Accepted Manuscript

Extraction, purification and characterization of pectin from

alternative sources with potential technological applications

Florina Dranca, Mircea Oroian

PII: S0963-9969(18)30519-2
DOI: doi:10.1016/j.foodres.2018.06.065
Reference: FRIN 7732
To appear in: Food Research International
Received date: 20 February 2018
Revised date: 25 June 2018
Accepted date: 28 June 2018

Please cite this article as: Florina Dranca, Mircea Oroian , Extraction, purification and
characterization of pectin from alternative sources with potential technological
applications. Frin (2018), doi:10.1016/j.foodres.2018.06.065

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Extraction, purification and characterization of pectin from alternative

sources with potential technological applications

Florina DRANCA*, Mircea OROIAN

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Faculty of Food Engineering, Stefan cel Mare University of Suceava

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Romania

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e-mail: florina.dranca@fia.usv.ro (Florina DRANCA)
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Abstract

Pectins are defined as a group of widely distributed plant cell wall polysaccharides

that contain galacturonic acid linked at both the 1 and 4 positions. The wide use of pectin as

an ingredient which imparts rheological and textural properties to various food products and

the development of applications beyond the food industry have brought about its increase in

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production and influenced research towards alternative sources and improving the overall

isolation process of pectic polysaccharides. In this context, this paper aims to give a complete

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perspective on the current state of pectin research by mainly focusing on recent research on

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the extraction of pectin from other feasible sources, on the post-extraction stages of pectin

recovery from plant materials (purification and fractionation), and, finally, on the
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advancements in the study of the physical, chemical, rheological, and functional properties of
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pectin.
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Keywords: pectin, sources, purification, fractionation, composition, analysis

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1. Introduction

Fruits, vegetables, and other plant-based foods contain a wide range of dietary

components essential to the human body and are rich in bioactive phytochemicals that may

provide desirable health benefits beyond basic nutrition (Liu, 2013). Plant foods are

particularly important as a source of dietary carbohydrates, providing almost all of the

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carbohydrate intake, and therefore much of the energy in the adult diet (Lovegrove et al.,

2015). Depending on the functional role, plant carbohydrates can be divided into two classes:

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storage carbohydrates (particularly starch) and cell wall carbohydrates. Given the fact that in

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plants starch-derived polysaccharides outweigh all others, the remainders are collectively

known as non-starch polysaccharides (NSPs) (BeMiller, 1996). NSPs constitute the major
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fraction of the plant cell wall in association and/or substituted with other polysaccharides, and
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they cover a great variety of biological functions and chemical structures (Kumar et al.,

2012). Structurally, NSPs are polysaccharides that contain up to several hundred thousand
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monosaccharide units joined through glycosidic linkages. The major plant cell wall NSPs are

cellulose, hemicelluloses, and pectins.

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Pectins represent a group of structurally heterogeneous polysaccharides widely

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distributed in primary cell walls and the middle lamella of higher plants (Luo et al., 2017).
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Pectic polysaccharides are vital structural components of plant cell walls, and they are often
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associated with other cell wall polysaccharides such as cellulose and hemicelluloses. The

diverse structural and macromolecular properties of pectins, such as galacturonan

methoxylation, galacturonic acid content, the composition of neutral sugars, and molecular

weight, are dependent on the pectin source and set the basis for multiple food and non-food

applications of this complex polysaccharide (Yoo et al., 2012). In the food sector, traditional

usage as a gelling agent, thickening agent, and stabilizer is being complemented by the

emerging utilization of pectin as a fat replacer and health-promoting functional ingredient

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(Ciriminna et al., 2016; Peng et al., 2014; Min et al., 2010). Non-food applications include

the use in the medical and pharmaceutical industries, where the health-promoting benefits

and bioactivities of pectin have shown potential for biomedical applications including drug

delivery, tissue engineering, and wound healing, as reviewed previously by Munarin, Tanzi,

& Petrini (2012). Each of the above-mentioned applications begins with the isolation of

pectin from the plant material. Generally, pectin is isolated from by-products of agro-foods.

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Pectin production dates back to the early 1900s when German producers of apple juice started

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to cook dried apple pomace, the main by-product of juice processing, and sold the extracted

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pectin as a gelling agent (Ciriminna et al., 2015). Apple pomace and citrus peel remain the

main sources for the production of commercial pectins, although other sources were
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considered due to rising demand and growing interest in valorizing side streams to obtain

pectins with diverse functional properties (Müller-Maatsch et al., 2016; Christiaens et al.,
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2015). Progress was also made in the extraction technologies with the emergence of novel

and effective techniques that inclined toward a cleaner process (Y. Yang et al., 2018).
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With an increasing number of studies that propose new by-products and agricultural

waste as sources for pectin extraction (Morales-Contreras et al., 2018; Sabater et al., 2018;
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Xu et al., 2018), a review of the potential for the capitalization of these raw materials is

demanded. One other main objective of this review is to assess the methods applied for the
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purification and fractionation of the extracted pectin prior to analyzing its composition and
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properties. It is important to be noted that the characterization of pectins extracted from

different sources is a target point of this paper as it is rarely thoroughly examined in other

reviews.
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2. Chemistry and function

Although the plant cell wall was first observed and defined by Robert Hooke (1665)

many centuries earlier, the knowledge regarding its architecture has increased considerably

since the 1900s. Detailed characterizations of the structure of the plant cell wall were

presented by Preston (1975) and Clarke & Knox (1979), who described the deposition of cell

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walls in three layers, primary wall, middle lamella, and secondary cell wall. An

oversimplified sketch of the plant cell wall, adapted from McCann & Roberts (1992) and

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modified, is presented in Fig. 1. Concerning the structural components, the growing plant cell

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wall consists of a mixture of polysaccharide and polyuronide components (Ponce et al., 2010;

Jansen et al., 1960). Both earlier determinations and subsequent studies agreed that the main
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components are cellulose, hemicelluloses, and pectin (Mankarios et al., 1980; Houwink &
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Roelofsen, 1954;Thimann & Bonner, 1933). Because of their multiple interaction properties,

pectins are considered key structural elements of the plant cell wall architecture. An in-depth
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understanding of the biological function should cover all the particularities related to the

chemistry of pectin, as its structural complexity imparts unique and diverse physical and
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biochemical properties. The chemistry, biosynthesis and functions of pectin have been
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reviewed multiple times recently, and therefore this section will be a summary of current
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knowledge.
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Prior to reporting on the progress made in the study of pectin chemistry and

biosynthesis, it is essential to emphasize that the term ‘pectin’ does not refer to a single

structural cell wall polymer, but it is rather attributed to a family of plant cell wall

polysaccharides and/or glycan domains that contain galacturonic acid residues linked at both

the 1 and 4 positions (Atmodjo, Hao, & Mohnen, 2013). To date, all research suggests that,

similar to other plant cell wall polysaccharides, pectin is synthesized in the Golgi apparatus.

Pectin synthesis is a complex process, involving a large number of unique enzymes as

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catalysts in the formation of each glycosidic linkage and in the modification of some glycosyl

residues in pectic chains. The biosynthetic enzymes required for the process include

glycosyltransferase (Scheible & Pauly, 2004; Bouton et al., 2002), methyltransferase

(Mouille et al., 2007), acetyltransferase (Pauly & Scheller, 2000), galacturonosyltransferase

(Sterling et al., 2006), glucuronosyltransferase (Iwai et al., 2002), arabinosyltransferase

(Harholt et al., 2006), and xylosyltransferase (Jensen et al., 2008). The mechanics of pectin

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synthesis have been extensively reviewed by Scheller et al. (2007), Mohnen (2008), Caffall &

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Mohnen (2009), Sila et al. (2009), Harholt et al. (2010), and Atmodjo, Hao, & Mohnen

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(2013), and therefore will not be further discussed.

Many structurally different regions were identified in the composition of pectins, and
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of these three classes of pectic polysaccharides have been extensively studied in primary cell

walls: homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II

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(RG-II) (Christiaens et al., 2016). The unsubtituted HGs are linear homopolymers consisting

of α-(1-4) linked D-galacturonic acid (GalA) residues. HGs are known to represent the
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‘smooth’ region of pectin, while RG-I, a segment rich in neutral sugars, is considered the

‘hairy’ region (De Vries et al., 1981). RG-I was found to possess a backbone containing
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diglycosyl repeats of [→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→] that has various side chains

attached to O-4 of the L-rhamnosyl residues (Willats et al., 2001; Lau et al., 1985; Darvill et
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al., 1980). Studies of the branched RG-I regions of apple pectin resulted in the isolation of
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xylogalacturonan (Schols et al., 1995), while apiogalacturonan, another HG analogue, was

determined in the cell wall of Lemna minor (Hart & Kindel, 1970). A structure which is often

described as a stretch of the HG backbone was found for RG-II, a complex polysaccharide

yielding different monosaccharides, including the rarely observed sugars apiose, 2-O-

methylxylose, and 2-O-methylfucose (Darvill, McNeil, & Albersheim, 1978).

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The basic structure of pectin and the structural variations observed in pectin segments

have been reviewed by many authors including Willats, Knox, & Mikkelsen (2006), Voragen

et al. (2009), Kumar et al. (2012), Zhang, Xu, & Zhang (2015), and, more recently, Chan et

al. (2017). A schematic representation of the composition of pectin elements, as proposed by

Hilz (2007), completed by including the representative side chains of RG-I (Caffall &

Mohnen, 2009), is given in Fig. 2. Some of these pectin elements show little change in the

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conformation of the polysaccharide chains in different plant species, while the well-preserved

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RG-II is the only pectic element that is not structurally diverse (O’Neill et al., 2004). This

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observation is broadly supported by many lines of scientific evidence, including the recent

study of the polysaccharides from Citrus unshiu peel, where Park et al. (2017) concluded that
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the structure of the purified RG-II was very similar to the one presented in Fig. 2. On the

other side, the detailed structure of RG-I from various plant materials is often very distinct
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and similar to the conformation presented in Fig. 2, mainly due to the high variability of its

side chains. Accordingly, non-ramified RG-I chains were found in the key areas for cell
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adhesion of the tomato pericarp tissue during its development (Guillon et al., 2017), while a

similar type I rhamnogalacturonan backbone with branches of β-1,4-D-galactan side chains

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occasionally substituted with α-L-Araf was identified in pumpkin residue (Zhao et al., 2017).

When compared to RG-I isolates from pumpkin residue, ginseng pectin displayed a greater
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variety of RG-I domains. From ginseng pectin Yu et al. (2010) isolated five RG-I domains,
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all containing galacturonic acid, rhamnose, galactose, and arabinose as main components.

Four of these had side chains of type I and probably type II arabinogalactans, and one

presented 4-O-methyl-β-D-glucuronic acid residues at non-reducing terminals.

A particularity in the structure of pectic substances is the esterification by methyl

groups (at C-6) and/or acetyl groups (at O-2 and/or O-3) of galacturonic acid residues on the

continuous poly-(GalA) chain of HG (Yapo & Koffi, 2013). The overall degree of
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substitution is known as degree of methylation (DM) and acetylation (DAc), respectively.

The degree of methylation, based on which pectins are divided into two categories – high-

methoxyl pectins (HMP, DM>50%) and low-methoxyl pectins (LMP, DM<50%) – is an

important parameter in choosing the most suitable pectin application (Łȩkawska-

Andrinopoulou et al., 2013).

Within the pectic polysaccharides group, several individual components have been

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identified and characterized, yet the assembly mechanism of these components in the plant

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cell wall is the subject of continuous research. Moreover, knowledge of the interactions

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established between pectic polysaccharides and the interconnections of pectin molecules with

other cell wall components is relatively limited. It is generally accepted that these interactions
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are particularly built upon cross-links between macromolecular components of the cell wall

and intercellular adhesion. A number of covalent and non-covalent interactions, which

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include ionic bridges, ferulic acid linkages, borate esters and uronyl esters, are considered to

be involved in intra- or intermolecular linkages. A comprehensive analysis of the formation,

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mechanisms, reaction conditions, and the macromolecular functionality of the pectic

polysaccharide cross-links (Fig. 3) can be found in the review published by Zaidel & Meyer
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(2012). Cross-linking of pectic polysaccharides is believed to have a major influence on both

the physical and macromolecular properties of plant materials and, consequently, it impacts
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their processing and quality. Alongside cross-linking, pectin biosynthesis, conversion

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reactions (enzymatic and non-enzymatic), and the solubility property of pectic

polysaccharides, are considered key mechanisms that dictate the structure-function

relationships of pectin. A rather common principle of research on the chemistry of pectin is

that the structural features govern its functional properties. Structure-function relations can be

studied in the context of pectin’s functionality within plant cell walls, and from the

perspective of selecting a suitable industrial application based on functional properties and

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physiological benefits. The review previously published by Sila et al. (2009) consists of a

comprehensive discussion involving all the aspects regarding structure-function relationships,

including their importance for the continuously evolving analysis methodology of pectins.

