Extraction, purification and characterization of pectin from alternative sources with potential technological applications

Accepted Manuscript Extraction, purification and characterization of pectin from alternative sources with potential technological applications

Florina Dranca, Mircea Oroian PII: DOI: Reference:

S0963-9969(18)30519-2 doi:10.1016/j.foodres.2018.06.065 FRIN 7732

To appear in:

Food Research International

Received date: Revised date: Accepted date:

20 February 2018 25 June 2018 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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ACCEPTED MANUSCRIPT Extraction, purification and characterization of pectin from alternative sources with potential technological applications

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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: [email protected] (Florina DRANCA)

ACCEPTED MANUSCRIPT 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

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the development of applications beyond the food industry have brought about its increase in production and influenced research towards alternative sources and improving the overall

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

materials

(purification

and

fractionation),

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recovery

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

ACCEPTED MANUSCRIPT 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

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particularly important as a source of dietary carbohydrates, providing almost all of the carbohydrate intake, and therefore much of the energy in the adult diet (Lovegrove et al.,

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

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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 they cover a great variety of biological functions and chemical structures (Kumar et al.,

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

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cellulose, hemicelluloses, and pectins.

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). Pectic polysaccharides are vital structural components of plant cell walls, and they are often 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

ACCEPTED MANUSCRIPT (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

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

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

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

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

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

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demanded. One other main objective of this review is to assess the methods applied for the purification and fractionation of the extracted pectin prior to analyzing its composition and 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.

ACCEPTED MANUSCRIPT 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

walls in three layers,

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presented by Preston (1975) and Clarke & Knox (1979), who described the deposition of cell primary wall, middle lamella, and secondary cell wall. An

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

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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 & Roelofsen, 1954;Thimann & Bonner, 1933). Because of their multiple interaction properties,

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

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chemistry of pectin, as its structural complexity imparts unique and diverse physical and biochemical properties. The chemistry, biosynthesis and functions of pectin have been

knowledge.

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reviewed multiple times recently, and therefore this section will be a summary of current

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

ACCEPTED MANUSCRIPT 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

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(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 &

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

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

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

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walls: homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) (Christiaens et al., 2016). The unsubtituted HGs are linear homopolymers consisting

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

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

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attached to O-4 of the L-rhamnosyl residues (Willats et al., 2001; Lau et al., 1985; Darvill et al., 1980). Studies of the branched RG-I regions of apple pectin resulted in the isolation of 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-Omethylxylose, and 2-O-methylfucose (Darvill, McNeil, & Albersheim, 1978).

ACCEPTED MANUSCRIPT 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 &

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

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

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

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

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similar type I rhamnogalacturonan backbone with branches of β-1,4-D-galactan side chains occasionally substituted with α-L-Araf was identified in pumpkin residue (Zhao et al., 2017).

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When compared to RG-I isolates from pumpkin residue, ginseng pectin displayed a greater variety of RG-I domains. From ginseng pectin Yu et al. (2010) isolated five RG-I domains, 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

ACCEPTED MANUSCRIPT 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 – highmethoxyl 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).

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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 established between pectic polysaccharides and the interconnections of pectin molecules with

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

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and intercellular adhesion. A number of covalent and non-covalent interactions, which include ionic bridges, ferulic acid linkages, borate esters and uronyl esters, are considered to

mechanisms,

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

and

the macromolecular functionality of the pectic

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

their

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the physical and macromolecular properties of plant materials and, consequently, it impacts processing

reactions

and

(enzymatic

polysaccharides,

are

quality.

and

Alongside cross-linking,

non-enzymatic),

considered

key

and

mechanisms

the that

pectin biosynthesis,

conversion

solubility

of

dictate

property the

pectic

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

ACCEPTED MANUSCRIPT 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

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

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

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

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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 high sugar content (>55%) (Kastner et al., 2014). On the other side, the gelation process of LMP (DM<50%) is governed by the cross-linking of HG chains via Ca2+ bridges, occurring

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after the addition of calcium ions to the pectin solution (Han et al., 2017). All the applications of pectin in the industry begin with the isolation of pectin from the plant material. At the 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

ACCEPTED MANUSCRIPT (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

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

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

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

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

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

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Citrus fruits are particularly rich in pectin and the large quantities that are processed and, consequently, the considerable amounts of wastes generated, have become the main 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, &

ACCEPTED MANUSCRIPT 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

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

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

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

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

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

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increase in pectin yield was correlated with increased temperature, extended extraction time, and reduced pH of the extracting solution. By studying the emulsifying properties it was 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

ACCEPTED MANUSCRIPT 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

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

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

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

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

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

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(around 82%) the extracted pectin was categorized as HMP. Two observations made by the authors are of particular relevance for the commercial production of pectin from tomato 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 2225% of the total dietary fiber (29.6%) (Stabnikova, Wang, & Ivanov, 2010), a lower content

ACCEPTED MANUSCRIPT 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

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

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

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

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

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

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level of 19-21% (w/w) pectin was determined (Banerjee et al., 2017). Petkowicz, Vriesmann, & Williams (2017) investigated the chemistry and rheological and emulsifying properties of 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

ACCEPTED MANUSCRIPT 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

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

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

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

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

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

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complete survey of pectic materials obtained from many diverse by-products belongs to Müller-Maatsch et al. (2016). The researchers selected 26 food waste streams according to 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

ACCEPTED MANUSCRIPT 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

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

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waste, pumpkin waste, and watermelon rinds.

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

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

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

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

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also greatly influenced by the choice of extraction technique and the purification method.

