Physicochemical and Functional Properties of Water Soluble Gum from Wrinkle Floweringquince (Chaenomeles Speciosa) Seeds

1. Introduction

Gums have evoked tremendous interests these years for its wide applications in food, pharmaceutical and cosmetic industries (Amin et al., 2007; Prajapati et al., 2013; Fathi et al., 2016a). There were various sources to obtain the hydrocolloids, such as plant exudates, tree exudates, sea weed extracts, bacteria and animal sources (Amid et al., 2012). Gums obtained from plant seeds are always found with specific functions, which are important sources for the natural hydrocolloids. Previous researches about the good thickening property of Lallenmantia royleana seed gum, the significant stabilization feature of quince seed gum, and the gel capacity of flaxseed gum are highly reported (Naji et al., 2014; Osano et al., 2014; Kirtil et al., 2016). Moreover, the excellent emulsifying and foaming ability of basil seed gum and sage seed gum were deeply studied (Razavi et al., 2015; Wang et al., 2017). The gums are widely applied as thickeners, stabilizers, emulsifiers, gelling agents, and packaging films by virtue of those important functionalities (Koocheki et al., 2009). In view of the wide applications and market demand, the desire of new seed gums attracts an increasing attention of research in recent years.

The Wrinkle Floweringquince (Chaenomeles speciose (Sweet) Nakai), a member of Rosaceae family, was ubiquitous in China (Du et al., 2013). Previous reports had proved that the pulps of Chaenomeles speciosa exhibited excellent properties in antioxidation, anti- influenza, anti-inflammatory and immunoregulatory, and therefore the fruit of Chaenomeles speciosa was widely applied in food industry and medicine (Liu et al., 2011; Xie et al., 2015). Facing the increasing market demand, the cultivated area of Chaenomeles speciosa was consequently increased rapidly these years. However, as the main industrial by-product, the seeds of which were still underutilized. Also, our previous study found that the chemical composition of Chaenomeles speciosa seeds contains a high proportion of carbohydrate, and poses the potential to provide new plant seed hydrocolloid. In addition, the physicochemical characteristics, chemical compositions and functional properties in gum varied from their sources (Lai et al., 2012). However, little attention has been paid to the gums derived from Chaenomeles speciose seeds. Therefore, the aim of this study is to investigate the chemical composition, molecular weight and functional characteristics of the Chaenomeles speciosa seed gum (CSG). The results of this study would be beneficial in high effective utilizing Chaenomeles speciosa seeds, and facilitating further study and application of the CSG in wider industry production.

2. Materials and Methods

2.1 Materials

The Chaenomles speciosa seeds were purchased from commercial market, and the seeds were produced from Sichuan, China. The pure monosaccharide standards of D-galactose (Gal), D-mannose (Man), D-glucose (Glc), D-xylose (Xyl), L-arabinose (Ara), D-fucose (Fuc), D-ribose (Rib), and D-rhamnose (Rha) were purchased from Aladdin Co., Ltd. (Shanghai, China). Standard dextrans of different molecular weight were bought from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other reagents were of analytical grade.

2.2 The CSG extraction and purification

The method used for extracting the CSG was referred to the previous study of Amin et al. (2007) with slight modification. The Chaenomeles speciosa seeds were powdered by a grinder (Sunbeam 7600, Australia). The Chaenomeles speciosa seed meal was then extracted by petroleum ether to remove the lipids. The 100 g defatted seed meal was mixed with 2 L distilled water at 80℃ and stirred for 2 h. The viscous aqueous solution was centrifuged at 8000 r/min for 20 min to remove any insoluble substances. The supernatant was collected and concentrated, and the concentrated solution was freeze dried to obtain the crude CSG.

The crude CSG was redissolved in distilled water. To remove the residual lipid and low molecular proteins, the gum solution was added slowly into four volumes of ethanol and stored at 4℃ overnight for precipitation. The precipitates were then collected by centrifugation and washed by acetone and petroleum ether in turn. The precipitates were finally lyophilized to obtain the purified CSG and sealed for further analysis.