3. Sources for pectin production: the potential of other vegetable wastes

Different types of polysaccharides and their derivatives recovered from plants and

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vegetables are currently used to obtain biopolymers with multiple uses in several distinct

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areas such as the health care, food, and polymer processing industries. In the food industry,

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the main use for the extracted pectin (labeled in the European Union as E440) is as a gelling

agent in the production of fruit-based products and fillings for bakery and confectionary
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products. Pectin is also applied in food production as a thickening, stabilizing, and

emulsifying agent. From the point of view of these applications, the most frequently
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exploited structure-function relationship is the one between the methoxylation of the

galacturonic acids in pectin backbone and pectin gelation. HMP (DM above 50%) form gels
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through hydrophobic interactions and hydrogen bonds under suitable conditions: pH≤3.5 and
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high sugar content (>55%) (Kastner et al., 2014). On the other side, the gelation process of
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LMP (DM<50%) is governed by the cross-linking of HG chains via Ca2+ bridges, occurring

after the addition of calcium ions to the pectin solution (Han et al., 2017). All the applications
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of pectin in the industry begin with the isolation of pectin from the plant material. At the
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present time, commercial production of pectin is limited to two major sources, apple pomace

and citrus peel. Both extraction sources represent by-products of juice manufacturing

operations. Since the first commercial production of liquid pectin from apple pomace,

documented in 1908 in Germany, the industry has seen rapid growth in Europe and North

America. Nowadays, the largest part of commercially available pectin originates from citrus

peel (85.5%), and only a small proportion is covered by extraction from apple pomace
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(14.0%) and sugar beet pulp (0.5%) (Ciriminna et al., 2015). Hence, the production is

centered in the European region and in citrus-producing countries of South America (Bhatia,

Sharma, & Alam, 2016).

Wastes resulting from apple and citrus processing are traditionally the main sources of

commercial pectin. Compared to citrus peel, apple pomace has the disadvantage of a lower

content of pectin. The difference in pectin content between apple varieties has been

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investigated along the years, and led to the understanding that winter (or late-season) varieties

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give the best pectin quality and extraction yields (National Institute of Industrial Research

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Board., 2010). The pectin content of dried pomace obtained from the commercial Granny

Smith apple was determined by Constenla, Ponce, & Lozano (2002), who reported that the
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extraction in nitric acid solution resulted in a maximum pectin yield of 4.2%. A newer study

by Kumar & Chauhan (2010) aimed to characterize the pectin from two different varieties of
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apple, Royal and Golden, cultivated in Himachal Pradesh, India. For both varieties maximum

extraction yields of pectin from pomace were obtained by using diluted citric acid and were
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as follows: 16.65% for Royal variety and 18.79% for Golden variety. As the harvest period

for Royal variety is mid-late season, while Golden apples are a late-season variety, the results
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of this study confirm that winter apples give higher extraction yields of pectin.

Citrus fruits are particularly rich in pectin and the large quantities that are processed
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and, consequently, the considerable amounts of wastes generated, have become the main
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sources for the production of commercial citrus pectin. In citrus peel the amount of pectin has

been estimated to account for as much as 25-30% of the dry weight (Ververis et al., 2007).

The literature provides a comprehensive overview of the pectin composition of several

varieties of citrus fruits including lime, lemon, orange, and grapefruit (Sharma et al., 2017).

Furthermore, research conducted to date has also explored the means by which the yields and

quality of pectin extracted from these sources can be improved. Naghshineh, Olsen, &
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Georgiou (2013) investigated the application of high hydrostatic pressure technology for

enzymatic extraction of pectin from lime peel and compared the extraction yields with those

obtained for acid and aqueous extraction. The latter two extraction methods gave extraction

yields of 13.4% (aqueous extraction) and 18.3% (acid extraction). Although no significant

effect on pectin characteristics was observed, pressure-induced enzymatic treatment

improved the pectin yield, which reached a maximum of 26.5%. Other recent studies reported

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on the use of enzymatic hydrolysis and membrane filtration for the extraction of pectin from

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lemon peels (Gómez et al., 2016), recovery of pectin from pomelo peel (Methacanon,

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Krongsin, & Gamonpilas, 2014), and the influence of acid type and pH on the extraction of

pectin from orange, lemon, lime, and grapefruit peel (Kaya et al., 2014).
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Another source considered for commercial pectin production was sugar beet pulp,

owing to its high pectin content (15-30%) on a dry weight basis and its availability in large
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quantities due to its high production in the sugar industry (Yapo et al., 2007a). The effects of

extraction parameters on the recovery of sugar beet pectin have been extensively studied over
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the years. Of recent studies, the work of Li et al. (2015) stands out as a comprehensive

analysis of the combined effect of pH, temperature, time, and liquid-to-solid ratio on the
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extraction process. The yield of sugar beet pulp pectin ranged from 6.3% to 23.0%; the

increase in pectin yield was correlated with increased temperature, extended extraction time,
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and reduced pH of the extracting solution. By studying the emulsifying properties it was
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concluded that sugar beet pectin could be used to prepare stable oil-in-water emulsions. Even

though it acts as an effective emulsifier, sugar beet pectin applications are limited by its poor

gelling properties, which have been attributed mainly to the high acetyl content (Ralet et al.,

2003) and its greater neutral sugars content (Guo X., Guo, Yu, & Kong, 2018). However, the

feruloyl groups on the arabinan side chains of RG-I provide a way for enzyme-catalyzed

oxidative cross-linking of sugar beet pectin to promote gelation. This can be accomplished
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via horseradish peroxidase (HRP, EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis. Research

conducted by Zaidel, Chronakis, & Meyer (2012) showed that the use of laccase over HRP to

catalyze gelation has two main advantages: it produces stronger gels at lower enzyme dosage

and a slower rate of gelation, and it does not require H2 O2 for the reaction.

Besides the sources presented above, the polysaccharide content of other residues of

fruit and vegetable processing that are generated in large quantities has been investigated in

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order to determine their suitability as profitable sources of commercial pectin (Table 1).

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Special attention was given to residues of the industrial processing of tomato and carrot, as

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these are important crops in various geographical areas. Tomato processing, mostly by the

canning industry, leads to the accumulation of large amounts of pomace (representing around
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4% of the fruit weight) composed of tomato peels, seeds, and a small amount of pulp.

Analysis of the composition of tomato pomace has concluded that the by-product contains
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7.55% pectin on a dry weight basis (Del Valle, Cámara, & Torija, 2006). The possibility of

utilizing tomato peel as a cheap and abundant source for pectin production was studied by
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Grassino et al. (2016) with the purpose of implementing a viable cyclical economy principle

for solving the main problem of waste disposal. Two different batches of dried tomato peel
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from a canning factory were used in the extraction. Based on the degree of esterification

(around 82%) the extracted pectin was categorized as HMP. Two observations made by the
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authors are of particular relevance for the commercial production of pectin from tomato
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waste: higher pectin yields are not necessarily correlated with higher pectin quality, and

sample origin has a considerable effect on pectin characteristics.

Similar to tomato, carrot is processed in large quantities in juice and canning factories,

and the resulting carrot pomace is usually discarded as an industrial waste or used as animal

feed. In the chemical composition of carrot pomace, pectins account for approximately 22-

25% of the total dietary fiber (29.6%) (Stabnikova, Wang, & Ivanov, 2010), a lower content
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when compared to the previously presented sources of pectin. However, the crop importance

in most geographical areas and the increased production of carrots have drawn interest in

investigating the suitability of carrot waste as a source for pectin isolation. Jafari et al. (2017)

studied the effects of process parameters (pH, temperature, heating time, and liquid/solid

ratio) on the extraction yield and degree of esterification of carrot pomace pectin. Extraction

yield ranged from 5.0 to 15.2% and the extracted pectin was classified as LMP. Emulsions

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prepared with carrot pectin had high stability, while 1% (w/v) carrot pectin solutions

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exhibited viscous and pseudoplastic behavior. The structural characteristics of carrot pectin

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were thoroughly investigated by Christiaens et al. (2015) in a study that evaluated the

extraction and properties of pectin from five vegetable waste streams including rejected
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carrots and carrot steam peels. Pectin from carrot steam peels was found to have a very high

level of linearity, a lower DM, and a better solubility than pectin extracted from rejected
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carrots. Because HMP and LMP are both applied as gelling agents in the food industry, it can

be concluded that both types of carrot wastes are suitable sources for pectin production.
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Watermelon rinds, which account for approximately one third of total fruit mass and

are usually discarded despite being edible (Al-Sayed & Ahmed, 2013), were proposed as
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another possible source of pectin. Regarding the pectin content, in wet watermelon rinds a

level of 19-21% (w/w) pectin was determined (Banerjee et al., 2017). Petkowicz, Vriesmann,
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& Williams (2017) investigated the chemistry and rheological and emulsifying properties of
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pectin extracted from fresh (FW) and lyophilized watermelon (LW) rinds in order to gain an

understanding about its potential for use in the food industry. Fresh watermelon rinds gave

higher yields of pectin than lyophilized rinds. FW and LW pectin had a high degree of methyl

esterification (~60%) and low molar mass. The relatively high viscosity of pectin solutions at

5% (w/w) indicated a suitable application as thickening agents, while the good foaming and
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emulsifying properties (compared to gum Arabic) suggested that it can be an efficient

emulsifier and stabilizing agent.

One more industrial waste introduced as source for pectin extraction is banana peel.

Since bananas are not only consumed in raw form, but are also processed into various

products, such as beverages, puree, jellies, chips, important volumes of banana peel are

discarded as waste causing environmental problems. It was estimated that banana peels

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account for about 30% of total weight of the fruit and contain a low amount of water-soluble

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pectin (Happi Emaga et al., 2008). Maran et al. (2017) reported on the optimization of the

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process parameters in pectin extraction from industrial banana waste. At optimum levels of

extraction parameters, the yield of pectin was approximately 9% (w/w). More research is
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needed regarding the chemistry and especially the properties of pectin from banana peel prior

to establishing whether or not this has potential in the food industry as a gelling, thickening,
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or stabilizing agent.

The previously outlined findings show that most of the research conducted to date is
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focused on singular fruit and vegetable residues as sources of commercial pectin, an

understanding that is actually validated by the literature. Of the few studies that investigate
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several sources of pectin and have been published up to this point, unarguably the most

complete survey of pectic materials obtained from many diverse by-products belongs to
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Müller-Maatsch et al. (2016). The researchers selected 26 food waste streams according to
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their exploitation potential, isolated the contained pectin and fully characterized it in terms of

uronic acid and other sugar composition, methylation, and acetylation degrees. The structure

of pectin extracted from these waste streams seemed generally well preserved when

compared to the original food material; notable exception to this observation were the

methylation and acetylation degrees that were often lowered either by processing and/or

enzymatic action. Finally, although the minimum requirement of 65% uronic acids prevented
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some of the plant residues to the considered sources for pectin extraction, it was noted that

these waste streams could be useful in other applications.

Important properties such as galacturonic acid content, degree of esterification,

molecular weight, neutral monosaccharides content of samples isolated from main sources of

commercial pectin and other vegetal sources are presented in Table 1. Among citrus pectin

sources, pomelo has shown a galacturonic acid content and degree of esterification

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(Methacanon et al., 2014) greater than other wastes commonly used in pectin extraction (e.g.

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orange peel). Properties which influence the use as a gelling agent, thickening agent and

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stabilizer, and which show promising applications, can be also noted for pectin from tomato

waste, pumpkin waste, and watermelon rinds.

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Any approach to selecting suitable pectin sources should take into account that pectin

structure and yield are highly diverse according to origin. Furthermore, for the same waste
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stream possible variations in pectin characteristics and content may be due to differences

between batches and countries. However, the information presented here offers a good insight
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into pectin composition and its modification after processing, both of which are valuable for

the industry when introducing new sources of commercial pectin. The possible uses of pectic
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polysaccharides, which derive from the data obtained in the previously described studies, are

also greatly influenced by the choice of extraction technique and the purification method.
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4. Extraction, purification and fractionation

The entire production process of pectin has been completely documented in the

literature and generally is comprised of three stages: a pretreatment, the extraction operation,

and a post-extraction stage. The purpose of the pretreatment, whether a drying, washing, or

blanching process, is to increase the stability of the raw material by inactivating bacteria and

enzymes that otherwise cause pectin degradation. On the industrial scale, the second stage of
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pectin production is the extraction, which combines the presence of a mineral acid (such as

hydrochloric acid, sulfuric acid, or nitric acid) with a heat treatment. The use of this

conventional acid extraction method raises some questions regarding resource management.

Because the prolonged length of the process, and especially the heating, is linked to increased

energy consumption, it is important to determine if the economic demands are justified by the

production of high-quality pectin or if it is possible to reduce the overall cost of the process

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without affecting the quality of the pectin. The same principle should be also considered in

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the context of an efficiency evaluation for alternative extraction techniques.

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4.1. Extraction techniques
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Pectin extraction is defined as a physical-chemical process which is comprised of

multiple stages of hydrolysis and extraction of pectin macromolecules from plant tissue and
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their solubilization into the bulk solvent, all of them occurring in a continuous manner under

the influence of different process parameters, mainly temperature, pH, and time (Methacanon
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et al., 2014). Pectin is a complex macromolecule, and the preservation of its structure, which

determines characteristics such as molecular weight, water solubility, and gelation properties,
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is connected to the extraction method applied in its isolation from the plant material. The

extraction and isolation of pectin from cell walls can be approached in various ways through
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the use of chemical, physical, as well as enzymatic treatments (Panouillé, Thibault, &
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Bonnin, 2006). Among the chemical methods, acid extraction seems to be the most widely

used in commercial pectin production.

4.1.1. Solvent extraction

The use of a suitable method, alongside a good understanding of the individual and

collective effect of process parameters is essential in order to maximize pectin yield and its
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quality. In the particular case of solvent extraction, the type of extraction solvent, its

concentration, and operation conditions such as pH, temperature, and extraction time impact

the release of pectin from the cell wall. The study of the parameters involved in solvent

extraction is not a novelty subject in the literature, as it has been widely studied through the

years.

