Solution properties of a heteropolysaccharide extracted from pumpkin (Cucurbita pepo, lady godiva)

Carbohydrate Polymers 132 (2015) 221–227

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Solution properties of a heteropolysaccharide extracted from pumpkin (Cucurbita pepo, lady godiva) Yi Song a,b , Jing Zhao a,c , Yuanying Ni a,c , Quanhong Li a,c,∗ a b c

National Engineering Research Center for Fruits and Vegetables Processing, Beijing 100083, China College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

a r t i c l e

i n f o

Article history: Received 17 January 2015 Received in revised form 13 June 2015 Accepted 20 June 2015 Available online 25 June 2015 Keywords: Pumpkin Polysaccharide Solution property

a b s t r a c t A water-soluble galactoglucofucomannan was extracted from pumpkin (Cucurbita pepo, lady godiva variety). GC–MS analysis indicated that the polysaccharide was composed of 1,6-linked-glucosyl, 1,2,6-linked-mannosyl, 1,3,6-linked-mannosyl, 1,2,6-linked-galactosyl, 1,2,6-linked-galactosyl, terminal fucosyl and terminal glucose. The solution properties of the polysaccharide were studied systematically by using size-exclusion chromatography combined with multi-angle laser light scattering, viscometry and dynamic light scattering at 25 ◦ C. The weight average molecular masses (Mw ), intrinsic viscosity [], radius of gyration (Rg ) and hydrodynamic radius (Rh ) were found to be 12.7 × 105 g/mol, 780 ml/g, 68 nm and 116 nm, respectively. The fraction dimension and value of  (Rg /Rh ) of the polysaccharide revealed that it existed in a sphere-like conformation in distilled water. The dependence of zero shear specific viscosity on the coil overlap parameter was analyzed using different models. Furthermore, degradation of samples upon autoclaving has been observed and quantified by intrinsic viscosity determination and SEC-MALLS. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction In common with many polysaccharides from different sources, plant polysaccharide has potentially important commercial applications, particularly in the pharmaceutical and food industries for controlling drug release and modifying texture, respectively (Lapasin, Delorenzi, Pricl, & Torriano, 1995). Moreover, it has been known for many years that some plant polysaccharides have nutritional and therapeutic benefits, notably in the treatment of metabolic disorders (Ellis, 1994) and reductions in the postprandial rise in blood glucose and insulin concentrations (Li, Fu, Rui, Hu, & Cai, 2005). However, the solubility of polysaccharides in water affects their potential clinical applications since intravenous administration of the micro-particulate form of insoluble glucans have been associated with a number of undesirable side effects including hepatosplenomegaly, microembolization and enhanced endotoxin sensitivity (Zhang, Cheung, Zhang, Chiu, & Ooi, 2004).

∗ Corresponding author at: College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua East Road, Beijing 100083, China. Tel.: +86 10 62738831. E-mail address: [email protected] (Q. Li). http://dx.doi.org/10.1016/j.carbpol.2015.06.061 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

Considerable researches in producing homogeneous solutions of polysaccharides have been reported using physical methods whereby supramolecular aggregates are affected by increasing the energy of the component polymer chains, including sonication, ␥irradiation, autoclaving and microwave (Tayal & Khan, 2000; King & Gray, 1993; Bello-Perezt, Roger, Baud, & Colonna, 1998). The technique of the autoclave has been employed in a comprehensive study of guar, locust bean, tara, detarium gums and tamarind seed polysaccharide (Picout, Ross-Murphy, Jumel, & Harding, 2002; Picout, Ross-Murphy, Errington, & Harding, 2003). The heating of samples in the autoclave is caused by the interaction of the temperature field and thermal stress with the chemical constituents of solutions. These interactions gradually generate heat because of molecular friction and excitation. Since some nutritional and therapeutic activities are related to the molecular weight and chain conformation of polysaccharides, it is essential to understand the solution properties for further research related to biochemistry or medical application (Szopinski, Kulicke, & Luinstra, 2015). Pumpkin (Cucurbita pepo) is one of the popular cultivated and underexploited crop, which has been accepted as a dietary constituent in China and received considerable attention in recent years because of the nutritional and health protective value of the proteins and oil, from the seeds as well as the polysaccharides from the fruits (Siegmund & Murkovic, 2004; Murkovic, Piironen, Lampi,

