meadows, Malaise, et al., 2017), emulsifiers

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(Dickinson, 2008; Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998) and in the design

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controlled delivery systems for carrying bioactive compounds (Ghasemi, Jafari, Assadpour, &

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Khomeiri, 2017, 2018; Mayya, Bhattacharyya, & Argillier, 2003; Raei, Shahidi, Farhoodi, Jafari,

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& Rafe, 2017; Xing, Cheng, Yi, & Ma, 2005). The level of complexation depends on the biopolymer mixing ratio, biopolymer molecular characteristics such as molecular weight,

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conformation, charge density, or environmental factors such as pH, ionic strength, solvent

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quality, mixing conditions etc. (Aryee & Nickerson, 2012; Elmer, Karaca, Low, & Nickerson, 2011; Niu, Su, Liu, Wang, Zhang, & Yang, 2014; Schmitt, Sanchez, Desobry-Banon, & Hardy,

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1998; Stone, Teymurova, Chang, Cheung, & Nickerson, 2015). The formed complexes can

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subsequently have unique functionality over the individual biopolymers themselves. Typically, complexes are formed through various pH-induced structure forming events associated with the initial electrostatic interactions between a protein and polysaccharide (denoted as pHc) and the formation of soluble complexes. This typically occurs near the isoelectric point of a protein, however can occur at pHs > pI where both biopolymers carry similar net charge in the case of when a highly charged polysaccharide is present (e.g., carrageenan, alginate and pectin) (Doublier, Garnier, Renard, & Sanchez, 2000). In the case of the latter, initial interactions are thought to originate between the anionic polysaccharide and

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positively charged patches on the protein’s surface (Niu, Su, Liu, Wang, Zhang, & Yang, 2014; Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998; Weinbreck, De Vries, Schrooyen, & De Kruif, 2003). Upon further acidification, soluble complexes grow in size and number via nucleation and growth kinetics to transform the solution from transparent to turbid. The point in which large changes in turbidity occurs is denoted by pH1, and the formation of insoluble complexes. Maximum biopolymer interaction occurs at pHopt, at which the electrical neutrality

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point (stoichiometric charge equivalence) of both biopolymers is reached. As the solution is

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further titrated with acid, charges on the polysaccharide begin to become protonated until the

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complete dissolution of complexes occurs (denoted as pH2) (De Kruif & Tuinier, 2001; Schmitt,

Weinbreck, Tromp, & De Kruif, 2004).

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Sanchez, Desobry-Banon, & Hardy, 1998; Turgeon, Beaulieu, Schmitt, & Sanchez, 2003;

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The present study examines the interactions between pea protein and pectin

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polysaccharides. Pea proteins are considered an emerging plant protein ingredient within the food industry due to its nutritional value, low cost, abundance and non-GM status. Pea protein

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isolates (PPI) are typically formed commercially with an alkaline extraction followed by

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isoelectric precipitation and spray drying. The isolates tend to be rich in salt-soluble globulin and water-soluble albumin proteins. Citrus pectin is an acidic polysaccharide comprised of ~90% α(1-4)-linked D-galacturonic acids with another ~10% having neutral sugars attached (Mohnen, 2008). Changes to the pectin’s structure by altering the degree of esterification (DE) (Li, Fang, Al-Assaf, Phillips, Yao, Zhang, et al., 2012; Ru, Wang, Lee, Ding, & Huang, 2012; Sperber, Schols, Stuart, Norde, & Voragen, 2009) and the distribution pattern of non-methyl esterified galacturonic acid (known as the degree of blockiness (DB) (Fraeye, Doungla, Duvetter, Moldenaers, Van Loey, & Hendrickx, 2009) can significantly affect their gelling abilities,

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complexing behavior with proteins and, other functional attributes (solubility, foaming and emulsification properties) (Klinchongkon, Khuwijitjaru, Adachi, Bindereif, Karbstein, & van der Schaaf, 2019; Lan, Chen, & Rao, 2018; Oduse, Campbell, Lonchamp, & Euston, 2017) . Proteinpectin complexes are highly utilized for the encapsulation and controlled delivery of various bioactive and nutraceuticals (Ghasemi, Jafari, Assadpour, & Khomeiri, 2017, 2018; Raei, Shahidi, Farhoodi, Jafari, & Rafe, 2017).

