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Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation
Journal Pre-proofs Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation Wei Xu, Yantao Liu, Fenglun Zhang, Fuhou Lei, Kun Wang, Jianxin Jiang PII: DOI: Reference:
S0141-8130(19)35504-7 https://doi.org/10.1016/j.ijbiomac.2019.09.161 BIOMAC 13440
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
16 July 2019 20 September 2019 25 September 2019
Please cite this article as: W. Xu, Y. Liu, F. Zhang, F. Lei, K. Wang, J. Jiang, Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.161
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© 2019 Published by Elsevier B.V.
Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation
Wei Xua, Yantao Liua, Fenglun Zhangb, Fuhou Leic, Kun Wanga, Jianxin Jianga,
a
Beijing Forestry University, MOE Engineering Research Center of Forestry Biomass Materials and Bioenergy, Beijing, 100083, China
b
Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing, 211111, China
c
GuangXi Key Laboratory of Chemistry and Engineering of Forest Products, College of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Nanning 530006, China
* Corresponding author: [email protected] Tel: +86-10-6233-8267
Graphical Abstract:
ABSTRACT: Gleditsia triacanthos polysaccharide, known as galactomannan, has not been exploited as a new functional material even though it possesses industrial potential in food and biomedicine. Galactomannans were recovered from the endosperm of seeds (15 weeks to 25 weeks after flowering) for deposition and maturation analysis. These 1
galactomannans were characterized by using Nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and monosaccharide composition analysis (particularly the mannose to galactose ratio) and molecular weight, solubility, and rheological measurements. The ratio of the three parts in mature seeds was as follows: endosperm (36.67%), hull (34.41%), and embryo (28.92%). The M/G ratio increased from 2.53 to 3.24 between 15 and 23 weeks and then decreased to 3.16 in 25 weeks, consistent with the trends of rheology and solubility. The molecular weight (1.28 × 106 g/mol) and intrinsic viscosity (882.53 mL/g) reached the maximum at 23 weeks and then decreased. Additionally, NMR and XRD showed that the M/G ratio did not change the basic chemical structure but caused slight changes in crystallinity. The purpose of the study was to reveal the changes in galactomannan structure, rheology, and solubility during G. triacanthos galactomannan deposition and maturation to facilitate exploration of its potential industrial applications.
Keywords: Gleditsia triacanthos; Galactomannans; M/G ratio; Viscosity; Molecular weight.
2
Highlights:
M/G ratios are increased first and then decreased. The variation in molecular weight coincides with the trend in M/G ratios. The decline in endosperm moisture is concomitant with galactomannan development. Solubility and viscosity depend on both M/G ratios and molecular weight. Changeable M/G ratios do not change the basic chemical structure.
3
1. Introduction Galactomannans are heterogenous and water-soluble polysaccharides found in numerous
endospermic
leguminous
seeds
[1],
consisting
of
a
linear
-(1→4)-D-mannan backbone partially substituted by -(1→6)-D-galactose side chains [2]. Galactomannans are non-toxic, biodegradable polysaccharides and are recognized as a “Safe Food” by the US Food and Drug Administration (FDA) [3]. Owing to their high viscosity and safety, galactomannans have been widely used in human food, animal feeding [4], and in biomaterial and biomedical industries as stabilizing agents, thickening agents, emulsifying agents, and gelling agents [2, 5]. The endosperm in leguminous seeds is mainly composed of galactomannan and also contains some other substances such as proteins, crude fiber, and fat, which need to be separated via an extraction process [6]. In general, galactomannan is prepared by extraction of endosperm powder. The extraction of galactomannans can be accomplished by one of two methods: alcohol precipitation and complexation. The former involves water extraction followed by ethanol precipitation [6] and the latter involves complexation with Cu2+ and Ba2+ salts followed by ethanol precipitation [6, 7]. Gleditsia triacanthos (G. triacanthos), a dioecious Gleditsia xylophyta, usually blossoms in late May or early June and matures in autumn [8]. G. triacanthos pods are 4
crescent-shaped, 1520 cm in length and their seeds are oval, mainly composed of the endosperm, hull, and embryo. Galactomannan development in leguminous plants has been divided into four widely spaced stages with a light microscopically: embryo development stage, galactomannan deposition stage, late galactomannan deposition stage, and galactomannan maturation stage [9]. And the specific time for each development stage is very different depending on the species and growth environment of leguminous plants [10]. A clear galactomannan biosynthesis model has been established to help us understand the relationship between galactomannan deposition and biosynthesis [11]. Specifically,
galactomannan
biosynthesis
is
catalyzed
cooperatively
by
mannosyltransferase and galactosyltransferase [2, 12]. Galactomannan biosynthesis is ongoing in the initial and intermediate galactomannan deposition stage. However, galactomannan biosynthesis, induced by the two transferases, ends due to a decrease in endosperm moisture in the late galactomannan deposition stage [13]. Simultaneously, the -(1→6) galactose side chains linked to the -(1→4) mannose backbone from G. triacanthos galactomannan are removed by -galactosidase due to increased -galactosidase activity [11]. Obviously, the galactomannan biosynthesis process regulated by several enzymes influences galactomannan deposition and galactose content, thus controlling the M/G ratio. The M/G ratio (the mannose to galactose ratio) is an important factor determining galactomannan physicochemical properties such as its solubility, rheology, and thermal behavior [14, 15]. Compared with galactomannan with lower 5
M/G ratios (1~1.8), galactomannan with higher M/G ratios (2.7~4.0) are insoluble in cold water and dissolve upon heating [15, 16, 17]. Moreover, higher M/G ratios contribute to higher viscosity due to intermolecular interactions [18]. In addition, the physicochemical properties and particularly the viscosity of galactomannans are also influenced by their molecular weight distribution. The M/G ratio changes differ during galactomannan deposition and maturation. For example, the M/G ratio in fenugreek and guar galactomannans remains constant at the galactomannan development stages. In contrast, the ratio in Senna galactomannans was reported to be about 2.3 at the beginning of galactomannan deposition and increased rapidly at the late galactomannan deposition stage [19]. Despite this, there has been no systematic study on the changes in the M/G ratio, physicochemical properties, and the relationship between them for G. triacanthos galactomannans during their deposition and maturation. The purpose of the present study was to compare the M/G ratios and physicochemical properties of G. triacanthos galactomannans at deposition and maturation stages.