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Kraushofer, & Sontag, 2004). In our previous work, the structure and hypoglycemic activity of pumpkin polysaccharide LGPP2-1 (Song, Li, Hu, Ni, & Li, 2011; Li et al., 2005) had been studied. LGPP2-1 and its acetylated derivatives also exhibited antioxidant and cytoprotective effect in vitro (Song et al., 2013). In the present study, we described the determination of the molecular properties of another kind of pumpkin polysaccharide using SEC combined with multiangle laser light scattering. Intrinsic viscosity was determined by capillary viscometry, and the hydrodynamic radii was measured by photon correlation spectroscopy. The zero shear viscosity was determined from shear flow measurements over a range of concentration. The monosaccharide composition and interglycosidic linkages of the polysaccharide was measured by GC–MS. Effects of heat/pressure treatment on the molecular mass distribution were also studied using the autoclave. The work provided valuable information for further understanding of solubility and chain conformation properties of water-soluble biopolymers. 2. Materials and methods

polysaccharide (LGPP2-1 and LGPP2-2). The yield and total sugar content of LGPP2-2 was estimated 5.23% and 61.6%, respectively. 2.3. Methylation analysis Methylation of LGPP2-2 (2 mg) was carried out using the method of Needs and Selvendran (1993). Complete methylation was confirmed by the disappearance of the hydroxyl peak (3200–3700 cm−1 ) in the IR spectrum. The permethylated product was hydrolyzed with 2 M TFA at 100 ◦ C for 4 h. Then the resulting hydrolysates were reduced with NaBH4 (20 mg) and acetylated with acetic anhydride. The methylated alditol acetates were analyzed by GC–MS using a Finnigan Trace GC 2000 equipped with a Finnigan DSQ mass spectrometer. The temperature program was as follows: injection temperature: 180 ◦ C; detector temperature: 180 ◦ C; column temperature programed from 150 ◦ C to 180 ◦ C at 10 ◦ C/min, then increasing to 260 ◦ C at 15 ◦ C/min and finally holding for 5 min at 260 ◦ C. The partially methylated alditol acetates were identified by their fragment ions in GC–MS and the molar ratios were estimated from the peak areas and the response factors.

2.1. Plant materials and chemicals 2.4. Determination of molecular mass Fresh pumpkin (C. pepo, lady godiva variety) fruits at the commercially mature stage were purchased from a local commercial market in Beijing, China. Fruits were selected for their uniformity in shape, weight and color. TFA, NaBH4 , acetic anhydride were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All the other chemicals used purchased from Sinopharm Chemical Reagent Beijing Co. Ltd., and were all of analytical grade. 2.2. Preparation of pumpkin polysaccharide solution Pumpkin polysaccharides LGPP2-2 were extracted by hot water following the method of Song et al. (2011). Briefly, the fresh pumpkins were pulped using the beater after peeling. Then the pumpkin puree was mixed with distilled water at the ratio of 1:4 and kept still in water bath at 85 ◦ C for 4 h. After centrifugation at 750 × g for 15 min, the residues were mixed with distilled water at the same ratio and let stand in water bath at 85 ◦ C for 3 h. The supernatants were mixed, concentrated to a quarter of the original volume by evaporation. After cooling, the proteins in the extract were removed by the Sevag reagent (Sevag:mixture = 1:3, v/v). Then the crude mixtures were precipitated by the addition of ethyl alcohol with the ratio of 1:1 and then precipitated at 4 ◦ C for 12 h. After centrifugation at 1200 × g for 20 min, the supernatants were precipitated from the filtrates by the addition of ethyl alcohol with the ratio of 4:1 to the final alcohol concentration 80% (v/v). The resulting precipitates were collected and washed with ethanol and lyophilized (termed LGPP2). The polysaccharide LGPP2 was re-dissolved in distilled water and applied to a DEAE Sepharose Fast Flow gel column (2.6 × 100 cm). One milliliter of 1 mg/mL crude LGPP2 was subjected to the gel column and eluted by filtered (0.45 ␮m membrane) distilled water and then by 0, 0.2, 0.4, 0.6 M NaCl at a flow rate of 4.0 mL/min. Each fraction with 1 mL of eluate was collected and determined by the phenol–sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Based on the colorimetric total carbohydrate test using the method mentioned above, the water fraction, appearing as a single peak, was further purified by the gel permeation chromatography on a column of Sephacryl S-300 High Resolution (2.6 × 100 cm). One milliliter of 1 mg/mL sample was subjected to the column and eluted by distilled water at a flow rate of 1.0 mL/min. Each fraction with 1 mL of eluate was collected, dialyzed and then lyophilized to get two purified pumpkin