2.3 Physicochemical analysis

2.3.1   Moisture and total ash contents

The moisture content of the gum was determined by referring to the AOAC method (AOAC, 1997). The total ash contents were detected by heating the gum sample in a muffle furnace at 550℃ for 3 h until constant weight was achieved (Razmkhah et al., 2016).

2.3.2   Total carbohydrate, protein and uronic acids contents

The total carbohydrate contents of the CSG were determined by phenol-sulfuric acid method, and it was calculated according to the established calibration curve using glucose as a standard (DuBois et al., 1956). To determine the contents of protein, an elemental analyzer (PerkinElmer, EA 2400 Ⅱ, USA) was employed to measure the nitrogen content, and the protein contents were obtained by multiplying the nitrogen content (%) by 6.25 (Kaushik et al., 2016). The quantification uronic acid referred to m-hydroxydiphenyl method and D-galactose employed as standard (Blumenkrantz et al., 1973).

2.4 Analysis of monosaccharide composition

The monosaccharide composition of the CSG was determined by gas chromatography (GC) analysis. First, about 10 mg gum sample was hydrolyzed by treating with 5 mL of 10% H₂SO₄ at 100℃ for 2 h, then excess barium carbonate was added into the hydrolysate to neutralize the acid thoroughly. The neutralized hydrolysate was centrifuged at 10 000 r/min for 5 min, and the supernatant was collected and evaporated to obtain the dried gum hydrolysate. Next 10 mg hydroxylamine hydrochloride and 0.6 mL of pyridine were added to the dried hydrolyzed gum sample and kept at 90℃ for 30 min. Then 0.6 mL of acetic anhydride was added inside and reacted for another 30 min under 90℃. The resulting acetylated mono-sugars were analyzed by gas chromatograph (7890A, Agilent Technologies, CA, USA) equipped with a HP-5 capillary column and a flame ionization detector. The following program was employed for gas chromatograph analysis: injection temperature: 250℃; detector temperature: 280℃; column temperature programed from 180℃ to 220℃ at 1.5℃/min, holding for 1 min, then increasing to 250℃ at 5℃/min and holding for 3 min. Nitrogen was employed as the carrier gas and the flow kept at 24 mL/min. The flow rate of hydrogen and air was 3.0 mL/min and 350 mL/min, respectively. Monosaccharide standards (D-galactose, D-mannose, D-glucose, D-xylose, L-arabinose, D-fucose, D-ribose, and D-rhamnose) were also acetylated and used as references.

2.5 Molecular weight measurement

The molecular weight of CSG was determined by gel permeation chromatography (GPC), which was performed on a high-performance liquid chromatography equipped with a differential refractive index detector, Ultrahydrogel guard column (6 mm×40 mm, Waters, Japan) and Ultrahydrogel column (7.8mm×300 mm, Waters, Japan). The gum sample was dissolved by distilled water and centrifuged at 10 000 r/min for 15 min to dedust, and then the supernatant was injected. Distilled water was employed as the mobile phase, and the flow rate was controlled at 1 mL/min. The calibration curve was established by injecting a series of standard dextrans.

2.6 The FT-IR analysis

The basic structural groups of the gum sample were determined by a Fourier transform infrared (FT-IR) spectroscopy (Nicolet iS10, Thermo Scientific) in the range of 4000–400 cm^–1.

2.7 Surface tension analysis

The surface tension of the gum solutions was detected by using an interface tensiometer (DCAT21, Dataphysics, German) at 25℃. Various concentrations of the CSG solution (0.01%, 0.05%, 0.10%, 0.25% and 0.50% (w/V)) were prepared, and the Du nouy ring method was employed for each determination after the calibration of the instrument was conducted by distilled water.

