Apple pomace from variety “Blanca de Asturias” as sustainable source of pectin: Composition, rheological, and thermal properties

Journal Pre-proof Apple pomace from variety “Blanca de Asturias” as sustainable source of pectin: Composition, rheological, and thermal properties B.E. Morales-Contreras, L. Wicker, W. Rosas-Flores, J.C. Contreras-Esquivel, J.A. Gallegos-Infante, D. Reyes-Jaquez, J. Morales-Castro PII:

S0023-6438(19)30983-1

DOI:

https://doi.org/10.1016/j.lwt.2019.108641

Reference:

YFSTL 108641

To appear in:

LWT - Food Science and Technology

Received Date: 21 June 2019 Revised Date:

22 August 2019

Accepted Date: 16 September 2019

Please cite this article as: Morales-Contreras, B.E., Wicker, L., Rosas-Flores, W., Contreras-Esquivel, J.C., Gallegos-Infante, J.A., Reyes-Jaquez, D., Morales-Castro, J., Apple pomace from variety “Blanca de Asturias” as sustainable source of pectin: Composition, rheological, and thermal properties, LWT Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108641. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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Apple pomace from variety “Blanca de Asturias” as sustainable source of pectin: composition, rheological, and thermal properties

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Morales-Contreras B. E.,1 Wicker L.,3 Rosas-Flores W.,1 Contreras-Esquivel J.C.,2 GallegosInfante J.A.,1 Reyes-Jaquez, D., Morales-Castro J.1

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Abstract

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Pectin derived from pomace from the apple variety “Blanca de Asturias” was isolated by acidic

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extraction and characterized as an alternative source of pectin. The influence of solid:liquid ratio

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and the extraction time on the chemical, rheological and thermal properties were evaluated. The

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molecular weight (Mw) decreased from 865 kDa to 590 kDa due to the hydrolysis reaction,

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effect that is favored at higher extraction times and to the increment in the solid:liquid ratio.

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Rheological studies suggest that the chains of apple pomace pectin (APP) disentangled during a

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short period of oscillation at high frequency and were related to inter- and intra-interaction at the

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junction zones. The composition and rheological characteristics of APP from the variety “Blanca

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de Asturias” exhibited strong potential as a thickener and gelling agent in the food industry.

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Keywords: apple pomace, composition, gels, pectin, rheological properties

Tecnológico Nacional de México/I. T. Durango. Posgrado en Ingeniería Bioquímica Felipe Pescador 1803, Nueva Vizcaya, 34080 Durango, Dgo., Mexico 2 Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila., Ing. J. Cardenas Valdez, República, Saltillo, Coah., Mexico 3 School of Nutrition and Food Sciences, Louisiana State University, Baton Rouge, LA 70808, USA Corresponding author: [email protected]

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1.

Introduction

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Pectin consists of three main pectic domains that include homogalacturonan (HG),

28

rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG- II). HG is a linear domain

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composed of GalA units randomly esterified with methoxyl groups and characterized as high

30

(HM) or low (LM) degree of methoxylation (DM) when the proportion of esterified GalA units

31

is greater or less than 50%, respectively. HMP forms gels under high concentrations of sugar and

32

low pH conditions, while LMP forms gels in the presence of ions. The HG may also be randomly

33

esterified with acetyl groups, affecting the gelling mechanism (Ralet, Crépeau, Buchholt, & 1

34

Thibault, 2003). RG-I is a branched domain formed by a backbone section interspersed with

35

GalA and rhamnose (Rha) units, with Rha as the site for branching with arabinose (Ara) and

36

galactose (Gal) chains. The RG-II domain has a complex branched structure composed of 12

37

sugars, including Rha, Ara, fucose (Fuc), apiose (Api), xylose (Xyl), Gal, 2-keto-3-deoxy-d-

38

lyxo-heptulosaric acid (Dha), and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) (Ridley,

39

O’Neill, & Mohnen, 2001). The knowledge of GalA, neutral sugar composition, Mw, and DM of

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different sources of pectin are fundamental factors, because these parameters govern pectin

41

behavior. In addition to external factors such as pH, solute presence, and ionic strength, the

42

physico-chemical properties of pectin define the functionality and possible application as a

43

gelling agent or stabilizer in the food, pharmaceutical, or cosmetic industries.