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

ACCEPTED MANUSCRIPT 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

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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

Pectin extraction is defined as a physical-chemical process which is comprised of

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

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

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

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extraction and isolation of pectin from cell walls can be approached in various ways through the use of chemical, physical, as well as enzymatic treatments (Panouillé, Thibault, & 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

ACCEPTED MANUSCRIPT 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.

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

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

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

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

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

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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

These differences in yield and pectin quality between acids can be explained by 2 separate phenomena: (a) when combined to increased temperatures and prolonged extraction time,

NU

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

mineral

acids,

under

identical

MA

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

conditions

(Maneerat,

Tangsuphoom,

&

ED

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

EP T

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

AC C

Pectin extraction from fruit and vegetable residues using weak organic acids has been intensively studied, suggesting that numerous data regarding the influence of operating 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

(2016) who evaluated the influence of pH (2-4), temperature (70-90 °C), and time (40-150 min) on pectin extraction from pomegranate peels with citric acid. Based on the results, it

NU

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

MA

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

ED

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

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

AC C

achieve a maximum yield and the desired characteristics of pectin. Numerous studies were aimed to optimize the process variables in the extraction of pectin from wastes such as sour 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.

ACCEPTED MANUSCRIPT 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,

PT

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

RI

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

SC

pectin production by Adetunji et al. (2017), who covered all the particularities from principles and operational issues to benefits and drawbacks. Important results regarding the

NU

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

MA

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

ED

summarized in Table 2. The potential of ultrasound-assisted extraction (UAE) of pectin was studied for plant sources such as from passion fruit peel (Freitas de Oliveira et al., 2016),

EP T

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

AC C

simultaneous effect of temperature and ultrasound treatment it is crucial to consider that ultrasounds cause a disintegration of the material, consequently affecting the separation of 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.

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

(Fishman et al., 2013) brought notable contribution to the knowledge regarding the use of microwaves in the extraction of pectin from plant materials. Some research has been

NU

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

MA

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

ED

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

EP T

sequential ultrasound-microwave-assisted extraction, Liew et al. (2016) reported that the combined extraction technique resulted in a higher GalA content because it allows a more

AC C

complete release of the pectin compound and therefore a better preservation of the GalA distribution from deeper plant matrix than a sole ultrasound or microwave extraction. The 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

result, different phenomena, including intracellular liquid release and diffusion of solutes, develop inside the cellular structure following the treatment (Vorobiev & Lebovka, 2009).

NU

Therefore, utilization of an electrical driving force as auxiliary energy leads to substantial improvements of mass transfer and extraction efficiency (Yamini, Seidi, & Rezazadeh,

MA

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

ED

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

EP T

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

AC C

closed loop. Through this setup the induced electric field in the system appears to be under the influence of alternating magnetic flux. Table 2 presents data regarding the investigated 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

noted that the ethanol concentration required for purification was dependent on pectin structure, and particularly the proportion of neutral side chains. Based on their results, the

NU

researchers indicated that at least a concentration of 75% ethanol is recommended to obtain a satisfactory yield of purified sugar beet pectin. However, the purity of pectin obtained only

MA

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

ED

compared alcoholic precipitation to the purification of pectin by ultrafiltration-diafiltration (Table 3) and reported that the use of the first technique resulted in more neutral sugars, more

EP T

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

AC C

(Kang et al., 2015), while metal ion-binding precipitation proved to be selective toward binding HG and RG regions and thereby is suitable to purify pectins from non-uronide 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

ACCEPTED MANUSCRIPT 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 The purification is based on the

PT

polysaccharides using protein (sodium caseinate).

RI

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

SC

steps: a first step where the charged pectins are precipitated using proteins at pH 3.5, and a second step in which the polymers are separated by the dissociation of the pectin-caseinate

NU

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

MA

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

ED

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

EP T

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

AC C

is substantially overshadowed by the drawback of requiring a large quantity of protein. When the objective of the research is to fully elucidate the composition of the 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

significant antioxidant activity and thymocyte and splenocyte activation. The beneficial effects of separate structures from the composition of pectin polysaccharides was also

NU

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

MA

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

ED

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

EP T

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

AC C

Adopting the high-throughput and fractionation techniques used by Nguema-Ona et al. (2012) to profile the different wall polymers present in the leaves of Nicotiana tabacum, 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

structure and

SC

pectin is polymolecular and polydisperse, meaning it is heterogeneous in both chemical molecular weight (BeMiller, 1986). In regards to chemical structure,

NU

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

MA

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

ED

polysaccharides. The variability in the structure of pectin chains, including the number of

EP T

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

AC C

Given the complexity of the multiblock pectin biopolymer, the analysis of the extracted whole macromolecule does not suffice in giving insight into the fine structure of 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.

ACCEPTED MANUSCRIPT

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

PT

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

RI

Regarding the effect of the neutral monosaccharides composition on the technological

SC

applications of pectin, research has shown a positive impact of this chemical parameter on the formation of pectin gels. A representative study focused on this matter is the research

NU

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

MA

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

ED

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

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In order to determine the neutral monosaccharides content, the polysaccharides must be first depolymerized into their constituent sugar residues (uronic acid and neutral

AC C

monosaccharides), which is commonly done by chemical or enzymatic hydrolysis. Most of the methods available for the determination of neutral monosaccharides involve the use of 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

ACCEPTED MANUSCRIPT 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

PT

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

RI

In several studies, high performance anion exchange chromatography with pulsed

SC

amperometric detection (HPAEC-PAD) was used to examine the sugar composition of pectin (Guo et al., 2018; Nagel et al., 2017; Christiaens et al., 2015; Kang et al., 2015). Kang et al.