2.8 Steady shear analysis

The determination of rheological characteristics of the CSG solutions was conducted by a rotational viscometer (Haake Mars, Thermo Scientific, USA) at 25℃. Different concentrations of the CSG solutions (1%, 4% and 8%) were prepared at 50℃, and stored at 4℃ overnight. The steady shear flow behavior was determined over a wide range shear rate from 10 s^–1 to 600 s^–1.

2.9 Emulsifying properties determination

The method to determine the emulsifying properties of the CSG was based on the report of Jindal et al. (2013). Briefly, 150 mL of various concentrations gum solutions (0.2%, 0.4%, 0.6%, 0.8% and 1% (w/V)) were firstly prepared, and 10 mL of commercial corn oil was gradually added inside with magnetic stirring at 1000 r/min. The mixture was further homogenized by high speed homogenizer (Ultra Tuttax T-18, IKA, Germany) at 15 000 r/min for 5 min to form coarse emulsions. The coarse emulsions were next sonicated by an ultrasonic processor (Ultrasonic processor FS-1200N, Shanghai) at 1200 W for 3 min. The emulsions were finally centrifuged at 2500 r/min for 10 min, and the emulsifying ability (EA) was calculated as Equation (1):

The emulsion stability (ES) of the prepared emulsions were determined by heating the emulsions in a water bath at 80℃ for 30 min, after cooling down to ambient temperature (23±3℃), and subsequently centrifuged at 2500 r/min for 10 min. The ES was evaluated as Equation (2):

2.10 Foaming properties evaluation

The foam ability (FA) and foam stability (FS) were evaluated referred to Alpizar-Reyes et al. 2017). Various concentrations gum solutions (0.2%, 0.4%, 0.6%, 0.8% and 1% (w/V)) were whipped by high speed homogenizer (Ultra Tuttax T-18, IKA, Germany) at 7800 r/min for 4 min. The FA and FS were calculated as equations (3) and (4), respectively:

2.11 Statistical analysis

All experiments were conducted in triplicate and the data were expressed as mean ± standard deviation (SD). Data analysis was performed on Microsoft Excel 2007, and P < 0.05 was regarded to be statistically significant.

3. Results and Discussion

3.1 Chemical composition

The chemical composition (moisture, total ash, protein, uronic acid, and total carbohydrate) of the CSG was shown in Table 1. The CSG consists of (63.80±2.50)% carbohydrate, (13.69±0.08)% protein, (10.30±1.02)% uronic acids, (6.80±0.16)% moisture and (3.18±0.65)% total ash. According to the results, the total carbohydrate content dominated the component of the CSG. The total carbohydrate content could be employed to evaluate the purity of gums (Fathi et al., 2016b). Accordingly, the amount of total carbohydrate of the CSG was lower than previous report about Australian chia seed gum (93.8%) (Timilsena et al., 2016), Prunus amygdalus (98.4%) (Bouaziz et al., 2015) and Prosopis juliflora seed gum (98.5%) (Rincón et al., 2014), but still approaching to be comparable to that of Prunus armeniaca (66.89%) (Fathi et al., 2016b). The monosaccharide composition of the CSG was determined by GC method, and the results revealed the presence of Rhamnose, Arabinose, Xylose and Glucose in the molar percentage of 29.77%, 10.54%, 18.55% and 15.84%, respectively. Monosaccharide composition of seed gums was pointed out to greatly affect the functional properties, such as, thermal, rheological, emulsifying and foaming properties (Samil, 2007). Besides, the application fields of the hydrocolloids were primarily affected by the heterogeneous monosaccharide composition (Razmkhah et al., 2016).