Materials and methods 2.1. Materials G. triacanthos pods from Beijing Forestry University (Beijing, China) were collected in 15, 17, 19, 21, 23 and 25 weeks after flowering (refer to below simply as WAF 15, WAF 17, WAF 19, WAF 21, WAF 23 and WAF 25). The length, width and fresh weight of pods were measured. The pods and seeds were manually separated to obtain hull, endosperm and embryo. The dry weight ratio of three parts and moisture 6
content in endosperm of G. triacanthos was determined after drying at 105 C for 24 h until constant weight reached. The rest of endosperm was firstly rolled and extruded by three-roll grinder and then smashed into white powders after drying. The powders (125 m mesh) were stored in a desiccator for further analysis. D-galactose, D-mannose and glucose were purchased from Sigma-Aldrich company. 2.2. G. triacanthos galactomannans extraction The crude G. triacanthos polysaccharides was extracted according to the method [20] with some modifications. Firstly, 1% (w/v) crude polysaccharides were extracted under continuous stirring at 70 C for 4 h. After centrifugation at 3000 × g for 10 min, the precipitate was dissolved in water and extracted at 70 C for 4 h. After centrifugation, the supernatant obtained was combined and precipitated with the same volume of absolute ethanol. Subsequently, the mixture was taken a place under the temperature at 4 C for 12 h. The precipitation after centrifugation was washed with absolute ethanol twice and lyophilized. The lyophilized galactomannans were smashed into white powders (125 m mesh) and called for extracted galactomannans. The insoluble substances have been removed from the extracted galactomannans after centrifugation. 2.3. M/G ratio determination of G. triacanthos The M/G ratio of crude polysaccharides and extracted galactomannans was determined according to the method [13, 21]. Samples (0.15g) were hydrolyzed using 72% H2SO4 at 30 C for 1 h followed by 4% H2SO4 at 121 C for 1 h [22]. Then hydrolysates obtained were neutralized with the CaCO3 and adsorbed by resin 7
(AG501-X8, Bio-Rad) to remove Ca2+. After centrifugation at 3000 × g for 10 min, the supernatant was filtered through a filter membrane (0.45 m). The M/G ratio was determined by High-Performance Liquid Chromatography (HPLC) (Agilent 1260, USA) using an Aminex HPX-87P column (300×7.8 mm; Bio-Rad Laboratories, USA) at 85 C with an evaporative light-scattering detector (ELSD). Ultra-pure water at 0.6 mL/min was used the mobile phase and nitrogen at 1.6 L/min was used as flowing gas, respectively. Calibration curves of D-galactose, D-mannose, glucose standards were used to determine the monosaccharides of crude polysaccharides and extracted galactomannans. 2.4. NMR (Nuclear magnetic resonance) spectra analysis It is necessary to degrade the extracted galactomannans before NMR determination because the extracted galactomannans have a higher molecular weight. 10 mg extracted galactomannans were dissolved in 0.55 mL of D2O (99.9%) and 0.55 mL of DCl (99.9%) at room temperature for an hour before determination. 1H NMR spectra of extracted galactomannans were obtained on Bruker Ascend 500 M NMR. Chemical shifts were calculated relative to sodium 2,2-dimethylsilapentane-5-sulphonate (DSS) as internal standard (0.00 ppm for 1H) [23]. The temperature of NMR spectrometer was 333 K, and the 1H NMR spectra was collected at a working frequency of 500 MHz. 2.5. Molecular weight analysis by SEC-MALLS The molecular weight distribution of extracted galactomannans was carried out on a size exclusion chromatography (Wyatt GPC/SEC-MALS system, Wyatt 8
Technology Corporation, Santa Barbara, CA, USA). The normalization of the detectors and determination of the inter-detector volume were performed with standard monodisperse dextran (MW = 50000Da, Mw / Mn = 1.36). Parameters including weight-average molecular weight (Mw), the number-average molecular weight (Mn), the intrinsic viscosity ([η]), the radius of gyration (Rg), the hydrodynamic radius (Rh) and the polydispersity index (ð = Mw / Mn) of extracted galactomannans were determined. TSK G5000PW column, TSK G3000PW column and OHPAK SB-G guard column were eluted with 0.1 M NaNO3. Extracted galactomannans were solubilized at 3 g/L in NaNO3 (0.1 M) under stirring at room temperature for 12h and the solution obtained was filtered through a 0.45 𝜇m filter before injection [24]. The refractive index increment (dn/dc) was 0.138 mL/g. Data were analyzed by the Astra version 5.3.4 software package. The fractal dimension (df) can be determined according to formula (1) and (2). 𝑅𝑔 = 𝑘1 × 𝑀𝑣𝑤
(1)
𝑑𝑓 = 1/𝑣
(2)
Where Mw is average molecular weight, Rg is gyration radius and df is fractal dimension. 2.6. XRD analysis Extracted G. triacanthos galactomannans were analyzed for X-ray diffraction (XRD) patterns. XRD diffractometer (D8 Advance, Bruker Co., Karlsruhe, Baden-Wurttemberg, Germany) (anode Cu-K, 45 kW, 40 mA) recorded the Wide-angle X-ray patterns, with a diffraction angle (2θ) range of 10 to 80 and 9
resolution of 0.02 at room temperature [18]. 2.7. Solubility and insoluble substances determination Solubility and insoluble substances were determined according to the method [25] with some modifications. The crude polysaccharides (0.15 g, dry basis) were dissolved in water (30 mL) at 30 C for 30 min and continuously stirred. The suspensions were then centrifuged at 3000 × g for 10 min. The insoluble substances were washed three times and dried at 105 C for 4 h until constant weight was obtained. The supernatant (10 mL) were dried at 105 C for 4 h until constant weight was obtained. The solubility was calculated by formula (3), and the insoluble substances were calculated by formula (4): Solubility (%) =
𝑤𝑠 × 300
(3)
𝑚0
Insoluble substances (%) =
𝑚1 × 100
(4)
𝑚0
Where 𝑤𝑠 is the dry weight of 10 mL supernatant, 𝑚1 is the dry weight of insoluble substances and 𝑚0 is the dry weight of crude G. triacanthos polysaccharides. 2.8. Rheological measurements of G. triacanthos galactomannan The apparent viscosity of extracted galactomannans from WAF 15 to WAF 25 was determined using Brookfield viscometer (Model LVDV-Ⅲ) with a spindle (No. SC4-31). The extracted galactomannans (0.4% w/v, dry basis) were dissolved by continuously stirring at 80 C for 30 min to achieve complete hydration [26] and then cooled to the room temperature before running the test. The shear stress (σ) and apparent viscosity were recorded with the shear rate (γ) increasing from 0 to 85 s-1 using the software (Rheocalc V3.2) at 25 C. All measurements were done in 10
triplicate. The rheology of GTS from WAF 15 to WAF 25 showed that shear rate versus shear stress were in good agreement with Power-law model (formula 5). 𝛾 = 𝑘2 × 𝛾𝑛
(5)
Where 𝛾 is the shear stress, 𝛾 is the shear rate, 𝑘2 is the consistency cofficient and 𝑛 is the flow behavior index.
3. Results 3.1. Physical parameter measurements of G. triacanthos To compare the physical parameters of G. triacanthos collected from WAF 15 to WAF 25, the size and fresh weight of each pod, the dry weight percentage of the three parts (endosperm, hull, and embryo) in seeds, and the endosperm moisture were measured. The results are shown in Figure 1. Figure 1a shows that the length and width of pods was constant whereas the fresh weight of pods decreased consistently from WAF 15 to WAF 25. As also seen from Figure 1a, the pods of G. triacanthos are crescent-shaped and their color gradually deepened from WAF 15 to WAF 25. Specifically, in the green, immature seed before WAF 19, galactomannan deposition may already begin [9]. The color of pods (Figure 1a) and seeds (Figure 1b) deepened gradually from WAF 21 to WAF 25, indicating that G. triacanthos galactomannans may be ripening [9]. As shown in Figure 1b, the endosperm moisture gradually decreased and the dry weight percentage of the three parts of seeds remained constant during galactomannan deposition and maturation. The ratio of the three parts in mature seeds was as follows: endosperm (36.67%), hull (34.41%), and embryo (28.92%). 11
3.2. M/G ratio of G. triacanthos galactomannan Figure 2 summarizes the M/G ratio and monosaccharide composition of crude polysaccharides (2a) and extracted galactomannans (2b). And the HPLC profile of crude polysaccharides and extracted galactomannans was provided in supplement (Figure S1). They were mainly composed of two monosaccharides: mannose and galactose. Compared to the crude polysaccharides, the content of mannose and galactose (M+G) in extracted galactomannans was higher, with trace amounts of glucose (Table S1 and Table S2), suggesting that the extraction process increased galactomannan purity. As shown in Figure 2b, the mannose content increased from 56.93% to 62.59% from WAF 15 to WAF 19. This may be a result of effective transport of the mannose to the end of the unreduced mannose chain by mannosyltransferase to prolong the mannose chain [11]. Subsequently the mannose content (63.39%) remained constant from WAF 19 to WAF 23. This result contributed to the ending of galactomannan deposition as result of loss of moisture in the endosperm (Figure 1b). Edwards et al. [13] also found that galactomannan deposition in fenugreek ended as the endosperm entered the desiccation phase. Finally, the mannose content decreased in WAF 25, which may be caused by a complex degradation mechanism involving several biochemical pathways [11, 27]. The galactose content gradually decreased from WAF 15 to WAF 25. These results suggest that α-galactosidase is capable of removing the galactosyl residues from the galactomannan side chains, which is consistent with previous
reports
[11].