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According to Ciriminna, Fidalgo, Delisi, Ilharco, & Pagliaro (2016), 85% of the worldwide

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pectin production is from citrus peels. In addition to citrus peel, 14% corresponds to apple

46

pomace (AP), and just a small amount is obtained from sugar beet. The global market for pectin

47

is estimated to increase annually by 8.56% in 2023 (abnewswire, 2018), therefore, the search for

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novel sources, such as agroindustrial byproducts or underutilized fruits as raw material for pectin

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is essential to contribute to a more sustainable, global supply. The apple variety “Blanca de

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Asturias” is used as a pollinator for the Golden and Red Delicious varieties, and it purportedly

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has high gelling capacity through its use in the making of products such as jellies and jams

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combined with other fruits such as guava and quince. The fruit of the variety “Blanca de

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Asturias” is not eating quality and is typically used for animal feed. In Durango Mexico, the third

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state with the largest apple production in the country, the total annual production of apple (Malus

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domestica) was 11 145.60 Ton in 2018, where approximately 20% corresponds to the “Blanca de

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Asturias” variety (SIAP, 2018). Scientific information concerning this apple variety is scarce,

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and only the antioxidant activity of the seeds (González-Laredo et al., 2007) have been studied.

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The main purpose of this research was to evaluate the commonly used “acidic extraction

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method” to evaluate suitability of apple variety “Blanca de Asturias” as a pectin source.

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Different solid:liquid ratios and different extraction times were used to establish the highest

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pectin yield and to characterize the effect on the chemical composition, and the changes on the

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rheological and thermal properties.

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2. Materials and Methods 2.1 Materials 2

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The apple variety “Blanca de Asturias” was used as the raw material to obtain APP. Fruit was

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harvested from a local orchard in Durango Mexico at mature stage. All chemicals used were

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purchased from Sigma-Aldrich Corp. (St. Louis, Missouri, USA) and EMD Millipore Corp.

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(Billerica, Massachusetts, USA).

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2.2 Proximate analysis of apple pomace (AP)

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The AP chemical composition including moisture, total solids, ash, and fat and protein content,

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was determined by 930.04, 930.05, 930.09, and 978.04, respectively, (AOAC Official Methods

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AOAC, 2007).

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2.3 Extraction of apple pomace pectin (APP)

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The apples were washed with tap water, cut in four parts and subjected to the juice extraction

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process in an electric heavy-duty extractor (Turmix de Mexico S.A. de C.V, Queretaro, Mexico).

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The AP was collected and steam-blanched for 5 min and dehydrated in a tray dryer (SEM 2

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Polinox, DF, Mexico) at 60°C for 6 h. The AP was ground in a blender (BEST02-E01, Oster,

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Chicago, USA) and sieved to particle size <6.3 mm. The extraction of APP was performed in

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two stages. First, a crude extract was obtained by acid hydrolysis using 0.1N hydrochloric acid

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(HCl) at 1:15, 1:20 and 1:25 ratio (dried AP:HCl), and 100°C during 20, 30, and 40 min

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respectively as presented in supplementary material (SM), (Figure 1 SM). Second, the APP was

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recovered as presented Figure 2 SM. The variables evaluated in the extraction process were the

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solid:liquid ratio and the extraction time (Table 1 SM). Extraction yield was determined

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gravimetrically and was reported in dry basis.

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2.4 Physicochemical characterization of APP 2.4.1

Galacturonic acid (GalA) content

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The colorimetric method was used for GalA determination (Filisetti-Cozzi & Carpita 1991).

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Briefly, 5 mg/mL of APP was dispersed in deionized water, diluted 1:15 and hydrolyzed in

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0.0125M sodium tetraborate in sulfuric acid (98%) by heating at 100°C for 20 min. After

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cooling, 3-hydroxybiphenyl in 0.5% NaOH was added and absorbance was measured at 520 nm

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(Genesys 10S, Thermo Scientific®, USA) against a blank of 0.5% NaOH. A standard curve was

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created by using GalA (Sigma-Aldrich) at concentrations between 10–120 µg/mL.

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2.4.2

Neutral sugar composition 3

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The neutral sugar composition was determined by gas chromatography-mass spectrometry (GC-

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MS) as described by Vicente, Ortugno, Powell, Greve, & Labavitch (2007), and the reduction

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and acetylation reactions were based on the method described by Blakeney, Harris, Henry, &

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Stone (1983). The alditol acetates were injected into a GC-MS system (7890 GC System, Agilent

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Technology, USA) and separated on a 30 m × 250 µm × 0.25 µm BD5ID capillary column (HP-

99

5ms Inert, 19091S-433UI, Agilent Technology, USA) and a mass selective detector (5977A

100

MSD, Agilent Technology, USA). For identification, MS spectra were used and compared with

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the standards, Rha, Fuc, Ara, Xyl, Man, Gal, and Glc, containing myo-inositol as an internal

102

standard. Finally, the quantification of the amount of each neutral sugar was calculated relative

103

to the myo-inositol internal standard.

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2.4.3

Soluble starch content

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The soluble starch content was determined by using the total starch HK assay kit according to

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manufacturer’s protocol (Megazyme International, Ireland).

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2.4.4

Protein content

108

The protein content of APP was determined by the BCA (bicinchoninic acid) protein assay kit

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(PierceTM, Thermo Scientific, USA) using bovine serum albumin as standard. The microplate

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was read at 562 nm in a microplate absorbance spectrophotometer (xMarkTM, Bio Rad, USA)

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(Karnik, Jung, Hawking, & Wicker, 2016).