The molecular weight and chain conformation of LGPP2-2 were determined by SEC combined with multi-angle laser light scattering ( = 690 nm, DAWN HELEOS II, Wyatt Technology Co., U.S.A) equipped with a Shodex Sugar KS-805 (8.0 × 300 mm) and an optilab refractometer (DAWN, Wyatt Technology Co., U.S.A). Fifty microlitres of sample solution was injected in each run, with the flow rate of 0.5 mL/min. The basic light scattering equation was as follows:



Kc 1 = R Mw

1+

 

 

162 S 2 32

z

2

· sin

 2

+ 2A2 C

(1)

where K was the optical constant equal to [42 n2 (dn/dc)2 ]/(4 NA ); c was the polysaccharide concentration (mg/mL); R was the Rayleigh ratio; k was the wavelength; n was the refractive index of the solvent; NA was the Avogadro’ number; A2 was the second virial coefficient. 2.5. Determination of the intrinsic viscosity The intrinsic viscosity [] was determined by the method of Ross-Murphy (1994). LGPP2-2 solutions of different concentrations (0.01–0.1 g/mL) were prepared by dispersing the sample in deionized water and filtered through a 0.45 ␮m membrane before measurements. Viscosity measurements were performed using a viscometer (CAV 2000 series, Cannon Instrument Company, PA, U.S.A.) at 25.0 ± 0.1 ◦ C. As the experiment was performed at low concentrations, the intrinsic viscosity was determined by Kraemer equation: lnr = [] − kK []2 C C

(2)

where kK was the Kraemer constant. 2.6. Determination of zero shear viscosity Shear flow measurements of the polysaccharide solutions prepared at different concentrations (0.1–3.0 g/mL) were carried out using the rheometer (ARES-G2, TA Instruments, New Castle, DE, U.S.A.) with the 50 mm diameter cone. The experiments were performed at 25.0 ± 0.1 ◦ C, typically in the shear rate range of

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Table 1 Molecular properties of pumpkin polysaccharide LGPP2-2. Mn × 105 (g/mol)

Mw × 105 (g/mol)

Mz × 105 (g/mol)

Rg (nm)

Dispersity

[] (mL/g)

KK

df

v

10.3 ± 0.6

12.7 ± 0.8

14.2 ± 1.6

68 ± 3

1.23 ± 0.2

780

1.4 ± 0.52

2.58

0.387

0.05–1000 s−1 with a reduction in rate at the higher concentration. Zero shear viscosity was determined as follows:

(Rg ) and is defined as the inverse of the exponent v (Hanselmann, Burchard, Ehrat, & Widmer, 1996):

 − ∞ 1 = 0 − ∞ 1 + (K)n

Rg ∼M v

(3)