These
enzymes 12
including
mannosyltransferase
and
α-galactosidase in galactomannan development have been intensively investigated [11, 13, 28]. In addition, the sum of mannose and galactose content (expressed as the sum of Man and Gal in Figure 2b) increased from WAF 15 (79.45%) to WAF 23 (83.55%) and the values then reduced to 81.19% in WAF 25. The M/G ratio is directly related to the degree of branching, determining a variety of physicochemical properties of galactomannan, including its solubility and viscosity [29]. From WAF 15 to WAF 25, the M/G ratios observed (Figure 2b) were close to those estimated by other authors, varying between 2.3 and 3.3 of galactomannan in the endosperm [19, 30]. The M/G ratio was about 2.53 in WAF 15 and increased substantially in WAF 23, which was in accordance with the M/G changes in Senna endosperm [13]. The M/G ratio was then reduced to 3.16 in WAF 25. As a result, the M/G ratio in WAF 23 was the highest (3.24), presenting regions of mannose with scarce branching. Conversely, the mannan backbone in guar gum was highly substituted and the M/G ratio was 1.50 [31]. Galactomannans with different M/G ratios can be used in different industrial fields. For example, galactomannans with a high M/G ratio (3.24) in WAF 23 are suitable for producing strong and flexible films with higher elastic modulus, tensile strength, and break elongation [32]. In contrast, galactomannans with low M/G (2.53) in WAF 15, could replace guar gum and be used as an strengtheners and stabilizers [17, 33]. 3.3. 1H-NMR spectra analysis The 1H-NMR spectra of extracted galactomannans were shown in Figure 3. There were two distinct peaks (5.04 ppm, 4.78 ppm) in the anomeric region of the 1H 13
spectra of the extracted galactomannans, which were α-galactopyranosyl and β-mannopyranosyl respectively [20]. The M/G ratio can be also obtained directly from the relative areas of the anomeric signals for 1H-NMR spectra as Cunhua et al. (2009) [23]. The ratios were 2.55, 2.82, 3.01, 3.18, 3.26, and 3.16 for WAF 15, WAF 17, WAF 19, WAF 21, WAF 23, and WAF 25. These values were in good agreement with the results obtained with HPLC analysis (Table S2). 3.4. Molecular weight analysis Molecular weight, gyration radius (Rg) and hydrodynamic radius (Rh) are summarized in Table 1; these affect the rheological and functional properties of galactomannans [34]. The PDI value (Mw / Mn) known as polydispersity index, is close to 1 for monodispersed polymers [35]. The PDI value ranging from 1.05 to 1.26 (Table 2), reflects the low polydispersity and relatively high purity of extracted G. triacanthos galactomannans. The weight average molecular weight (g/mol) and the [η] values from WAF 15 to WAF 25 were lower than the values of G. triacanthos harvested in Portugal (1.62 ×106 Da and 1042 mL/g) [36]. These differences can be attributed to the dependence of Mw and [η] values on both the seed growing conditions, extraction, and purification methods. Furthermore, the values of Mw and the [η] were increased from WAF 15 (0.98×106 g/mol, 729.96 mL/g) and reached the maximum (1.28×106 g/mol, 882.53 mL/g) in WAF 23, followed by a decrease in WAF 25 (1.12×106 g/mol, 726.14 mL/g). The former increase further confirming that galactomannan deposition was underway from WAF 15 to WAF 23. The decreased values of Mw and [η] in WAF 25 may be attributed to a complex degradation 14
including some biochemical pathways [11, 26]. Galactomannan with different molecular weights can be used for different industrial applications. For example, galactomannans with high molecular weight (1.28×106 g/mol) in WAF 23 were used as a thickening agent [37]. In contrast, galactomannans with low molecular weight (0.98×106 g/mol) in WAF 15 could be used to produce manno-oligosaccharides which are regarded as dietary fiber and prebiotics [21]. Furthermore, the structure factor (ρ = Rg / Rh) for G. triacanthos was revealed to be 1.801.88 by the SEC-MALS system, which confirmed that GTSP was in random coil conformation in the diluted solution [38]. The vg values of GTSP were between 0.5 and 1, further confirming the random coil shape of GTSP in dilute solutions [39]. The fractal dimension (df) is a measure of the compactness of a polymer structure and the larger its value, the more compact is the polymer structure [38, 40]. The df values (close to 1.93) was highest in WAF 23, and galactomannan compactness was caused by the highest M/G ratio in WAF 23 (Figure 2). 3.5. X-ray diffraction analysis of GTSP The X-ray diffractograms of extracted galactomannans from WAF 15 to WAF 25 are shown in Figure 4. The results indicate that extracted galactomannans presents low overall crystallinity. The crystalline regions of extracted galactomannans were observed at the angle (2θ) 20.2 ° from WAF 15 to WAF 25, indicating that extracted galactomannans underwent negligible changes in the XRD curve during galactomannan deposition and maturation. However, the crystallinity of extracted galactomannans showed a slight difference. The increased crystallinity of extracted 15
galactomannans from WAF 15 (30.9%) to WAF 19 (32.2%) may be attributed to the lengthened mannose main chain caused by mannosyltransferase (Figure 2b) and more aggregated molecule chains to increase crystallinity. The crystallinity of samples from WAF 19 to WAF 23 then remained constant because of the end of transport by mannosyltransferase due to moisture loss (Figure 1b). Furthermore, the crystallinity of samples decreased in WAF 25 (31.2%), possibly due to lower molecular weight (Table 1). Kong et al. [41] also found that lower crystallinity of a biopolymer is actually related to its lower molecular weight. Furthermore, reduction in the Mw of galactomannan chains (Table 1) did not form a new crystalline structure and did not affect the amorphous nature of galactomannans, which was supported by previous reports [42]. 3.6. Solubility and Insoluble substances From WAF 15 to WAF 23, there was a general trend showing a decrease in solubility and an increase in insoluble substances in the gums (Figure 5). This low solubility might be due to the high M/G ratio (Figure 2a), which allows compact packing of polysaccharides [43]. The low solubility of polysaccharides is also related to their high molecular weight (Table 1) and the high viscosity of polysaccharides occurs from resistance to flow (Figure 6) [44]. Compared to high molecular weight polysaccharides, low molecular weight polysaccharides are more soluble in water. From WAF 15 to WAF 23, the molecular weight and intrinsic viscosity increase gradually (Table 1), resulting in a decreased solubility. This trend was inverted in WAF 25 with 32.89% insoluble substances, which is 16
significantly lower than the 42.20% insoluble substances in WAF 23. This may result from the low molecular weight in WAF 25 (Table 1). Furthermore, a decrease in M/G (Figure 2a) results in an increase in solubility in WAF 25 [43]. The total recovery yield (the sum of solubility and insoluble substance) is close to 100%, indicating that the results are reliability. 3.7. Rheological analysis The rheological properties of extracted galactomannans from WAF 15 to WAF 25 were compared. As shown in Table 2, the coefficients of determination (R2) were higher than 90.6 for all tested samples, indicating the flow properties of GTSP fitted the power law model [45]. The k values for the consistency coefficient increased from WAF 15 to WAF 23, causing an increase in intermolecular interactions [46]. In addition, the flow behavior index (n) was in the range of 0.80 to 0.86, indicating that the solutions of extracted galactomannans from WAF 15 to WAF 25 were typical pseudoplastic fluids [47]. The power law model designates that the pseudoplasticity of the fluid increases with a decrease in n values. The pseudoplasticity of the fluid was the highest in WAF 15, WAF 17, and WAF 23, which may be caused by the predominance of hydrogen bonds from the water medium [26]. As shown in Figure 6a, the extracted galactomannans solutions displayed shear thinning behavior, which further confirmed their pseudoplastic behavior. This phenomenon could be caused by the interactions between galactomannan chains. With increasing shear rate, the GTSP molecule chain was gradually unfolded due to increasing shear stress (Fig. 6b). The continued shear stress breaks towards linkages, 17
resulting in an apparent loss of viscosity [48]. The viscosity of galactomannan solutions increased from WAF 15 to WAF 23 due to the increase in M/G ratios (Figure 2b), combined with the increase in molecular weight (Table 1). In general, higher viscosity can represent a greater amount of β-(1→4)-mannan chains per unit carbohydrate weight due to the slight decrease in galactose content (Figure 2b). Mallet et al. [49] also found that the increasing viscosity may be due to the higher M/G ratio of galactomannans, in combination with a somewhat greater molecular weight. However, the viscosity of galactomannan decreased since WAF 25, which was caused by a decrease in M/G ratio and molecular weight in WAF 25, which was consistent with Jian, et al. [26]. 4. Conclusions The fresh weight of G. triacanthos pods and the endosperm moisture was decreased, whereas the weight ratio of the three parts in seeds (endosperm, hull, and embryo) remained constant during G. triacanthos galactomannan deposition and maturation. NMR and XRD analysis demonstrated that no changes in chemical structure but showed the slight changes in crystallinity. The M/G, apparent viscosity, and molecular weight increased from WAF 15 to WAF 23 firstly, and then decreased at WAF 25, causing changes in solubility. Based on the different physicochemical properties of G. triacanthos galactomannans during their deposition and maturation, their industrial applications could be developed further. Declaration of interest: None. 18
Acknowledgments This research was financially supported by the National Key R&D Program of China (2016YFD0600803), the Natural Science Foundation of China (31670579), and the Specific research project of Guangxi for research bases and talents (AD18126005). References [1] C. Sandolo, P. Matricardi, F. Alhaique, T. Coviello, Dynamo-mechanical and rheological characterization of guar gum hydrogels, Eur. Polym. J. 43 (2007) 3355-3367. [2] K.S. Dhugga, R. Barreiro, B. Whitten, K. stecca, Jan Hazebroek, G.S. Randhawa, M. Dolan, A.J. Kinney, D. tomes, S.Nichols, P. Anderson, Guar seed -mannan synthase is a member of the cellulose synthase super gene family, Science, 303 (2004) 363-366. [3] N.M. Siqueira, B. Paiva, M. Camassola, E.Q. Rosenthal-Kim, K.C. Garcia, F.P. Santos,
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Figures and captions Figure 1a. Length, width, fresh weight, and photographs of each Gleditsia triacanthos pod collected from WAF 15 to WAF 25 (weeks after flowering).