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2.4.5

Degree of methoxylation (DM) by Fourier-transform infrared (FTIR) spectroscopy

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The APP samples were tested on a Tensor 27 FTIR spectrometer (BI021703, Bruker, UK) with a

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Pike Miracle diamond/ZnSe ATR cell as a sampling accessory. The spectra were measured in the

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region of 4,000 to 650 cm-1 with 64 scans per reading. The data were collected and analyzed on

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OPUS Software (Version 7.2, Bruker, UK). The DM was calculated according to Karnik et al.,

118

(2016) (equation 1).

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DM = (

120

Abs1690cm−1

Abs1690cm−1 + Abs1550cm−1

2.4.6

)100

Equation (1)

Molecular weight (Mw) 4

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The Mw was determined by size-exclusion chromatography with multi-angle light scattering

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(MALS) and differential refractive index (dRI) detectors (Wyatt Technology, Goleta, CA, USA).

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The separating column used was an Aquagel PL-OH, 7.5 × 300 mm and a PL guard column

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Aquagel-OH, 7.5 × 50 mm. An aliquot of 3 mg/mL of APP dispersions within buffer solution

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(10 mM sodium phosphate, 100 mM sodium nitrate, pH 7.0) was prepared and hydrated

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overnight. After the samples were filtered through 0.45 µm Acrodisc syringe filters

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(polyethersulfone membrane, WhatmanTM, UK), they were transferred to 2.5 mL vials for

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injection (Jung & Wicker, 2012). The data were processed by using the Astra software version

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6.1 (Wyatt Technology Corp.); a dn/dc value of 0.131 was used and average Mw and

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polydispersity index were determined.

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2.5 Rheological characterization of APP 2.5.1 Flow behavior

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To determine the critical concentration (C*), APP dispersions were prepared with 1:25/40

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sample at 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, and 3% (w/v) in deionized water by magnetic stirring for 12

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h at 24°C. The flow behavior was evaluated according to Morales-Contreras, Rosas-Flores,

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Contreras-Esquivel, Wicker, & Morales-Castro (2018) with an increasing shear rate range from

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0.1 to 1 400 s-1 using a Discovery Hybrid Rheometer 3 (TA Instruments, USA). A 40 mm

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parallel plate geometry (SST ST Sand-Blast ARG2), and a 1,000 µm measurement gap was used.

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The data were fitted to the rheological power law model (equation 2) using the Trios software

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version 3.3.0.4055 (TA Instruments, USA). ,

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Equation (2)

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where τ is the shear stress, K is the consistency index,

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behavior index.

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From the concentration versus viscosity (K values for each concentration) plot, two zones with

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different behaviors were identified, which were adjusted by linear regression and by solving the

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equations system, and the intersection point between both lines was determined as C* (1.57%

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w/v). Once C* was obtained, the remaining APP dispersions were prepared at 2% (w/v). These

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were dispersed in deionized water by magnetic stirring for 12 h at room temperature. The flow

is the shear rate, and n is the flow

5

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behavior of 2% (w/v) APP dispersions were analyzed by the same methodology described above.

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Once obtained, the data were fitted to the power law model (equation 2). 2.5.2

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Activation energy (Ea)

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The equation described by Steffe (1996) was used to determine Ea. See equation 3, which relates

153

the shear stress to the temperature.

,

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Equation

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(3)

156

where σ is the shear stress as a function of temperature and shear rate, KT is the consistency as

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function of temperature and shear rate, Ea is the activation energy in kJ/mol, R is the universal

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gas constant (8.314 × 10-3 kJ/mol K), T is the absolute temperature (K),

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n

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To determine Ea by means of this equation, the flow behavior of 2% (w/v) APP dispersions was

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obtained as previously described (section 2.11.1). The data obtained were adjusted to the power

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law model (equation 2). Ea values were calculated using equation 4 by plotting ln K versus 1/T.

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 E  1 ln K = ln KT +  a    ,  R T

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where K is the consistency index, KT is the consistency index in function to the temperature and

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shear rate, Ea is the activation energy in kJ/mol, R is the universal gas constant (8.314 × 10-3

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kJ/mol K), and T is the absolute temperature (K).

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is the shear rate, and

is the average value of the flow behavior index from different temperatures.

2.5.3

Equation (4)

Viscoelastic characterization and gelling point (GP)

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To determine the GP of APP, the samples were prepared according to Morales-Contreras et al.