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In previous work, we chemically modified high methoxy citrus pectin by alkaline

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hydrolysis to alter its DE and blockiness, and then investigated its complexing behavior with

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PPI. The process resulting in changes to the molecular weight of the pectin chains, which

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impacted its interactions with PPI. The random distribution of non-methyl esterified galacturonic acids obtained via the alkaline de-esterified pectins created more rigid and stiffer chains, which

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enhanced its interaction with PPI by shifting the critical pH values associated with structure

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forming events to a higher value (Pillai, Stone, Guo, Guo, Wang, & Nickerson, 2019). However, that study lacked an understanding of the impact of localized charge density on the pectin chains

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on its complexing behavior. By enzymatically de-esterifying the pectin using plant PME

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(pPME), the number of blocks of adjacent non-methyl esterified galacturonic acid units is expected to increase (i.e., the localized charge density) without altering its molecular weight (Hunter & Wicker, 2005; Kastner, Einhorn-Stoll, & Drusch, 2019). The overall goal of the present study is to examine the effect of various levels of pPME-modification of citrus pectin on its DE value, and the impact that has on its ability to form complexes with PPI. It is hypothesized that increased extent of PME de-esterification (results increased number of blockiness) of pectin may decreases their electrostatic attractive interaction with PPI and favors strong electrostatic repulsion. Complexation was examined as a function of biopolymer mixing ratio and pH, with

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the resulting effects on protein solubility accessed at the pI of PPI. Information arising from this work could lead to the development of mixed PPI-pectin ingredients with improved functionality (i.e., solubility) over PPI alone.

2. MATERIALS AND METHODS

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2.1 Materials Yellow peas (CDC Meadow; certified seed grade) used in this study were grown in

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North Battleford, SK, Canada (2014). Commercial unstandardized high methoxy pectin (Citrus

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pectin GENUR Pectin type B rapid set Z) was kindly donated by CP Kelco (Lille Skenseved,

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Denmark). Crude Valencia PME extract was obtained from Valencia orange pulp following the

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Kim, Teng & Wicker (2005). Proximate analysis was carried out according to AOAC Official Methods (AOAC, 2003) 925.10 (moisture), 923.03 (ash), 920.85 (lipid) and 920.87 (crude

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protein), whereas carbohydrate content was calculated based on percent difference from 100%.

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The pectin was assumed to be devoid of any significant amount of protein or lipid.

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2.2 Preparation of pea protein isolates Pea protein isolate was produced from milled defatted flour as described in detail by (Warnakulasuriya, Pillai, Stone, & Nickerson, 2018).

2.3 Demethylation of pectin by PME hydrolysis The enzyme de-esterification of pectin was performed using a crude PME extracted in the lab from the Valencia orange pulp. The crude PME extract from Valencia orange pulp was obtained according to the method by (Kim, Teng, & Wicker, 2005) with some minor

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modifications. Briefly, 40 g of frozen pulp were weighed, 160 mL of 0.1M NaCl, 0.25 M Tris buffer, pH 8.0, 0.02% NaN3 were added. The mixture was homogenized for 2 min at 4°C, the pH was adjusted to 8.0 manually with 2 M NaOH, after that, the extract was filtered through Miracloth (CalBiochem, La Joya, USA) and then centrifuged at 8 000 g (Sorvall RC6 Plus, Thermo Scientific, USA), at 4°C for 20 min. Finally, the supernatant was dialyzed (Dialysis membrane, Spectra/Por 1, Spectrum, USA) overnight with distilled water, and it was changed

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tree times. After dialysis the extract was transfer to a bottle and storage at 4°C, until use. The

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PME activity was determined by a pH stat titrator (902 Titrando, Metrohm, Herisau,

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Switzerland) at 30C with 1% high methoxyl pectin (Citrus pectin type B rapid set-Z, CP Kelco

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Company, Lille Skensved, Denmark) dispersed in 0.1 M NaCl at a set point pH of 7.5 during 30

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min time interval. The unit of PME activity was defined as mol of ester hydrolyzed/mLmin at 30C (U/mL). The crude Valencia PME extract activity obtained was 33.6 U/mL.

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Pectin was de-esterified by enzyme hydrolysis (by means of PME) using a previously published method (Hunter & Wicker, 2005). To obtain pectin having different DE, 1% (w/v)

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pectin dispersion in 0.1M NaCl was prepared and stirred overnight at room temperature. Prior to

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the modification, the pectin solution was stabilized at 30 °C, then the pH was adjusted to 7.5 with 0.1N NaOH. Predetermined amount of PME (2.77, 5.35 and 7.97 mL to 500 mL reaction mixture) were added to the reaction mixture, and the reaction was kept at pH 7.5 (by controlled addition of 0.05M NaOH) for 30 min. Immediately after 30 min, to the reaction mixture, boiling ethanol (95%) at 1:4 ratio of pectin solution to ethanol, was added and the mixture was boiled for another 10 min to quench further enzymes present in the reaction mixture. The reaction mixture was further cooled to room temperature and washed again with 95% ethanol. The finally washed precipitate was dried at room temperature (3 days drying at room temperature), and were named

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as MP42, MP37 and MP33, respectively. A control pectin sample was subjected to the same procedure, except for the pH adjustment, and was labelled as NP.