Figure 1b. Dry weight percentage of the three parts (endosperm, hull, embryo) of each Gleditsia triacanthos pods, the endosperm moisture and the photographs from 26
WAF 15 to WAF 25 (weeks after flowering).
Figure 2. Monosaccharide compositions of Gleditsia triacanthos galactomannan (2a crude polysaccharides, 2b extracted galactomannans) from WAF 15 to WAF 25 (weeks after flowering).
Figure 3. the 1H spectra of extracted galactomannans from WAF 15 to WAF 25 (M: mannose, G: galactose).
Figure 4. X-ray diffractograms of extracted Gleditsia triacanthos galactomannans from WAF 15 to WAF 25.
Figure 5. Solubility and insoluble substances (%) of crude Gleditsia triacanthos galactomannans from WAF 15 to WAF 25.
Figure 6. Apparent viscosity (6a) and flow curve (6b) of the 0.4% (w/v) extracted G. triacanthos galactomannans solutions at 25 C.
27
Figure 1a
28
Figure 1b
29
Figure 2
30
WAF 15 WAF 21
G-1
WAF 17 WAF 23
WAF 19 WAF 25
M-1
1:3.16
1:3.26
1:3.18
1:3.01
1:2.82
1:2.55
5.2
5.0
4.8
4.6
4.4
4.2
4.0
Chemical shift (p.p.m.)
Figure 3
31
3.8
3.6
3.4
WAF 13-30.9% WAF 15-31.6% WAF 17-32.2% WAF 19-32.4% WAF 21-32.2% WAF 23-31.2%
10
20
30
40
Diffraction angle 2θ（degree)
Figure 4
32
50
60
100
solubility
80
insoluble substance 90
70 80
60 50
70 40 60
30 20
50 10 40
0
WAF 15
WAF 17
WAF 19
WAF 21
Sampling time
Figure 5
33
WAF 23
WAF 25
Total recovery yield (%)
Solubility and insoluble substance (%)
90
Figure 6
34
Tables Table 1. Molecular weight of extracted G. triacanthos galactomannans from WAF 15 to WAF 25 by SEC MALLS. Parameter aM
WAF 15
WAF 17
WAF 19
WAF 21
WAF 23
WAF 25
0.98±0.16
1.12±0.12
1.09±0.12
1.21±0.14
1.28±0.21
1.12±0.16
1.05±0.01
1.12±0.02
1.17±0.01
1.11±0.06
1.26±0.01
1.14±0.02
92.7±3.16
83.8±2.06
96.4±3.22
92.9±3.16
91.6±3.33
90.3±3.10
(nm)
51.5±1.60
46.4±1.16
51.4±1.11
49.9±1.60
50.5±1.26
48.3±1.61
(Rg/Rh)
1.80±0.16
1.81±0.12
1.88±0.12
1.86±0.14
1.81±0.26
1.87±0.16
729.96±
750.26±
764.66±
788.11±
882.53±
726.14±
5.16
5.23
6.10
6.66
6.12
6.34
0.89
0.87
0.89
0.77
0.52
0.78
1.11
1.14
1.16
1.29
1.93
1.28
w (10
6 g/mol)
bPDI
c
Rg (nm)
dR h
eρ
f[η]
(mL/g) gv
g
hd
f
a
Mw: average molecular weight. b PDI: polydispersity index. c Rg: gyration radius.
d
Rh: hydrodynamic radius. e ρ: the structure sensitive. f [η]: intrinsic viscosity.
gv
g:
vg = 1/ df. hdf: fractal dimension.
35
Table. 2. Power law parameters for extracted G. triacanthos galactomannans dispersions (0.4% w/v) from WAF 15 to WAF 25. Sampling Time WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
k (Pa.sn)
n
R2
0.113±0.002
0.80±0.00
98.1±0.1
0.133±0.003
0.80±0.02
98.1±0.1
0.137±0.001
0.86±0.01
96.8±0.4
0.157±0.002
0.86±0.01
95.3±0.2
0.261±0.019
0.80±0.01
95.2±0.4
0.125±0.005
0.83±0.02
98.2±0.1
Where k is the consistency coefficient, (R2) is the coefficient of determination and n is the flow behavior index.
36
Supplemental files:
Figure. S1. the HPLC profile of crude polysaccharides (a) and extracted galactomannans (b) from WAF 15 to WAF 25 (M: mannose, G: galactose).
37
Table. S1. Monosaccharide composition of crude Gleditsia triacanthos polysaccharides from WAF 15 to WAF 25 (M: mannose, G: galactose, M+G: the sum content of mannose and galactose). Sampling time
Mannose
Galactose
Glucose
M+G
M/G
WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
52.64±0.05 56.28±0.07 57.27±0.80 57.62±0.30 58.47±0.19 56.94±0.14
22.11±0.30 19.48±0.02 19.30±0.08 19.22±0.02 18.37±0.01 18.31±0.05
2.78±0.05 3.9±0.07 3.06±0.09 2.08±0.00 1.17±0.09 4.28±0.14
74.75±0.15 75.76±0.49 76.57±0.27 76.84±0.33 76.84±0.18 75.25±0.11
2.37±0.00 2.79±0.06 2.97±0.03 3.00±0.01 3.18±0.01 3.11±0.05
38
Table. S2. Monosaccharide composition of extracted Gleditsia triacanthos galactomannans from WAF 15 to WAF 25 (M: mannose, G: galactose, M+G: the sum content of mannose and galactose). Sampling time
Mannose
Galactose
Glucose
M+G
M/G
WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
56.93±0.11 60.64±0.27 62.59±0.18 63.39±0.15 63.85±0.01 61.66±0.04
22.52±0.09 21.64±0.03 20.65±0.05 19.96±0.06 19.70±0.02 19.53±0.04
0.68±0.01 0.86±0.07 0.97±0.08 0.00±0.05 0.00±0.01 1.99±0.04
79.45±0.02 82.28±0.35 83.25±0.67 83.35±0.21 83.55±0.01 81.19±0.00
2.53±0.02 2.80±0.02 3.03±0.02 3.18±0.00 3.24±0.00 3.16±0.01
39
S0141-8130(19)35504-7 https://doi.org/10.1016/j.ijbiomac.2019.09.161 BIOMAC 13440
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
16 July 2019 20 September 2019 25 September 2019
Please cite this article as: W. Xu, Y. Liu, F. Zhang, F. Lei, K. Wang, J. Jiang, Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.161
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© 2019 Published by Elsevier B.V.
Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation
Wei Xua, Yantao Liua, Fenglun Zhangb, Fuhou Leic, Kun Wanga, Jianxin Jianga,
a
Beijing Forestry University, MOE Engineering Research Center of Forestry Biomass Materials and Bioenergy, Beijing, 100083, China
b
Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing, 211111, China
c
GuangXi Key Laboratory of Chemistry and Engineering of Forest Products, College of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Nanning 530006, China
* Corresponding author: [email protected] Tel: +86-10-6233-8267
Graphical Abstract:
ABSTRACT: Gleditsia triacanthos polysaccharide, known as galactomannan, has not been exploited as a new functional material even though it possesses industrial potential in food and biomedicine. Galactomannans were recovered from the endosperm of seeds (15 weeks to 25 weeks after flowering) for deposition and maturation analysis. These 1
galactomannans were characterized by using Nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and monosaccharide composition analysis (particularly the mannose to galactose ratio) and molecular weight, solubility, and rheological measurements. The ratio of the three parts in mature seeds was as follows: endosperm (36.67%), hull (34.41%), and embryo (28.92%). The M/G ratio increased from 2.53 to 3.24 between 15 and 23 weeks and then decreased to 3.16 in 25 weeks, consistent with the trends of rheology and solubility. The molecular weight (1.28 × 106 g/mol) and intrinsic viscosity (882.53 mL/g) reached the maximum at 23 weeks and then decreased. Additionally, NMR and XRD showed that the M/G ratio did not change the basic chemical structure but caused slight changes in crystallinity. The purpose of the study was to reveal the changes in galactomannan structure, rheology, and solubility during G. triacanthos galactomannan deposition and maturation to facilitate exploration of its potential industrial applications.
Keywords: Gleditsia triacanthos; Galactomannans; M/G ratio; Viscosity; Molecular weight.
2
Highlights:
M/G ratios are increased first and then decreased. The variation in molecular weight coincides with the trend in M/G ratios. The decline in endosperm moisture is concomitant with galactomannan development. Solubility and viscosity depend on both M/G ratios and molecular weight. Changeable M/G ratios do not change the basic chemical structure.
3
1. Introduction Galactomannans are heterogenous and water-soluble polysaccharides found in numerous
endospermic
leguminous
seeds
[1],
consisting
of
a
linear
-(1→4)-D-mannan backbone partially substituted by -(1→6)-D-galactose side chains [2]. Galactomannans are non-toxic, biodegradable polysaccharides and are recognized as a “Safe Food” by the US Food and Drug Administration (FDA) [3]. Owing to their high viscosity and safety, galactomannans have been widely used in human food, animal feeding [4], and in biomaterial and biomedical industries as stabilizing agents, thickening agents, emulsifying agents, and gelling agents [2, 5]. The endosperm in leguminous seeds is mainly composed of galactomannan and also contains some other substances such as proteins, crude fiber, and fat, which need to be separated via an extraction process [6]. In general, galactomannan is prepared by extraction of endosperm powder. The extraction of galactomannans can be accomplished by one of two methods: alcohol precipitation and complexation. The former involves water extraction followed by ethanol precipitation [6] and the latter involves complexation with Cu2+ and Ba2+ salts followed by ethanol precipitation [6, 7]. Gleditsia triacanthos (G. triacanthos), a dioecious Gleditsia xylophyta, usually blossoms in late May or early June and matures in autumn [8]. G. triacanthos pods are 4
crescent-shaped, 1520 cm in length and their seeds are oval, mainly composed of the endosperm, hull, and embryo. Galactomannan development in leguminous plants has been divided into four widely spaced stages with a light microscopically: embryo development stage, galactomannan deposition stage, late galactomannan deposition stage, and galactomannan maturation stage [9]. And the specific time for each development stage is very different depending on the species and growth environment of leguminous plants [10]. A clear galactomannan biosynthesis model has been established to help us understand the relationship between galactomannan deposition and biosynthesis [11]. Specifically,
galactomannan
biosynthesis
is
catalyzed
cooperatively
by
mannosyltransferase and galactosyltransferase [2, 12]. Galactomannan biosynthesis is ongoing in the initial and intermediate galactomannan deposition stage. However, galactomannan biosynthesis, induced by the two transferases, ends due to a decrease in endosperm moisture in the late galactomannan deposition stage [13]. Simultaneously, the -(1→6) galactose side chains linked to the -(1→4) mannose backbone from G. triacanthos galactomannan are removed by -galactosidase due to increased -galactosidase activity [11]. Obviously, the galactomannan biosynthesis process regulated by several enzymes influences galactomannan deposition and galactose content, thus controlling the M/G ratio. The M/G ratio (the mannose to galactose ratio) is an important factor determining galactomannan physicochemical properties such as its solubility, rheology, and thermal behavior [14, 15]. Compared with galactomannan with lower 5
M/G ratios (1~1.8), galactomannan with higher M/G ratios (2.7~4.0) are insoluble in cold water and dissolve upon heating [15, 16, 17]. Moreover, higher M/G ratios contribute to higher viscosity due to intermolecular interactions [18]. In addition, the physicochemical properties and particularly the viscosity of galactomannans are also influenced by their molecular weight distribution. The M/G ratio changes differ during galactomannan deposition and maturation. For example, the M/G ratio in fenugreek and guar galactomannans remains constant at the galactomannan development stages. In contrast, the ratio in Senna galactomannans was reported to be about 2.3 at the beginning of galactomannan deposition and increased rapidly at the late galactomannan deposition stage [19]. Despite this, there has been no systematic study on the changes in the M/G ratio, physicochemical properties, and the relationship between them for G. triacanthos galactomannans during their deposition and maturation. The purpose of the present study was to compare the M/G ratios and physicochemical properties of G. triacanthos galactomannans at deposition and maturation stages.
Materials and methods 2.1. Materials G. triacanthos pods from Beijing Forestry University (Beijing, China) were collected in 15, 17, 19, 21, 23 and 25 weeks after flowering (refer to below simply as WAF 15, WAF 17, WAF 19, WAF 21, WAF 23 and WAF 25). The length, width and fresh weight of pods were measured. The pods and seeds were manually separated to obtain hull, endosperm and embryo. The dry weight ratio of three parts and moisture 6
content in endosperm of G. triacanthos was determined after drying at 105 C for 24 h until constant weight reached. The rest of endosperm was firstly rolled and extruded by three-roll grinder and then smashed into white powders after drying. The powders (125 m mesh) were stored in a desiccator for further analysis. D-galactose, D-mannose and glucose were purchased from Sigma-Aldrich company. 2.2. G. triacanthos galactomannans extraction The crude G. triacanthos polysaccharides was extracted according to the method [20] with some modifications. Firstly, 1% (w/v) crude polysaccharides were extracted under continuous stirring at 70 C for 4 h. After centrifugation at 3000 × g for 10 min, the precipitate was dissolved in water and extracted at 70 C for 4 h. After centrifugation, the supernatant obtained was combined and precipitated with the same volume of absolute ethanol. Subsequently, the mixture was taken a place under the temperature at 4 C for 12 h. The precipitation after centrifugation was washed with absolute ethanol twice and lyophilized. The lyophilized galactomannans were smashed into white powders (125 m mesh) and called for extracted galactomannans. The insoluble substances have been removed from the extracted galactomannans after centrifugation. 2.3. M/G ratio determination of G. triacanthos The M/G ratio of crude polysaccharides and extracted galactomannans was determined according to the method [13, 21]. Samples (0.15g) were hydrolyzed using 72% H2SO4 at 30 C for 1 h followed by 4% H2SO4 at 121 C for 1 h [22]. Then hydrolysates obtained were neutralized with the CaCO3 and adsorbed by resin 7