where  was the viscosity, ∞ was the infinity shear viscosity, 0 was the zero shear viscosity,  was the shear stress, n was the power law exponent and K was the arbitrary constant. 2.7. Determination of hydrodynamic radius LGPP2-2 was diluted with appropriate amounts of distilled water to achieve concentrations in the range of 0.025–0.1% g/mL. Measurements were made on each at 25 ◦ C using a Zetasizer Nano ZSP instrument (Malvern Instruments, Malvern, U.K.). Samples were filtered through 3 ␮m membrane and distilled water was used as solvent. 2.8. Effects of autoclave treatment on pumpkin polysaccharide 0.1 g/mL LGPP2-2 solutions were subjected to up to four 15 min autoclave cycles at 121 ◦ C using a electric autoclave (Betastar, Honey Brook, PA, U.S.A.) for molecular weight and intrinsic viscosity measurement. For each sample, the molecular mass distribution and intrinsic viscosity were determined as for the untreated polymer. Fifty microlitres of sample solution was injected in each run, with the flow rate of 0.5 mL/min. 3. Results and discussion 3.1. Methylation analysis The monosaccharide composition and interglycosidic linkages between monosaccharide residues of the pumpkin polysaccharide LGPP2-2 were investigated by methylation analysis. As a result of it, 2,3,4-tri-O-methylfucose, 2,3,4-tri-O-methylgalactose, 3,4-di-O-methylgalactose, 3,6-di-O-methylgalactose, 3,4-di-Omethylmannose and 2,3,4,6-tetra-O-methylglucose were detected (Table S1). The composition indicated that LGPP2-2, a galactoglucofucomannan, might present as part of hemicelluloses in the cell wall of pumpkin, of which the detailed structure varies widely between different species and cell types (Scheller & Ulvskov, 2010).

df =

1

v

(4) (5)

Fig. 1 shows the plot of (S2 )z 1/2 (Rg ) versus Mw of the polysaccharide. After a calibration period, the v value and df value was calculated to be 0.387 and 2.58 for pumpkin polysaccharide according to Eqs. (4) and (5), respectively. The value of 2.58 is characteristic of a particle having an internal structure between the hard sphere (df = 3.0) and the fully swollen branched macromolecule in a thermodynamically good solvent (df = 2.0) (Hanselmann et al., 1996). A fractional dimension of df = 2.5 is predicted for branched clusters, which are not swollen (Bauer & Burchard, 1993). Scherrenberg et al. (1998) have found that the value of df for some dendrimers is 3, suggesting a uniform distribution of segments in the space pervaded by the polymer. The results confirmed that pumpkin polysaccharide LGPP2-2 existed as a compact conformation of sphere-like structure in water aqueous solution (Trappe, Bauer, Weissmüller, & Burchard, 1997). 3.3. Intrinsic viscosity The [] was first determined by extrapolation of the straight line to the y-axis in a plot where the reduced viscosity (s /C) is drawn again the polysaccharide concentration (Fig. 2A). It was estimated to be 7.8 dL/g (780 mL/g) (Table 1), for polysaccharide concentrations between 0.01 and 0.1 g/mL. Furthermore, the viscosity (r = /0 ) relative to that of the solvent (water) lay in the range of 1.1 to 2.1, which indicated that the solution was essentially a Newtonian (Fig. 2B). The high value of [] resulted in a low C* (C* = 1/[] = 1/7.8 g/dL = 0.128 g/dL), which meant that solutions of polysaccharide had significant viscosity effects at relatively low concentrations. The Kraemer coefficient (kK ) determined from the viscometry measurement was ∼1.4. Similar results have been reported for aqueous solutions of galactomannan from locust bean gum, guar galactomannan, cassia and tara gums (Daas, Grolle, Van Vliet, Schols, & De Jongh, 2002; Gaisford, Harding, Mitchell, & Bradley, 1986; Doublier & Launay, 1981). The high values indicated a poor solvent environment. In this unfavorable local environment, large fraction of the polysaccharide tended to assemble into large

3.2. Molecular properties Molecular properties and R.I. response of the pumpkin polysaccharide LGPP2-2 were shown in Table 1 and Fig. S1. The number average molecular weight (Mn ), which was the statistical average molecular weight of all the polymer chains in the sample, was 10.3 × 105 g/mol, while the z-average molecular weight (Mz ), which represented the third moment or third power average molecular weight, was 14.2 × 105 g/mol. The dispersity index (1.23), which was defined by Mw /Mn , was used as a measure of the broadness of a molecular weight distribution of a polymer. The weight average radius of gyration (68 nm), indicated the stiffness of the molecule. Furthermore, relevant structural information of the polysaccharide could be obtained by investigating the fractal dimension (df ) value, which could be determined from the Mw dependences of (S2 )z 1/2

Fig. 1. log (S2 )z 1/2 versus log Mw for pumpkin polysaccharide LGPP2-2 at 25 ◦ C.