NU

(2015) reported on the use of ultrafiltration prior to quantification, in order to remove impurities such as pigments, salts, acids, and saccharides from the extracted pectin sample.

MA

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

ED

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

EP T

chromatographic analysis preceded by a combined acid hydrolysis and methanolysis is a convenient method for obtaining the composition of monosaccharides. Another technique

AC C

examined for the determination of monosaccharides present in pectin was centrifugal partition chromatography. Ward et al. (2015) evaluated the efficiency of a highly polar twophase 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.

ACCEPTED MANUSCRIPT

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

PT

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

RI

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

other

method

requires

different

types

of

SC

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

activities

such

as

pectolytic,

NU

hemicellulolytic, and carbohydratases for an efficient degradation of pectin. Despite this drawback, enzymatic hydrolysis is considered a better technique for the hydrolysis of pectin.

galacturonic

colorimetric

acid.

Among

methods

are

available

MA

Several

spectrophotometric

for

analytical

the

quantitative

techniques,

analysis

of

carbazole-H2 SO4

ED

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

EP T

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

AC C

of galacturonic acid. In recent studies, the carbazole method was used to determine the galacturonic acid content of polysaccharides from Opuntia microdasys var. rufida cladodes (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.

ACCEPTED MANUSCRIPT (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

PT

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

RI

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

methods

to

the carbazole reaction are the

SC

assay stimulates any neutral sugar molecules present to form additional color. Similar m-hydroxydiphenyl sulfuric acid

assay

NU

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

MA

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

ED

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

EP T

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

AC C

method that avoids the use of concentrated acids commonly needed in the colorimetric determination with m-hydroxydiphenyl was proposed by Anthon & Barrett (2008) who 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,

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

and rapid analytical method based on converting GalA in the polysaccharide chain into the stable neutral sugar Gal (preventing decomposition of urinate residues) prior to hydrolysis

NU

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

MA

proposed method was higher, and the results showed good linearity, high precision, and high

ED

sensitivity.

5.1.3. Glycosidic linkage conformation

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For a detailed structural characterization of pectin polysaccharides, the investigation into monosaccharide composition can be completed with a determination of the positions of

AC C

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

ACCEPTED MANUSCRIPT 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

PT

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

RI

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

SC

reported in some studies, including those belonging to Wu et al. (2015), Zhang et al. (2016) and Wang et al. (2017), showed that correct identification of derivatives and the sugars they

NU

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

MA

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

ED

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

EP T

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

13

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

AC C

linkage and side chain location by 1D and 2D NMR was also reported in structural studies of arabinan-rich pectic polysaccharides from Abies sibirica L. (Shakhmatov et al., 2014), watersoluble 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

ACCEPTED MANUSCRIPT Petkowicz, 2018; Shakhmatov et al., 2014), while the correlation peak C-1/H-1 at 99.1102.2/5.03 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

PT

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

RI

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

SC

of esterification influences physical properties such as surface tension, emulsification capabilities (Lutz et al., 2009) and gel formation (Yoo et al, 2006). In regards to gelation,

NU

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

MA

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

ED

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

EP T

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

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

ACCEPTED MANUSCRIPT 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-

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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 pectin is limited and mostly involves quantification using chromatographic techniques such

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

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

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substitution levels in pumpkin pectin extracted by microwave heating. A different approach to this matter was taken by Müller-Maatsch et al. (2014) who developed a 1 H NMR (proton

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

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applied to pectic polysaccharides originating from a wide range of sources and having different physical properties and can detect small amounts of methanol, acetic acid, and 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 FTRaman and FT-IR, as described by Synytsya et al. (2003). According to their observations,

ACCEPTED MANUSCRIPT the very intense Raman band at 857 cm−1 is sensitive to the state of uronic carboxyls and Oacetylation, 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

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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

As was mentioned before, the degree and pattern of methyl-esterification influences

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

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

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

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powders (Einhorn-Stoll, 2018). Studies showed that, when compared to pectin samples with randomly distributed carboxyl groups, HMP with partly blockwise distributed free carboxyl 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

ACCEPTED MANUSCRIPT 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

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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 polymers from the polygalacturonase digest.

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

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

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

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deesterification. Lack of specificity for the characterization of DB was also indicated by Sousa et al. (2015a) who took a multivariate analysis approach with the purpose to analyze a

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

ACCEPTED MANUSCRIPT composition on the compressive mechanical properties of low-ester pectin gels it was observed that preparations with molecular weight distributions possessing broad lowmolecular-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

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

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emulsification properties of citrus pectin Schmidt et al. (2015) reached the conclusion that

improve emulsion stability. The

molecular

weight

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pectins with a reduced molecular weight do neither significantly reduce droplet sizes nor

distribution

of

pectins

is

generally

determined

by

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chromatographic techniques. Early analytical methods were developed on citrus pectin samples and were based on the use of gel permeation chromatography (GPC) (Harding et al.,

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

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Han, & Li (2011). The researchers combined GPC with a HPLC instrument equipped with an Ultrahydrogel column. This method was later employed to determine the molecular weight of 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

ACCEPTED MANUSCRIPT 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

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(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). The average molar weight of pectin samples can also be estimated using the Mark-

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Houwink equation (Eq. 1), as described by Arslan (1995): η = K × Ma (1)

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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. [η]= lim C→0 (

ηr-1 C

) (2)

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where ηr is relative viscosity and C (g∙L−1 ) is pectin concentration. Based on the Mark-Houwink equation and using a Cannon-Fenske capillary 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.