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(2018) as follows: 15 mL of APP dispersions (1% w/w) were prepared in deionized water by

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magnetic stirring overnight at 25°C. Next, refined sugar was added until a soluble solid content

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of 30% (w/w) was reached, followed by the addition of 90 mg of CaCl2 and 70 mg of -

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gluconolactone. Before gelling was initiated, the solutions were transferred to the rheometer, and

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40 mm parallel plate geometry (SST ST SMART-SWAP) and a 1,000 µm measurement gap

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were used. Linear viscoelastic region (LVR) was determined to 2% of strain through an 6

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amplitude sweep at 25ºC and constant angular frequency (10 rad/s). All measurements were

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performed in range with the LVR. Initially, storage (G’) and loss (G”) modulus were monitored

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during 3,600 s in a time sweep measure in the quest to let -gluconolactone induce the gel

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formation (data no shown). Second, the temperature sweeps were determined from 90ºC to 4ºC,

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the cooling rate was set to 3°C/min, and GP was determined as the cross over point of the

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modulus. Finally, the frequency sweeps were performed from 0.1 to 100 rad/s at 25°C.

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2.6 Thermal characterization of APP 2.6.1

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Differential scanning calorimetry (DSC)

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The thermal properties of APP were determined using a TA Q2000 DSC (TA Instruments,

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USA). The APP powders were placed into aluminum pans, sealed, and scanned over the range

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from 20°C to 300°C with a heating rate of 10°C/min. An empty aluminum pan was used as

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reference. The melting temperature (Tm) and melting enthalpy (∆Hm) was determined through the

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thermograms analysis using the Universal Analysis 2000 software (TA Instruments, New Jersey,

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USA).

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2.7 Data analysis

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A full factorial experimental design was used to analyze the solid:liquid ratio (1:15, 1:20, and

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1:25) and the extraction time (20, 30, and 40 min) as shown in Table 2, SM. All experiments

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were performed in triplicate. Least significant differences (LSD) using the Tukey methodology

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was performed in the Minitab Software version 17.1 (Minitab Inc., PA, USA) with p<0.05.

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3. Results and Discussion 3.1 Proximate analysis of AP

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The proximate analysis of AP is presented in Table 1. No earlier reports of the composition of

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AP from the variety “Blanca de Asturias” exist. The total pectin content from AP is 33.5% ± 4.1,

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similar to that of citrus peel; thus, it can be considered as a good source of pectin. According to

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Lopes da Silva & Rao (2006), the total pectin content for different plant matrices, including

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apple peel, ranges from 15% to 20%, while for citrus peel, the pectin content varies between

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30% and 35%. The yield values obtained here indicate that AP from the variety “Blanca de

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Asturias” is an excellent source of pectin, comparable to citrus pectin.

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3.2 Extraction yield 7

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The factor that has a greater effect on APP extraction yield was the extraction time, while the

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lower effect is the solid:liquid ratio, and practically no effect of the interaction of both factors

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was observed (Figure 1). According to the main effect analysis (Figure 3 SM) and data from

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Table 2, this effect is positive on the yield response; thus, at longer extraction time and higher

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solid:liquid ratio, higher yield is obtained. Lowest yield values were obtained at 20 min, while

209

yield increases with solid:liquid ratio—33.7, 35.5, and 36.0 mg APP/g AP. The effect of

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solid:liquid ratio can be explained by an increase in solvent volume that favors dissolution of the

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polysaccharides, causing a concentration difference in the medium and the interior of the tissue

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material, thus improving the extraction process. At longer extraction times, the yield increased,

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favored by temperature (100°C) and hydrolysis of glycosidic bonds in cellulose and

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hemicellulose chains, as previously reported (Raji, Khodaiyan, Rezaei, Kiani, & Hosseini, 2017).

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Under these extraction conditions, the hydrolysis of cell wall compounds present in plant

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matrices is favored, contributing to a higher mass transfer of the soluble polysaccharides to the

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extraction medium. The extraction method applied shows higher yield compared to data reported

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by Wang, Chen & Lü, (2014) for pectin extracted from AP by subcritical water (10.05-13.33 mg

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pectin/g AP) and to pectin obtaining by enzymatic and acidic method from AP (10-20 mg

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pectin/g AP), reported by Wikiera, Mika, Starzyńska-Janiszewska, & Stodolak, (2016). In

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addition, for pectin acidic extraction from AP, with HCl, citric acid, oxalic acid and sulfuric acid,

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values for pectin yield ranged from 3.50 to 14.32 mg pectin/g AP (O’Shea et al., 2015, cited by

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Perussello, Zhang, Marzocchella & Tiwari, 2018), lower values than the results obtained for APP

224

of the present study.

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3.3 Physicochemical characterization of APP

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The APP GalA content ranged between 38.0 and 47.1 g/100 g APP, and the extraction time and

227

the solid:liquid ratio had a significant effect (p<0.05) on this parameter for the two milder

228

treatments (1:15/20 and 1:15/30) and also to the more severe treatment (1:25/40). The main

229

effect test (Figure 4 SM) shows that the solid:liquid ratio has a positive effect on the GalA

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content, which means that as the solid:liquid ratio increased, the GalA content increased as well.