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the polymer led to the increase of kK value, and thus result in the very large values of the kK (above 0.8) (Lefebvre & Doublier, 2005). 3.4. Shear flow measurements

Fig. 2. (A) Estimating intrinsic viscosity of pumpkin polysaccharide from plots s /C versus C(%); (B) Estimating intrinsic viscosity of pumpkin polysaccharide from plots ln (r )/C versus C(%).

particles to minimize interfacial contact. This assembling process upon addition of poor solvent was expected to take place at relatively fast rate and occurred in a random fashion, preventing the chains to organize. The unorganized arrangement did not allow the backbones to overlap, leaving electronic properties of the chains unaltered (Traiphol et al., 2007). Lefebvre and Doublier (2005) also suggested that knowledge of kK value might be more complex because some polymer coils could interpenetrate and shrink as concentration increases. This concentration-dependent aggregation of

A high intrinsic viscosity [] probably means that the viscosity depends on the shear rate of the test. Likewise, the extrapolation to zero concentration will also depend on shear rate. The zero shear viscosity is the intrinsic viscosity that should be used in molecular weight calculations. If [] is not too high, the measured value of [] at some non-zero shear rate can probably be assumed to be a good estimate of the zero shear viscosity. The response of the polysaccharide solutions to shear rate experiments were studied over a wide range of concentration (0.1–3.0 g/mL). Fig. 3 shows the shear viscosity versus shear rate range 10−2 to 103 s−1 on a double logarithmic scale. No shear rate viscosity dependence () was discovered when concentrations were lower than 0.3 g/mL. But obvious shear-thinning behavior was noticed when solutions of concentration were above level of 0.3 g/mL. It is useful to combine the zero shear specific viscosities from shear flow measurements and viscosities against the coil overlap parameter, c[], which could be defined as a measure of the hydrodynamic volume of the polymer in the solution. As Fig. 4A shown, the data was considered simply as two power law regimes. The polymer concentration at the intersection of the lines defined the critical overlap concentration, C* (0.282 g/dL). C* is generally accepted as the upper limit of dilute solution behavior, above which the influence of overlapping molecular domains becomes significant. For most random coil polysaccharides, C* coincides with an overlap parameter of approximately 4 g/dL, whereas values considerably lower (∼2.5 g/dL) have been reported for galactomannans (Morris, Cutler, Ross-Murphy, Rees, & Price, 1981; Ratcliffe, Williams, Viebke, & Meadows, 2005). Close inspection of Fig. 4A revealed that, for LGPP2-2, C* [] was calculated to be 1.9, similar to that reported for a galactomannan obtained from the seeds of Mimosaceae spp. and Amorphophallus konjac (Ratcliffe et al., 2005; Ganter & Reicher, 1999), although no specific value for C* commented that significant coil overlap was apparent when c[] was greater than unity. The gradient for the lines were 1.3 for the dilute regime and 4.1 for the semi-dilute regime. The exponent in the dilute regime compared more generally for random coil polysaccharides (Morris et al., 1981). For random coil polymers, the exponent in the concentrated regime always fall in the range of 3.3 ± 0.3. However, rheological properties of some polysaccharide have been found to depart from those of typical “random coil”

Fig. 3. Shear rate dependence of viscosity for different concentrations of pumpkin polysaccharides.

Y. Song et al. / Carbohydrate Polymers 132 (2015) 221–227

225

Fig. 5. Determination of D0 for pumpkin polysaccharide LGPP2-2.

3.5. Rh from dynamic light scattering The diffusion coefficients were measured for a range of LGPP2-2 concentrations and extrapolated to zero concentration (Fig. 5). Rh was then calculated from D0 using the Stokes–Einstein equation (Sun, 1994): D0 =

Fig. 4. (A) Variation in the zero-shear specific viscosity, log  of pumpkin polysaccharide LGPP2-2 with the coil overlap parameter, log C[]; (B) Illustration of curve fitting for the variation in the zero-shear specific viscosity, log  of pumpkin polysaccharide LGPP2-2 with the coil overlap parameter, log C[].