2.4 Determination of degree of methyl esterification (DE) The degree of methyl esterification of pectin samples (NP, MP42, MP37 and MP33) were examined on a Tensor 27 FTIR spectrometer (BI021703, Bruker, UK) with a Pike Miracle

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diamond/ZnSe ATR cell as sampling accessory. The spectra were measured in the region of

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4000 to 650 cm-1 with 50 scans per reading. The data was collected and analyzed on OPUS

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Software (Version 7.2, Bruker, UK). DE was calculated (Eq.2) according to Shpigelman and

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Kyomugasho method with minor modification (Kyomugasho, Christiaens, Shpigelman, Van Loey, & Hendrickx, 2015; Shpigelman, Kyomugasho, Christiaens, Van Loey, & Hendrickx,

1

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Abs1690cm 1

Abs1690cm  Abs1550 cm

1

)

(1)

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DE %  100  (

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2014).

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2.5 Determination of critical pHs (pHc, pHϕ1 and pHϕ2) by turbidimetric analysis Interactions between PPI and the pectin (NP, MP42, MP37 and MP33) were examined according to Warnakulasuriya et al. (2018) during a pH (8.0-1.5) - acid turbidimetric titration. The total biopolymer concentration of 0.05% (w/w) was used with the following PPI-pectin mixing ratios, 1:1, 2:1, 4:1, 8:1, 10:1 and 15:1, with the addition of 20:1, 25:1 and 30:1 ratios for only the PPI-MP37 and PPI-MP33 mixtures. Critical pH values of complex formation were graphically determined as described in (Liu, Low, & Nickerson, 2009). All measurements were carried out in triplicate.

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2.6 Determination of molecular weight (MW) The molecular weight (MW) of the modified pectin was determined by size-exclusion chromatography with Multi-Angle Light Scattering (MALS), and differential Refractive Index (dRI) detectors (Wyatt Technology, Goleta, CA, USA). The separating column used was an Aquagel PL-OH, 7.5x300mm, and a PL guard column Aquagel-OH, 7.5 x 50 mm. 3mg/mL of

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pectin dispersion in buffer (10 mM sodium phosphate, 100 mM sodium nitrate, pH 7.0) were

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prepared and hydrated overnight; after, the samples were filtered through 0.45µm acrodisc

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syringe filters (Polyethersulfone membrane, Whatman TM, UK), and were transferred to the 2.5

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mL vials for the subsequent injection (Corredig, Kerr, & Wicker, 2000). The analysis was performed at a flow rate of 0.5 ml/min in a mobile phase of 10 mM sodium phosphate, 100mM

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sodium nitrate, pH 7.0 buffer solution. Data were processed by using the Astra software version

MW calculation.

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2.7 Surface charge

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6.1 (Wyatt Technology Corp.). 0.13 was used as dn/dc value. The Zimm model was used for

Surface charge (zeta potential) was determined using a Zetasizer Nano ZS90 (Malvern Instruments, Westborough, MA, USA) for individual and mixed PPI-pectin solutions (0.05% w/w) as a function of pH (8.0-1.5) according to Warnakulasuriya et al. (2018). Measurements were made only the optimal mixing ratios as identified in Section 2.5. All measurements were carried out in triplicates and reported as the mean value ± one standard deviation.

2.8 Statistics

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A one-way analysis of variance (ANOVA) test was used to assess statistical differences within critical pH values (pHc, pH1, pHopt, pH2) and maximum OD as a function of mixing ratios. Also, a student T-test was used to assess changes in solubility for samples at their optimal mixing ratio relative to the homogeneous PPI solution. All statistical analysis was performed using Microsoft Excel 2013.

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3. RESULTS AND DISCUSSIONS

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3.1 Physicochemical properties

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Pea protein isolate was found to be comprised of 88.8% protein, 0.5% lipid, 5.3% ash,

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0.8% carbohydrate (determined by difference) and 4.5% moisture, on a wet weight basis (w.b.). Table 1 gives the physiochemical properties of native (NP; DE 63%) and modified high

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methoxyl citrus pectin (labelled MP42, MP37 and MP33 to reflect ‘modified pectin’ and their

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respective DE value). The native pectin was found to comprise of 78.9% (w.b.) carbohydrates and 3.9% ash, whereas the modified pectin had slightly lower carbohydrate levels ranging

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between 69.7 and 72.1% (w.b.), and higher ash levels ranging between 9.4-9.5% (w.b.). The

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increased ash content for the enzyme modified samples is may be due to the usage of 0.1 M NaCl for the modification reaction. Large changes in MW of the pectin were not observed indicating that depolymerization or -elimination during PME de-esterification was negligible. A small significant decrease in molecular weight was observed for MP42 (174 kDa) and MP 37 (178 kDa) compare to the native form (188 kDa), which was attributed to the de-esterification procedure used in the study which had removed some of the methyl ester groups from pectin. An increase in molecular weight was found in the case of MP33 (253 kDa) relative to NP (although not significant), which was thought to be caused by the formation of aggregates through