(AG501-X8, Bio-Rad) to remove Ca2+. After centrifugation at 3000 × g for 10 min, the supernatant was filtered through a filter membrane (0.45 m). The M/G ratio was determined by High-Performance Liquid Chromatography (HPLC) (Agilent 1260, USA) using an Aminex HPX-87P column (300×7.8 mm; Bio-Rad Laboratories, USA) at 85 C with an evaporative light-scattering detector (ELSD). Ultra-pure water at 0.6 mL/min was used the mobile phase and nitrogen at 1.6 L/min was used as flowing gas, respectively. Calibration curves of D-galactose, D-mannose, glucose standards were used to determine the monosaccharides of crude polysaccharides and extracted galactomannans. 2.4. NMR (Nuclear magnetic resonance) spectra analysis It is necessary to degrade the extracted galactomannans before NMR determination because the extracted galactomannans have a higher molecular weight. 10 mg extracted galactomannans were dissolved in 0.55 mL of D2O (99.9%) and 0.55 mL of DCl (99.9%) at room temperature for an hour before determination. 1H NMR spectra of extracted galactomannans were obtained on Bruker Ascend 500 M NMR. Chemical shifts were calculated relative to sodium 2,2-dimethylsilapentane-5-sulphonate (DSS) as internal standard (0.00 ppm for 1H) [23]. The temperature of NMR spectrometer was 333 K, and the 1H NMR spectra was collected at a working frequency of 500 MHz. 2.5. Molecular weight analysis by SEC-MALLS The molecular weight distribution of extracted galactomannans was carried out on a size exclusion chromatography (Wyatt GPC/SEC-MALS system, Wyatt 8
Technology Corporation, Santa Barbara, CA, USA). The normalization of the detectors and determination of the inter-detector volume were performed with standard monodisperse dextran (MW = 50000Da, Mw / Mn = 1.36). Parameters including weight-average molecular weight (Mw), the number-average molecular weight (Mn), the intrinsic viscosity ([η]), the radius of gyration (Rg), the hydrodynamic radius (Rh) and the polydispersity index (ð = Mw / Mn) of extracted galactomannans were determined. TSK G5000PW column, TSK G3000PW column and OHPAK SB-G guard column were eluted with 0.1 M NaNO3. Extracted galactomannans were solubilized at 3 g/L in NaNO3 (0.1 M) under stirring at room temperature for 12h and the solution obtained was filtered through a 0.45 𝜇m filter before injection [24]. The refractive index increment (dn/dc) was 0.138 mL/g. Data were analyzed by the Astra version 5.3.4 software package. The fractal dimension (df) can be determined according to formula (1) and (2). 𝑅𝑔 = 𝑘1 × 𝑀𝑣𝑤
(1)
𝑑𝑓 = 1/𝑣
(2)
Where Mw is average molecular weight, Rg is gyration radius and df is fractal dimension. 2.6. XRD analysis Extracted G. triacanthos galactomannans were analyzed for X-ray diffraction (XRD) patterns. XRD diffractometer (D8 Advance, Bruker Co., Karlsruhe, Baden-Wurttemberg, Germany) (anode Cu-K, 45 kW, 40 mA) recorded the Wide-angle X-ray patterns, with a diffraction angle (2θ) range of 10 to 80 and 9
resolution of 0.02 at room temperature [18]. 2.7. Solubility and insoluble substances determination Solubility and insoluble substances were determined according to the method [25] with some modifications. The crude polysaccharides (0.15 g, dry basis) were dissolved in water (30 mL) at 30 C for 30 min and continuously stirred. The suspensions were then centrifuged at 3000 × g for 10 min. The insoluble substances were washed three times and dried at 105 C for 4 h until constant weight was obtained. The supernatant (10 mL) were dried at 105 C for 4 h until constant weight was obtained. The solubility was calculated by formula (3), and the insoluble substances were calculated by formula (4): Solubility (%) =
𝑤𝑠 × 300
(3)
𝑚0
Insoluble substances (%) =
𝑚1 × 100
(4)
𝑚0
Where 𝑤𝑠 is the dry weight of 10 mL supernatant, 𝑚1 is the dry weight of insoluble substances and 𝑚0 is the dry weight of crude G. triacanthos polysaccharides. 2.8. Rheological measurements of G. triacanthos galactomannan The apparent viscosity of extracted galactomannans from WAF 15 to WAF 25 was determined using Brookfield viscometer (Model LVDV-Ⅲ) with a spindle (No. SC4-31). The extracted galactomannans (0.4% w/v, dry basis) were dissolved by continuously stirring at 80 C for 30 min to achieve complete hydration [26] and then cooled to the room temperature before running the test. The shear stress (σ) and apparent viscosity were recorded with the shear rate (γ) increasing from 0 to 85 s-1 using the software (Rheocalc V3.2) at 25 C. All measurements were done in 10
triplicate. The rheology of GTS from WAF 15 to WAF 25 showed that shear rate versus shear stress were in good agreement with Power-law model (formula 5). 𝛾 = 𝑘2 × 𝛾𝑛
(5)
Where 𝛾 is the shear stress, 𝛾 is the shear rate, 𝑘2 is the consistency cofficient and 𝑛 is the flow behavior index.
3. Results 3.1. Physical parameter measurements of G. triacanthos To compare the physical parameters of G. triacanthos collected from WAF 15 to WAF 25, the size and fresh weight of each pod, the dry weight percentage of the three parts (endosperm, hull, and embryo) in seeds, and the endosperm moisture were measured. The results are shown in Figure 1. Figure 1a shows that the length and width of pods was constant whereas the fresh weight of pods decreased consistently from WAF 15 to WAF 25. As also seen from Figure 1a, the pods of G. triacanthos are crescent-shaped and their color gradually deepened from WAF 15 to WAF 25. Specifically, in the green, immature seed before WAF 19, galactomannan deposition may already begin [9]. The color of pods (Figure 1a) and seeds (Figure 1b) deepened gradually from WAF 21 to WAF 25, indicating that G. triacanthos galactomannans may be ripening [9]. As shown in Figure 1b, the endosperm moisture gradually decreased and the dry weight percentage of the three parts of seeds remained constant during galactomannan deposition and maturation. The ratio of the three parts in mature seeds was as follows: endosperm (36.67%), hull (34.41%), and embryo (28.92%). 11
3.2. M/G ratio of G. triacanthos galactomannan Figure 2 summarizes the M/G ratio and monosaccharide composition of crude polysaccharides (2a) and extracted galactomannans (2b). And the HPLC profile of crude polysaccharides and extracted galactomannans was provided in supplement (Figure S1). They were mainly composed of two monosaccharides: mannose and galactose. Compared to the crude polysaccharides, the content of mannose and galactose (M+G) in extracted galactomannans was higher, with trace amounts of glucose (Table S1 and Table S2), suggesting that the extraction process increased galactomannan purity. As shown in Figure 2b, the mannose content increased from 56.93% to 62.59% from WAF 15 to WAF 19. This may be a result of effective transport of the mannose to the end of the unreduced mannose chain by mannosyltransferase to prolong the mannose chain [11]. Subsequently the mannose content (63.39%) remained constant from WAF 19 to WAF 23. This result contributed to the ending of galactomannan deposition as result of loss of moisture in the endosperm (Figure 1b). Edwards et al. [13] also found that galactomannan deposition in fenugreek ended as the endosperm entered the desiccation phase. Finally, the mannose content decreased in WAF 25, which may be caused by a complex degradation mechanism involving several biochemical pathways [11, 27]. The galactose content gradually decreased from WAF 15 to WAF 25. These results suggest that α-galactosidase is capable of removing the galactosyl residues from the galactomannan side chains, which is consistent with previous
reports
[11].