In regard to extraction solvent, an ideal choice should feature the following desirable

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characteristics: the solvent has a high capacity for the solute separated into it, it is selective,

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can dissolve the specific component to a large extent while having a minimum capacity for

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other components, is chemically stable, renewable, and has a low viscosity, which eases

pumping and transportation (Oroian & Escriche, 2015). Accordingly, when studying the
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extraction efficiency of a solvent, not only the pectin yields are of relevance, but also its

structure and chemical composition because these are known to govern its applications.
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Commonly used solvents are diluted strong mineral acids. Kalapathy & Proctor (2001)

investigated the effect of hydrochloric acid strength (0.05, 0.1, 0.2, and 0.3 N) on the yield
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and purity of pectin extracted from soy hull. It was reported that the highest yields (26 and

28%) were obtained when the acid strength was 0.05 and 0.1 N, and that a further increase in
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acid strength caused a decrease of pectin yield. Unlike the strong effect on extraction yield,

strength of acid did not affect pectin purity. Besides solvent strength, the influence of solvent
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type on pectin extraction was also studied. Begum et al. (2014) carried out a comparison of
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extraction solvents (ammonium oxalate, diluted sulfuric acid, and sodium

hexametaphosphate) for the isolation of pectic substances from jackfruit. It was found that

acid extraction gave the lowest extraction yield (8.94%); in contrast, extraction with sodium

hexametaphosphate gave the highest yield (15.14%), but the isolated pectin had high ash

content and the lowest solubility. Comparison between extraction solvents was extended to

analyzing the efficiency of mineral acid extraction against the extraction of pectin with
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organic acids. Kliemann et al. (2009) investigated the extractability of pectin from passion

fruit waste using three kinds of acid, citric, hydrochloric, and nitric acids. The best extraction

yield was obtained with citric acid, and the extracted pectin was rich in anhydrogalacturonic

acid and had a low DM. The high efficiency of citric acid in pectin extraction was also

confirmed by Chan & Choo (2013) when compared to the pectin yields determined for

hydrochloric acid, and by Yang, Mu, & Ma (2018) following an investigation on the effects

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of the extraction of potato pectins with hydrochloric, sulfuric, nitric, citric, and acetic acids.

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These differences in yield and pectin quality between acids can be explained by 2 separate

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phenomena: (a) when combined to increased temperatures and prolonged extraction time,

strong acid solutions (e.g. hydrochloric acid) could enhance pectin hydrolysis and hence
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result in a more degraded, shorter pectin chain; (b) the additional extraction activity of citric

acid on chelator-solubilised pectin fraction leads to increased yields when compared to

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mineral acids, under identical extraction conditions (Maneerat, Tangsuphoom, &

Nitithamyong, 2017; Jamsazzadeh Kermani et al., 2014). Considering that strong mineral
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acids are corrosive, are deemed a potential threat to health, and are linked to possible

increased costs of waste treatment, the extraction using citric acid and other weak organic
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acids may bring a major advantage to the overall process of pectin isolation.

Pectin extraction from fruit and vegetable residues using weak organic acids has been
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intensively studied, suggesting that numerous data regarding the influence of operating
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conditions on pectin yield and quality is available. Alba, Laws, & Kontogiorgos (2015)

designed an isolation protocol to extract pectin from okra with citric acid and studied the

influence of the extraction pH (6.0 or 2.0) on the composition and physicochemical properties

of the polysaccharides. Extraction with citric acid adjusted to pH 6.0 resulted in higher pectin

yield compared to that at pH 2.0. The extraction conditions determined some structural

variations between samples: pectin polysaccharides extracted at pH 2.0 had a lower content
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of neutral sugars and galacturonic acid, while pectin obtained by extraction at pH 6.0 had

lower DM and DAc. Influence of pH (2.0, 3.3, or 4.5), alongside that of the extraction time

(30, 75, or 120 min) on pectin yield and composition was also studied by Liew, Chin, &

Yusof (2014) in a citric acid extraction process. The maximum extraction yield was found at

75 min and the lowest pH; of these two factors, pH showed a greater influence on pectin

yield. On the other hand, the degree of esterification was significantly affected by extraction

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time. A more complete analysis of the involved factors was conducted by Pereira et al.,

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(2016) who evaluated the influence of pH (2-4), temperature (70-90 °C), and time (40-150

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min) on pectin extraction from pomegranate peels with citric acid. Based on the results, it

was noted that harsh extraction conditions (low pH, high temperatures, and prolonged
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extraction) determined higher pectin yields and higher galacturonic acid contents, while

causing a decrease in the degree of methylation. This conclusion regarding the combined
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positive impact of low pH values, high temperature and prolonged extraction time on the

extraction yield of pectin is corroborated in numerous studies, as it can be deduced from the
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data presented in Table 2. Alongside studying the influence of operating conditions on pectin

yield and quality, the various factors affecting pectin extraction from plant sources, including
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extraction temperature, pH, time, and liquid/solid ratio (LSR) are optimized in order to

achieve a maximum yield and the desired characteristics of pectin. Numerous studies were
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aimed to optimize the process variables in the extraction of pectin from wastes such as sour
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orange peel (Hosseini, Khodaiyan, & Yarmand, 2016), durian rinds (Maran, 2015), lime peel

(Andersen et al., 2017), and Valencia orange peels (Casas-Orozco et al., 2015), for the latter

two plant sources the process being designed for an industrial-scale extraction. Important

findings of research on pectin extraction conducted with different solvents in specific

conditions are summarized in Table 2.

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4.1.2. Novel extraction techniques

The development of green chemistry has impacted the isolation step of the analysis of

macromolecules from plants and, as a result, in the last few years environment-friendly

techniques have emerged as an alternative to the traditional acid extraction method. Current

research aims at optimizing cleaner extraction techniques such as enzyme-assisted extraction,

microwave-assisted extraction, ultrasound-assisted extraction, subcritical water extraction,

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and induced electric field extraction. The first four techniques have been recently reviewed in

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pectin production by Adetunji et al. (2017), who covered all the particularities from

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principles and operational issues to benefits and drawbacks. Important results regarding the

application of these techniques in pectin isolation and the influence of extraction parameters
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are presented in Table 2. The conditions of enzyme-assisted extraction were studied in

several researches including the works of Dominiak et al. (2014), Jeong et al. (2014), Wikiera
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et al. (2015a), Wikiera et al. (2015b), and Wikiera et al. (2016), some of them being

summarized in Table 2. The potential of ultrasound-assisted extraction (UAE) of pectin was

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studied for plant sources such as from passion fruit peel (Freitas de Oliveira et al., 2016),

grapefruit peel (Wang et al., 2015; Xu et al., 2014), mango peel (Wang et al. (2016), and
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jackfruit peel (Moorthy et al., 2017). It should be mentioned here that when studying the

simultaneous effect of temperature and ultrasound treatment it is crucial to consider that

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ultrasounds cause a disintegration of the material, consequently affecting the separation of

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solid and liquid phases. This may translate into a lower pectin yield of the ultrasound-assisted

heating extraction by comparison with a conventional heating process. To avoid erroneous

conclusions regarding the effect of temperature and sonication on extraction yields, a second

UAE is recommended in order to completely dissolve the pectin previously absorbed in the

residue.
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As in the case of the previous extraction techniques, that are presented in Table 2,

microwave-assisted extraction (MAE) has been applied to extract pectin from wastes of fruit

and vegetable processing, including unutilized pumpkin biomass (Košťálová, Aguedo, &

Hromádková, 2016), the waste peels of papaya (Maran & Prakash, 2015) and mango (Maran

et al., 2015), tangerine peels (Chen et al., 2016), and Opuntia ficus indica cladodes (Lefsih et

al., 2017). Along the years the work of Fishman and collaborators on the extraction of pectin

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from orange albedo (Fishman et al., 1999), lime (Fishman et al., 2006), and sugar beet pulp

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(Fishman et al., 2013) brought notable contribution to the knowledge regarding the use of

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microwaves in the extraction of pectin from plant materials. Some research has been

conducted in the last years with the purpose of studying the use of microwave-assisted
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extraction and ultrasound-assisted extraction as complementary techniques for the isolation of

pectin from vegetable sources. Bagherian et al. (2011) described the results of a MAE of
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pectin from grapefruit peel where a preliminary ultrasonic heating of grapefruit solution was

introduced. They observed that this pretreatment provided a higher pectin yield, especially in
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the case of intermittent sonication. Regarding the composition of pectin extracted through a

sequential ultrasound-microwave-assisted extraction, Liew et al. (2016) reported that the

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combined extraction technique resulted in a higher GalA content because it allows a more

complete release of the pectin compound and therefore a better preservation of the GalA
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distribution from deeper plant matrix than a sole ultrasound or microwave extraction. The
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combination of microwave and ultrasound treatment alongside the use of “in situ” water,

which was recycled and used as solvent, allowed Boukroufa et al. (2015) to obtain valuable

compounds (pectin, essential oil, and polyphenols) from orange peels waste in a shorter time.

Citrus waste, such as flavedo of Citrus junos (Ueno et al., 2008), Citrus junos peel (Tanaka et

al., 2012), and citrus peel (Wang, Chen, & Lü, 2014), was also used as a source for the

subcritical water extraction of pectin; the same extraction technique was applied for the
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isolation of pectin from apple pomace (Wang, Chen, & Lü, 2014) and sugar beet pulp (Chen,

Fu, & Luo, 2015), as shown in Table 2.

Another novel technique applied for the isolation of pectin from plant materials is

induced electric field-assisted extraction. Application of induced electric field for the

extraction of targeted compounds is based on the use of inductive methodology, which

represents an alternating magnetic flux creating an alternating voltage in accordance to

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Faraday’s law of induction. The induced electric field acts upon the biological tissue and, as a

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result, different phenomena, including intracellular liquid release and diffusion of solutes,

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develop inside the cellular structure following the treatment (Vorobiev & Lebovka, 2009).

Therefore, utilization of an electrical driving force as auxiliary energy leads to substantial

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improvements of mass transfer and extraction efficiency (Yamini, Seidi, & Rezazadeh,

2014). Yang et al. (2016) developed an experimental system to extract pectin from orange
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peel waste that, unlike other electric field-assisted techniques, avoids the use of powered

electrodes. The experimental system contains a power source, circulating water bath, a ferrite
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o-core, primary coil, glass spiral (supporting the tube of the secondary coil), glass chamber,

sample, and solution inlet, and also a sand filter. In the process the extractant (dilute
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hydrochloric acid) acts as a secondary coil connected to the glass chamber which forms a

closed loop. Through this setup the induced electric field in the system appears to be under
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the influence of alternating magnetic flux. Table 2 presents data regarding the investigated
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effect of excitation voltage, frequency, temperature, and pH on pectin yield.

4.2. Purification and fractionation of pectin polysaccharides

In early approaches to the study of pectin it was considered that the presence of

adventitious substances (such as sugars and acids) must be excluded as far as possible and a

definition of what constitutes “pure” pectin was necessary. In this context, the following
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methods of pectin purification were acknowledged: precipitation, dialysis, ionic exchange,

nitration, as well as combined methods (Lampitt et al., 1947). Precipitation is a method

generally employed to purify the pectin contained in an aqueous extract through subsequent

washings with alcohol or acetone. Alcohol precipitation is frequently used at both laboratory

and industrial scales because it gives satisfactory pectin yields at advantageous costs. Guo et

al. (2016) proved that a stepwise ethanolic precipitation is a more effective method for the

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purification of sugar beet pectin (SBP) than a one-step ethanolic precipitation. They also

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noted that the ethanol concentration required for purification was dependent on pectin

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structure, and particularly the proportion of neutral side chains. Based on their results, the

researchers indicated that at least a concentration of 75% ethanol is recommended to obtain a

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satisfactory yield of purified sugar beet pectin. However, the purity of pectin obtained only

through alcoholic precipitation was found to be lower when compared to the composition of
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pectin purified by ultrafiltration and metal ion-binding precipitation. Yapo et al. (2007b)

compared alcoholic precipitation to the purification of pectin by ultrafiltration-diafiltration

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(Table 3) and reported that the use of the first technique resulted in more neutral sugars, more

proteins, and more ash, but less galacturonic acids in sugar beet pectin. In contrast,
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ultrafiltration has been reported to effectively remove impurities such as pigments and salts

(Kang et al., 2015), while metal ion-binding precipitation proved to be selective toward
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binding HG and RG regions and thereby is suitable to purify pectins from non-uronide
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contaminants (Guo et al., 2015). Although it presents a great advantage of a better selectivity

towards adventitious compounds, metal ion-binding precipitation is likely to generate, at an

industrial scale, a large amount of effluents that demand treatment prior to their discharge in

order to avoid environmental damage. By considering this main drawback, Yapo (2009)

concluded that it would be more advantageous to precede alcohol precipitation with an

industrially-practical membrane procedure (such as ultrafiltration-diafiltration) for an

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effective removal of pectin contaminants that assures the compositional quality and gelling

properties of the final pectin product.

Apart from the techniques presented above (Table 3), the literature provides other new

methods that have been developed with the purpose of achieving a better purification of

pectin. Garna et al. (2011) proposed a technique for the purification of electrically charged

polysaccharides using protein (sodium caseinate). The purification is based on the

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electrostatic interactions taking place between the two polymers and is comprised of two

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steps: a first step where the charged pectins are precipitated using proteins at pH 3.5, and a

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second step in which the polymers are separated by the dissociation of the pectin-caseinate

complexes and the precipitation of caseinate at a pH close to its isoelectric point (pH 4.6).
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The feasibility of the process was verified through application on commercial pectin from

apple pomace, and the results indicated that this purification method is very effective for the
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recovery of charged polysaccharides. Happi Emaga et al. (2012) then applied this technique

to purify apple pomace pectin and evaluated its efficiency against purification by ethanol
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precipitation. It was observed that, under certain conditions, ethanol caused the precipitation

of other compounds, but allowed total precipitation of pectin when compared to caseinate.
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The use of caseinate has the advantage of a higher specificity for the charged polymer, which

is substantially overshadowed by the drawback of requiring a large quantity of protein.