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chelation with buffer salts during the molecular weight analysis. A similar phenomena was reported by several researchers (Hunter & Wicker, 2005; Yoo, Fishman, Savary, & Hotchkiss, 2003), where separation issues of aggregates were noted using filter sizes as low as 0.05 µm, resulting in higher molecular weights being reported. Polydispersity for NP and the enzyme modified pectins (MP42, MP37 and MP33) were similar, ~1.38, which supports that there is no drastic depolymerization or oligomerization of pectin with enzyme treatment. Unlike the alkaline

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de-esterification reported by (Pillai, Stone, Guo, Guo, Wang, & Nickerson, 2019), the PME de-

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esterification did not substantially alter the pectin’s molecular size (approximately similar MW)

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however increased the number of non-methyl esterified galacturonic acid on the pectin chain and

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the subsequently their stiffness (due to rigid nature from galacturonic acid) with respect to the extent of de-esterification. Although not measured in the present study, the degree of blockiness

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of pectin is known to increase with increasing PME (Kastner, Einhorn-Stoll, & Drusch, 2019),

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and > NP.

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indicating that for the present study DB would increased in the order of MP33 > MP37 > MP42

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3.2 Associative phase behaviour of PPI-pectin mixtures Changes to the optical density (OD) during an acid pH titration in solutions of PPI alone and admixtures of PPI and pectin [(a) NP, (b) MP42, (c) MP37 and (d) MP33] as a function of biopolymer mixing ratio is given in Figure 1. Homogenous PPI displayed a bell-shaped turbidity profile beginning at pH ~6.5, reaching a maximum OD value of 0.59 at ~pH 4.2 near its isoelectric point (pH 4.8) where aggregation is the highest and solubility the lowest, and then ending at pH ~2.5. In contrast, all pectin treatments had no discernible OD reading over the complete pH range (data not shown). The addition of pectin (regardless of the treatment) caused

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a shift in the bell- shaped profile towards lower pH, and skewed to the left. This effect became more pronounced as the pectin concentration within the PPI: pectin ratio, especially at the 4:1, 2:1 and 1:1 ratio where a significant amount of inter- and intra-chain repulsion is thought to occur between pectin chains in solution that would inhibit protein-protein aggregation. In the case of PPI: NP, the mixing ratios 10:1, 8:1 and 4:1 reached higher OD at their peak than that of PPI alone suggesting a stoichiometric equivalent ratio may have been reached. All other ratios,

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and all other PPI: modified pectin admixtures had lower ODmax than the PPI control (Figure 1).

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The reduced OD in the modified pectin is hypothesized because of the greater amount of

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carboxyl sites obtained via PME de-esterification and the blockwise pattern of carboxyl groups

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on the pectin backbone, both of which would create a greater amount of repulsion between chains the would initially limit the formation of protein aggregates.

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The effect of biopolymer mixing ratio on the critical pH values (pHc, pH1, pHopt, pH2)

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associated with complexation in admixtures of PPI and pectin is given in Figure 2. Irrespective of the biopolymer ratio or pectin treatment, the formation of soluble complex formation occurred

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at pH ~6.5, which was greater than the pI of PPI. This was previously been reported in PPI-

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pectin mixtures (Pillai, Stone, Guo, Guo, Wang, & Nickerson, 2019; Warnakulasuriya, Pillai, Stone, & Nickerson, 2018), and is thought to be associated with the electrostatic attraction between carboxyl sites and positively charged patches on the surface of PPI (De Kruif & Tuinier, 2001; Niu, Su, Liu, Wang, Zhang, & Yang, 2014; Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998; Tolstoguzov, 2003; Weinbreck, De Vries, Schrooyen, & De Kruif, 2003). For all other critical pHs [pHϕ1, pHopt and pHϕ2], they shifted to higher pHs as the amount of pectin within the ratio declined (i.e., the PPI: pectin ratio increased) due to a greater amount of PPI-PPI aggregation (Liu, Low et al. 2009). In the case of pH1 and pHopt, a plateau was reached at

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mixing ratios of 8:1 for both PPI: NP and PPI: MP42, however the same plateau wasn’t reached until a 20:1 ratio for both PPI: MP37 and PPI: MP33 due to the greater repulsion within the system. At these ratios, the pH1 and pHopt were found to be at 4.2 and 3.6, respectively for PPI: NP; 4.1 and 3.5 for PPI: MP42; 5.0 and 3.8 for PPI: MP37 and 5.1 and 3.8 for PPI: MP33 (Figure 2). The critical pH where complexes disassociated at these same mixing ratios were found to be 2.5, 2.4, 2.4 and 2.4 for PPI: NP, -MP42, -MP37 and –MP33, respectively, although