Table  1.  Chemical composition of Chaenomles speciosa seed gum (CSG)

Composition	CSG
Moisture (%)	6.80±0.16
Total ash (%)	3.18±0.65
Protein (%)	13.69±0.08
Uronic acid (%)	10.30±1.02
Total carbohydrate (%)	63.80±2.50
Monosaccharides
Rhamnose (%)	29.77±0.66
Arabinose (%)	10.54±0.43
Xylose (%)	18.55±0.28
Glucose (%)	15.84±0.34
Molecular weight (u)	9.85×106
Note: values are mean±SD of triplicate determination.

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The amount of uronic acid of gums could imply the relative amounts of acidic polysaccharides. Uronic acid content of the CSG (10.30%) was higher than that of Ocimum bacilicum L. (6.51%) (Naji-Tabasi et al., 2016) and close to Albizia procera (10.27%) (Pachuau et al., 2012), however lower than those of anghouzeh gum (17.8%) (Milani et al., 2012) and xanthan gum (21.5%) (Melton et al., 1976). Given the comparatively low content of uronic acid of the CSG compared to anghouzeh gum and xanthan gum, we could infer that the CSG had a lower negative charge than these gums.

Previous research from Pachuau et al. (2012) indicated that the protein content was an important indication to distinguish the source of the various gums. Furthermore, it had also been proved that some functional characteristics like film formation capacity, stabilizing and emulsifying abilities were closely related to the protein content of the gums. As shown in Table 1, the CSG consisted of a high amount of protein content (13.69±0.08)% compared with the prior reports from Prosopis chilensis seed gum (2.5%–5.16%), cress seed gum (2.45%), and Camelina seed gum (12.3%) (Estévez et al., 2004; Karazhiyan et al., 2009; Li et al., 2016). It was necessary to point out that the presence of protein of CSG indicated the ability to reduce the interfacial tensions, hence posed the potential to increase the emulsifying capacity.

3.2 Molecular weight

The molecular weight of hydrocolloids greatly affected their functional properties, and therefore the applications. The weight average molecular weight of the CSG was determined by gel permeation chromatography (GPC) method, result turned to be 9.85×10⁶ u. It was a high value when compared with the Chaenomeles sinensis seed gum (1.4×10⁶ u, fraction 2) (Wang et al., 2018), xanthan gum (4.05×10⁶ u) (Viturawong et al., 2008), Prunus persica gum (5.61×10⁶ u) (Simas-Tosin, et al., 2009), and close to those of cress seed gum (9.28×10⁶ u) (Razmkhah et al., 2016) and Prunus cerasus gum (1.119×10⁷ u) (Fathi et al., 2016a), but lower than the previous report of ghatti gum (8.94×10⁷ u) (Kaur et al., 2009). Polysaccharides with high molecular exhibited a low tendency to be adsorbed at the air-water interface, however they could signally enhance the stability of protein foams for the thickening or gelling features (Martinez et al., 2005). Additionally, the functional properties of the polymers, such as thickening and gelling capacity, could be greatly affected by the molecular weight, and the hydrocolloids with higher molecular weight could contribute higher viscosity-enhancing characteristic (Hamidabadi et al., 2017). Therefore, there was a potential for the CSG to serve as the thickening agent due to its high molecular weight.

3.3 The FT-IR analysis

The FT-IR spectrum was typically used to identify the basic functional groups of the gums. As shown in Fig. 1, the FT-IR spectrum demonstrated the identical peaks of the CSG sample. The broad and intense characteristic band presented ranging from 3500 cm^–1 to 3000 cm^–1 was related to the free hydroxyl groups stretching bonds in vapor phase and bonded O—H bands of carboxylic acid (Silverstein et al., 1962). The absorption peak observed around 2928 cm^–1 could be attributed to C—H stretching, symmetric and asymmetric of free sugar, and a possibility for the doubles overlapping with O—H (Kacuráková, 2000). The absorption at 1649 cm^–1 indicated the presence of COO groups and valence vibration, and the adsorption at 1414 cm^–1 was referred to bonding vibration of —CH₂ and —CH₃. The peak at 1249 cm^–1 and 1045 cm^–1 was responsible for the uronic acid and o-acetyl groups (Khondka, 2009). Furthermore, the peak at 1045 cm^–1 might correspond to the C—N (Fathi et al., 2016a), which indicated the existence of protein content in the CSG. The wave number ranged between 800 cm^–1 and 1200 cm^–1 were considered as the finger print region to identify the structure differences among the gums from various species (Nep et al., 2011). A weak absorption peak found at 847 cm^–1 was the evidence to prove the presence of α-glycosidic linkages in the structure of the CSG (Kacuráková, 2000).