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When the objective of the research is to fully elucidate the composition of the
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extracted pectin, purification is usually performed together with a fractionation which can be

achieved through various techniques. For example, Lin et al. (2016) fractionated the crude

polysaccharide obtained from flowers of Lonicera japonica by anion-exchange

chromatography performed on a DEAE-cellulose52 column, where a stepwise elution with

water gave six different fractions; the major fraction (LJ-02) was further purified with a

Sephacryl S-200HR column. Fractionation allowed the evaluation of antipancreatic cancer

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activity, which concluded in the finding that a RG-I polysaccharide from Lonicera japonica

flowers could be a potential novel therapeutic agent against pancreatic cancer. To elucidate

the structure of an immune-enhancing pectic polysaccharide from the aqueous extract of

green bean pods, Patra et al. (2012) subjected the crude polysaccharide to purification and

fractionation by gel permeation chromatography on a column of Sepharose 6B and water as

an eluant using a fraction collector. The isolated water-soluble pectin contained a [→4)-α-D-

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GalpA6Me-(1→4)-α-D-GalpA6Me-(1→] backbone with branching at C-2 and showed

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significant antioxidant activity and thymocyte and splenocyte activation. The beneficial

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effects of separate structures from the composition of pectin polysaccharides was also

investigated by Li et al. (2016) who fractionated the hydrolysate from orange peel via
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membrane separation into three different molecular weight fractions that had prebiotic and

antimicrobial properties. When the three fractions were compared, it was noted that one of
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them had glucose as the main component, presented higher galacturonic acid content, and

also contained high amounts of arabinose and galactose, which ranked third and fourth
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among components. Membrane separation, more exactly ultrafiltration, was also employed in

the fractionation of crude extract from star fruit, which led to elucidating the structure of
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pectic type II arabinogalactans from its composition (Leivas, Iacomini, & Cordeiro, 2016).

Adopting the high-throughput and fractionation techniques used by Nguema-Ona et

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al. (2012) to profile the different wall polymers present in the leaves of Nicotiana tabacum,
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Moore et al. (2014) reported on the profile of main cell wall polysaccharides of grapevine

leaves. In both studies the alcohol-insoluble residue was chemically and enzymatically

fractionated. The enzymatic fractionation procedure was performed with glycosyl hydrolases

(endopolygalacturonase, EC 3.2.1.15) and two different xyloglucan-specific endoglucanases

(EC 3.2.1.151). The two procedures caused a sequential degradation of the recovered residue
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which ended in the structural reveal of the major sub-networks, pectin and hemicellulose,

from grapevine leaves.

5. Characterization of pectin composition and properties

The analysis of pectic carbohydrates isolated from the cell walls of various plants

presents a major challenge because of the variation and the complexity of non-uronide

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compounds associated with these carbohydrates. Similar to most other polysaccharides,

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pectin is polymolecular and polydisperse, meaning it is heterogeneous in both chemical

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structure and molecular weight (BeMiller, 1986). In regards to chemical structure,

galacturonic acid and neutral sugars are the major constituents of pectin chains. The number
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and percentage of individual monomeric units varies from molecule to molecule in any pectin

sample, while the distribution of molecular weights is determined by source and the
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conditions of isolation and other treatments applied following the recovery of pectin

polysaccharides. The variability in the structure of pectin chains, including the number of
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esterified methoxyl groups to galacturonic acid, the differences in molecular weight, and the
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intrinsic properties of pectin determine its physical properties and contribute to the
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commercial interest for this soluble fiber (Luz Fernandez, 2001).

Given the complexity of the multiblock pectin biopolymer, the analysis of the
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extracted whole macromolecule does not suffice in giving insight into the fine structure of
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pectin. Knowledge on pectin structure has been largely obtained from chemical-enzymatic

analyses which involve different extraction protocols for sequentially isolating and

characterizing the polymer (Sila et al., 2009). To reveal its structural characteristics, pectin

biopolymer is usually degraded into oligosaccharides that are further fractionated to isolate

structural elements. Analytical techniques (Table 4) used to study and quantify the structural

elements of pectin are discussed below.

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5.1. Monosaccharide composition and linkage pattern

5.1.1. Sugar composition

Accurate analysis of the carbohydrate composition is particularly important when

studying the structure of pectin polysaccharides, and it is often desired for monitoring the

extraction and purification process and the quality parameters of the final pectin product.

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Regarding the effect of the neutral monosaccharides composition on the technological

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applications of pectin, research has shown a positive impact of this chemical parameter on the

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formation of pectin gels. A representative study focused on this matter is the research

conducted by Sousa et al. (2015b) on the effect of the specific reduction in neutral sugars
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concentration (through controlled enzymatic debranching) on the rheological properties of

high-sugar HM citrus pectin gels. As explained by the authors, following the decrease in
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temperature, chain mobility, and thus the velocity at which new hydrophobic interactions are

formed in the initial stage of gelling, the neutral monosaccharides side chains play a key role
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in further tightening the gelling network.

In order to determine the neutral monosaccharides content, the polysaccharides must

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be first depolymerized into their constituent sugar residues (uronic acid and neutral

monosaccharides), which is commonly done by chemical or enzymatic hydrolysis. Most of

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the methods available for the determination of neutral monosaccharides involve the use of
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chromatographic techniques. A simple chromatographic method was described by Blakeney

et al. (1983) and is based on the determination of alditol acetate derivatization products of the

monosaccharides contained in the pectin sample by gas chromatography. In a first step,

monosaccharides are reduced with a solution of sodium borohydride in dimethyl sulfoxide;

an acetylation step then follows, where 1-methylimidazole is added as the catalyst. The

resulting alditol acetates are completely separated in a chromatographic system equipped

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with a glass-capillary column. This method was employed in newer research on the chemical

analysis of pectin from various sources including apple and citrus (Kaya et al., 2014; Wang,

Chen, & Lü, 2014). Other variations of the gas chromatographic method involve flame

ionization detection (GC-FID) (Garna et al., 2007) or mass spectrometric detection (GC-MS)

(Colodel & De Oliveira Petkowicz, 2018; Müller-Maatsch et al., 2016), which is one of the

most frequently used methods for the analysis of neutral monosaccharide composition.

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In several studies, high performance anion exchange chromatography with pulsed

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amperometric detection (HPAEC-PAD) was used to examine the sugar composition of pectin

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(Guo et al., 2018; Nagel et al., 2017; Christiaens et al., 2015; Kang et al., 2015). Kang et al.

(2015) reported on the use of ultrafiltration prior to quantification, in order to remove

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impurities such as pigments, salts, acids, and saccharides from the extracted pectin sample.

Willför et al. (2009) conducted a comparison between commonly used methods for
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hydrolysis and subsequent analysis of the released monosaccharides: gas chromatography

(using capillary columns and flame ionization detection and/or mass spectrometric detection),
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HPAEC-borate technique, and HPAEC-PAD. Based on the results it was concluded that gas

chromatographic analysis preceded by a combined acid hydrolysis and methanolysis is a

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convenient method for obtaining the composition of monosaccharides. Another technique

examined for the determination of monosaccharides present in pectin was centrifugal

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partition chromatography. Ward et al. (2015) evaluated the efficiency of a highly polar two-
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phase system containing ethanol and aqueous ammonium sulfate for the separation of neutral

monosaccharides (L-rhamnose, L-arabinose, D-galactose, and D-galacturonic acid) of

hydrolyzed sugar beet pectin. Dimethyl sulfoxide was selected as an effective phase system

modifier improving monosaccharide separation; in the combined form ethanol:dimethyl

sulfoxide:aqueous ammonium sulfate (0.8:0.1:1.8, v:v:v) the system enabled the separation of

monosaccharides by centrifugal partition chromatography in an ascending mode.

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5.1.2. Galacturonic acid content

In order to analyze the galacturonic acid content, pectic substances must be first

hydrolyzed to uronic acid and neutral monosaccharides. Several procedures have been

developed to hydrolyze pectin, most of them involving the use of concentrated acids or

enzymes. All these methods were reported to present some drawbacks and call for

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improvement; acid hydrolysis requires prolonged treatment and can cause a degradation of

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galacturonic acid which ultimately leads to a low recovery (Garna et al., 2004), while the

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other method requires different types of enzyme activities such as pectolytic,

hemicellulolytic, and carbohydratases for an efficient degradation of pectin. Despite this

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drawback, enzymatic hydrolysis is considered a better technique for the hydrolysis of pectin.

Several colorimetric methods are available for the quantitative analysis of

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galacturonic acid. Among spectrophotometric analytical techniques, carbazole-H2 SO4

reaction is one of the early methods of determination which have been described and later
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modified by various other authors. The method was first proposed by Dische (1947) and is

based on the color reaction between the degradation products of pectin hydrolysis with
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concentrated acid and carbazole; the resulting coloration is proportional to the concentration

of galacturonic acid. In recent studies, the carbazole method was used to determine the
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galacturonic acid content of polysaccharides from Opuntia microdasys var. rufida cladodes
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(Jouini et al., 2018) and pectin extracted from waste grapefruit peels (Wang et al., 2017) A

modification of Dische's carbazole reaction for uronic acid in the presence of borate was later

described by Bitter & Muir (1962) who noted that the advantages of this method were the

increased sensitivity, greater reproducibility, and reduction of interference by chloride ion

and oxidants. This modified carbazole assay was used by Yeoh, Shi, & Langrish (2008) for

the determination of galacturonic acid content of pectin from orange peels, by Zhang et al.
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(2013) in the analysis of physicochemical properties of polysaccharides obtained from

Flammulina velutipes, and by Romdhane et al. (2017) to analyze the uronic acid content of

polysaccharides from watermelon rinds. In their research, Yeoh, Shi, & Langrish (2008)

considered the removal of neutral carbohydrates from the sample a requirement in ensuring

that this method is an accurate colorimetric assay to quantify the amount of pectin. The

presence of non-uronide carbohydrates (such as starch, cellulose, and neutral sugars) in the

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pectin extract was shown to interfere in the analysis of galacturonic acid, since the carbazole

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assay stimulates any neutral sugar molecules present to form additional color. Similar

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methods to the carbazole reaction are the m-hydroxydiphenyl sulfuric acid assay

(Blumenkrantz & Asboe-Hansen, 1973) and the 3,5-dimethylphenol sulfuric acid reaction
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(Scott, 1979). Both methods are characterized by a high specificity for uronides and less

sensitivity to the presence of neutral sugars; an exception was observed for the m-
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hydroxydiphenyl sulfuric acid assay when non-uronide carbohydrates were contained by the

sample in high concentrations (Sila et al., 2009). The m-hydroxydiphenyl reaction, which has
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become a common method for the determination of uronic acids, was widely applied in the

last years for the analysis of pectin from various plant sources (Abid et al., 2017;
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Chaharbaghi, Khodaiyan, & Hosseini, 2017; Vasco-Correa & Zapata Zapata, 2017). A

method that avoids the use of concentrated acids commonly needed in the colorimetric
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determination with m-hydroxydiphenyl was proposed by Anthon & Barrett (2008) who
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described it as a simple procedure for determining the galacturonic acid that relies on

enzymatic pectin hydrolysis and colorimetric quantification (using arsenic containing the

Nelson reagent). Application of the determination procedure was evaluated for both soluble

and insoluble pectins from apple, oranges, and several tomato products.

The literature also contains several gas and liquid chromatographic techniques that

were developed to determine the content of uronic acid in pectin (Garleb, Bourquin, & Fahey,
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1991; Ford, 1982; Jones & Albersheim, 1972); although these techniques indicated promising

applications, they resulted in lower quantification. An accurate analysis of uronic acids

without any derivatization was obtained by Garna et al. (2006) using HPAEC-PAD. When

the HPAEC-PAD assay was preceded by a combined chemical and enzymatic hydrolysis

with Viscozyme L9, a major advantage for detection, namely the liberation of galacturonic

acid without any degradation, was observed. Moreover, the method is selective and sensitive

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and provides better accuracy and repeatability. Burana-osot et al. (2010) developed a simple

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and rapid analytical method based on converting GalA in the polysaccharide chain into the

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stable neutral sugar Gal (preventing decomposition of urinate residues) prior to hydrolysis

with trifluoroacetic acid and analysis of the hydrolysate by HPAEC with fluorescence
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detection. By comparison to HPAEC-PAD, the galacturonic acid content determined with the

proposed method was higher, and the results showed good linearity, high precision, and high
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sensitivity.
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5.1.3. Glycosidic linkage conformation

For a detailed structural characterization of pectin polysaccharides, the investigation

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into monosaccharide composition can be completed with a determination of the positions of

the glycosidic linkages by which the monosaccharide units are connected in polysaccharides.
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The distribution pattern of glycosidic linkages in the RG backbone determines the

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technological applications through the changes in pectin-water interactions, given the fact

that the cleavage of these linkages, as side reaction of pectin demethoxylation, can lead to

reduced water sorption of the resulting LMP (Einhorn-Stoll, 2018).