ACCEPTED MANUSCRIPT 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 the localization of esterified

groups,

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distribution pattern, and profile of the neutral monosaccharides.

the molecular weight

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degree of esterification,

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In the particular case of fluorescence microscopy, the progress in the application of this technique prompted the increase of research on the use of fluorescent markers with

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

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

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

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

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antibodies, research on the spatial distribution and the relative amount of acidic and methylated pectin present in various plant tissues was made possible (Casero & Knox, 1995; 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,

ACCEPTED MANUSCRIPT 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 subnanometerscale images of individual molecules, AFM has proved to be a useful tool for the

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

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

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of largely un-aggregated chains: a major fraction (67%) was polysaccharide-protein complexes containing a single protein molecule attached to one end of the polysaccharide

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

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

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Another technique that found great application in recent studies on pectin extracted from various plant materials is scanning electron microscopy (SEM). Liew, Chin, & Yusof (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

ACCEPTED MANUSCRIPT 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.,

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2004).

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5.6. Other methods

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More information regarding pectin structure (amorphous or crystalline) can be obtained by X-ray diffraction (XRD) analysis (Jiang et al., 2018; Sharma et al., 2015).

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

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

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

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

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

ACCEPTED MANUSCRIPT 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

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

scanning calorimetry,

thermogravimetry,

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esterified pectin, which makes it less responsive to calcium addition. Using differential and

differential thermogravimetry (DTG) in a

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

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chemically (demethoxylation and amidation) and mechanically (disaggregation). Their results showed that thermal behavior of pectin is considerably influenced by the physical state,

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

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

ACCEPTED MANUSCRIPT 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

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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

5.6.2. Rheological characterization solutions

of

pectin

show

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Aqueous

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films (Moreira et al., 2014).

pseudoplastic

non-thixotropic

behavior,

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

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

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

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have non-Newtonian flow (Iagher, Reicher, & Ganter, 2002); however, it is important to be mentioned that the actual critical concentration at which the solution transforms from 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

ACCEPTED MANUSCRIPT 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

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

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

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

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

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

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5.6.3. Functional properties Pectin is a valuable functional ingredient with food applications that include the use as 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

ACCEPTED MANUSCRIPT 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

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

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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, probably due to the high oil content in the formulation, the emulsions stabilized by HMP-

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zein, showed good stability and lower percent of creaming index. The gelling properties of pectin have been extensively studied along the years and are

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the main focus of numerous investigations on the structure-function relationships of pectin in food. For HMP, which form gel structures through hydrogen bonding and hydrophobic

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

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was confirmed by Jiang et al. (2012) in a study on the properties of pectins extracted from Akebia trifoliate var. australis peel. Textural analysis (hardness, springiness, adhesiveness, 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

ACCEPTED MANUSCRIPT gels, the work of (X. Yang et al., 2018) aimed to explore the effects of a wide pH range (3.59.5) 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

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

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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 The ubiquitous cell wall component pectin found applications as emulsifier, gelling,

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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 pectin research carried out with the purpose to isolate pectin from various plant materials and to elucidate its structure in order to assign possible applications. A predominant focus of

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recent research is to investigate the composition and the properties of pectin extracted from alternative sources that, in most part, seem to be represented by wastes of the fruit and 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)

ACCEPTED MANUSCRIPT 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

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

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MA

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polysaccharides, and particularly those that dictate its use in the food industry.

ACCEPTED MANUSCRIPT References Abid, M., Cheikhrouhou, S., Cuvelier, G., Leverrier, C., Renard, C. M. G. C., Attia, H., & Ayadi, M. A. (2017). Rheological properties of pomegranate peel suspensions: The effect of fibrous material and low-methoxyl pectin at acidic pH. Food Hydrocolloids, 62, 174–181. https://doi.org/10.1016/j.foodhyd.2016.08.008

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Ayadi, M. A. (2017). Characterization of pectins extracted from pomegranate peel and

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Adetunji, L. R., Adekunle, A., Orsat, V., & Raghavan, V. (2017). Advances in the pectin production process using novel extraction techniques: A review. Food Hydrocolloids,

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62, 239–250. https://doi.org/10.1016/j.foodhyd.2016.08.015 Al-Sayed, H. M. A., & Ahmed, A. R. (2013). Utilization of watermelon rinds and sharlyn

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melon peels as a natural source of dietary fiber and antioxidants in cake. Annals of

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Agricultural Sciences, 58(1), 83–95. https://doi.org/10.1016/j.aoas.2013.01.012 Alba, K., Laws, A. P., & Kontogiorgos, V. (2015). Isolation and characterization of acetylated LM-pectins extracted from okra pods. Food Hydrocolloids, 43, 726–735.

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https://doi.org/10.1016/j.foodhyd.2014.08.003 Andersen, N. M., Cognet, T., Santacoloma, P. A., Larsen, J., Armagan, I., Larsen, F. H., … Huusom, J. K. (2017). Dynamic modelling of pectin extraction describing yield and functional characteristics. Journal of Food Engineering, 192, 61–71. https://doi.org/10.1016/j.jfoodeng.2016.08.006 Anthon, G. E., & Barrett, D. M. (2004). Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase. Journal of Agricultural and Food Chemistry, 52(12), 3749–3753.

ACCEPTED MANUSCRIPT https://doi.org/10.1021/jf035284w Anthon, G. E., & Barrett, D. M. (2008). Combined enzymatic and colorimetric method for determining the uronic acid and methylester content of pectin: Application to tomato products. Food Chemistry, 110(1), 239–247. https://doi.org/10.1016/j.foodchem.2008.01.042

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Atmodjo, M. a., Hao, Z., & Mohnen, D. (2013). Evolving Views of Pectin Biosynthesis. Annual Review of Plant Biology, 64(1), 747–779. https://doi.org/10.1146/annurev-

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Bagherian, H., Zokaee Ashtiani, F., Fouladitajar, A., & Mohtashamy, M. (2011).