Figure  1.  The FT-IR spectrum of Chaenomeles speciosa seed gum (CSG)

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3.4 Surface tension analysis

The surface tension characteristics of the CSG were determined at different concentrations ranged from 0% to 0.5% and the result was demonstrated in Fig. 2. The surface tension of the CSG decreased significantly at 0.25% (w/V) (60.2 mN/m) when compared with the air/distilled water surface tension (72.78 mN/m). Hydrocolloid gums were generally not considered to have a strong surface activity, however the existence of protein imparted them the capacity to decrease the surface tension (Funami et al., 2007). As it could be observed, the surface tension of the CSG dispersion decreased as the concentration increased ranged between 0% and 0.25%. However, as the concentration increased further and more than 0.25%, the surface tension exhibited a tendency to increase. Similar result could be observed from locust bean gum and fenugreek gum (Brummer et al., 2003; Chaires-Martínez et al., 2008). The increase of surface tension of gums at higher concentrations could be mainly contributed by the excessive viscosity and gelation, which made the surface tension measurement a problem by du Nouy ring method. The surface tension of the CSG at 0.5% (65.2 mN/m) was comparable with some commercial gums like carrageenan at 0.5% (65 mN/m) (Pachuau et al., 2012). According to the surface tension measurements, the surface active CSG could serve as emulsifying and foaming agents.

Figure  2.  Surface tension of CSG (20.0℃)

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3.5 Steady shear property

The viscosity was an important index to evaluate the quality of hydrocolloids when served as thickener and stabilizer in pharmaceutical and food industries (Pawar et al., 2015). The apparent viscosity characteristics of the CSG at concentrations of 1%, 4% and 8% were exhibited in Fig. 3. The apparent viscosity was greatly influenced by the CSG concentrations and shear rate. The viscosity increased as the concentration increased, and it was mainly related to the higher proportion of solid contents which always resulted in the increase of viscosity (Maskan et al., 2000). It was clear that the highest concentration (8%) showed the most pronounced pseudoplastic flow behavior. The apparent viscosity of the CSG solutions at 8% decreased sharply as the shear rate increased, revealing the distinct non-Newtonian shear-thinning behavior of the sample. The viscosity decreased as the shear rate increased could be mainly contributed by the disentanglement of macromolecular chains and the alignment of microstructure in the shear flow direction which resulted in less interaction between adjacent polymer chains (Dakia et al., 2008; Chandra et al., 2015). For the CSG solution at 4%, the pseudoplastic flow behavior was not so obvious as that of the 8% sample. When the concentration reached 1%, the CSG solution showed a Newtonian flow behavior. Moreover, according to the research reported by Verardo et al. (2008), the polysaccharide solutions with shear-thinning behavior imparted the food products with less slimy mouth feel characteristic and easier to swallow. Hence, to obtain a better mouth feel, a higher concentration of the CSG should be used in food system.