The most common methods for carbohydrate linkage analysis are exoglycosidase (EC

3.2.1.37) digestion, methylation analysis (both involving GC techniques), mass spectroscopy,

and nuclear magnetic resonance (NMR) spectroscopy (Bertozzi et al., 2009). Of these
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analytical approaches, methylation analysis is viewed as the most important single method

which quantifies all the modes of linkage of the monosaccharide residues in the

polysaccharide (BeMiller, 1996). The general procedure, as described by Sims & Bacic

(1995), is based on the methylation-induced cleavage of the glycosidic linkages in the native

oligosaccharide and the conversion of the resulting partially methylated individual

monosaccharide residues to alditol acetates (partially methylated alditol acetates, PMAAs)

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that are separated on a GC column. The results of the quantification of PMAAs by GC-MS

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reported in some studies, including those belonging to Wu et al. (2015), Zhang et al. (2016)

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and Wang et al. (2017), showed that correct identification of derivatives and the sugars they

are derived from requires both retention time and mass fragmentation data (Sims et al., 2018).
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A drawback of this method is that glycosidic linkages adjacent to uronic acids are not

detectable as alditol acetate unless they are prereduced to their neutral sugar counterparts
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(Pettolino et al., 2012).

Glycosyl linkage analysis has been applied to elucidate the structure of pectin from
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various plant matrixes, including the recent use in the analysis of pectin from flowers of

Lonicera japonica conducted by Lin et al. (2016), who used methylation in combination with
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NMR analysis (1 H NMR, 13

C NMR spectra and 2D spectra). Identification of glycosidic

linkage and side chain location by 1D and 2D NMR was also reported in structural studies of
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arabinan-rich pectic polysaccharides from Abies sibirica L. (Shakhmatov et al., 2014), water-
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soluble polysaccharides in papaya during ripening (John et al., 2018), and pectins from Tilia

tomentosa (Georgiev et al., 2017). Characteristic signals in 1D and 2D NMR provide

information related to pectin structure. For example, in regards to the assignments of 1 H and
13
C NMR chemical shifts, the predominant signal at 100.4-105.2/4.60-4.63 ppm was assigned

to C-1/H-1 of galacturonic acid β(1→4)-linked (Kienteka, Corrêa-Ferreira, & De Oliveira

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Petkowicz, 2018; Shakhmatov et al., 2014), while the correlation peak C-1/H-1 at 99.1-

102.2/5.03 was due to 1,4-α-D-GalpA residues (John et al., 2018; Shakhmatov et al., 2014).

5.2. Degree of substitution

The functional properties of pectin in food, the reactivity towards calcium and other

cations and, therefore, its potential for cross-linking is mostly dependent on the amount of

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non-esterified GalA subunits and their distribution pattern within the HG chain. The degree

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of esterification influences physical properties such as surface tension, emulsification

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capabilities (Lutz et al., 2009) and gel formation (Yoo et al, 2006). In regards to gelation,

pectin with a degree of methylation above 50% can form gels at low pH and high sugar
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concentrations, whereas for pectin with a methyl esterification degree below that level the

gelation is dictated by the reaction with calcium (May, 1990). Acetylation, like methylation,
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is well known to strongly alter pectin associative properties by decreasing the affinity of HG

domains for cations (Ralet, Lerouge, & Quéméner, 2009; Renard & Jarvis, 1999).
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Various analytical methods have been described in the literature to determine the

degree of esterification of pectin samples isolated from diverse plant materials. Most methods
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are based on using alkaline hydrolysis to release methoxyl groups from the galacturonic acid

by incubation with sodium hydroxide solution; the methanol content can then be determined
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using chromatography, spectrophotometric methods or FT-IR spectroscopy. Klavons &

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Bennett (1986) improved the colorimetric method proposed by Wood & Siddiqui (1971) by

shortening the oxidation time through the use of alcohol oxidase as replacement for

potassium permanganate. In a later study, Anthon & Barrett (2004) found alcohol oxidase

(EC 1.1.3.13) in conjunction with Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole)

more sensitive to the released methanol. Compared to spectrophotometric techniques, the

major advantage of chromatography is that it allows the separation of impurities prior to the
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quantification. Efficient separation and good reproducibility was reported for head-space gas

chromatography (Huisman, Oosterveld, & Schols, 2004) and ion-exclusion chromatography

(Luzio & Cameron, 2013). Another technique validated as a method for the routine analysis

of the degree of methyl-esterification was Fourier transform infrared (FT-IR) spectroscopy.

Kyomugasho et al. (2015) showed that when using FT-IR for the analysis of DM of a protein-

rich sample, such as pectin from broccoli, the peak deconvolution is vital to correct

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interferences due to the presence of proteins and to improve the determination accuracy.

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Research on the simultaneous determination of methylation and acetylation degree of

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pectin is limited and mostly involves quantification using chromatographic techniques such

as HPLC (Levigne et al., 2002) and GC-MS (Savary & Nuñez, 2003). By performing a base-
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hydrolysis of esters and acidification of pectin samples, followed by headspace solid-phase

microextraction and, finally, analysis of the resulting methanol and acetic acid by GC-MS, as
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described by Savary & Nuñez (2003), Yoo et al. (2012) determined the methyl and acetyl

substitution levels in pumpkin pectin extracted by microwave heating. A different approach

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to this matter was taken by Müller-Maatsch et al. (2014) who developed a 1 H NMR (proton

nuclear magnetic resonance) method that allowed the detection of methylation, acetylation,
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and feruloylation degrees of pectin after an alkaline saponification. This method can be

applied to pectic polysaccharides originating from a wide range of sources and having
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different physical properties and can detect small amounts of methanol, acetic acid, and
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ferulic acid. Reports on the use of the 1 H NMR method indicated a sharp singlet at 3.75 ppm,

which corresponds to protons in the methoxy group of esterified galacturonic acid, and

chemical shifts at 2.01-2.17 ppm, attributed to the acetyl groups binding at O-2 and O-3 of

galacturonic acid residues (Grassino et al., 2016; Gopi et al., 2014). Simultaneous

determination of methylation and acetylation degree of pectin can be also achieved using FT-

Raman and FT-IR, as described by Synytsya et al. (2003). According to their observations,
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the very intense Raman band at 857 cm−1 is sensitive to the state of uronic carboxyls and O-

acetylation, decreasing to the minimum of 850 cm−1 with the degree of substitution by methyl

groups and increasing (to max. 862 cm−1 ) with acetylation. Low intensity Raman bands at

1164, 1184 and 1471 cm−1 were attributed to acetylation of pectin samples (Kumar &

Chauhan, 2010). Other research that must be mentioned here is the environmentally friendly

method for the automated determination of pectin DE using the µSI-LOV system developed

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by Naghshineh et al. (2016). The determination proved to be fairly simple, precise,

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reproducible, and economical.

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5.3. Degree of blockiness
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As was mentioned before, the degree and pattern of methyl-esterification influences

the functional properties of pectin; because of its effect on gel-forming and rheological
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properties, the commercial implications of DE have been extensively studied. As a way to

describe the percent of non-esterified GalA units present in pectin, expressed as the
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blockwise distribution of the free carboxyl groups, Daas et al. (1999) introduced the term

degree of blockiness (DB). The degree of blockiness is an important parameter for the
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retention of water in pectin gels and influences, to a lower extent, the water uptake of pectin

powders (Einhorn-Stoll, 2018). Studies showed that, when compared to pectin samples with
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randomly distributed carboxyl groups, HMP with partly blockwise distributed free carboxyl
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groups displayed increased water uptake and later dissolution (Einhorn-Stoll et al., 2015).

Daas et al. (1999) described a method used to determine the degree of blockiness, which was

based on the analysis of the oligomers released after pectin digestion with

endopolygalacturonase (EC 3.2.1.15) from Kluyveromyces fragiles, their separation and

quantification using HPAEC at pH 5. Determination of DB by HPAEC-PAD method (Daas,

Voragen, & Schols, 2000; Daas et al., 1999) was important for the investigation of pectin
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rheological behavior and the study on the effect of pectin properties on coacervates formation

with pea protein isolate (Warnakulasuriya et al., 2018; Sousa et al., 2015b). A similar

procedure for the separation of oligomers, involving digestion of pectin with enzymes,

followed by analysis using capillary electrophoresis (CE) for the determination of the degree

of blockiness of several commercial pectins, was reported by Guillotin et al. (2007). The CE

method was found to present two main advantages by comparison to other quantification

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methods available up to that point: the very low amount of sample required, especially when

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compared to HPAEC and the possibility to simultaneously analyze the oligomers and

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polymers from the polygalacturonase digest.

While the above-mentioned methods give good results, other techniques found in the
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literature have proved not to be accurate for the determination of DB. When applying 1 H

NMR for the quantification of the degree of blockiness of pectin, Winning et al. (2007)
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observed that the H-1 signal strongly covariated with random deesterification, but not with

blockiness. As a result, it was concluded that the proposed method was better suited for the
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prediction of random deesterification rather than the determination of the degree of block

deesterification. Lack of specificity for the characterization of DB was also indicated by

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Sousa et al. (2015a) who took a multivariate analysis approach with the purpose to analyze a

set of pectin samples probed with 14 different monoclonal antibodies. In their study the
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development of partial least squares models allowed prediction of DM values in an accurate

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form, but at the same time was not good enough for the prediction of DB.

5.4. Molecular weight

Early research on the application as gelling, thickening, and stabilizing agents showed

that the performance of pectin is critically dependent on the average molecular weight and the

distribution of molecular weights. For example, by studying the effect of chemical

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composition on the compressive mechanical properties of low-ester pectin gels it was

observed that preparations with molecular weight distributions possessing broad low-

molecular-weight tails yielded poor gelling properties (Kim, Rao, & Smit, 1978). A method

to increase pectin calcium sensitivity, while preserving its molecular weight that has been

evaluated, is enzymatic modification. The analysis conducted by Hotchkiss et al. (2002) on

the deesterification of citrus pectin with a purified salt-independent PME revealed no

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reduction in average molecular weight, in contrast to alkali deesterification that caused a

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rapid reduction of both molecular weight and intrinsic viscosity. The same major influence of

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molecular weight was not uncovered in emulsification experiments. By researching the

emulsification properties of citrus pectin Schmidt et al. (2015) reached the conclusion that
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pectins with a reduced molecular weight do neither significantly reduce droplet sizes nor

improve emulsion stability.

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The molecular weight distribution of pectins is generally determined by

chromatographic techniques. Early analytical methods were developed on citrus pectin

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samples and were based on the use of gel permeation chromatography (GPC) (Harding et al.,

1991; Berth et al., 1990). A more recent application of GPC to determine the molecular
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weight of a purified fraction of polysaccharide from mulberry leaves was described by Ying,

Han, & Li (2011). The researchers combined GPC with a HPLC instrument equipped with an
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Ultrahydrogel column. This method was later employed to determine the molecular weight of
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samples of citrus and apple pectin (Wang, Chen, & Lü, 2014). Other variations of the

chromatographic method involved the determination by high-performance size-exclusion

chromatography (HPSEC) equipped with two columns in series (Lim et al., 2012) or a

TSKGel column (Lira-Ortiz et al., 2014) and a refractive index detector (HPSEC-RID), high

performance size exclusion chromatography coupled to a multi-angle laser light scattering

detector (HPSEC-MALLS) (Jung & Wicker, 2014), high performance gel filtration
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chromatography with refractive index detector (Hua et al., 2018), and a HPLC system

equipped with a TSKgel column coupled on-line with three detectors – a differential

refractometer that measures the refractive index, a right-angle laser light-scattering detector,

and a differential viscometer detector (Combo et al., 2013). Of these chromatographic

methods, HPSEC coupled to MALLS or RID work well for pectin from various sources

(Kienteka et al., 2018; Liu et al., 2017; Karnik et al., 2016; Kang et al., 2015). A recent

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evaluation of the evaporative light scattering (ELS) and refractive index detectors coupled to

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HPSEC and comparison in terms of molecular weight estimation on commercial pectin

samples, in a wide range (0.342–805 kDa), led to the conclusion that HPSEC-ELS gives

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better results for low molecular weight compounds (Muñoz-Almagro et al., 2018).
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The average molar weight of pectin samples can also be estimated using the Mark-

Houwink equation (Eq. 1), as described by Arslan (1995):

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η = K × Ma (1)

where K (L∙g−1 ) and a are constants (K=0.0436 and a=0.78), M (g∙mol−1 ) is the
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molecular weight, and η (L∙g−1 ) is the intrinsic viscosity defined according to Eq. 2.

ηr-1
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[η]= lim C→0 ( ) (2)

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where ηr is relative viscosity and C (g∙L−1 ) is pectin concentration.

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Based on the Mark-Houwink equation and using a Cannon-Fenske capillary

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viscometer for the measurement, Venzon et al. (2015) determined the molecular weight of

modified pectin extracted from orange pomace, while Urias-Orona et al. (2010) applied the

relationship between intrinsic viscosity and molecular weight to characterize chickpea husk

pectin.
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5.5. Microscopic analysis

With the development of microscopic techniques more information about the structure

of the cell wall and its components could be collected. The visualization of pectin isolates

from various plant materials can provide data regarding the content of galacturonic acid and

the distribution of GalA residues, the position of glycosidic linkages in pectic chains, the

degree of esterification, the localization of esterified groups, the molecular weight

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distribution pattern, and profile of the neutral monosaccharides.