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Wang and Lü (2014) attributed this positive effect to the diminishment of the solid load in the

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liquid phase. In another hand, increasing extraction time from 20 to 30 min had no effect on

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Ga1A content, but when extraction time was higher (40 min) Ga1A content increased 8

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significantly. According to Yapo, (2009) and Morales-Contreras, Contreras-Esquivel, Wicker,

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Ochoa-Martínez, & Morales-Castro, (2017) this is due to by hydrolysis effect, since at longer

236

exposure at high temperature as 100°C, the degradation of compounds as hemicellulose or

237

galactans present in the cell wall is favored. Once hydrolyzed these are removed by precipitation;

238

therefore, by the end of the extraction process, the proportion of GalA increases significantly.

239

With respect to the neutral sugar composition, Glc was the major sugar in APP (Table 2). Gal

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was the second most predominant with almost 10 times less concentration that ranged from 5.8

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to 6.9 g/100 g APP. Man, Xyl and Ara were present at about 10 times less than Gal and

242

concentration range from about 0.3 to 0.7 g/100 g APP. Only traces of Rha were observed,

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suggesting that the APP sample structures are more linear. The Glc and Gal content was not

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affected by the extraction process conditions. In contrast, the Ara, Xyl, and Man content

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presented some extraction effect without a clear trend. A possible explanation could be

246

differences in hydrolysis resistance of sugar, where some fractions of polysaccharides such

247

pectin are more susceptible to hydrolysis, depending on the monomer composition, while others,

248

such as Glc, are more resistant than Rha and Man (Wikiera, Mika, Starzyńska-Janiszewska, &

249

Stodolak, 2015).

250

The soluble starch content for APP ranged between 13.9 and 18.7 g/100 g APP (Table 2),

251

depending on the extraction conditions. At longer extraction times and at the same solid/liquid

252

ratio, soluble starch decreased as extraction time increased (p<0.05). The presence of starch in

253

polymers as APP can be explained by a possible co-extraction of residual starch present in the

254

corresponding source as reported for potato pulp pectin extracted under similar conditions

255

(Yang, Mu, & Ma, 2018).

256

The protein content for the APP samples was between 5.1 and 7.2 g/100 g APP (Table 2).

257

Significant changes were observed in the protein content of some of the APP samples; similar

258

changes were observed by Ma, Yu, Zheng, Wang, & Bao, (2013), who attributed this behavior to

259

the denaturation of proteins under these conditions, which in turn causes a lower recovery of the

260

protein during ethanol precipitation. The interest in the protein content in pectin resides in the

261

close relationship established between the emulsifying capacity of pectin and the protein content.

262

For mango pectin, at higher protein content (3.44–5.94 g/100 g mango pectin), pectin shows a

9

263

higher emulsifying capacity (Wang et al., 2016), which suggests a possibly emulsifying capacity

264

of APP.

265

The DM of APP ranged between 61% and 63% (Table 2) (p>0.05). These values are slightly

266

lower than those reported by Canteri-Schemin, Ramos-Fertonani, Waszczynskyj, & Wosiacki

267

(2005) for pectin from AP. With this DM, the APP is classified as HMP. The Mw and

268

polydispersity data for APP were significantly different (p < 0.05) by extraction conditions

269

(Table 2). The increase in the extraction time and the solid:liquid ratio has a negative effect on

270

Mw. The samples obtained at the mildest treatment (1/15:20) have the highest Mw (865 kDa),

271

while the sample obtained under the more severe treatment (1/25:40) has the lowest value for

272

Mw (590 kDa). Since the acid extraction process is considered a destructive extraction method

273

and HCl, a mineral acid, has strong hydrolysis power, the Mw is negatively affected under more

274

harsh conditions. Accordingly, at longer extraction time, the extent of degradation of the polymer

275

expands, visible by a decline in Mw. In addition, a relationship between Mw and polydispersity

276

index was found, the samples with the higher Mw had a lower polydispersity index value and

277

vice versa. Studies have been reported that when a lower polydispersity index is presented, a

278

more homogeneous Mw distribution is present (Zhang et al., 2013). Typical polydispersity

279

values for commercial citrus, apple, or sugar beet pectins are less than 1.5. With polydispersity

280

values up to 3.1, APP samples exhibited a more heterogeneous Mw. According to Corredig,

281

Kerr, & Wicker, (2000) a molecular weight range is between 8-800 kDa; Moreover, Wikiera,

282

Mika, Starzyńska-Janiszewska, & Stodolak, (2016) reported a Mw range between 331-899 kDa

283

for apple pomace pectin extracted by enzymatic and acidic extraction method.