These
enzymes 12
including
mannosyltransferase
and
α-galactosidase in galactomannan development have been intensively investigated [11, 13, 28]. In addition, the sum of mannose and galactose content (expressed as the sum of Man and Gal in Figure 2b) increased from WAF 15 (79.45%) to WAF 23 (83.55%) and the values then reduced to 81.19% in WAF 25. The M/G ratio is directly related to the degree of branching, determining a variety of physicochemical properties of galactomannan, including its solubility and viscosity [29]. From WAF 15 to WAF 25, the M/G ratios observed (Figure 2b) were close to those estimated by other authors, varying between 2.3 and 3.3 of galactomannan in the endosperm [19, 30]. The M/G ratio was about 2.53 in WAF 15 and increased substantially in WAF 23, which was in accordance with the M/G changes in Senna endosperm [13]. The M/G ratio was then reduced to 3.16 in WAF 25. As a result, the M/G ratio in WAF 23 was the highest (3.24), presenting regions of mannose with scarce branching. Conversely, the mannan backbone in guar gum was highly substituted and the M/G ratio was 1.50 [31]. Galactomannans with different M/G ratios can be used in different industrial fields. For example, galactomannans with a high M/G ratio (3.24) in WAF 23 are suitable for producing strong and flexible films with higher elastic modulus, tensile strength, and break elongation [32]. In contrast, galactomannans with low M/G (2.53) in WAF 15, could replace guar gum and be used as an strengtheners and stabilizers [17, 33]. 3.3. 1H-NMR spectra analysis The 1H-NMR spectra of extracted galactomannans were shown in Figure 3. There were two distinct peaks (5.04 ppm, 4.78 ppm) in the anomeric region of the 1H 13
spectra of the extracted galactomannans, which were α-galactopyranosyl and β-mannopyranosyl respectively [20]. The M/G ratio can be also obtained directly from the relative areas of the anomeric signals for 1H-NMR spectra as Cunhua et al. (2009) [23]. The ratios were 2.55, 2.82, 3.01, 3.18, 3.26, and 3.16 for WAF 15, WAF 17, WAF 19, WAF 21, WAF 23, and WAF 25. These values were in good agreement with the results obtained with HPLC analysis (Table S2). 3.4. Molecular weight analysis Molecular weight, gyration radius (Rg) and hydrodynamic radius (Rh) are summarized in Table 1; these affect the rheological and functional properties of galactomannans [34]. The PDI value (Mw / Mn) known as polydispersity index, is close to 1 for monodispersed polymers [35]. The PDI value ranging from 1.05 to 1.26 (Table 2), reflects the low polydispersity and relatively high purity of extracted G. triacanthos galactomannans. The weight average molecular weight (g/mol) and the [η] values from WAF 15 to WAF 25 were lower than the values of G. triacanthos harvested in Portugal (1.62 ×106 Da and 1042 mL/g) [36]. These differences can be attributed to the dependence of Mw and [η] values on both the seed growing conditions, extraction, and purification methods. Furthermore, the values of Mw and the [η] were increased from WAF 15 (0.98×106 g/mol, 729.96 mL/g) and reached the maximum (1.28×106 g/mol, 882.53 mL/g) in WAF 23, followed by a decrease in WAF 25 (1.12×106 g/mol, 726.14 mL/g). The former increase further confirming that galactomannan deposition was underway from WAF 15 to WAF 23. The decreased values of Mw and [η] in WAF 25 may be attributed to a complex degradation 14
including some biochemical pathways [11, 26]. Galactomannan with different molecular weights can be used for different industrial applications. For example, galactomannans with high molecular weight (1.28×106 g/mol) in WAF 23 were used as a thickening agent [37]. In contrast, galactomannans with low molecular weight (0.98×106 g/mol) in WAF 15 could be used to produce manno-oligosaccharides which are regarded as dietary fiber and prebiotics [21]. Furthermore, the structure factor (ρ = Rg / Rh) for G. triacanthos was revealed to be 1.801.88 by the SEC-MALS system, which confirmed that GTSP was in random coil conformation in the diluted solution [38]. The vg values of GTSP were between 0.5 and 1, further confirming the random coil shape of GTSP in dilute solutions [39]. The fractal dimension (df) is a measure of the compactness of a polymer structure and the larger its value, the more compact is the polymer structure [38, 40]. The df values (close to 1.93) was highest in WAF 23, and galactomannan compactness was caused by the highest M/G ratio in WAF 23 (Figure 2). 3.5. X-ray diffraction analysis of GTSP The X-ray diffractograms of extracted galactomannans from WAF 15 to WAF 25 are shown in Figure 4. The results indicate that extracted galactomannans presents low overall crystallinity. The crystalline regions of extracted galactomannans were observed at the angle (2θ) 20.2 ° from WAF 15 to WAF 25, indicating that extracted galactomannans underwent negligible changes in the XRD curve during galactomannan deposition and maturation. However, the crystallinity of extracted galactomannans showed a slight difference. The increased crystallinity of extracted 15
galactomannans from WAF 15 (30.9%) to WAF 19 (32.2%) may be attributed to the lengthened mannose main chain caused by mannosyltransferase (Figure 2b) and more aggregated molecule chains to increase crystallinity. The crystallinity of samples from WAF 19 to WAF 23 then remained constant because of the end of transport by mannosyltransferase due to moisture loss (Figure 1b). Furthermore, the crystallinity of samples decreased in WAF 25 (31.2%), possibly due to lower molecular weight (Table 1). Kong et al. [41] also found that lower crystallinity of a biopolymer is actually related to its lower molecular weight. Furthermore, reduction in the Mw of galactomannan chains (Table 1) did not form a new crystalline structure and did not affect the amorphous nature of galactomannans, which was supported by previous reports [42]. 3.6. Solubility and Insoluble substances From WAF 15 to WAF 23, there was a general trend showing a decrease in solubility and an increase in insoluble substances in the gums (Figure 5). This low solubility might be due to the high M/G ratio (Figure 2a), which allows compact packing of polysaccharides [43]. The low solubility of polysaccharides is also related to their high molecular weight (Table 1) and the high viscosity of polysaccharides occurs from resistance to flow (Figure 6) [44]. Compared to high molecular weight polysaccharides, low molecular weight polysaccharides are more soluble in water. From WAF 15 to WAF 23, the molecular weight and intrinsic viscosity increase gradually (Table 1), resulting in a decreased solubility. This trend was inverted in WAF 25 with 32.89% insoluble substances, which is 16
significantly lower than the 42.20% insoluble substances in WAF 23. This may result from the low molecular weight in WAF 25 (Table 1). Furthermore, a decrease in M/G (Figure 2a) results in an increase in solubility in WAF 25 [43]. The total recovery yield (the sum of solubility and insoluble substance) is close to 100%, indicating that the results are reliability. 3.7. Rheological analysis The rheological properties of extracted galactomannans from WAF 15 to WAF 25 were compared. As shown in Table 2, the coefficients of determination (R2) were higher than 90.6 for all tested samples, indicating the flow properties of GTSP fitted the power law model [45]. The k values for the consistency coefficient increased from WAF 15 to WAF 23, causing an increase in intermolecular interactions [46]. In addition, the flow behavior index (n) was in the range of 0.80 to 0.86, indicating that the solutions of extracted galactomannans from WAF 15 to WAF 25 were typical pseudoplastic fluids [47]. The power law model designates that the pseudoplasticity of the fluid increases with a decrease in n values. The pseudoplasticity of the fluid was the highest in WAF 15, WAF 17, and WAF 23, which may be caused by the predominance of hydrogen bonds from the water medium [26]. As shown in Figure 6a, the extracted galactomannans solutions displayed shear thinning behavior, which further confirmed their pseudoplastic behavior. This phenomenon could be caused by the interactions between galactomannan chains. With increasing shear rate, the GTSP molecule chain was gradually unfolded due to increasing shear stress (Fig. 6b). The continued shear stress breaks towards linkages, 17
resulting in an apparent loss of viscosity [48]. The viscosity of galactomannan solutions increased from WAF 15 to WAF 23 due to the increase in M/G ratios (Figure 2b), combined with the increase in molecular weight (Table 1). In general, higher viscosity can represent a greater amount of β-(1→4)-mannan chains per unit carbohydrate weight due to the slight decrease in galactose content (Figure 2b). Mallet et al. [49] also found that the increasing viscosity may be due to the higher M/G ratio of galactomannans, in combination with a somewhat greater molecular weight. However, the viscosity of galactomannan decreased since WAF 25, which was caused by a decrease in M/G ratio and molecular weight in WAF 25, which was consistent with Jian, et al. [26]. 4. Conclusions The fresh weight of G. triacanthos pods and the endosperm moisture was decreased, whereas the weight ratio of the three parts in seeds (endosperm, hull, and embryo) remained constant during G. triacanthos galactomannan deposition and maturation. NMR and XRD analysis demonstrated that no changes in chemical structure but showed the slight changes in crystallinity. The M/G, apparent viscosity, and molecular weight increased from WAF 15 to WAF 23 firstly, and then decreased at WAF 25, causing changes in solubility. Based on the different physicochemical properties of G. triacanthos galactomannans during their deposition and maturation, their industrial applications could be developed further. Declaration of interest: None. 18
Acknowledgments This research was financially supported by the National Key R&D Program of China (2016YFD0600803), the Natural Science Foundation of China (31670579), and the Specific research project of Guangxi for research bases and talents (AD18126005). References [1] C. Sandolo, P. Matricardi, F. Alhaique, T. Coviello, Dynamo-mechanical and rheological characterization of guar gum hydrogels, Eur. Polym. J. 43 (2007) 3355-3367. [2] K.S. Dhugga, R. Barreiro, B. Whitten, K. stecca, Jan Hazebroek, G.S. Randhawa, M. Dolan, A.J. Kinney, D. tomes, S.Nichols, P. Anderson, Guar seed -mannan synthase is a member of the cellulose synthase super gene family, Science, 303 (2004) 363-366. [3] N.M. Siqueira, B. Paiva, M. Camassola, E.Q. Rosenthal-Kim, K.C. Garcia, F.P. Santos,
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galactomannan
hydrolysates:
Rheological
behavior
and
physicochemical
characterization, Carbohydr Polym. 206 (2019) 573-582. [43] L.S. Sciarini, F. Maldonado, P.D. Ribotta, G.T. Pérez, A.E. León, Chemical composition and functional properties of Gleditsia triacanthos gum, Food Hydrocol. 23 (2009) 306-313. [44] F. Rashid, S. Hussain, Z. Ahmed, Extraction purification and characterization of galactomannan from fenugreek for industrial utilization, Carbohydr Polym. 180 (2017) 88-95. [45] E.A. Reyes, A.R. Guerrero, R.G. Rivera, V.V. Guerrero, J.C. Olivares, C.P. Alonso, Rheological properties of tamarind seed mucilage obtained by spray-drying as a novel source of hydrocolloid, Int. J. Biol. Macromol. 107 (2017) 817-824. [46] A. Koocheki, A.R. Taherian, A. Bostan, Studies on the steady shear flow behavior and functional properties of lepidium perfoliatum seed gum, Food Res Int. 50 (2013) 446-456. [47] M. Wu, D. Li, L. Wang, Y. Zhou, Z. Mao, Rheological property of extruded and enzyme treated flaxseed mucilage, Carbohydr Polym. 80 (2010) 460-466. [48] V. Singh, R. Sethi, A. Tiwari, Structure elucidation and properties of a non-ionic galactomannan derived from the Cassia pleurocarpa seeds, Int. J. Biol. Macromol. 44 (2009) 9-13. [49] AB.V. McCleary, I. Mallet, N.K. Matheson, Galactomannan changes in developing Gleditsia triacanthos seeds, Phytochemistry. 26 (1987) 1889-1894. 25
Figures and captions Figure 1a. Length, width, fresh weight, and photographs of each Gleditsia triacanthos pod collected from WAF 15 to WAF 25 (weeks after flowering).