polysaccharides. In particular, an abrupt change of slope is obtained at exponent of 2.5, in comparison with exponent of 4 for most other disordered coils, and the subsequent slope is unusually high (∼4.5 in comparison with ∼3.3). This anomalous behavior has been rationalized in terms of intermolecular association (hyperentanglement) between unsubstituted regions of glycan chains, in addition to normal topological entanglement (Morris et al., 1981; Goycoolea, Morris, & Gidley, 1995). Tuinier, Zoon, Cohen Stuart, Fleer, and de Kruif (1999) reported that for a range of polysaccharides, the dependence of the zero shear specific viscosity on the coil overlap parameter could be described as follows: sp0 = []0 c +

1 ([]0 c)7/2 25

(6)

As depicted in Fig. 4B, the broken line represented a similar deviation from the log sp versus log c[] master curve reported for galactomannans (Morris et al., 1981), which was attributed to the presence of non-specific segment–segment interactions. An alternative curve fit was also illustrated in Fig. 4B according to the Martin equation: sp0 = c[]0 eKc[]0

(7)

The constant K expressed the balance of polymer–polymer and polymer–solvent interactions (Kasaai, Charlet, & Arul, 2000). The power law exponent in the more concentrated regime was 4.08, which was analogous to that reported in the discussion of Fig. 4B.

kT 6Rh

(8)

where k was the Boltzmann constant (1.38 × 10−23 m2 kg/s2 /K), T was the absolute temperature (25 ◦ C, 298.15 K) and  was the viscosity of the solvent (0.8949 × 10−3 Pa s). For LGPP2-2, a combined extrapolation of both data sets gave a value for Rh of 116 nm, which was larger than that of the radius of gyration. For a given polymer solution, the ratio Rg /Rh () depends on its polymer architecture and conformation. Zimm theory predicted that values for  of 1.6 represented a linear monodisperse polymer in good solvent, and values for  of 0.77 implied a hard sphere (Rubinstein & Colby, 2003). The  of LGPP2-2 was found to be ∼0.59. Similar values have been observed for various polysaccharides including ␤-D(1,3)(1,4)-glucan and tamarind seed polysaccharide (Burchard & Schulz, 1995; Grimm, Krüger, & Burchard, 1995). The low value of  indicated a strictly lateral aggregation in the case of tricarbanilate. The value was attributed not to a hard sphere architecture but rather an aggregate comprising a core of aggregated chains surrounding by hairy dangling chains and would be in agreement with the suggested fringed micelle in a highly aggregated state. 3.6. Effect of autoclave treatment The molecular mass distribution of LGPP2-2 following multiple autoclave treatment was presented in Fig. 6. The associated trends in weight average molecular weight and dispersity were listed in Table 2. Collectively, the data showed a progressive reduction in molecular mass with each subsequent autoclave treatment, which agreed well with that noted for autoclaving studies of oat ␤-glucan and guar gum (Picout, Ross-Murphy, Errington, & Harding, 2001). Both [] and Mw reductions decrease with each successive treatment. Mitchell, Hill, Jumel, Harding, and Aidoo (1992) showed that viscosity loss on autoclave of certain galactomannans could be reduced by treatment with antioxidants. This suggested that oxidative–reductive depolymerization might play an important role in the polysaccharide hydrolysis. On the other hand, the decrease in viscosity was a result of intramolecular hydrophobic associations and variation of Mw led to simultaneous variation of electrostatic and hydrophobic interactions (Nichifor, Stanciu, Ghimici, & Simionescu, 2011). Studies on solvents also revealed

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of LGPP2-2 were calculated from the experimental data of weight average molecular masses (Mw ), intrinsic viscosity [], radius of gyration (Rg ) and hydrodynamic radius (Rh ). Results showed that LGPP2-2 existed in a sphere-like conformation in distilled water in view of its fractal dimension. Furthermore, the polysaccharide had an internal structure between a hard sphere and a swollen branched macromolecule. Furthermore, the molecular mass distribution of LGPP2-2 following multiple autoclave treatment was investigated. Both [] and Mw reductions decrease with each successive treatment, which suggested that oxidative–reductive depolymerization might play an important role in the polysaccharide hydrolysis. In general, the whole work provided information of solution characteristics and chain conformation for a heteropolysaccharide extracted from pumpkin. Acknowledgement Fig. 6. Variation in the molecular mass distribution of LGPP2-2 following autoclave treatment. Symnols represent molecular weights (molar mass); lines represent the mRIU responses.