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the plateau was reached at slightly lower ratios (Figure 2). Max OD was found to occur at pHopt

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3.6, 3.5, 3.9 and 3.9 at ratios of 8:1 for PPI: NP, 8:1 for PPI: MP42, 25:1 for PPI: MP37 and 25:1

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for PPI: MP33, respectively (Figure 2E). The maximum OD obtained for these mixtures were

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0.66 (PPI-NP), 0.54 (PPI-MP42), 0.49 (PPI-MP37) and 0.48 (PPI-MP33). After this ratio, OD was found to decline indicating the domination of

protein aggregation over protein-

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polysaccharide complexation (Aryee & Nickerson, 2012). Similar trends in critical pHs were

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reported by Pillai et al. (Pillai, Stone, Guo, Guo, Wang, & Nickerson, 2019) in a PPI-pectin (alkaline modified) mixture; by Warnakulasuriya et. al (Warnakulasuriya, Pillai, Stone, &

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Nickerson, 2018) in PPI-commercial citrus pectin mixture, and by Aryee et. al (Aryee &

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Nickerson, 2012) in lentil protein–gum Arabic mixture. Figure 3 represents the surface charge measurements of homogenous (A) and mixed (B) biopolymer systems at their optimum mixing ratio as a function of pH. Charge neutralization occurred at pH 4.8 for PPI, whereas all pectins were negatively charged over the majority of pH until ~pH 1.5 where they became neutral. PME modification of the pectin resulted in higher charge than the NP, which then increased as the level of modification increased due to an increased number of carboxyl sites (Figure 3A). Complexation of PPI and pectin resulted a shift to net neutrality point of PPI-pectin admixtures to lower pHs than the pI of PPI (pH 4.8), and

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occurred at ~pH 4.0, 3.9, 4.2 and 4.3, respectively for PPI: NP, -MP42, -MP37 and -MP33, respectively.

3.2 Effect of pectin characteristics on coacervation The DE, GalA content, molecular weight, stiffness of the pectin molecule and the pattern of the carboxyl groups or blockiness on the pectin chain have a big effect on its ability to form

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complexes with proteins (Pillai, Stone, Guo, Guo, Wang, & Nickerson, 2019; Warnakulasuriya,

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Pillai, Stone, & Nickerson, 2018). Previously it was shown that even small differences in the

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molecular structure of modified pectin can have an impact in determining their functional

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properties (Fraeye, Doungla, Duvetter, Moldenaers, Van Loey, & Hendrickx, 2009; Kastner, Einhorn-Stoll, & Drusch, 2019). Figure 4A and B shows the effect of DE on ODMax (A) and on

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associated critical pHs (B). In the case of the former, a relatively linear trend was observed with

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DE where OD was lowest at DE 33 (ODMax = 0.48) and highest for DE 63 (ODMax = 0.66) (Figure 4A). This trend is because of the increased repulsive forces of pectin molecules which

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was hypothesized due to the increased blockiness obtained via PME de-esterification. In the case

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of critical pH values at the optimal ratio (i.e., 8:1 for PPI: NP, 10:1 for PPI: MP42 and 25:1 for PPI: MP37/MP33), both pHc and pH2 remained independent of DE. In contrast, for pH1 and pHopt, lower DE values (DE 33-37) were found to be similar and at higher pHs than that systems with higher DE value pectin (DE 42-63). Previously it was reported that the pectin with high DE (high methoxy pectin) and high DB (large number of blocks) can enhance the complexation with PPI to produce maximum OD at their optimum mixing ratio (Warnakulasuriya, Pillai, Stone, & Nickerson, 2018). This may be due to the large number of methoxy groups on the high methoxy

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pectin chains which may shield the reactive sites on the pectin chain and drive attractive over repulsive forces.

3.3. Solubility The solubility (at pH 4.5) of PPI and the PPI: pectin mixtures at their optimal mixing ratio [8:1, 8:1, 25:1 and 25:1 for PPI complexed with NP, MP42, MP37 and MP33, respectively]

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near the isoelectric point of PPI was chosen to observe any negative or positive effects, and

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given in Figure 5. PPI displayed low solubility (~2.7%) at this pH because of minimal repulsion

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present and the tendency for proteins to aggregate (Doublier, Garnier, Renard, & Sanchez, 2000;

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Klemmer, Waldner, Stone, Low, & Nickerson, 2012). PPI: MP33 and PPI: MP37 should similar results (solubility 2.8%) to PPI (p>0.05), whereas PPI: NP and PPI: MP42 were found to have

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significantly higher values of 16.5% and 19.9%, respectively (p<0.05) (Figure 5). Finding

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suggest that lower DE values (33 and 37) of the pectin more effectively inhibited the aggregation of proteins or protein-pectin complexes than higher DE values (Warnakulasuriya, Pillai, Stone,

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& Nickerson, 2018). Previously it was reported that the addition of high methoxy pectin

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(DE~78%) enhanced the solubility of PPI to 45% compared to the low methoxy pectin (DE~29%), which didn’t change the solubility of PPI (Warnakulasuriya, Pillai, Stone, & Nickerson, 2018).