284 285

3.4 Rheological measurement 3.4.1 Flow behavior

286

Changes in the viscosity of APP (1:25/40) dispersions at different concentrations are depicted

287

(Figure 2A). Two regions can be observed: the first one at lower concentrations (0.1%, 0.25%,

288

0.5%, and 1.0% w/v) and the second one for the higher concentrations (1.5%, 2.0%, and 3.0%

289

w/v). The two regions were adjusted to a linear regression, resulting in

290

y = 0.0081x + 0.0109

Equation (5)

291

y = 0.2103x − 0.3079

Equation (6)

10

292

The system equations 5 and 6 were solved by an algebraic method, and the intersection point was

293

determined as x=1.57, y=0.023, where the x values corresponded to C*. According to Williams

294

& Phillips (2009), above C*, the viscosity of the polymer dispersions increases considerably

295

because a transition occurs from “dilute region” to “semi-dilute region.” At this point, the

296

polymer coils and interpenetrate. The flow behavior under these conditions is important in the

297

functionality of the pectin as thickeners since these must accomplish an increase in the viscosity

298

of the food systems without necessarily modifying other product characteristics. The flow

299

behavior of APP dispersions (Figure 2B) were fitted to the power law model (equation 2) for

300

comparison purposes (Table 3). The viscous nature of the APP dispersions can be described by

301

the consistency index (K) and the flow behavior index (n), which are not significantly different

302

(p>0.05) since all APP dispersions have a shear-thinning behavior, n<1. The shear-thinning

303

behavior can be attributed to the long and more disordered chains of the polymer at low shear

304

rate, where the chains have more interaction between each other, and as the shear rate increases,

305

these interactions become weaker, causing the decrease in viscosity. The viscosity and flow

306

characteristics of APP dispersions indicate that they can be used as a potential food thickener and

307

stabilizer.

308

3.4.2

Activation energy (Ea)

309

The effect of different extraction conditions on the Ea is observed for 2% (w/w) APP dispersions

310

(Figure 3). The solid/liquid ratio and the extraction time exert a negative effect on the Ea, and the

311

most severe and the mildest treatments are significantly different (p<0.05). Most likely, changes

312

in the Mw occur because of the extraction process of APP, particularly the 1:15/20 sample

313

(mildest treatment), which presented the highest Ea value (17.71 kJ/mol) and highest Mw (775

314

kDa), while the 1:25/40 (strongest treatment) presented the lowest Ea (8.01 kJ/mol), which also

315

corresponded to lower Mw (530 kDa). High values for Ea indicate more inter- and intra-

316

interactions between polymer chains, meaning that this type of interactions is favored as Mw

317

increases. The Ea values of APP dispersions provide key information for potential applications in

318

the food industry for calculating the energy expenditure during processing operations such as

319

grinding, agitating, and pumping.

320

3.4.3

Viscoelastic characterization and gelling point (GP)

11

321

The crossover between G’ and G” modulus marks the point where the sol-gel transition occurs

322

(Figure 4A). As observed in Figure 4B, APP gels have a GP temperature between 60°C and

323

90°C, where the lowest temperature corresponds to the most severe extraction treatment, and the

324

highest temperature corresponds to the mildest extraction treatment. The gelling process under

325

this range of temperatures corresponds to HMP mechanism, where the structuring is promoted by

326

the presence of sugar since it increases the hydrophobic interaction between methoxyl groups.

327

A broad look at the above results shows evidence that extraction conditions have a deep effect on

328

neutral sugar composition and Mw, parameters that reflect the role of the neutral side chains of

329

APP in gel structure formation. It has been established that the presence of neutral side chains

330

promotes the entanglement and interaction of the polymer molecules until tighter conformation is

331

reached, facilitating hydrophobic interactions and hydrogen bonding (Sousa et al., 2015). This

332

agrees with the values of GalA/NS ratio for APP (Table 2) since the lower values of the

333

solid:liquid ratio correspond to the higher GP temperature and the higher values of the

334

solid:liquid ratio correspond to the lower GP temperatures. Indeed, the Mw has some effect on

335

the gelling mechanism of APP since as the Mw decreases the GP temperature decreases. The low

336

Mw of the APP, that is, shorter chains, results in that the junction zones are formed from shorter

337

segments, and the structuration process is more difficult.

338

A frequency sweep is presented (sample 1:25/30) in Figure 5. APP gel samples behave as gel-

339

like when G’>G”. According to Ngouémazong et al. (2012), when a high-frequency dependence

340

is observed, a network relaxation phenomenon is present, which is consistent with “weak gels.”

341

Furthermore, gel-like behavior over 0.1–100 rad/s confirms the structuring of a physical gel

342

(Yuliarti & Othman, 2018). However, it is important to emphasize that at high frequency (up to

343

about 5 rad/s), the gel-sol transition point was detected, attributed to the molecular chains

344

disentangling during a short period of oscillation at high frequency. This disentangling was

345

observed in a previous work with husk tomato pectin gels (Morales-Contreras et al., 2018) and is

346

related to the interactions that occur in the junction zones because all these inter- and intra-

347

interactions as hydrogen bonds and hydrophobic interactions are weak for the HMP.