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In the particular case of fluorescence microscopy, the progress in the application of

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this technique prompted the increase of research on the use of fluorescent markers with

defined excitation and emission spectra as specific labels of cellular functions (Sila et al.,
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2009). When analyzing cell wall components, specific staining or immunolabeling techniques

are generally used. Van Der Veen & Van Den Ent (1994) applied immunolocalization with a
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fluorescent pectin probe, specific for a block of at least 16 subsequent homogalacturonic

units, and they showed that the middle lamella of Populus deltoids (eastern cottonwood)
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contains pectin polysaccharides. In the same study, staining with ruthenium red indicated the

presence of pectin in both ray parenchyma cell walls and the middle lamella area of Populus
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deltoids and Pinus sylvestris (Scots pine). With the introduction of JIM5 and JIM7

antibodies, research on the spatial distribution and the relative amount of acidic and
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methylated pectin present in various plant tissues was made possible (Casero & Knox, 1995;
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Knox et al., 1990). Immunocytochemical localization of pectin by JIM5 and JIM7

monoclonal antibodies has shown great enhancement of specificity in detection. This

conclusion was corroborated by Hafrén, Daniel, & Westermark (2000) who immunolocalized

HGs with low and high degrees of methyl esterification in the cambium, differentiating

xylem and mature xylem of Pinus sylvestris. Outside plant material, JIM5 antibody has been

successfully used for in situ localization of pectin in yogurt and model milk gels (Arltoft,
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Madsen, & Ipsen, 2007). A set of monoclonal antibodies (LM18, LM19, and LM20) with a

better defined HG epitope was later developed by Verhertbruggen et al. (2009).

Atomic force microscopy (AFM) is a powerful imaging technology with an increased

number of applications in the study of pectin isolates. Because it can produce subnanometer-

scale images of individual molecules, AFM has proved to be a useful tool for the

characterization of complex samples. By studying the images obtained following an AFM

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analysis of isolated polymers, some researchers reported on the structure and degree of

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branching of pectin. Notable studies include visualization of the structures of pectin

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molecules isolated from unripe tomato, which concluded in the finding that the complex

biopolymer consists of HGs held together by RG-I regions (Round et al., 2010). AFM was
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also used in the analysis of pectin isolated from sugar beet tissue, and it revealed the presence

of largely un-aggregated chains: a major fraction (67%) was polysaccharide-protein

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complexes containing a single protein molecule attached to one end of the polysaccharide

chains, and a small fraction (33%) of these was extended stiff polysaccharide chains (Kirby,
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MacDougall, & Morris, 2008). Besides information regarding the branching of pectin chains,

atomic force microscopy was successfully employed as a mean to distinguish between pectins
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from different peach cultivars (Yang et al., 2009).

Another technique that found great application in recent studies on pectin extracted
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from various plant materials is scanning electron microscopy (SEM). Liew, Chin, & Yusof
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(2014) used this microscopic technique to elucidate the morphological changes of pectin

samples which were extracted by an acidic extraction method from passion fruit peel, while

Zouambia et al. (2014) performed a SEM analysis on alcohol-insoluble solids before and

after extraction in order to visualize the effect of heating mode on the destruction of plant

tissue used for the extraction of pectin. By combining scanning electron microscopy with

atomic force microscopy, Zhongdong el al. (2006) researched the process of pectin extraction
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from orange skin assisted by microwave energy. Morphological analysis using scanning

electron microscopy was reported for samples of pectin from pomelo peel (Liew et al., 2016)

and raw and treated jackfruit peel (Moorthy et al., 2017), and also for biodegradable matrices

composed of a pectin network reinforced by a poly(lactide-co-glycolide) network (Liu et al.,

2004).

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5.6. Other methods

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More information regarding pectin structure (amorphous or crystalline) can be

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obtained by X-ray diffraction (XRD) analysis (Jiang et al., 2018; Sharma et al., 2015).

Similar to all polymers, pectin presents some degree of crystallinity, which has been defined
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as the fraction of a polymer that consists of regions showing three-dimensional order (Riley,

2012). Based on the peaks displayed on the diffraction patterns, Ponmurugan et al. (2017)
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observed a parallel crystalline nature (sharp and narrow diffraction peaks) in both commercial

pectin and pectin extracted from sunflower waste, while Kumar & Chauhan (2010) concluded
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that pectin extracted from the commercial Royal apple was more crystalline in nature than

pectin extracted from Golden apple variety. Another analytical technique that can be used to
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differentiate between pectin samples is gel electrophoresis. Polysaccharide analysis using

carbohydrate gel electrophoresis (PACE) is a method proposed for studying the blockwise or
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nonblockwise distribution of methylation of galacturonic acid residues by analyzing the

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endopolygalacturonase-digest products (Goubet et al., 2005; Goubet, Morriswood, & Dupree,

2003). Other analytical methods used to investigate the properties and behavior of pectin

samples are discussed below.

5.6.1. Thermal analysis

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Based on the fact that carbohydrates show first-order phase transitions (such as

melting and crystallization) and state transitions (such as gelatinization and glass transition),

thermal analysis proved to be a very useful and rapid screening method for the

characterization of pectin (Einhorn-Stoll, Kastner, & Senge, 2012; Roos, 2003). Thermal

analysis techniques, such as dielectric analysis, differential scanning calorimetry (DSC), and

thermogravimetry analysis (TGA), can provide insight into the physicochemical properties of

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the biopolymer. Lin, Yuen, & Varner (1991) used differential scanning calorimetry to study

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the phase transition of cell wall preparations, and they reached the conclusion that the mature

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region of soybean hypocotyls either has more calcium in the wall or has more methyl-

esterified pectin, which makes it less responsive to calcium addition. Using differential
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scanning calorimetry, thermogravimetry, and differential thermogravimetry (DTG) in a

combined simultaneous thermal analysis, Einhorn-Stoll, Kunzek, & Dongowski (2007) aimed
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to investigate the thermal behavior of highly methoxylated citrus pectins that were modified

chemically (demethoxylation and amidation) and mechanically (disaggregation). Their results

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showed that thermal behavior of pectin is considerably influenced by the physical state,

resulting both from different raw materials and modifications of the molecular structure
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(different substituents or decreasing molecular weight). In a later study Einhorn-Stoll &

Kunzek (2009) concluded that the characteristic degradation temperatures along with other
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parameters of thermal analysis give important information on the stability, homogeneity,

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molecular interactions, conformation, and conformational changes, the latter two assumed to

be detectable by thermal analysis to a certain extent.

Thermal analysis (DSC and TGA with DTG) was evaluated alongside other analytical

methods (chemical analysis, color measurement, SEM, and FT-IR spectroscopy) for its

potential to be used as a screening method for the characterization of changes occurring in

pectin during storage. For this purpose, Einhorn-Stoll, Kastner, & Drusch (2014) examined
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citrus pectin samples that were stored and then analyzed for alterations in molecular

parameters, surface morphology, color, as well as behavior in thermal analysis. The

combination of DSC and TGA proved to be a valuable instrument for the detection of

differences in stored pectins, as DTG peaks indicated two similar changes in pectin samples,

namely reduced homogeneity (mainly by depolymerization) and maximum degradation

velocity after storage. Other uses of DSC were to examine the effects of extraction

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temperature and raw material on the thermodynamic properties of pectin (Wang, Chen, & Lü,

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2014), and in combined TG-DSC to characterize and evaluate pectin and mefenamic acid

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films (Moreira et al., 2014).
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5.6.2. Rheological characterization

Aqueous solutions of pectin show pseudoplastic non-thixotropic behavior,

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independent of the degree of methoxylation, but directly dependent on the concentration. In

other words, the pseudoplasticity of pectin solutions decreases with decreasing concentration
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(Visser & Voragen, 1996). The linear relationship observed between shear stress and shear

rate of pectin solutions indicates Newtonian behavior, but only below a certain concentration
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(Fig. 4). According to the literature, at concentrations higher than 3% (v/v) pectin solutions

have non-Newtonian flow (Iagher, Reicher, & Ganter, 2002); however, it is important to be
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mentioned that the actual critical concentration at which the solution transforms from
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Newtonian to shear-thinning behavior depends on the molecular weight of pectin (Chan et al.,

2017).

Lira-Ortiz et al. (2014) studied the rheological properties alongside the chemical

characteristics of pectic polysaccharides extracted from the peel of prickly pear fruit (Opuntia

albicarpa Scheinvar ‘Reyna’) with the purpose to assess their potential as new food

hydrocolloids. The aqueous pectin systems in the range of 5-20 g/kg exhibited shear-thinning
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behavior over the shear rate range examined, while the viscosity values were higher when

compared to citrus pectin dispersion. The high viscosities and shear-thinning behavior

exhibited by the aqueous dispersions were mainly explained by the high molecular weight of

the polysaccharide. Moreover, the authors noted that the likely branched structure of prickly

pear fruit pectin, mainly formed by side chains of oligosaccharides, suggests that the shear

flow behavior of pectin in aqueous dispersions was due to increasing physical entanglements

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among chains as polymer concentration increased, which were then disrupted at higher

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shearing, thus yielding shear-thinning behavior. The influence of pectin chain branching was

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also confirmed by Sousa et al. (2015b) who studied the effects of controlled enzymatic

debranching on structural and rheological properties of HM citrus pectin, and the possible
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implications of RG-I side chains. They reported that for all the analyzed concentrations

debranched pectin solutions displayed lower viscosities. In the particular case of pomegranate
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Abid et al. (2017) observed that the rheological properties of peel gels result from a strong

synergism between fibrous material and pectins. Moreover, mechanical treatment of fibrous
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material suspensions was found to also significantly affect gel strength improvement,

probably due to the decrease of particle size and the heat induced by mechanical treatment.
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5.6.3. Functional properties

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Pectin is a valuable functional ingredient with food applications that include the use as
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gelling agent in jams and jellies, emulsifying agent in various applications such as flavor,

mineral, and vegetable oils emulsions, effective stabilizer in fruit juices and acidified milk

drinks, and as fat replacer in ice creams and spreads (Begum et al., 2017; Naqash et al.,

2017). Among pectins, sugar beet pectin has shown excellent emulsifying properties mainly

due to many factors such as the highly branched polysaccharide structures, high content of

hydrophobic acetyl groups on the GalA chain, and protein moiety, which plays an important
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role. Karnik & Wicker (2018) compared the stability of emulsions prepared with protein rich

and protein poor fractions of SBP by measuring the particle size, steady stress controlled

tests, and visual analysis using dark field microscopy, and observed that the protein poor

fraction, with smaller particle size, but higher DE and molecular weight was a more effective

emulsifier than the pectin fraction rich in protein. The emulsion stability was also studied by

Juttulapa et al. (2017) with the purpose of analyzing the effect of using high-pressure

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homogenization for the preparation of an emulsion containing pectin and zein. The use of this

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technique was based on the hypothesis that high-pressure homogenization can cause a

decrease of droplet size below 1 μm, improving the shelf-life of the emulsions by reducing

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the creaming rate. Although in the case of this study the droplet size decrease only slightly,
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probably due to the high oil content in the formulation, the emulsions stabilized by HMP-

zein, showed good stability and lower percent of creaming index.

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The gelling properties of pectin have been extensively studied along the years and are

the main focus of numerous investigations on the structure-function relationships of pectin in

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food. For HMP, which form gel structures through hydrogen bonding and hydrophobic

interactions, the gelation mechanism is promoted by low pH and water activity, as well as the
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presence of a high sucrose concentration. In positive influence of low pH on pectin gelation

was confirmed by Jiang et al. (2012) in a study on the properties of pectins extracted from
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Akebia trifoliate var. australis peel. Textural analysis (hardness, springiness, adhesiveness,
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chewiness, gumminess, cohesiveness, and resilience) performed on the pectin gels showed a

decrease of gelling properties with the decrease of pH from 4 to 2. In the case of LMP,

gelation is promoted by intrinsic factors such as high GalA content, molecular weight, and

DB, and is influenced by some extrinsic factors including pectin concentrations, temperature,

pH, type and concentration of soluble solids, and type and concentration of divalent cations

(Kastner, Einhorn-Stoll, & Drusch, 2017). Of the recent research on the formulation of LMP
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gels, the work of (X. Yang et al., 2018) aimed to explore the effects of a wide pH range (3.5-

9.5) on LM apple pectin gelation by analyzing the gel strengths, rheological properties,

thermal stabilities, and crystalline structures. The results of pectin gelation analysis indicated

an increase of gel strength and gelling rate with the increase of pH from 3.5 to 8.5, due to the

high amount of dissociated carboxylic groups, and a decrease of gel strength and gelling rate

for the pH increase to 9.5, attributed to the decreasing molecular weight of the pectin.

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Furthermore, it was noted that the incorporation of Ca 2+ into pectin caused a reduction of the

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thermal decomposition rate and the crystalline degree. For deesterified pectins, the

dependence of pectin gelation on pH and Ca 2+ was found to vary mainly with the change in

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molecular weight and degree of methylation (Hua et al., 2018).
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6. Concluding remarks and future perspectives
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The ubiquitous cell wall component pectin found applications as emulsifier, gelling,

thickening and stabilizing agent that go beyond the food industry and include various uses in
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the medical and pharmaceutical industries. The last few years have seen with an increase in
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pectin research carried out with the purpose to isolate pectin from various plant materials and
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to elucidate its structure in order to assign possible applications. A predominant focus of

recent research is to investigate the composition and the properties of pectin extracted from
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alternative sources that, in most part, seem to be represented by wastes of the fruit and
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vegetable processing industries. Based on the content of pectin and its composition, some of

the representative sources that have shown great potential for commercial production of

pectin are pomelo peel and the wastes from tomato and carrot processing, mostly those

generated by the canning industry. To understand their applicability in commercial pectin

production and to establish whether or not these can be considered sustainable pectin sources,

studies on a designed pilot scale process, such as those published by Andersen et al. (2017)
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and Casas-Orozco et al. (2015), are necessary. An important particularity that also needs to

be considered in future studies is the geographical distribution and the variability in the

quantity of these plant wastes.