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Comparisons between conventional, microwave- and ultrasound-assisted methods for extraction of pectin from grapefruit. Chemical Engineering and Processing: Process

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Intensification, 50(11–12), 1237–1243. https://doi.org/10.1016/j.cep.2011.08.002 Banerjee, J., Singh, R., Vijayaraghavan, R., MacFarlane, D., Patti, A. F., & Arora, A. (2017). Bioactives from fruit processing wastes: Green approaches to valuable chemicals. Food Chemistry, 225, 10–22. https://doi.org/10.1016/j.foodchem.2016.12.093 Begum, R., Aziz, M. G., Uddin, M. B., & Yusof, Y. A. (2014). Characterization of Jackfruit (Artocarpus Heterophyllus) Waste Pectin as Influenced by Various Extraction Conditions. Agriculture and Agricultural Science Procedia, 2, 244–251. https://doi.org/10.1016/j.aaspro.2014.11.035

ACCEPTED MANUSCRIPT Begum, R., Yusof, Y. A., Aziz, M. G., & Uddin, M. B. (2017). Structural and functional properties of pectin extracted from jackfruit (Artocarpus heterophyllus) waste: Effects of drying. International Journal of Food Properties, 20(1), 190–201. https://doi.org/10.1080/10942912.2017.1295054 BeMiller, J. N. (1986). An introduction to pectins: structure and properties. ACS Symposium

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ACCEPTED MANUSCRIPT Table 1 Physicochemical properties of pectin: main sources of commercial pectin vs. other sources Molecu lar weight

Neutral monosaccha ride content

Reference s

58.6% (Granny Smith) 67.14% (Royal variety)

52.51% (Royal variety) 76.4% (Granny Smith)

331899 kDa

14.3-31.1%

Constenla , Ponce, & Lozano (2002); Kumar & Chauhan (2010); Wikiera et al. (2016)

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Degree of esterificat ion

13.426.3% 21.95 % 24.2%

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16.65 % 18.79 % 19.8%

Galacatur onic acid content

68.88% (mandarin orange) 91.6% (lime)

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Citrus peel

Granny Smith variety Royal variety Golden variety Pomace utilized in commer cial producti on of pectin Lime peel Mandari n orange peel Orange peel Sour orange peel Grapefru it peel Pomelo pectin

29.1%

27.34 % 27.6337.52 %

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Apple pomace

Yield of pectin recove rya 4.2%

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Plant source for pectin isolation

37.5% (sour orange) 82.2% (lime)

342.7 kDa (lime) 918 kDa (pomel o)

1.33% (pomelo) 9.3% (lime) Ara, 0.1% (lime) 0.16% (pomelo) Fuc, 1.64% (pomelo) 4.1% (lime) Gal, 0.8% (mandarin orange) 3.25% (pomelo) Glc, 0.62% (mandarin orange) 1.5% (lime) Rha, 0.18% (mandarin orange) 0.7% (lime) Xyl

Naghshin eh, Olsen, & Georgiou (2013); Dominiak et al. (2014); Methacan on, Krongsin, & Gamonpil as (2014); Wang, Chen, & Lü (2014); Boukrouf a et al. (2015); Wang et al. (2015); Hosseini, Khodaiya

ACCEPTED MANUSCRIPT n, & Yarmand (2016); Liew et al. (2016) 311 kDa

Tomato pomace Dried tomato peel

7.55%

78.4% (CBP)

76.9288.98%

n.d.

Rejected carrots Carrot pomace Carrot steam peels

8.9%

32.6%

24.3% 6.4% Ara, 13.2% Gal, 0.6% Glc, 4.4% Rha, 0.2% Xyl

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DM=52% , DAc=28. 1%

2.9% Ara, 3.85% Gal, 0.7% Glc, 3.8% Man, 1.4% Rha, 2.3% Xyl

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72.4%

Waterme lon rinds

Mango peel

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62-69%

53-77% (WSP)

114 kDa (rejecte d carrots) – 1460 kDa (carrot steam peels)

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515.2% 9%

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Carrot waste

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Tomato waste

2324.87 %

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Sugar beet pulp

1921%

68.7% (LW) 74.2% (FW)

DM: 61.5% (LW) 63% (FW)

3.451 × 104 g/mol (FW), 4.039 × 104 g/mol (LW)

17.15 %

29.3553.35%

DM: 85.4388.38%

378.42858 kDa

8-11.9% Ara, 0.120.18% Fuc, 13-24.4% Gal, 40.950.6% Glc, 1.6-1.9% Man, 1.73.2% Rha, 0.3% Xyl (in WSP) 0.6-0.7% Ara, 0.10.2% Fuc, 20.2-22.6% Gal, 1.4-4% Glc, 0.40.6% Man, 2.4-2.9% Rha, 0.5% Xyl n.d.

Li et al. (2015); Chen, Fu, & Luo (2015); Guo et al. (2016) Del Valle, Cámara, & Torija (2006); Grassino et al. (2016); MüllerMaatsch et al. (2016) Christiae ns et al. (2015); Jafari et al. (2017)

Banerjee et al. (2017); Petkowic z, Vriesman n, & Williams (2017) Wang et al. (2016)

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66.6568.7%

DE=60.3 6%, DM=45.9 4%

n.d.