348 349

3.5 Thermal characterization of APP 3.5.1 DSC

12

350

Table 2 shows the average Tm ranged between 105.64°C and 113.81°C (p>0.05) and ∆Hm ranged

351

between 99.4 J/g and 107.7 J/g (p>0.05). Biopolymers as pectin can be classified as

352

thermoplastics, thermosets, and elastomers; those that are amorphous or semi-crystalline

353

polymers can become soft or melt during heating (Gregorova, 2013). According to the statistical

354

analysis, the APP samples had a stable structure in thermal terms under the extraction conditions

355

evaluated since no significant differences were observed. From an application point of view, it is

356

important to know the Tm value since it is defined as the temperature at which the thermal energy

357

in a solid material is enough to overcome the intermolecular forces of attraction in the crystalline

358

lattice so that the lattice breaks down and the material becomes liquid (Vithanage et al., 2010),

359

and with this information, it is possible to identify in what kind of products APP can be used. In

360

addition, the values of both Tm and ∆Hm as measure of an endothermic phenomenon, indicate the

361

amount of energy needed to remove the absorbed water (Wang, Chen, & Lü, 2014). From the

362

values for Tm and ∆Hm, it can be inferred that the extraction conditions studied did not affect

363

significantly the water retention capacity in the APP samples.

364 365 366

4. Conclusion

367

pectin, managing to obtain 83.8 mg APP/g AP with the higher levels for the solid:liquid ratio and

368

time (1:25/40) evaluated. The DM for all the APP samples was between 61% and 63%, being

369

APP classified as HMP. The extraction conditions caused significant changes in the APP

370

structure. Mw was the most affected parameter since it decreased from 865 kDa (softest

371

treatment) down to 590 kDa (strongest treatment), which was attributed at the removal of

372

branched sections since the neutral sugars such as Ara, Xyl, and Man were decreased at the same

373

conditions. These changes in the APP composition had a great impact on the GP temperature,

374

decreasing from 90°C to 60°C. The rheological studies show that all the APP samples were

375

capable of structuring and disentangling during a short period of oscillation at high frequency,

376

which is related to the interactions that occur in the junction zones, originated by inter- and intra-

377

interactions as hydrogen bonds and hydrophobic. The 2% (w/v) APP dispersions presented a

378

pseudoplastic behavior, and the Ea values were not significantly affected. APP obtained from the

379

variety “Blanca de Asturias” under the acidic extraction process had characteristics in terms of

The AP from the variety “Blanca de Asturias” has shown potential as a sustainable source of

13

380

its composition and rheological behavior, that resembles the techno-functional capacity of

381

commercial pectins, and it can be used in the food industry as a thickener or gelling agent.

382 383

Acknowledgments

384 385

The authors appreciate the funding provided by Tecnológico Nacional de México, TecNM,

386

through grant number 6259.17, to the Cocyted grant in the program of “Apoyos Institucionales

387

para el Financiamiento de Proyectos de Investigación”, Durango 2017 and to the National

388

Council of Science and Technology, Conacyt, for the scholarship number 276296 from México,

389

granted to Blanca E. Morales-Contreras to pursue PhD studies. The partial support of the LSU

390

AgCenter is gratefully acknowledged.

391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

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16

Table 1. The proximate analysis results of apple pomace (AP) from var. “Blanca de Asturias”. Parameter Moisture Total solids Ash Fat Protein Total pectin Results are expressed in dry basis Average of three replicates ± SD.

Content (%) 8.5 ± 0.3 91.4 ± 0.3 Trace 0.6 ± 0.2 4.8 ± 0.2 33.5 ± 4.1

Table 2. Yield, physico-chemical, and thermal characterization of apple pomace pectin (APP) samples. Yield GalA Rha Ara Xyl Man Glc Gal GalA/NS Protein Soluble Starch Mw

1:15/20 33.7c ±8.4 38.5b ±3.7 Trace 0.4b,c ±0.03 0.4c ±0.03 0.7a,b ±0.08 48.6a ±1.8 5.8a ±0.5 0.63 6.4a,b ±0.2 18.2a,b ±0.5

1:15/30 47.8b,c ±2.9 38.0b ±2.6 Trace 0.3d,e ±0.02 0.3c,d ±0.02 0.6a,b,c ±0.07 43.1a ±4.2 6.4a ±0.4 0. 65 6.7a,b ±0.4 15.5b,c ±0.4

1:15/40 53.5b,c ±10.3 40.4a,b ±1.4 Trace 0.5b ±.03 0.6a,b ±0.06 0.7a ±0.09 42.6a ±4.5 6.8a ±0.3 0.59 6.9a,b ±0.3 15.3b,c ±0.7