The present work also reviewed the purification and fractionation techniques and the

methods used in the analysis of physicochemical properties, therefore giving complete insight

into the current state of research on all the particularities of pectin characterization.

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Concerning this matter, it is important to highlight that various techniques have been applied

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in the analysis of pectin, and therefore continuous improvement is expected to be made in the

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analytical methods for determining the composition and properties of these cell wall

polysaccharides, and particularly those that dictate its use in the food industry.
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domains from ginseng pectin. Carbohydrate Polymers, 79(4), 811–817.

AC

https://doi.org/10.1016/j.carbpol.2009.08.028

Zaidel, D. N. A., Chronakis, I. S., & Meyer, A. S. (2012). Enzyme catalyzed oxidative

gelation of sugar beet pectin: Kinetics and rheology. Food Hydrocolloids, 28(1), 130–

140. https://doi.org/10.1016/j.foodhyd.2011.12.015

Zaidel, D. N. A., & Meyer, A. S. (2012). Biocatalytic cross-linking of pectic polysaccharides

for designed food functionality: Structures, mechanisms, and reactions. Biocatalysis and
 ACCEPTED MANUSCRIPT

Agricultural Biotechnology, 1(3), 207–219. https://doi.org/10.1016/j.bcab.2012.03.007

Zhang, M., Wang, G., Lai, F., & Wu, H. (2016). Structural characterization and

immunomodulatory activity of a novel polysaccharide from Lepidium meyenii. Journal

of Agricultural and Food Chemistry, 64(9), 1921–1931.

https://doi.org/10.1021/acs.jafc.5b05610

Zhang, W., Xu, P., & Zhang, H. (2015). Pectin in cancer therapy: A review. Trends in Food

PT
Science & Technology, 44(2), 258–271. https://doi.org/10.1016/j.tifs.2015.04.001

RI
Zhang, Z., Lv, G., He, W., Shi, L., Pan, H., & Fan, L. (2013). Effects of extraction methods

SC
on the antioxidant activities of polysaccharides obtained from Flammulina velutipes.

Carbohydrate Polymers, 98(2), 1524–1531.

NU
https://doi.org/10.1016/j.carbpol.2013.07.052

Zhao, J., Zhang, F., Liu, X., St. Ange, K., Zhang, A., Li, Q., & Linhardt, R. J. (2017).
MA

Isolation of a lectin binding rhamnogalacturonan-I containing pectic polysaccharide

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ED

https://doi.org/10.1016/j.carbpol.2017.01.067

Zhongdong, L., Guohua, W., Yunchang, G., & Kennedy, J. F. (2006). Image study of pectin
T
EP

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C

Zouambia, Y., Youcef Ettoumi, K., Krea, M., & Moulai-Mostefa, N. (2017). A new approach
AC

for pectin extraction: Electromagnetic induction heating. Arabian Journal of Chemistry,

10(4), 480–487. https://doi.org/10.1016/j.arabjc.2014.11.011

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Table 1 Physicochemical properties of pectin: main sources of commercial pectin vs. other

sources

Plant source for Yield Galacatur Degree of Molecu Neutral Reference

pectin isolation of onic acid esterificat lar monosaccha s
pectin content ion weight ride content
recove
rya
Apple Granny 4.2% 58.6% 52.51% 331- 14.3-31.1% Constenla
pomace Smith (Granny (Royal 899 , Ponce,

PT
variety Smith) - variety) - kDa & Lozano
Royal 16.65 67.14% 76.4% (2002);
variety % (Royal (Granny Kumar &

RI
Golden 18.79 variety) Smith) Chauhan
variety % (2010);

SC
Pomace 19.8% Wikiera
utilized et al.
in (2016)
commer
NU
cial
producti
on of
MA

pectin
Citrus Lime 13.4- 68.88% 37.5% 342.7 1.33% Naghshin
peel peel 26.3% (mandarin (sour kDa (pomelo) - eh, Olsen,
Mandari 21.95 orange) - orange) - (lime) - 9.3% (lime) &
ED

n orange % 91.6% 82.2% 918 Ara, 0.1% Georgiou

peel (lime) (lime) kDa (lime) - (2013);
Orange 24.2% (pomel 0.16% Dominiak
T

peel o) (pomelo) et al.

Sour 29.1% Fuc, 1.64% (2014);
EP

orange (pomelo) - Methacan

peel 4.1% (lime) on,
Grapefru 27.34 Gal, 0.8% Krongsin,
C

it peel % (mandarin &

orange) - Gamonpil
AC

Pomelo 27.63-
pectin 37.52 3.25% as (2014);
% (pomelo) Wang,
Glc, 0.62% Chen, &
(mandarin Lü
orange) - (2014);
1.5% (lime) Boukrouf
Rha, 0.18% a et al.
(mandarin (2015);
orange) - Wang et
0.7% (lime) al.
Xyl (2015);
Hosseini,
Khodaiya
 ACCEPTED MANUSCRIPT

n, &
Yarmand
(2016);
Liew et
al. (2016)

Sugar 23- 72.4% DM=52% 311 24.3% Li et al.

beet pulp 24.87 , kDa 6.4% Ara, (2015);
% DAc=28. 13.2% Gal, Chen, Fu,
1% 0.6% Glc, & Luo
4.4% Rha, (2015);

PT
0.2% Xyl Guo et al.
(2016)
Tomato Tomato 7.55% 78.4% 76.92- n.d. 2.9% Ara, Del

RI
waste pomace (CBP) 88.98% 3.85% Gal, Valle,
Dried 32.6% 0.7% Glc, Cámara,

SC
tomato 3.8% Man, & Torija
peel 1.4% Rha, (2006);
2.3% Xyl Grassino
et al.
NU
(2016);
Müller-
Maatsch
MA

et al.
(2016)

Carrot Rejected 8.9% 62-69% 53-77% 114 8-11.9% Christiae

ED

waste carrots (WSP) kDa Ara, 0.12- ns et al.

Carrot 5- (rejecte 0.18% Fuc, (2015);
pomace 15.2% d 13-24.4% Jafari et
T

Carrot 9% carrots) Gal, 40.9- al. (2017)

steam – 1460 50.6% Glc,
EP

peels kDa 1.6-1.9%

(carrot Man, 1.7-
steam 3.2% Rha,
C

peels) 0.3% Xyl

(in WSP)
AC

Waterme 19- 68.7% DM: 3.451 × 0.6-0.7% Banerjee

lon rinds 21% (LW) - 61.5% 104 Ara, 0.1- et al.
74.2% (LW) - g/mol 0.2% Fuc, (2017);
(FW) 63% (FW), 20.2-22.6% Petkowic
(FW) 4.039 × Gal, 1.4-4% z,
104 Glc, 0.4- Vriesman
g/mol 0.6% Man, n, &
(LW) 2.4-2.9% Williams
Rha, 0.5% (2017)
Xyl
Mango 17.15 29.35- DM: 378.4- n.d. Wang et
peel % 53.35% 85.43- 2858 al. (2016)
88.38% kDa
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Passion 10- 66.65- DE=60.3 n.d. n.d. Kliemann

fruit peel 12.67 68.7% 6%, et al.
% DM=45.9 (2009);
4% Freitas de
Oliveira
et al.
(2016)
Banana 9% 40.2- DM: 49- 87-248 1-5.3% Ara, Happi
peel 71.8% 80%, kDa 1.6-5.7% Emaga et
DAc: 1.2- Gal, 0.1- al.
5.7% 0.24% Rha (2008);

PT
Maran et
al. (2017)
Pumpkin 7.4% 63.3- DM=18% 139- 4.1-4.6% Košťálov

RI
waste 73.8% , 289 Ara, 0.4- á,
DAc=3% kDa 0.6% Fuc, Aguedo,

SC
7.2-9% Gal, &
6.6-11.2% Hromádk
Glc, 0.8% ová
Man, 4.2- (2016);
NU
4.6% Rha, Müller-
0.9-4.3% Maatsch
Xyl et al.
MA

(2016)
a
on a dry weight basis
ED

n.d. – not determined, WSP – water soluble pectin, FW – fresh watermelon rinds, LW –

lyophilized watermelon rinds, CBP - calcium-bound pectin

T
C EP
AC
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Table 2 Extraction techniques and their effects on pectin yield and quality

Extraction Plant source Operating Effect on yield and References

technique conditions quality
Solvent Cocoa husks Solvent: water, The highest pectin Chan & Choo
extraction citric acid or yield (7.62%) was (2013)
hydrochloric acid obtained using
pH: 2.5 or 4.0 citric acid (pH 2.5,
Temperature: 50 or 95°C, 3.0 h), while
95°C the highest uronic
Time: 1.5 or 3.0 h acid content in

PT
pectin (65.20%)
resulted by using
water (95°C, 3.0

RI
h).
Extraction with

SC
citric acid
produced pectin
with a wider DM
range.
NU
Sour orange Solvent: water The yield of pectin Hosseini,
peel Liquid/solid ratio: extracted at the Khodaiyan, &
20:1, 30:1 or 40:1 optimal condition Yarmand
MA

(v/w) (95°C, 90 min, and (2016)

Temperature: 75, liquid/solid ratio
85 or 95°C of 25:1) was
Time: 30, 60 or 90 18.35%. GalA
ED

min content and DE of

the extracted
pectin ranged from
T

57% to 83%, and

17-30.5%,
EP

respectively.
Durian rinds Solvent: water Under optimal Maran (2015)
Solid/liquid ratio: conditions
C

1:5-1:15 (g/mL) (solid/liquid ratio

pH: 2-3 of 1:10 g/mL, pH
AC

Temperature: 75- of 2.8, 43 min,

95°C 86°C) the pectin
Time: 20-60 min yield reached
9.1%.
Lime peel Solvent: Higher pectin Andersen et al.
water:nitric acid solution (2017)
pH: 1.5, 2.3 or 3.1 concentrations
Temperature: 60, were obtained at
70 or 80°C the lower pH
values. Increased
temperature and
especially acidity
caused a faster
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decrease of DE,
effect that was
particularly
significant during
extractions at
pH=1.5.
Enzyme- Apple pomace Enzymes: Polygalacturonic Wikiera et al.
a
assisted xylanase and acid was (2015b)
extraction multicatalytic completely
preparation hydrolyzed after
Celluclast 2.5 h incubation

PT
Acid used in with 2 M TFA at
extraction: 120°C. Prolonged
trifluoroacetic acid extraction was

RI
2M positively
Temperature: 100 correlated with an

SC
or 120°C increase of GalA
Time: 1, 1.5, 2, released.
2.5, 3 or 4 h Efficient release of
neutral sugars was
NU
performed at
100°C for 2.5 h.
Apple pomace Enzymes: endo- Treatment with Wikiera et al.
MA

xylanase and endo- endo-xylanase (2016)

cellulaseb resulted in the
Solid/liquid ratio: highest pectin
1 g/15 mL yield (19.8%) and
ED

Enzyme dose: 50 very high DM

U/g (73.4%). Pectin
pH: 5.0 extracted by endo-
T

Temperature: 40°C cellulase treatment

Time: 10 h was characterized
EP

by the high GalA

content (70.5%).
Simultaneous use
C

of both enzymatic
preparations
AC

resulted in a 10.2%
extraction yield,
and a pectin rich in
galacturonic acid
(74.7%).
Rapeseed cake Commercial The optimized Jeong et al.
enzymes: conditions for an (2014)
Celluclast and improved pectin
Alcalase yield (6.85%)
Enzyme/rapeseed without significant
cake ratio: 1:50 to loss of GalA were
1:65 (v/w) a 1:50
Celluclast/Alcalase enzyme/RSC
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ratio: 0:5, 1:4, 2:3, ration with a

3:2, 4:1 or 5:0 Celluclast/Alcalase
(v/v) ratio of 1:4 for a
Time: 90, 180, 270 min
270, 360 or 450 hydrolysis time.
min Enzymes indicated
different functions:
Alcalase led to the
destruction of
protein-
carbohydrate

PT
complexes, while
Celluclast slightly
cleaved some

RI
linkages of
carbohydrate.

SC
Lime peel Enzymes: Laminex C2K Dominiak et al.
Laminex C2K, preparation proved (2014)
Multifect B, to the most
GC220, and effective, as in
NU
GC880 optimum
pH: 3.5-6.5 conditions (4 h
Temperature: 40- treatment at pH
MA

70°C 3.5, 50°C) gave a

high yield (23%)
and a pectin with
good composition
ED

and properties
(gelling,
stabilization).
T

Ultrasound- Passion fruit Extraction solvent: The highest pectin Freitas de

assisted peel 1.0 mol/L HNO 3 , yield (12.67%) Oliveira et al.
EP

extraction pH 2.0, was obtained at (2016)

peel/solvent ratio 85°C and a power
of 1:30 (g/mL) intensity of 664
C

Temperature: 45- W/cm2 . Despite

85°C the fact that pectin
AC

Power intensity: isolation reached

132.8-664.0 the highest level,
W/cm2 the isolate did not
Time and displayed the best
frequency composition, as
(constant): 10 min, the GalA content
20 kHz and DE showed
minimum values.
Mango peel Extraction solvent: Extraction yield of Wang et al.
citric acid, pH 2.5, pectins varied (2016)
peel/solvent ratio greatly with the
of 1:40 (g/mL) increase in
Time and temperature, from
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frequency 2.09% (at 20°C) to

(constant): 15 min, 17.15% (at 85°C).
20 kHz A significant
Temperature: 20 or influence of
85°C temperature was
also observed for
GalA content
(increase from
29.35% to
53.35%) and
molecular weight

PT
(increase from
(378.4 kDa to
2320 kDa).