This work was supported by National Natural Science Foundation of China (no. 31301525). Appendix A. Supplementary data

Table 2 Effects on molecular weight, dispersity and intrinsic viscosity of autoclave treatment for pumpkin polysaccharide LGPP2-2. Autoclave regime

Mw × 105 (g/mol)

Control 1 × 15 min exposure

12.7 8.6 8.6 8.1 6.3 6.9 6.5 5.4 5.1 5.2 2.1 2.5 2.5

2 × 15 min exposure

3 × 15 min exposure

4 × 15 min exposure

± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.2 0.5 0.3 0.2 0.5 0.6 0.4 0.3 0.4 0.7 0.3 0.4

Dispersity 1.23 1.27 1.31 1.25 1.22 1.24 1.25 1.28 1.26 1.24 1.27 1.23 1.30

± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.4 0.3 0.4 0.2 0.5 0.4 0.4 0.2 0.6 0.1 0.3 0.3

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carbpol.2015.06.061

[] (mL/g) 780 540

460

420

380

that, in the case of water, the stability of the hydrogen bonds decreased with increasing temperature (at 300–400 ◦ C, they have disappeared completely) due to the increasing influence of the entropy loss associated with the formation of bonds (Hakem, Boussaid, Benchouk-Taleb, & Bockstaller, 2007; Postorino, Tromp, Ricci, Soper, & Neilson, 1993). Besides, a temperature rise caused a restructuring of the hydrogen bonded network, since the stability of hydrogen bonds decreased, accompanied by a drop (of about 20%) in the number of OH groups that were closely associated (Hakem et al., 2007). Furthermore, the physicochemical properties of large biopolymers were also associated with pH of medium. When pH of medium changed, hydroxyl groups became ionized, converting neutral polysaccharides to polyelectrolytes. This would have the effect of expanding molecular dimensions by intramolecular electrostatic repulsion, and would, therefore, be expected to change the viscosity of solutions of independent disordered coils. Meanwhile, intermolecular associations between the constituent chains would also be destabilized. 4. Conclusions A heteropolysaccharide LGPP2-2 was extracted from pumpkins (C. pepo, Lady godvia) and purified by the ion exchange chromatography and gel permeation chromatography. The yield and total sugar content of LGPP2-2 was estimated 5.23% and 61.6%, respectively. GC–MS analysis revealed that LGPP2-2 was a galactoglucofucomannan. The macromolecular solution properties