4. CONCLUSION High methoxy citrus pectin was de-esterified using plant pectin methyl esterase (PME) to produce PME modified pectin with varied block degree of esterification, and were investigated for the use of protein-polysaccharide interaction with pea protein isolate (PPI). The pHs

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associated to different structure forming events (pHc, pHϕ1, and pHopt) shifted to higher values with respect to an increase in biopolymer concentration and also with the increased extent of PME modification. Maximum number of coacervates were formed when PPI was complexed with non-modified pectin (NP), which indicated that increased extent of PME de-esterification has suppressed the coacervation of enzyme de-esterified pectin with PPI. The coacervation yield at the optimum mixing ratio for the PPI-pectin mixtures were in the order PPI: NP > PPI: MP42

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> PPI: MP37 > PPI: MP33 at ratios 8:1, 8:1, 25:1 and 25:1 PPI-pectin, respectively, indicating

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that the increased charge density of pectin suppresses the complexation greatly. Maximum

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interactions for the biopolymer mixtures occurred at pH 3.6 (PPI: NP/MP42) and 3.9 (PPI:

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MP37/MP33). The PME de-esterified pectins possessed similar molecular weight and found that the critical pHs [pHopt and pH1] of biopolymer mixtures has shifted significantly to higher pHs

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with the increase of presumed local charge density of pectin (MP33>MP37>MP42>NP). The

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addition of NP and MP42 to PPI greatly increased protein solubility at pH 4.5 (isoelectric point) compared to the other pectin suggesting the by tailoring the pectin’s DE without altering pectin’s

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molecular weight could represent a means of improving PPI’s functionality. Findings from this

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study will lead to the improved utilization pea protein as food and /or biomaterial ingredients.

5. ACKNOWLEDGEMENTS Financial support for this research was provided by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-05661).

6. CONFLICT OF INTEREST STATEMENT None of the authors has any conflict of interest with this research.

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7. REFERENCES Aryee, F. N., & Nickerson, M. T. (2012). Formation of electrostatic complexes involving mixtures of lentil protein isolates and gum Arabic polysaccharides. Food Research International, 48(2), 520-527. Corredig, M., Kerr, W., & Wicker, L. (2000). Molecular characterization of commercial pectins

of

by separation with linear mix gel permeation columns in-line with multi-angle light

ro

scattering detection. Food hydrocolloids, 14(1), 41-47.

-p

De Kruif, C., & Tuinier, R. (2001). Polysaccharide protein interactions. Food hydrocolloids,

re

15(4), 555-563.

Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by protein–

lP

polysaccharide interactions. Soft matter, 4(5), 932-942.

na

Doublier, J.-L., Garnier, C., Renard, D., & Sanchez, C. (2000). Protein–polysaccharide interactions. Current opinion in colloid & interface science, 5(3), 202-214.

ur

Elmer, C., Karaca, A. C., Low, N. H., & Nickerson, M. T. (2011). Complex coacervation in pea

Jo

protein isolate–chitosan mixtures. Food Research International, 44(5), 1441-1446. Fraeye, I., Doungla, E., Duvetter, T., Moldenaers, P., Van Loey, A., & Hendrickx, M. (2009). Influence of intrinsic and extrinsic factors on rheology of pectin–calcium gels. Food hydrocolloids, 23(8), 2069-2077. Ghasemi, S., Jafari, S. M., Assadpour, E., & Khomeiri, M. (2017). Production of pectin-whey protein nano-complexes as carriers of orange peel oil. Carbohydrate Polymers, 177, 369377.

Journal Pre-proof

18

Ghasemi, S., Jafari, S. M., Assadpour, E., & Khomeiri, M. (2018). Nanoencapsulation of dlimonene within nanocarriers produced by pectin-whey protein complexes. Food hydrocolloids, 77, 152-162. Hunt, J. N., Feldman, K. E., Lynd, N. A., Deek, J., Campos, L. M., Spruell, J. M., Hernandez, B. M., Kramer, E. J., & Hawker, C. J. (2011). Tunable, high modulus hydrogels driven by ionic coacervation. Advanced Materials, 23(20), 2327-2331.

of

Hunter, J. L., & Wicker, L. (2005). De‐ esterification of pectin by alkali, plant and fungal

ro

pectinmethylesterases and effect on molecular weight. Journal of the Science of Food

-p

and Agriculture, 85(13), 2243-2248.

re

Kastner, H., Einhorn-Stoll, U., & Drusch, S. (2019). Influence of enzymatic and acidic

hydrocolloids, 89, 207-215.

lP

demethoxylation on structure formation in sugar containing citrus pectin gels. Food

na

Kim, Y., Teng, Q., & Wicker, L. (2005). Action pattern of Valencia orange PME deesterification of high methoxyl pectin and characterization of modified pectins.

ur

Carbohydrate research, 340(17), 2620-2629.