RI
Jack fruit peel Extraction solvent: Optimal conditions Moorthy et al.
distilled water) for the extraction (2017)

SC
Liquid-solid ratio: were: liquid-solid
10:1-20:1 (mL/g) ratio of 15:1 mL/g,
pH: 1-2 pH of 1.6,
Sonication time: sonication time of
NU
15-30 min 24 min, and
Extraction temperature of
temperature: 50- 60°C. Under this
MA

70°C conditions the

pectin yield was
14.5%
Grapefruit Emitter surface: 13 Heating Xu et al.
ED

peel mm or 25 mm significantly (2014)

Power density: improved the
0.20, 0.27, 0.33, extractability and
T

0.40, 0.47 or 0.53 extraction rate of

W/mL pectin, leading to
EP

Duty cycle: 33%, higher yield

40%, 50%, 60%, (26.74%) in
70% or 80% shorter extraction
C

Temperature: 30, time (51.79 min).

40, 50, 60, 70 or The optimized
AC

80°C parameters were:

Solid-liquid ratio: ultrasound power
1/30, 1/40, 1/50, density 0.40
1/60 or 1/70 W/mL, duty cycle
(g/mL) 50%, temperature
Sonication time: 60°C, S/L 1/50
10, 20, 30, 40, 50 g/mL.
or 60 min
Microwave- Waste papaya Microwave power: All the process Maran &
assisted peel 320, 480 or 640 W variables had Prakash (2015)
extraction pH: 1, 2 or 3 significant effect
Time: 20, 100 or on pectin yield.
180 s The optimal
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Solid-liquid ratio: conditions for

1:5, 1:15 or 1:25 reaching a
g/mL maximum pectin
yield (25.41%)
were: microwave
power of 512 W,
pH of 1.8, time of
140 s and solid-
liquid ratio of 1:15
g/mL.
Waste mango Microwave power: For all process Maran et al.

PT
peel 160, 320 or 480 W parameters a (2015)
pH: 2, 3 or 4 similar influence
Time: 60, 120 or was observed: the

RI
180 s increase in their
Solid-liquid ratio: level was

SC
1:10, 1:20 or 1:30 positively
g/mL correlated with the
pectin yield up to a
certain point,
NU
beyond which their
effect on pectin
extraction was
MA

negative.
The maximum
pectin yield
(28.86%) could be
ED

obtained at a
microwave power
of 413 W, pH of
2.7, time of 134 s
T

and solid-liquid
EP

ratio of 1:18 g/mL.

Tangerine Microwave power: The optimal Chen et al.
peels 600, 700 or 800 W extraction (2016)
C

Temperature: 40, parameters were:

50 or 60°C microwave power
AC

Time: 30, 40 or 50 704 W, 52.2°C

s extraction
temperature, and
extraction time of
41.8 min. Under
these conditions
the experimental
yield of pectin was
19.9±0.2%.
Opuntia ficus Microwave power: The optimum Lefsih et al.
indica 200, 400 or 600 W conditions to (2017)
cladodes pH: 1.5, 2.25 or 3 obtain a maximum
Time: 1, 2 or 3 pectin recovery of
 ACCEPTED MANUSCRIPT

min 12.56% were 2.16

Solid-liquid ratio: min, pH 2.26, 517
2:20, 2:35 or 2:50 W microwave
g/mL power and 2
g/30.66 mL of
solid-liquid ratio.
FTIR analysis
indicated a GalA
content of 34.4%
and no alterations
in the chemical

PT
structure of pectin
following
microwave

RI
treatment.
Subcritical Apple pomace Solid to liquid The highest yield Wang, Chen,

SC
water and citrus peel ratio of 1:30 g/mL, (21.95%) of citrus & Lü (2014)
extraction extraction time of pectin (68.88%
5 min (constant) GalA content) was
Extraction obtained at 120°C,
NU
temperature: and the highest
130°C, 150°C or yield of apple
170°C C for apple pectin (16.68%)
MA

pomace; 100°C, was gained at

120°C or 140°C 150°C (GalA
for citrus peel content of
40.13%).
ED

Differential
scanning
calorimetry
analysis showed
T

that the
EP

endothermic
property of pectin
was affected by
C

extraction
temperature while
AC

the exothermic
property was only
affected by its
constituents and
raw material.
Sugar beet Temperature: Increase in all Chen, Fu, &
pulp 110°C, 120°C or parameters Luo (2015)
130°C enhanced the
Extraction time: extraction up to a
20, 30 or 40 min certain level past
Liquid/solid ratio: which a decrease
30, 40 or 50 (w/w) of pectin recovery
Extraction was recorded.
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pressure: 8, 10 or The optimum

12 MPa extraction
conditions to
obtain a maximum
yield of 24.63%
(with 59.12%
GalA and 21.66%
arabinose content)
were as follows:
L/S ratio of 44.03,
120.72°C

PT
extraction
temperature,
extraction time of

RI
30.49 min and
extraction pressure

SC
of 10.70 MPa.
Induced Orange peel Solvent: diluted An increase in Yang et al.
electric field- waste hydrochloric excitation voltage (2016)
assisted Acid caused
NU
extraction pH: 2, 3 or 6.8 enhancement of
Excitation voltage: pectin yield,
0-300 V opposite to the
MA

Frequency: 20-200 increase in

kHz frequency that had
Temperature: 20- a negative impact
80°C on the extraction.
ED

Lower pH also
provided a higher
pectin yield.
The electrical
T

effect was found to

EP

predominate at
temperatures
below 45°C, and
C

the joint electrical

and thermal effects
AC

governed the
extraction at
temperatures from
45 to 65°C.
a
- EC 3.2.1.8, b- EC 3.2.1.4
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Table 3 Methods of pectin purification: comparative view and new approaches

Method Procedure Effect on extract References

Alcohol precipitation A first washing of The technique gave a Yapo et al. (2007b)
with washing crude pectin with higher pectin yield,
60% (twice), 70% and the isolate
(twice) and 80% contained more
ethanol, followed neutral sugars,
by a final washing proteins, and ash but
step with 96% had a galacturonic
ethanol (twice). acid content lower

PT
The final gel was than a pectin sample
recovered in purified by
water, freeze- ultrafiltration.

RI
dried, vacuum-
dried at 40°C.

SC
Stepwise ethanol- Precipitation with Pectin fractions rich Guo et al. (2016)
precipitation an equal volume in neutral sugars were
of ethanol in precipitated at
elevated relatively high ethanol
NU
concentration from concentrations.
50% to 80% (v/v) The stepwise
followed by precipitation is more
MA

centrifugation, selective with respect

ethanol washing to pectin structural
and lyophilization. features and surface
properties than a one-
ED

step process.
Ultrafiltration in Purification was Pectin isolated from Yapo et al. (2007b)
combination with performed using the crude aqueous
T

diafiltration hollow fiber extract by the 10kD

tangential flow membrane procedure
EP

filtration contained more GalA

membranes and less neutral
(polysulfone, 0.5 sugars, suggesting a
C

mm i.d, 615 cm2 ) higher purity of the

of 10 and 50 kD pectin extract.
AC

MWCOs.
Ultrafiltration The ultrafiltration Most of the salts and Kang et al. (2015)
was conducted pigments were
under 15 bar at removed from the
40°C using a sample which also
membrane with a had a low protein and
surface area of acid-insoluble ash
1.77 m2 and a content.
molecular weight
cut-off of 8000
Da.
Metal precipitation Precipitation of Pectin purified Yapo (2009)
the extract with a through this method
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solution of had a higher

CuSO 4 ∙5H2 O galacturonic acid
followed by content and a low
centrifugation, neutral sugars, protein
filtration and and ash content.
washing with HCl
and ethanol to
eliminate Cu2+
ions.
Copper precipitation Precipitation of Significantly higher Guo et al. (2015)
pectin with yield and GalA

PT
aqueous CuSO 4 content and lower
completed with a neutral sugar and
dispersion of protein content by

RI
EDTA solution in comparison to the un-
the filtration cloth precipitated sample.

SC
for the extraction
of copper ions
from the copper-
pectin complexes.
NU
Purification using Precipitation takes Whatever the Happi Emaga et al.
protein (sodium place after the precipitation (2012)
caseinate) addition of conditions, the purity
MA

caseinate (pH 3.5; of extracts, expressed

10 g/l); to separate as GalA content, was
pectins from lower in pectin
sodium caseinate purified by caseinate
ED

the pH is increased than in that purified

to 6.5 by adding by ethanol (96%)
1M NaOH, NaCl precipitation.
T

is added after the However, pectins

dissolution of the purified by caseinates
EP

pellet, followed by are characterized by a

0.1M HCl to lower total content of
decrease pH to 4.6 neutral sugars.
C

(precipitation of
caseinate).
AC
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Table 4 Analytical methods for pectin characterization

Parameter Analytical method References

Monosaccharide composition
Galacturonic acid content
Hydrolysis, derivatization
(alditol acetates) and
quantification by GC and
mass spectrometric detection
TFA hydrolysis, HPAEC-
PAD assay
Hydrolysis, carbazole-H2 SO 4
reaction and
Blakeney et al. (1983);
Müller-Maatsch et al.
(2016); Colodel &
Petkowicz (2018)
Kang et al. (2015); Nagel et
al. (2017); Guo et al. (2018)
Dische (1947); Wang et al.
(2017); Jouini et al. (2018)

PT
spectrophotometric detection
Hydrolysis, m- Blumenkrantz & Asboe-
hydroxydiphenyl sulfuric acid Hansen (1973); Abid et al.

RI
assay and spectrophotometric (2017); Chaharbaghi,
detection Khodaiyan, & Hosseini

SC
(2017); Vasco-Correa &
Zapata Zapata (2017)
Combined TFA-enzymatic Garna et al. (2006); Burana-
NU
hydrolysis, HPAEC-PAD osot et al. (2010)
assay;
TFA hydrolysis and analysis
by HPAEC-FID
MA

Glycosidic linkage Methylation- induced cleavage Sims & Bacic (1995); Wu et

conformation of the glycosidic linkages, al. (2015); Zhang et al.
conversion to PMAAs, GC- (2016); Wang et al. (2017)
MS assay
ED

Methylation, identification of Lin et al. (2016); Georgiev et

glycosidic linkage and side al. (2017); John et al. (2018)
chain location by 1D and 2D
T

NMR
Degree of substitution Hydrolysis and acidification Savary & Nuñez (2003);
EP

of pectin, headspace solid- Yoo et al. (2012)

phase microextraction
(Carboxen-PDMS fiber
C

assembly), separation of
AC

methanol and acetic acid by

GC and detection using
electron impact MS with
selected ion monitoring
Hydrolysis of pectin in Müller-Maatsch et al.
NaOH/D2 O, direct 1 H NMR (2014); Grassino et al.
analysis (2016)
Simultaneous determination Synytsya et al. (2003);
of methylation and Kyomugasho et al. (2015)
acetylation degree of pectin
by FT-IR;
Analysis of the degree of
methyl-esterification by FT-
IR with peak deconvolution
 ACCEPTED MANUSCRIPT

for protein-rich samples

Determination of methylation Synytsya et al. (2003);

and acetylation degree by FT- Kumar & Chauhan (2010)
Raman
Detection using a µSI-LOV Naghshineh et al. (2016)

PT
system (includes a VIS–NIR-
diode array
spectrophotometer) with

RI
ultra-pure deionized water as
carrier

SC
Degree of blockiness Pectin digestion, separation Daas et al. (1999); Daas,
and quantification using Voragen, & Schols (2000);
HPAEC-PAD at pH 5 Sousa et al. (2015b)
Determination by capillary Guillotin et al. (2007)
NU
electrophoresis using
phosphate as buffer in an
automatic system equipped
MA

with a UV detector
Molecular weight High performance size Lira-Ortiz et al. (2014); Jung
exclusion chromatography & Wicker (2014)
with refractive index detector
ED

(HPSEC-RID) and a TSK-Gel

column;
High performance size
T

exclusion chromatography
coupled to a multi-angle laser
EP

light scattering detector

(HPSEC-MALLS)
Microscopic analysis In situ analysis of pectin Hafrén, Daniel, &
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structure/location of specific Westermark (2000); Arltoft,

domains by fluorescence Madsen, & Ipsen (2007)
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microscopy combined with

immunolocalisation by
monoclonal antibodies JIM5
and JIM7
AFM for the analysis of
specific regions of pectin
following acid hydrolysis
Analysis of morphological
changes/optical sectioning by
SEM
Other methods Analysis of pectin structure
(amorphous or crystalline) by
XRD performed on solid
Kirby, MacDougall, &
Morris (2008); Round et al.
(2010)
Liew, Chin, & Yusof (2014);
Zouambia et al. (2014)
Kumar & Chauhan (2010);
Sharma et al. (2015);
Ponmurugan et al. (2017);
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samples (powder form) Jiang et al. (2018)

Enzymatic digestion of
pectin, analysis of the
blockwise/nonblockwise
distribution of methylation of
GalA by PACE
Goubet, Morriswood, &
Dupree (2003); Goubet et al.
(2005)

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Highlights

 Recently, an increasing number of studies proposed new sources for pectin extraction.
 The potential of these plant wastes needs to be reviewed in order to establish sustainability.
 Purification and fractionation of extracted pectin are essential steps of the isolation process.
 An overview of the methods used for the analysis of pectin composition and properties is
provided.

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