Gelation of Nicandra physalodes (Linn.) Gaertn. polysaccharide induced by calcium hydroxide: A novel potential pectin source

Abstract

In this study, a novel gelling polysaccharide with a yield of ~6.7% (dry basis) was obtained from Nicandra physalodes (Linn.) Gaertn. seeds, and subsequent structural analysis suggested that Nicandra physalodes (Linn.) Gaertn. polysaccharide (NPGP) was composed of galacturonic acid (87.8%), rhamnose (3.7%), glucose (3.6%), arabinose (1.8%), galactose (1.8%), and a small amount of xylose (1.3%). The weight average molar weight (Mw) and number average molecular weight (Mn) of NPGP were determined as ~ 79.6 kDa and ~23 kDa, respectively. Also, it was found that NPGP had a low methoxylation degree (DM) of ~28%. These results indicate that NPGP can be grouped into the family of low methoxyl pectins (LMP). Subsequently, we further investigated the gelling properties of NPGP, and found that NPGP exhibited varying sensitivities to different types of calcium sources, with Ca(OH)₂ being especially effective in inducing NPGP gelation. At a fixed NPGP concentration (0.5%, w/v), an increased amount of Ca(OH)₂ addition induced a stronger gel, with the greatest gel hardness being observed at 0.06% (w/v) of Ca(OH)₂ concentration. However, further Ca(OH)₂ addition decreased the gel hardness. Furthermore, it was also observed that NPGP gels had relatively good heating stability but poor freeze-thawing stability, which could be greatly enhanced by incorporating appropriate amounts of sucrose. Overall, these findings highlight the potential commercial value of developing NPGP as a novel pectin resource.

Introduction

Due to non-toxicity, extensive sources, and health-promoting properties, pectins have been extensively used in the food industry as thickening agent, gelling agent, emulsifying agent, etc., to confer numerous food products with improved functionality (Chan, Choo, Young, & Loh, 2017; Liu et al., 2020; Wang, Zhang, Zhang, et al., 2020; Zhu et al., 2020). Essentially, pectin is a water-soluble polysaccharide that mostly exists in the middle lamellae of higher plants and can be obtained through various extraction techniques, including enzyme extraction, acid extraction, microwave-assisted extraction, and metal ion precipitation, and so on (Adetunji, Adekunle, Orsat, & Raghavan, 2017; Chan et al., 2017; Guo, Meng, Zhu, Zhang, & Yu, 2015; Hosseini, Khodaiyan, & Yarmand, 2016; Wang et al., 2016). Even if identical sources are used for pectin extraction, different extraction methods can often lead to a slight difference in pectin structure (Kumar et al., 2020). Despite this, it has been widely accepted that pectin molecules as a whole consist of three regions, i.e., homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) (Chan et al., 2017). HG is commonly composed of several hundreds of α-d-galacturonic acid units bonded one another by α-1,4-linked-glycosidic bonds, and thus HG region is also known as “smooth region” (Cameron, Kim, Galant, Luzio, & Tzen, 2015). RG-I region is constituted by up to 100 or more repeating α-1,2-linked-l-rhamnose-α-1,4-d-galacturonic acid disaccharide units, where 20–80% of rhamnose residues are substituted with neutral sugar side chains, predominantly including galactose and arabinose (Chan et al., 2017). RG-II is typically described as a stretch of HG backbone consisting of approximately seven to nine 1 → 4 linked α-d-galacturonic acid residues with four heteropolymeric side-chains attached (Chan et al., 2017). RG-I and RG-II regions are usually described as “hairy regions”. Moreover, because the galacturonic acid (GalA) units that constitute the HG region can be partly methoxylated, pectins can be thus classified into high methoxyl pectins (HMP) and low methoxyl pectins (LMP) (Cao, Lu, Mata, Nishinari, & Fang, 2020). For HMP, more than 50% of the carboxylic groups of the GalA units are methylated (DM > 50%), whereas in LMP, less than 50% of the carboxylic groups are methylated (DM < 50%). These structural characteristics collectively determine the functionality of pectins (Fraeye et al., 2009, 2010).

