Extraction and Purification of Pectin from Agro-Industrial Wastes

1. Introduction

Pectins are complex branched polysaccharides present in the primary cell wall of plants [1]. It is a highly valued food ingredient commonly used as a gelling agent and stabilizer [2]. It is usually extracted by chemical or enzymatic methods from fruits [3]. Pectin is considered as the most complex macromolecule in nature, since it can be composed of up to 17 different monosaccharides containing more than 20 different linkages [4].

Pectins are enriched with repeated units of methyl ester galacturonic acid [4]. They form chemically stable and physically strong skeletal tissues of plants when combined with proteins and other polysaccharides [5]. They are usually produced in the initial stages of primary cell wall growth and make one third of the cell wall in both monocots and dicots [6]. Pectin is significantly reduced or absent in non-extendable secondary cell walls and is the only major class of plant polysaccharide largely limited to primary cell walls [7]. Pectin imparts strength and flexibility to the cell wall, apart from number of fundamental biological functions such as signalling, cell proliferation, differentiation, cell adhesion and maintaining turgor pressure of cell [8]. Pectins are involved in regulating mobility of water and plant fluids through the rapidly growing parts [6]. It also influences the texture of fruit and vegetables [9]. Apple pomace and orange peel are the two major sources of commercial pectin due to the poor gelling behaviour of pectin from other sources [6].

Pectin is one of the most important polysaccharides due to its increasing demand in the global market, reaching a total production capacity of around 45–50 Million tonnes per annum. While the demand in 2011 was approximately 140–160 Million tonnes per annum, earning the interest of industry in this complex polysaccharide processing [10]. Pectins have received considerable attention as a high fibre diet that benefits health by reducing cholesterol and, serum glucose levels and acting as anticancer agents [11]. Pectins have shown promising results as drug carriers for oral drug delivery and are widely used for various bio-medical applications [5]. In addition, pectin has been described as an emerging prebiotic with the ability to modulate colon microbiota [12]. Considering above properties and applications, pectin has gained immense priority in the global biopolymer market with great potential and opportunities for future developments.

2. Structure and properties of pectin

One of the most abundant macromolecules present in the primary cell wall of the plants is pectin; their presence is detected in the matrix as well as in the middle lamellae [7]. Pectin is highly rich with galacturonic acid (GalA), that forms the backbone of three more domains found along with pectin that are homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) [13]. About 70% of pectin is mainly composed of galacturonic acid (GA) [14]. Pectin is made of three polysaccharides that are covalently linked together, thus forming pectin networks in the cell wall matrix and the middle lamellae [15, 16].

Homogalacturonan (HG) takes up about 60–65% of the total pectin [3, 17], with a backbone of alpha-1,4-linked GalA residues, these GalA residues are methyl esterified which has an important role in the physical properties of pectin [4]. The presence of HG is seen to be present in approximately 100 GalA residues, but there are cases when its detected interspersed within other pectin polysaccharide [14]. On the other hand rhamnogalacturonan-I (RG-I) backbone which contributes 20–35% of pectin is composed of repeated and alternating groups ofl-rhamnosyl andd-galacturonosyl residues [18]. There can be as many repeats as 300 of this disaccharide in case of sycamore cells, which are cultured in suspension [3, 16, 19]. The rhamnosyl residues have side chains of sugars which are mainly consisting of either galactosyl or arabinosyl residues [5]. The GalA residue of RGI unlike HGA are mostly not methyl esterified [20].

Rhamnogalacturonan-II (RG-II) is one of the highly conserved and complex structure which consist of distinct regions within HG, which makes up about 10% of the pectin [3], they have side chains of four different types with a particular sugar residue like aceric acid, apiose-3-deoxy-lyxo-2-heptulosaric acid, and 3-deoxy-manno-2-octulosonic acid. The HG residues along with nine of the GalA residues are attached to these side chains [3, 5]. There are other substituted HG residues that make up pectin such as xylogalacturonan and apiogalacturonan whose expression is restriction. Even a minor mutation in R-II structure can lead to defects in the plant growth like dwarfism, thus suggesting its importance for normal growth of plant [3]. RG-I being highly branched in nature thus, called as the hairy region of pectin on the other hand HGA domain are known as the smooth region [7]. It is generally believed and noticed that there is covalent linkage within the pectin polysaccharides and pectin degrading enzymes are needed to separate and isolate HG, RG-I and RG-II from each other [21, 22]. Due to their similarity in HG and RG-II backbone structure composing of 1-4-linked alpha-D-GalA residues, they are likely to be linked covalently but there are no reports of RG-I to be covalently linked with HG [23].

3. Properties of pectin

4. Extraction and purification of pectin from agrowaste

Pectin is a high molecular weight polysaccharide that is present in almost all plants and help in maintaining the integrity of cell structure. Pectin is used in food industries to increase the viscosity of food products such as beverages, jams and jellies. It also has implications in pharmaceutical industry, especially in drug formulations, as an excipient due to its characteristics in release kinetics. Due to increased demand for pectin in food, pharmaceutical and therapeutic applications, thus, require efficient extraction processes. In order to increase the yield of pectin, various extraction methods have been adapted to obtain insoluble pectin present in the middle lamellae of plant cells, one of them being heating in acidic medium that makes insoluble pectin as soluble. Ripening of fruits also converts insoluble pectin into soluble pectin. Pectin can be extracted from various kinds of fruits, but the most commercial form of pectin is extracted from the peels of citrus fruits by alcohol precipitation [9, 25]. Citrus fruits contain 0.5–3.5% pectin which is largely present in the peel of fruits [26].