Figure  3.  Effect of different concentrations on apparent viscosity of CSG

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3.6 Emulsifying property

The emulsifying properties of natural hydrocolloids had been widely studied, and the gums derived from plant seeds always demonstrated desirable emulsifying characteristics, such as Alyssum homolocarpum seed gum, flaxseed gum, fenugreek seed gum (Garti et al., 1997; Koocheki et al., 2009; Wang et al., 2011). Generally, polysaccharides were not considered as surface active agents due to its hydrophilic feature, however the presence of proteins combined with polysaccharides could greatly improve the emulsifying ability (EA) of gums (Soleimanpour et al., 2013; Najafi et al., 2016). As shown in Fig. 4, the CSG demonstrated excellent EA, and this might owe to the protein content and rheological modification (Goycoolea et al., 1997). There were many factors that can contribute to the different emulsion abilities among various seed gums, such as structural features, chemical compositions, protein content and molecular weight. Considering the excellent EA, it could be due to the high molecular weight of the CSG, as higher molecular weight could lead to better emulsifying ability (Razmkhah et al., 2016). Furthermore, the protein fraction of gums was an important factor that could greatly affect the EA. The co-existence of hydrophobic and hydrophilic groups of protein ensured it amphiphilic characteristic which was crucial to the emulsifying ability (Najafi et al., 2016). The good emulsifying ability of the CSG therefore might be related to its protein fraction.

Figure  4.  Emulsifying ability (EA) and emulsifying stability (ES) of CSG

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With regarding to the ES, the CSG exhibited a great performance in keeping the emulsions stable at 80℃ for 30 min. The high ES could be explained by the large molecular weight of polysaccharides, which were typically used as thickening agents to enhance the viscosity of emulsion system and therefore retarded the flocculation, creaming and sedimentation (Bouyer et al., 2012). In addition, protein fraction of gums could adsorb rapidly onto the surface of newly formed droplets to provide a protective coating and could also reduce the surface tension to stabilize the emulsions. Furthermore, the proteins could protect the emulsions from flocculation and coalescence through spatial and electrostatic repulsion (Soleimanpour et al., 2013).

3.7 Foaming property

The foaming properties of gums played an important role in its applications of food systems (Naji et al., 2014). The foaming properties of the CSG were demonstrated in Fig. 5, and the CSG showed an outstanding performance in both FA and FS at each concentration. The foaming properties of gums might be influenced by its chemical structures, molecular weight and protein content (Amid et al., 2013). Generally, the carbohydrate content of gums was not considered as a good foaming agent since it lacked the ability to adsorb at the interface, whereas the protein fraction played a vital role in forming and stabilizing the dispersed gas phase. The ability of the protein to form bubbles mainly depended on its chemical structures, as the hydrophilic groups of protein resulted in the attraction of water phase, however the hydrophobic groups were arranged towards air phase (Amid et al., 2013). Hence, the excellent foaming ability of the CSG could be owed to the high proportion of protein content (13.69%, in Table 1).

Figure  5.  Foaming ability (FA) and foaming stability (FS) of CSG

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The CSG showed a great performance in foaming stability due to a high level in protein content as shown in Fig 5. Rodriguezpatino et al. (2007) reported the protein fraction could reduce the interfacial tension and consequently gave rise to the formation of stable foam. From another point of view, the presence of unfolded protein was another important factor influencing the FS. As stated by Lomakina and Mikova (Lomakina et al., 2011), the unfolded protein dispersed in the liquid-air interface could facilitate the formation of cohesive and viscoelastic film around the foams, and thus could protect the air bubbles against the adversely thermal and mechanical effects.

4. Conclusions

The CSG was mainly composed of carbohydrate, and the natural hydrocolloid showed a high molecular weight (9.85×10⁶ u). This gum comprised rhamnose, arabinose, xylose and glucose with a respective ratio of 29.77%, 10.54%, 18.55% and 15.84%. The CSG contained a high protein content (13.96%), and the hydrophobic groups of the protein enhanced the emulsifying and foaming properties of the CSG. This imparted the potential of the CSG to be used as emulsifier and foaming agent in industry. Moreover, the CSG solution at a high concentration (8%) showed a pronounced pseudoplastic behavior, indicating its possibility to be utilized as a release retarding excipient in food and medicine. However, the structure feature and cytotoxicity of the CSG were still unclear, and these would be carried out in our following work.