Jo

Klemmer, K., Waldner, L., Stone, A., Low, N., & Nickerson, M. (2012). Complex coacervation of pea protein isolate and alginate polysaccharides. Food Chemistry, 130(3), 710-715. Klinchongkon, K., Khuwijitjaru, P., Adachi, S., Bindereif, B., Karbstein, H. P., & van der Schaaf, U. S. (2019). Emulsifying properties of conjugates formed between whey protein isolate and subcritical-water hydrolyzed pectin. Food hydrocolloids, 91, 174-181. Kyomugasho, C., Christiaens, S., Shpigelman, A., Van Loey, A. M., & Hendrickx, M. E. (2015). FT-IR spectroscopy, a reliable method for routine analysis of the degree of

Journal Pre-proof

19

methylesterification of pectin in different fruit-and vegetable-based matrices. Food Chemistry, 176, 82-90. Lalevée, G., David, L., Montembault, A., Blanchard, K., Meadows, J., Malaise, S., Crépet, A., Grillo, I., Morfin, I., & Delair, T. (2017). Highly stretchable hydrogels from complex coacervation of natural polyelectrolytes. Soft matter, 13(37), 6594-6605. Lan, Y., Chen, B., & Rao, J. (2018). Pea protein isolate–high methoxyl pectin soluble complexes

ro

concentrations. Food hydrocolloids, 80, 245-253.

of

for improving pea protein functionality: Effect of pH, biopolymer ratio and

-p

Li, X., Fang, Y., Al-Assaf, S., Phillips, G. O., Yao, X., Zhang, Y., Zhao, M., Zhang, K., & Jiang,

re

F. (2012). Complexation of bovine serum albumin and sugar beet pectin: Structural transitions and phase diagram. Langmuir, 28(27), 10164-10176.

lP

Liu, S., Low, N. H., & Nickerson, M. T. (2009). Effect of pH, salt, and biopolymer ratio on the

na

formation of pea protein isolate− gum arabic complexes. Journal of Agricultural and Food Chemistry, 57(4), 1521-1526.

ur

Mayya, K. S., Bhattacharyya, A., & Argillier, J. F. (2003). Micro‐ encapsulation by complex

Jo

coacervation: influence of surfactant. Polymer International, 52(4), 644-647. Mohnen, D. (2008). Pectin structure and biosynthesis. Current opinion in plant biology, 11(3), 266-277. Niu, F., Su, Y., Liu, Y., Wang, G., Zhang, Y., & Yang, Y. (2014). Ovalbumin–gum arabic interactions: effect of pH, temperature, salt, biopolymers ratio and total concentration. Colloids and Surfaces B: Biointerfaces, 113, 477-482.

Journal Pre-proof

20

Oduse, K., Campbell, L., Lonchamp, J., & Euston, S. R. (2017). Electrostatic complexes of whey protein and pectin as foaming and emulsifying agents. International Journal of Food Properties, 20(sup3), S3027-S3041. Pillai, P. K. S., Stone, A. K., Guo, Q., Guo, Q., Wang, Q., & Nickerson, M. T. (2019). Effect of alkaline de-esterified pectin on the complex coacervation with pea protein isolate under different mixing conditions. Food Chemistry, 284, 227-235.

of

Raei, M., Shahidi, F., Farhoodi, M., Jafari, S. M., & Rafe, A. (2017). Application of whey

-p

biological macromolecules, 105, 281-291.

ro

protein-pectin nano-complex carriers for loading of lactoferrin. International journal of

re

Ru, Q., Wang, Y., Lee, J., Ding, Y., & Huang, Q. (2012). Turbidity and rheological properties of bovine serum albumin/pectin coacervates: effect of salt concentration and initial

lP

protein/polysaccharide ratio. Carbohydrate Polymers, 88(3), 838-846.

na

Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure and technofunctional properties of protein-polysaccharide complexes: a review. Critical reviews in food

ur

science and nutrition, 38(8), 689-753.

Jo

Shpigelman, A., Kyomugasho, C., Christiaens, S., Van Loey, A. M., & Hendrickx, M. E. (2014). Thermal and high pressure high temperature processes result in distinctly different pectin non-enzymatic conversions. Food hydrocolloids, 39, 251-263. Sperber, B. L., Schols, H. A., Stuart, M. A. C., Norde, W., & Voragen, A. G. (2009). Influence of the overall charge and local charge density of pectin on the complex formation between pectin and β-lactoglobulin. Food hydrocolloids, 23(3), 765-772.