Pectin has been isolated from various plants such as apple [27], citrus peel, carrots [28], sugar beet pulp [29, 30], sunflower heads [31], papaya [32] and oranges [33]. The most commonly used method for extracting pectin from plant tissue is by heating the plant sample in acidified water. The addition of extra chelating agents such as EDTA to the extraction mixture helps in easy release of pectin from cell wall. Care should be taken not to perform a long period of direct heating as it may lead to the thermal degradation of the polymer. Extraction process of pectin is carried out under reflux using acidified water at 97°C for 30 min. The hot acid extract was then filtered using a cheese cloth to remove the pulp. The filtrate was then cooled to 4°C and precipitated using double the volume of ethanol. The solvent precipitate mixture is then mixed till the pectin floats and removed by using cheese cloth followed by drying [27].

Pectin is also extracted from dried sugar beet pulp after treating with acidified medium followed by purification through alcohol precipitation. Xin Huang et al., slightly modified the traditional method, where the sample was diluted with deionized water and was made acidic (pH −1.2) by using HCl. The sample was then heated to 90°C for 3 h and cooled to 40°C (pH −4.5) with 25 g/100 g ammonia. The mixture was then filtered using a Buchner funnel and pectin was precipitated using ethanol [29]. The ethanol is removed by squeezing with nylon cloth and washed several times followed by drying.

The carrot pomace is also used for pectin extraction by treating with hot aqueous citric acid solution adjusted to the desired pH. The pectin yield was maximum at the following optimum conditions: pH −1.3; temperature 90°C; heating time 79.8 min. Under these conditions, the extraction yield of carrot pomace pectin was found to be 16.0%. The extract mixture was then allowed to cool, filtered and precipitated by using ethanol in the ratio 2:1 [28]. Dried papaya peel can be used in pectin extraction where the majority of the lipids, and soluble pigments are removed by treating with ethanol and acetone. It is repeatedly homogenised with 95% ethanol and filtered until the filtrate becomes clear. The final filtration was done with the residue homogenised in acetone and dried overnight to obtain the alcohol insoluble residue (AIR). The majority of the pectin in the papaya AIR is present as chelator soluble pectin (CSP) followed by sodium carbonate soluble pectin (SSP) and water-soluble pectin (WSP). The WSP fraction is first obtained from the AIR by boiling it in water and filtering it. The remaining residue is treated with 0.05 M cyclohexane trans-1,2-diamine tetra-acetic acid (CDTA) in 0.1 M potassium acetate (pH 6.5) for 6 h at 28°C and filtered to give the CSP fraction. The residue is then treated with 0.05 M sodium carbonate solution having 0.02 M NaBH₄ for 16 h at 4°C, and subsequently for 6 h at 28°C. The solution when filtered gives the SSP fraction of the AIR [32].

Agro-industrial wastes can be used as the raw material for the production of industrial low and high methoxy pectin. The alcohol insoluble material (AIM) produced from dried agrowaste by boiling it with 3 volumes of ethanol for 25 min and continuous washing with 70% ethanol to remove impurities such as pigments, free sugars, etc. Sunflower heads also act as potential sources for pectin extraction. The heads are washed by hot distilled water through a mesh or cheese cloth and the pectin was precipitated by addition of 1 M nitric acid at 1:5 acid:filtrate ratio [34]. The mixture was maintained for 1 h at 5°C and was washed six times in ethanol solvent at 1:2 gel:solvent ratio to remove the impurities and to increase pH by removing the acid [31]. The washed pectin gel can be dried in a vacuum oven at 55°C for 16 h. The dried pectin flakes are ground into a powder for further use (Table 1).

5. Characterization

Large amounts of fruit wastes that are being generated can be disposed effectively by manufacturing beneficial by-products like pectin. Pectin is used to increase foaming power of gases, as agglutinator, as filler in pharmaceutical preparations and also in food industry. The use of pectin for different purposes depends on its characters like acetyl value, degree of esterification, uronic acid content and methoxyl content, etc. [68]. The amount of anhydrouronic acid gives the purity of pectin which is not less than 65% for pectin that is used commercially [69].

6. Applications

Pectin being a great inert, biodegradable and biocompatible complex, is widely used in various fields such as in textiles, food industries as gelling agents, pharmaceuticals and other products as well [70]. Pectin are used as biomaterials in gene delivery [71], application in oral drug delivery [72], as edible coating for food packaging [25], biomass yield and bio refinery [21, 22]. It also has applications in tissue engineering as scaffolds [73], in paper and textile industries for the preparation of ultracentrifugation membrane [74].

7. Conclusion

Pectin is one of the most extensively studied natural biodegradable polymer. In spite of its availability in a large number of plant species, commercial sources of pectin are very limited. There is, therefore, a need to explore other sources of pectin or modify the existing sources to obtain pectin of desired quality attributes. Current knowledge of the molecular basis of pectin has helped us to understand some aspects of this complex polysaccharide. Extensive studies must be carried out to find out more about the biological pathways to devise various efficient means of pectin extraction that are scalable and can be commercialized. The large variety of applications as well as the increasing number of studies on pectin suggests that the potential of pectin as novel and versatile biomaterial will be even more significant in the future. As the research and development continues in pectin-based products, we expect to see many innovative and exciting applications.

Acknowledgments

The authors would like to thank Fr. Jobi, Head of the Department of Life Sciences, for providing laboratory facilities and supporting this work.

Conflict of interest

The authors would like to declare that there was no conflict of interest in this work.