Banana peel

9%

40.271.8%

DM: 4987-248 80%, kDa DAc: 1.25.7%

1-5.3% Ara, 1.6-5.7% Gal, 0.10.24% Rha

Pumpkin waste

7.4%

63.373.8%

DM=18% , DAc=3%

4.1-4.6% Ara, 0.40.6% Fuc, 7.2-9% Gal, 6.6-11.2% Glc, 0.8% Man, 4.24.6% Rha, 0.9-4.3% Xyl

on a dry weight basis

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139289 kDa

n.d.

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Passion fruit peel

Kliemann et al. (2009); Freitas de Oliveira et al. (2016) Happi Emaga et al. (2008); Maran et al. (2017) Košťálov á, Aguedo, & Hromádk ová (2016); MüllerMaatsch et al. (2016)

n.d. – not determined, WSP – water soluble pectin, FW – fresh watermelon rinds, LW –

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lyophilized watermelon rinds, CBP - calcium-bound pectin

ACCEPTED MANUSCRIPT Table 2 Extraction techniques and their effects on pectin yield and quality Effect on yield and quality The highest pectin yield (7.62%) was obtained using citric acid (pH 2.5, 95°C, 3.0 h), while the highest uronic acid content in pectin (65.20%) resulted by using water (95°C, 3.0 h). Extraction with citric acid produced pectin with a wider DM range. The yield of pectin extracted at the optimal condition (95°C, 90 min, and liquid/solid ratio of 25:1) was 18.35%. GalA content and DE of the extracted pectin ranged from 57% to 83%, and 17-30.5%, respectively. Under optimal conditions (solid/liquid ratio of 1:10 g/mL, pH of 2.8, 43 min, 86°C) the pectin yield reached 9.1%. Higher pectin solution concentrations were obtained at the lower pH values. Increased temperature and especially acidity caused a faster

References Chan & Choo (2013)

Solvent: water Liquid/solid ratio: 20:1, 30:1 or 40:1 (v/w) Temperature: 75, 85 or 95°C Time: 30, 60 or 90 min

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Sour orange peel

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Cocoa husks

Operating conditions Solvent: water, citric acid or hydrochloric acid pH: 2.5 or 4.0 Temperature: 50 or 95°C Time: 1.5 or 3.0 h

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Plant source

Durian rinds

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Extraction technique Solvent extraction

Lime peel

Solvent: water Solid/liquid ratio: 1:5-1:15 (g/mL) pH: 2-3 Temperature: 7595°C Time: 20-60 min Solvent: water:nitric acid pH: 1.5, 2.3 or 3.1 Temperature: 60, 70 or 80°C

Hosseini, Khodaiyan, & Yarmand (2016)

Maran (2015)

Andersen et al. (2017)

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Wikiera et al. (2015b)

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Wikiera et al. (2016)

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Apple pomace

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Apple pomace

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Enzymeassisted extraction

decrease of DE, effect that was particularly significant during extractions at pH=1.5. Enzymes: Polygalacturonic a xylanase and acid was multicatalytic completely preparation hydrolyzed after Celluclast 2.5 h incubation Acid used in with 2 M TFA at extraction: 120°C. Prolonged trifluoroacetic acid extraction was 2M positively Temperature: 100 correlated with an or 120°C increase of GalA Time: 1, 1.5, 2, released. 2.5, 3 or 4 h Efficient release of neutral sugars was performed at 100°C for 2.5 h. Enzymes: endoTreatment with xylanase and endo- endo-xylanase cellulaseb resulted in the Solid/liquid ratio: highest pectin 1 g/15 mL yield (19.8%) and Enzyme dose: 50 very high DM U/g (73.4%). Pectin pH: 5.0 extracted by endoTemperature: 40°C cellulase treatment Time: 10 h was characterized by the high GalA content (70.5%). Simultaneous use of both enzymatic preparations resulted in a 10.2% extraction yield, and a pectin rich in galacturonic acid (74.7%). Commercial The optimized enzymes: conditions for an 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

Rapeseed cake

Jeong et al. (2014)

ACCEPTED MANUSCRIPT ration with a Celluclast/Alcalase ratio of 1:4 for a 270 min hydrolysis time. Enzymes indicated different functions: Alcalase led to the destruction of proteincarbohydrate complexes, while Celluclast slightly cleaved some linkages of carbohydrate. Laminex C2K preparation proved to the most effective, as in optimum conditions (4 h treatment at pH 3.5, 50°C) gave a high yield (23%) and a pectin with good composition and properties (gelling, stabilization). The highest pectin yield (12.67%) was obtained at 85°C and a power intensity of 664 W/cm2 . Despite the fact that pectin isolation reached the highest level, the isolate did not displayed the best composition, as the GalA content and DE showed minimum values. Extraction yield of pectins varied greatly with the increase in temperature, from

Enzymes: Laminex C2K, Multifect B, GC220, and GC880 pH: 3.5-6.5 Temperature: 4070°C

Dominiak et al. (2014)

Passion fruit peel

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Ultrasoundassisted extraction

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Lime peel

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ratio: 0:5, 1:4, 2:3, 3:2, 4:1 or 5:0 (v/v) Time: 90, 180, 270, 360 or 450 min

Mango peel

Extraction solvent: 1.0 mol/L HNO 3 , pH 2.0, peel/solvent ratio of 1:30 (g/mL) Temperature: 4585°C Power intensity: 132.8-664.0 W/cm2 Time and frequency (constant): 10 min, 20 kHz Extraction solvent: citric acid, pH 2.5, peel/solvent ratio of 1:40 (g/mL) Time and