Because of structural differences, HMP and LMP have different gelation mechanisms. HMP gelation requires a relatively low pH value (e.g., pH ~3.5) and a high concentration of sucrose as co-solute (typically 65% sucrose) (Chan et al., 2017). Low pH values are necessary to minimize the electrostatic repulsion between pectin molecules via suppressing the dissociation of carboxyl groups of GalA units, while a high content of sucrose acts to reduce water activity thereby promoting pectin chain-chain associations (Chan et al., 2017). In contrast, the formation of LMP gels is predominantly governed by ionic cross-linking between two sequences of dissociated carboxyl groups from different pectin chains (known as the “egg-box” model and characterized by the junction zones formed by the ordered, side-by-side associations of GalA units) (Cao et al., 2020). It has been reported that, pectin gelation (for both HMP and LMP) can be affected by pectin structures and extrinsic factors (Fraeye et al., 2009; Ngouémazong, Kabuye, et al., 2012; Ngouémazong, Kabuye, et al., 2012). In terms of LMP, the higher the GalA content, the lower the DM value, the higher the DB_abs value (which is known as absolute degree of “blockiness”, defined as the amount of non-esterified mono-, di- and tri- GalA liberated from degradation of pectin by endopolygalacturonase relative to the total amount of GalA present in the pectin), the stronger the LMP gel (Fraeye et al., 2009, 2010). Unlike HMP, LMP gelation can occur at a wide pH range, and the co-solute is not required. Instead, LMP gelation requires the presence of divalent cations to create the ionic crosslinks between pectin chains, where Ca²⁺ is the most used divalent cation because of its safety (Han et al., 2017; Yang, Nisar, Liang, et al., 2018). Theoretically, the maximum number of calcium cross-linkages can be achieved when the Ca²⁺ concentration is adequately high to bind all dissociated carboxyl groups, which can be quantified as R value (defined as the stoichiometric ratio of two-fold Ca²⁺ concentration to the dissociated carboxyl group concentration, namely R = 2[Ca²⁺]/[COO⁻]) (Ngouémazong, Kabuye, et al., 2012). At R < 1, an increasing Ca²⁺ concentration is anticipated to promote LMP gelation, as characterized by an improved sol-gel transition temperature and a higher gel strength (Cao et al., 2020); whereas at R ≫ 1, increasing Ca²⁺ concentration has been reported to negatively contribute to LMP gelation, leading to either precipitates or inhomogeneous gel structure and ultimately weakening the gel strength (Han et al., 2017; Ventura, Jammal, & Bianco-Peled, 2013). Based on these properties, LMP has received a great attention in the food industry, especially in formulating reduced-sugar jam-like products (Chan et al., 2017).

Nicandra physalodes (Linn.) Gaertn. (NPG) is an erect annual herb that belongs to Solanaceae and initially originated from South America but now has been widely planted in China for medicinal or ornamental uses (Liu et al., 2017; Zhang et al., 2018). NPG is branch-spreading and the stem has a height of 1.5–2.0 m, with yellow globose berries (1–2 cm in diameter) enclosed by bluish flower (Bisoi, Biswal, & Satapathy, 2018). The seeds in the berries are a main byproduct of NPG (with each hectare producing approximately 600 kg of the seeds), and they contain a layer of abundant pectic polysaccharide within the episperm. After fully soaking, NPG polysaccharide (NPGP) can be readily dissolved in the water by manually rubbing out the swollen episperm. When adding a small amount of lime water (calcium hydroxide) and holding the NPGP solution at room temperature for ~ 20–30 min, a homogeneous, soft, and elastic gel can be obtained. This method has been traditionally adopted in many China's areas to make “ice jelly”, which is a very popular street food in summer. However, we find that the relevant studies focused on NPGP are rather limited, which promotes the impetus and necessity to systemically study the gelling properties of NPGP. On the other hand, despite the fact that some commercial pectin types such as citrus pectin, apple pectin, sugar beet pectin and also their modified pectins are available in the market (Dranca & Oroian, 2018), it is still a good motivation for food scientists to search for new pectin sources with better functionality to meet the increasing demands from food industry (Pan, Zhou, Liu, & Wang, 2021; Vriesmann & de Oliveira Petkowicz, 2017; Wang, Jiang, Ren, Shen, & Xie, 2019). As a result, in this work, we first obtained NPGP by water extraction and ethanol precipitation technique, and then revealed the basic structural information of the polysaccharide. Subsequently, the gelling properties of NPGP were investigated at a fixed polysaccharide concentration (0.5%, w/v) and varying Ca(OH)₂ addition amounts (0.02–0.1%, w/v). Moreover, the effects of sucrose addition on NPGP gels were also considered. It is expected that our results can contribute to the development of NPGP as a novel hydrocolloid resource with potential commercial value.