Figure 1b. Dry weight percentage of the three parts (endosperm, hull, embryo) of each Gleditsia triacanthos pods, the endosperm moisture and the photographs from 26
WAF 15 to WAF 25 (weeks after flowering).
Figure 2. Monosaccharide compositions of Gleditsia triacanthos galactomannan (2a crude polysaccharides, 2b extracted galactomannans) from WAF 15 to WAF 25 (weeks after flowering).
Figure 3. the 1H spectra of extracted galactomannans from WAF 15 to WAF 25 (M: mannose, G: galactose).
Figure 4. X-ray diffractograms of extracted Gleditsia triacanthos galactomannans from WAF 15 to WAF 25.
Figure 5. Solubility and insoluble substances (%) of crude Gleditsia triacanthos galactomannans from WAF 15 to WAF 25.
Figure 6. Apparent viscosity (6a) and flow curve (6b) of the 0.4% (w/v) extracted G. triacanthos galactomannans solutions at 25 C.
27
Figure 1a
28
Figure 1b
29
Figure 2
30
WAF 15 WAF 21
G-1
WAF 17 WAF 23
WAF 19 WAF 25
M-1
1:3.16
1:3.26
1:3.18
1:3.01
1:2.82
1:2.55
5.2
5.0
4.8
4.6
4.4
4.2
4.0
Chemical shift (p.p.m.)
Figure 3
31
3.8
3.6
3.4
WAF 13-30.9% WAF 15-31.6% WAF 17-32.2% WAF 19-32.4% WAF 21-32.2% WAF 23-31.2%
10
20
30
40
Diffraction angle 2θ（degree)
Figure 4
32
50
60
100
solubility
80
insoluble substance 90
70 80
60 50
70 40 60
30 20
50 10 40
0
WAF 15
WAF 17
WAF 19
WAF 21
Sampling time
Figure 5
33
WAF 23
WAF 25
Total recovery yield (%)
Solubility and insoluble substance (%)
90
Figure 6
34
Tables Table 1. Molecular weight of extracted G. triacanthos galactomannans from WAF 15 to WAF 25 by SEC MALLS. Parameter aM
WAF 15
WAF 17
WAF 19
WAF 21
WAF 23
WAF 25
0.98±0.16
1.12±0.12
1.09±0.12
1.21±0.14
1.28±0.21
1.12±0.16
1.05±0.01
1.12±0.02
1.17±0.01
1.11±0.06
1.26±0.01
1.14±0.02
92.7±3.16
83.8±2.06
96.4±3.22
92.9±3.16
91.6±3.33
90.3±3.10
(nm)
51.5±1.60
46.4±1.16
51.4±1.11
49.9±1.60
50.5±1.26
48.3±1.61
(Rg/Rh)
1.80±0.16
1.81±0.12
1.88±0.12
1.86±0.14
1.81±0.26
1.87±0.16
729.96±
750.26±
764.66±
788.11±
882.53±
726.14±
5.16
5.23
6.10
6.66
6.12
6.34
0.89
0.87
0.89
0.77
0.52
0.78
1.11
1.14
1.16
1.29
1.93
1.28
w (10
6 g/mol)
bPDI
c
Rg (nm)
dR h
eρ
f[η]
(mL/g) gv
g
hd
f
a
Mw: average molecular weight. b PDI: polydispersity index. c Rg: gyration radius.
d
Rh: hydrodynamic radius. e ρ: the structure sensitive. f [η]: intrinsic viscosity.
gv
g:
vg = 1/ df. hdf: fractal dimension.
35
Table. 2. Power law parameters for extracted G. triacanthos galactomannans dispersions (0.4% w/v) from WAF 15 to WAF 25. Sampling Time WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
k (Pa.sn)
n
R2
0.113±0.002
0.80±0.00
98.1±0.1
0.133±0.003
0.80±0.02
98.1±0.1
0.137±0.001
0.86±0.01
96.8±0.4
0.157±0.002
0.86±0.01
95.3±0.2
0.261±0.019
0.80±0.01
95.2±0.4
0.125±0.005
0.83±0.02
98.2±0.1
Where k is the consistency coefficient, (R2) is the coefficient of determination and n is the flow behavior index.
36
Supplemental files:
Figure. S1. the HPLC profile of crude polysaccharides (a) and extracted galactomannans (b) from WAF 15 to WAF 25 (M: mannose, G: galactose).
37
Table. S1. Monosaccharide composition of crude Gleditsia triacanthos polysaccharides from WAF 15 to WAF 25 (M: mannose, G: galactose, M+G: the sum content of mannose and galactose). Sampling time
Mannose
Galactose
Glucose
M+G
M/G
WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
52.64±0.05 56.28±0.07 57.27±0.80 57.62±0.30 58.47±0.19 56.94±0.14
22.11±0.30 19.48±0.02 19.30±0.08 19.22±0.02 18.37±0.01 18.31±0.05
2.78±0.05 3.9±0.07 3.06±0.09 2.08±0.00 1.17±0.09 4.28±0.14
74.75±0.15 75.76±0.49 76.57±0.27 76.84±0.33 76.84±0.18 75.25±0.11
2.37±0.00 2.79±0.06 2.97±0.03 3.00±0.01 3.18±0.01 3.11±0.05
38
Table. S2. Monosaccharide composition of extracted Gleditsia triacanthos galactomannans from WAF 15 to WAF 25 (M: mannose, G: galactose, M+G: the sum content of mannose and galactose). Sampling time
Mannose
Galactose
Glucose
M+G
M/G
WAF 15 WAF 17 WAF 19 WAF 21 WAF 23 WAF 25
56.93±0.11 60.64±0.27 62.59±0.18 63.39±0.15 63.85±0.01 61.66±0.04
22.52±0.09 21.64±0.03 20.65±0.05 19.96±0.06 19.70±0.02 19.53±0.04
0.68±0.01 0.86±0.07 0.97±0.08 0.00±0.05 0.00±0.01 1.99±0.04
79.45±0.02 82.28±0.35 83.25±0.67 83.35±0.21 83.55±0.01 81.19±0.00
2.53±0.02 2.80±0.02 3.03±0.02 3.18±0.00 3.24±0.00 3.16±0.01
39