Freitas de Oliveira et al. (2016)

Wang et al. (2016)

ACCEPTED MANUSCRIPT 2.09% (at 20°C) to 17.15% (at 85°C). A significant influence of temperature was also observed for GalA content (increase from 29.35% to 53.35%) and molecular weight (increase from (378.4 kDa to 2320 kDa). Extraction solvent: Optimal conditions distilled water) for the extraction 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 15-30 min 24 min, and Extraction temperature of temperature: 5060°C. Under this 70°C conditions the pectin yield was 14.5% Emitter surface: 13 Heating mm or 25 mm significantly Power density: improved the 0.20, 0.27, 0.33, extractability and 0.40, 0.47 or 0.53 extraction rate of W/mL pectin, leading to Duty cycle: 33%, higher yield 40%, 50%, 60%, (26.74%) in 70% or 80% shorter extraction Temperature: 30, time (51.79 min). 40, 50, 60, 70 or The optimized 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 power: All the process 320, 480 or 640 W variables had pH: 1, 2 or 3 significant effect Time: 20, 100 or on pectin yield. 180 s The optimal

Moorthy et al. (2017)

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Grapefruit peel

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Jack fruit peel

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frequency (constant): 15 min, 20 kHz Temperature: 20 or 85°C

Microwaveassisted extraction

Waste papaya peel

Maran & Prakash (2015)

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Microwave power: 160, 320 or 480 W pH: 2, 3 or 4 Time: 60, 120 or 180 s Solid-liquid ratio: 1:10, 1:20 or 1:30 g/mL

Maran et al. (2015)

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Waste mango peel

conditions for reaching a maximum pectin yield (25.41%) were: microwave power of 512 W, pH of 1.8, time of 140 s and solidliquid ratio of 1:15 g/mL. For all process parameters a similar influence was observed: the increase in their level was positively correlated with the pectin yield up to a certain point, beyond which their effect on pectin extraction was negative. The maximum pectin yield (28.86%) could be obtained at a microwave power of 413 W, pH of 2.7, time of 134 s and solid-liquid ratio of 1:18 g/mL. The optimal extraction parameters were: microwave power 704 W, 52.2°C extraction temperature, and extraction time of 41.8 min. Under these conditions the experimental yield of pectin was 19.9±0.2%. The optimum conditions to obtain a maximum pectin recovery of

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Solid-liquid ratio: 1:5, 1:15 or 1:25 g/mL

Microwave power: 600, 700 or 800 W Temperature: 40, 50 or 60°C Time: 30, 40 or 50 s

Opuntia ficus indica cladodes

Microwave power: 200, 400 or 600 W pH: 1.5, 2.25 or 3 Time: 1, 2 or 3

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Tangerine peels

Chen et al. (2016)

Lefsih et al. (2017)

ACCEPTED MANUSCRIPT 12.56% were 2.16 min, pH 2.26, 517 W microwave 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 structure of pectin following microwave treatment. The highest yield (21.95%) of citrus pectin (68.88% GalA content) was obtained at 120°C, and the highest yield of apple pectin (16.68%) was gained at 150°C (GalA content of 40.13%). Differential scanning calorimetry analysis showed that the endothermic property of pectin was affected by extraction temperature while the exothermic property was only affected by its constituents and raw material. Increase in all parameters enhanced the extraction up to a certain level past which a decrease of pectin recovery was recorded.

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Solid to liquid ratio of 1:30 g/mL, extraction time of 5 min (constant) Extraction temperature: 130°C, 150°C or 170°C C for apple pomace; 100°C, 120°C or 140°C for citrus peel

Wang, Chen, & Lü (2014)

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Apple pomace and citrus peel

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Subcritical water extraction

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min Solid-liquid ratio: 2:20, 2:35 or 2:50 g/mL

Sugar beet pulp

Temperature: 110°C, 120°C or 130°C Extraction time: 20, 30 or 40 min Liquid/solid ratio: 30, 40 or 50 (w/w) Extraction

Chen, Fu, & Luo (2015)

ACCEPTED MANUSCRIPT The optimum 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 extraction temperature, extraction time of 30.49 min and extraction pressure of 10.70 MPa. An increase in Yang et al. excitation voltage (2016) caused enhancement of pectin yield, opposite to the increase in frequency that had a negative impact on the extraction. Lower pH also provided a higher pectin yield. The electrical effect was found to predominate at temperatures below 45°C, and the joint electrical and thermal effects governed the extraction at temperatures from 45 to 65°C.

Solvent: diluted hydrochloric Acid pH: 2, 3 or 6.8 Excitation voltage: 0-300 V Frequency: 20-200 kHz Temperature: 2080°C

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Orange peel waste

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Induced electric fieldassisted extraction

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pressure: 8, 10 or 12 MPa

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- EC 3.2.1.8, b- EC 3.2.1.4

ACCEPTED MANUSCRIPT Table 3 Methods of pectin purification: comparative view and new approaches References Yapo et al. (2007b)

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Effect on extract The technique gave a higher pectin yield, and the isolate contained more neutral sugars, proteins, and ash but had a galacturonic acid content lower than a pectin sample purified by ultrafiltration.

Pectin fractions rich in neutral sugars were precipitated at relatively high ethanol concentrations. The stepwise precipitation is more selective with respect to pectin structural features and surface properties than a onestep process. Pectin isolated from the crude aqueous extract by the 10kD membrane procedure contained more GalA and less neutral sugars, suggesting a higher purity of the pectin extract.

Guo et al. (2016)

Most of the salts and pigments were removed from the sample which also had a low protein and acid-insoluble ash content.