Journal Pre-proof

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Stone, A. K., Teymurova, A., Chang, C., Cheung, L., & Nickerson, M. T. (2015). Formation and functionality of canola protein isolate with both high-and low-methoxyl pectin under associative conditions. Food Science and Biotechnology, 24(4), 1209-1218. Tolstoguzov, V. (2003). Some thermodynamic considerations in food formulation. Food hydrocolloids, 17(1), 1-23. Turgeon, S., Beaulieu, M., Schmitt, C., & Sanchez, C. (2003). Protein–polysaccharide

of

interactions: phase-ordering kinetics, thermodynamic and structural aspects. Current

ro

opinion in colloid & interface science, 8(4), 401-414.

-p

Warnakulasuriya, S., Pillai, P. K., Stone, A. K., & Nickerson, M. T. (2018). Effect of the degree

re

of esterification and blockiness on the complex coacervation of pea protein isolate and commercial pectic polysaccharides. Food Chemistry, 264, 180-188.

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Weinbreck, F., De Vries, R., Schrooyen, P., & De Kruif, C. (2003). Complex coacervation of

na

whey proteins and gum arabic. Biomacromolecules, 4(2), 293-303. Weinbreck, F., Tromp, R., & De Kruif, C. (2004). Composition and structure of whey

ur

protein/gum arabic coacervates. Biomacromolecules, 5(4), 1437-1445.

Jo

Xing, F., Cheng, G., Yi, K., & Ma, L. (2005). Nanoencapsulation of capsaicin by complex coacervation of gelatin, acacia, and tannins. Journal of applied polymer science, 96(6), 2225-2229. Yoo, S.-H., Fishman, M. L., Savary, B. J., & Hotchkiss, A. T. (2003). Monovalent salt-induced gelation of enzymatically deesterified pectin. Journal of Agricultural and Food Chemistry, 51(25), 7410-7417.

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Figure and table captions

Figure 1.

Mean turbidity curves for PPI-pectin (NP, A; MP42, B; MP37, C; MP33, D) mixtures and a homogenous PPI solution as a function of pH and biopolymer mixing ratio (n=3).

Figure 2.

Critical pH values associated with the formation of soluble (pHc) and insoluble

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complexes (pHϕ1), maximum interactions (pHopt), and complex dissolution (pHϕ2),

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maximum optical density at pHopt in PPI-pectin (NP, A; MP42, B; MP37, C;

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MP33, D; optical density, E) mixtures as a function of biopolymer mixing ratio.

Figure 3.

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Data represent the mean ± one standard deviation (n= 3). Surface charge (zeta potential, mV) of homogenous and mixed PPI-pectin

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biopolymer solutions ((PPI-pectin: NP, MP42, 8:1; MP37, 25:1; MP33, 25:1) as a

Figure 4.

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function of pH. Data represent the mean  one standard deviation (n= 3). Effect of pectin degree of methyl esterification and on (A) the maximum optical

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density; (B) critical pH values associated with complex coacervation of PPI-

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pectin mixtures at optimum mixing ratio (PPI-pectin: NP, 8:1; MP42, 8:1; MP37, 25:1; MP33, 25:1). Data represent the mean ± one standard deviation (n= 3). Figure 5.

Solubility of homogenous PPI solutions and mixed PPI-pectin solutions (PPIpectin: NP, 8:1; MP42, 8:1, MP37, 25:1; MP33, 25:1; 1.0% w/w) at pH 4.5. Data represent the mean  one standard deviation (n= 3).

Table 1.

Physicochemical properties of unmodified and alkaline modified pectin.

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None

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Table 1. Physicochemical properties of unmodified and alkaline modified pectin. Parameters

NP

MP42

MP37

MP33

Moisture (%)

17.4 ± 0.5a

18.9 ± 1.0b

20.6 ± 0.1b

20.8 ± 0.2bc

Ash (%)

3.9 ± 0.5a

9.5 ± 0.6b

9.4 ± 1.0b

9.4 ± 0.8bc

Carbohydrate (%)

78.9± 0.4a

72.1± 0.2b

69.9±1.1b

69.7±1.0bc

Degree of esterification (%)

63 ± 1.1a

42 ± 1.5b

37 ± 1.0c

33 ± 2.7dc

174 ± 0.9b

178 ± 6.6b

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Weight-average molecular weight (Mw) 188 ± 5.8a

35.4ac

(kDa)

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Means with difference letters within a row indicate significantly different values (p < 0.05).

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Highlights

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-pectin mixing ratios increased as DE values increased.

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was enhanced with the addition of pectin (DE 42%).

- 42%.

±

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5