1:20/20 35.5c ±7.3 40.7a,b ±2.4 Trace 0.4b,c,d ±0.04 0.3d ±0.02 0.5c,d ±0.03 48.9a ±3.5 6.2a ±0.5 0.66 6.4a,b ±0.3 17.7a,b ±0.5

1:20/30 51.8b,c ±3.8 41.7a,b ±3.3 Trace 0.6a ±0.06 0.4c,d ±0.03 0.5b,c,d ±0.05 47.4a ±1.9 6.5a ±0.6 0.56 6.3a,b ±0.3 16.4a,b,c ±1.2

1:20/40 63.8a,b ±5.8 42.1a,b ±4.1 Trace 0.4b,c,d,e ±0.01 0.6b ±0.05 0.3e ±0.03 45.1a ±0.6 5.8a ±0.3 0.68 6.6a,b ±0.1 13.9c ±1.2

1:25/20 36.0c ±10.8 39.2a,b ±1.6 Trace 0.3e ±0.02 0.4c ±0.04 0.4d,e ±0.04 47.3a ±4.0 6.0a ±0.4 0.64 5.1c ±0.04 18.7a ±1.4

1:25/30 65.7a,b ±8.3 38.8a,b ±1.6 Trace 0.4c,d,e ±0.02 0.6a,b ±0.06 0.4d,e ±0.04 43.7a ±4.6 6.3a ±0.3 0.68 6.0b,c ±0.2 14.2c ±1.3

1:25/40 83.8a ±2.0 47.1a ±4.1 Trace 0.4b,c ±0.04 0.7a ±.06 0.6a,b,c ±0.04 41.5a ±2.5 6.9a ±0.7 0.74 7.2a ±0.6 14.3c ±1.3

865a ±7.5 708e ±3.4 683e,f ±2.6 850a,b ±18.9 817c ±5.6 661f ±6.1 836b,c ±6.7 757d ±2.1 590g ±6.09 b a,b a,b b a,b a,b b a,b Polydispersity 2.2 ±0.1 2.7 ±0.1 2.7 ±0.07 2.3 ±0.1 2.7 ±0.01 2.7 ±0.03 2.3 ±0.09 2.6 ±0.3 3.1a ±0.5 a a a a a a a a 62 ±0.8 62 ±1.2 62 ±1.2 61 ±0.4 61 ±2.1 63 ±1.8 62 ±1.1 62 ±1.6 63a ±0.9 DM a a a a a a a a 110.9 ±4.0 112.1 ±2.7 106.8 ±4.3 112.9 ±1.9 105.6 ±0.2 108.9 ±1.1 112.3 ±3.9 113.2 ±8.8 113.8a ±4.0 Tm a a a a a a a a 99.9 ±1.5 104.2 ±8.6 103.7 ±8.8 109.8 ±6.6 104.2 ±7.1 98.9 ±7.0 107.7 ±4.1 99.4a±8.1 105.8 ±8.7 ∆ Hm Average of three replicates ± SD. Different letter in the same row are significantly different (Tukey, p <0.05). Yield is expressed as mg APP/g AP. GalA, Rha, Ara, Xyl, Man, Glu, Gal, Protein, and soluble starch are expressed as g/100 g APP sample, Mw is expressed in kDa, DM in %, Tm in °C, and ∆Hm in J/g.

Table 3. Power Law model parameters (K, and n) for 2% w/v APP dispersions. Sample 1:15/20 1:15/30 1:15/40 1:20/20 1:20/30 1:20/40 1:25/20 1:25/30 1:25/40

K (Pa·s)n 0.058±0.006a 0.070±0.013a 0.045±0.005a 0.054±0.012a 0.048±0.007a 0.056±0.017a 0.049±0.015a 0.056±0.023a 0.034±0.009a

n 0.901±0.024a 0.897±0.015a 0.934±0.009a 0.922±0.019a 0.930±0.012a 0.916±0.023a 0.931±0.026a 0.931±0.044a 0.955±0.023a

R2 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999

Average of three replicates ± SD. Different letter in the same column are significantly different (Tukey, p <0.05)

Figure 1. Pareto chart standardized effect. Response in yield (mg APP/g AP), α=0.05.

Figure 2. A) Changes in the viscosity of APP (Sample 1:25/40) dispersions, respect to different concentrations. B) Stress and apparent viscosity versus shear rate for 2% w/v APP dispersion (Sample 1:25/30).

Figure 3. Activation energy of 2% w/v APP dispersions.

Figure 4. A) Temperature sweep for APP gel (Sample 1:15/20). B) Extraction conditions effect on gelling point for APP gels.

Figure 5. Frequency sweep for APP gel (Sample 1:25/30).

Highlights •

High methoxylated pectin was isolated from apple pomace variety “Blanca de Asturias”

•

Compositional and rheological properties were studied

•

Molecular weight and gelling point were the main parameters affected by the acidic extraction process

•

Pectin obtained from apple pomace variety “Blanca de Asturias” can be used as thickener and gelling agent in food systems