Kang et al. (2015)

Pectin purified through this method

Yapo (2009)

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Purification was performed using hollow fiber tangential flow filtration membranes (polysulfone, 0.5 mm i.d, 615 cm2 ) of 10 and 50 kD MWCOs. The ultrafiltration was conducted under 15 bar at 40°C using a membrane with a surface area of 1.77 m2 and a molecular weight cut-off of 8000 Da. Precipitation of the extract with a

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Ultrafiltration in combination with diafiltration

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Stepwise ethanolprecipitation

Procedure A first washing of crude pectin with 60% (twice), 70% (twice) and 80% ethanol, followed by a final washing step with 96% ethanol (twice). The final gel was recovered in water, freezedried, vacuumdried at 40°C. Precipitation with an equal volume of ethanol in elevated concentration from 50% to 80% (v/v) followed by centrifugation, ethanol washing and lyophilization.

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Method Alcohol precipitation with washing

Ultrafiltration

Metal precipitation

Yapo et al. (2007b)

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Significantly higher yield and GalA content and lower neutral sugar and protein content by comparison to the unprecipitated sample.

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Purification using protein (sodium caseinate)

had a higher galacturonic acid content and a low neutral sugars, protein and ash content.

Whatever the precipitation conditions, the purity of extracts, expressed as GalA content, was lower in pectin purified by caseinate than in that purified by ethanol (96%) precipitation. However, pectins purified by caseinates are characterized by a lower total content of neutral sugars.

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Copper precipitation

solution of CuSO 4 ∙5H2 O followed by centrifugation, filtration and washing with HCl and ethanol to eliminate Cu2+ ions. Precipitation of pectin with aqueous CuSO 4 completed with a dispersion of EDTA solution in the filtration cloth for the extraction of copper ions from the copperpectin complexes. Precipitation takes place after the addition of caseinate (pH 3.5; 10 g/l); to separate pectins from sodium caseinate the pH is increased to 6.5 by adding 1M NaOH, NaCl is added after the dissolution of the pellet, followed by 0.1M HCl to decrease pH to 4.6 (precipitation of caseinate).

Happi Emaga et al. (2012)

ACCEPTED MANUSCRIPT Table 4 Analytical methods for pectin characterization References 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) Blumenkrantz & AsboeHansen (1973); Abid et al. (2017); Chaharbaghi, Khodaiyan, & Hosseini (2017); Vasco-Correa & Zapata Zapata (2017) Garna et al. (2006); Buranaosot et al. (2010)

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Galacturonic acid content

Analytical method Hydrolysis, derivatization (alditol acetates) and quantification by GC and mass spectrometric detection TFA hydrolysis, HPAECPAD assay Hydrolysis, carbazole-H2 SO 4 reaction and spectrophotometric detection Hydrolysis, mhydroxydiphenyl sulfuric acid assay and spectrophotometric detection

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Parameter Monosaccharide composition

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Combined TFA-enzymatic hydrolysis, HPAEC-PAD assay; TFA hydrolysis and analysis by HPAEC-FID Methylation- induced cleavage of the glycosidic linkages, conversion to PMAAs, GCMS assay Methylation, identification of glycosidic linkage and side chain location by 1D and 2D NMR Hydrolysis and acidification of pectin, headspace solidphase microextraction (Carboxen-PDMS fiber assembly), separation of methanol and acetic acid by GC and detection using electron impact MS with selected ion monitoring Hydrolysis of pectin in NaOH/D2 O, direct 1 H NMR analysis Simultaneous determination of methylation and acetylation degree of pectin by FT-IR; Analysis of the degree of methyl-esterification by FTIR with peak deconvolution

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Glycosidic linkage conformation

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Degree of substitution

Sims & Bacic (1995); Wu et al. (2015); Zhang et al. (2016); Wang et al. (2017) Lin et al. (2016); Georgiev et al. (2017); John et al. (2018)

Savary & Nuñez (2003); Yoo et al. (2012)

Müller-Maatsch et al. (2014); Grassino et al. (2016) Synytsya et al. (2003); Kyomugasho et al. (2015)

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Synytsya et al. (2003); Kumar & Chauhan (2010)

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Determination of methylation and acetylation degree by FTRaman Detection using a µSI-LOV system (includes a VIS–NIRdiode array spectrophotometer) with ultra-pure deionized water as carrier Pectin digestion, separation and quantification using HPAEC-PAD at pH 5 Determination by capillary electrophoresis using phosphate as buffer in an automatic system equipped with a UV detector High performance size exclusion chromatography with refractive index detector (HPSEC-RID) and a TSK-Gel column; High performance size exclusion chromatography coupled to a multi-angle laser light scattering detector (HPSEC-MALLS) In situ analysis of pectin structure/location of specific domains by fluorescence 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 Analysis of pectin structure (amorphous or crystalline) by XRD performed on solid

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Degree of blockiness

Lira-Ortiz et al. (2014); Jung & Wicker (2014)

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Microscopic analysis

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Molecular weight

Daas et al. (1999); Daas, Voragen, & Schols (2000); Sousa et al. (2015b) Guillotin et al. (2007)

Other methods

Hafrén, Daniel, & Westermark (2000); Arltoft, Madsen, & Ipsen (2007)

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);

ACCEPTED MANUSCRIPT Jiang et al. (2018) Goubet, Morriswood, & Dupree (2003); Goubet et al. (2005)

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samples (powder form) Enzymatic digestion of pectin, analysis of the blockwise/nonblockwise distribution of methylation of GalA by PACE

ACCEPTED MANUSCRIPT Highlights

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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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