References Bauer, J., & Burchard, W. (1993). Determination of scaling properties of randomly branched polycyanurates by combined SEC/LALLS/VISC. Macromolecules, 26, 3103–3107. Bello-Perezt, L. A., Roger, P., Baud, B., & Colonna, P. J. (1998). Macromolecular features of starches determined by aqueous high-performance size exclusion chromatography. Journal of Cereal Science, 27, 267–278. Burchard, W., & Schulz, L. (1995). Functionality of the ␤(1,4)glycosidic linkage in polysaccharides. Macromolecular Symposia, 99, 57–69. Daas, P. J. H., Grolle, K., Van Vliet, T., Schols, H. A., & De Jongh, H. H. J. (2002). Toward the recognition of structure–function relationships in galactomannans. Journal of Agricultural and Food Chemistry, 50, 4282–4289. Doublier, J. L., & Launay, B. (1981). Rheology of galactomannan solutions: Comparative study of guar gum and locust bean gum. Journal of Texture Studies, 12, 151–172. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. Ellis, P. R. (1994). Polysaccharide gums: Their modulation of carbohydrate and lipid metabolism and role in the treatment of diabetes mellitus. In G. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilities for the food industry 7 (pp. 207–216). Oxford: Oxford University Press. Gaisford, S. E., Harding, S. E., Mithcell, J. R., & Bradley, T. D. (1986). A comparison between the hot and cold water soluble fractions of two locust bean gum samples. Carbohydrate Polymers, 6, 423–442. Ganter, J. L. M. S., & Reicher, F. (1999). Water-soluble galactomannans from seeds of Mimosaceae spp. Bioresource Technology, 68, 55–62. Grimm, A., Krüger, E., & Burchard, W. (1995). Solution properties of ␤-D(1,3)(1,4)-glucan isolated from beer. Carbohydrate Polymers, 27, 205–214. Goycoolea, F. M., Morris, E. R., & Gidley, M. J. (1995). Viscosity of galactomannans at alkaline and neutral pH: Evidence of ‘hyperentanglement’ in solution. Carbohydrate Polymers, 27, 69–71. Hakem, I. F., Boussaid, A., Benchouk-Taleb, H., & Bockstaller, M. R. (2007). Temperature, pressure, and isotope effects on the structure and properties of liquid water: A lattice approach. Journal of Chemical Physics, 127, 224106. Hanselmann, R., Burchard, W., Ehrat, M., & Widmer, H. M. (1996). Structural properties of fractionated starch polymers and their dependence on the dissolution process. Macromolecules, 29, 3277–3282. Kasaai, M. R., Charlet, G., & Arul, J. (2000). Master curve for concentration dependence of semi-dilute solution viscosity of chitosan homologues: The Martin equation. Food Research International, 33, 63–67. King, K., & Gray, R. (1993). The effect of gamma irradiation on guar gum, locust bean gum, gum tragacanth and gum karaya. Food Hydrocolloids, 6, 559–569. Lapasin, R., Delorenzi, L., Pricl, S., & Torriano, G. (1995). Flow properties of hydroxypropyl guar gum and its long chain hydrophobic derivatives. Carbohydrate Polymers, 28, 195–202. Lefebvre, J., & Doublier, J. L. (2005). Rheological behaviour of polysaccharide aqueous systems. In S. Dimitriu (Ed.), Polysaccharides: Structural diversity and functional versatility. USA: Marcel Dekker. Li, Q. H., Fu, C. L., Rui, Y. K., Hu, G. H., & Cai, T. Y. (2005). Effects of protein-bound polysaccharide isolated from pumpkin on insulin in diabetic rats. Plant Foods for Human Nutrition, 60, 1–4.

Y. Song et al. / Carbohydrate Polymers 132 (2015) 221–227 Mitchell, J. R., Hill, S. E., Jumel, K., Harding, S. E., & Aidoo, M. (1992). In G. O. Philips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilizers for the food industry 6 (p. 303). Oxford, UK: Pergamon Press. Morris, E. R., Cutler, A. N., Ross-Murphy, S. B., Rees, D. A., & Price, J. (1981). Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions. Carbohydrate Polymers, 1, 5–21. Murkovic, M., Piironen, V., Lampi, A. M., Kraushofer, T., & Sontag, G. (2004). Changes in chemical composition of pumpkin seeds during the roasting process for production of pumpkin seed oil (Part 1: Non-volatile compounds). Food Chemistry, 84, 359–365. Needs, P. W., & Selvendran, R. R. (1993). Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydrate Research, 245, 1–10. Nichifor, M., Stanciu, M. C., Ghimici, L., & Simionescu, B. C. (2011). Hydrodynamic properties of some cationic amphiphilic polysaccharides in dilute and semi-dilute aqueous solutions. Carbohydrate Polymers, 83, 1887–1894. Picout, D. R., Ross-Murphy, S. B., Errington, N., & Harding, S. E. (2003). Pressure cell assisted solubilization of xyloglucans: Tamarind seed polysaccharide and detarium gum. Biomacromolecules, 4, 799–807. Picout, D. R., Ross-Murphy, S. B., Errington, N., & Harding, S. E. (2001). Pressure cell assisted solution characterization of polysaccharides. 1. Guar gum. Biomacromolecules, 2, 1301–1309. Picout, D. R., Ross-Murphy, S. B., Jumel, K., & Harding, S. E. (2002). Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum. Biomacromolecules, 3, 761–767. Postorino, P., Tromp, R. H., Ricci, M. A., Soper, A. K., & Neilson, G. W. (1993). The interatomic structure of water at supercritical temperatures. Nature, 366, 668–670. Ratcliffe, I., Williams, P. A., Viebke, C., & Meadows, J. (2005). Physicochemical characterization of konjac glucomannan. Biomacromolecules, 6, 1977–1986. Ross-Murphy, S. B. (1994). Rheological methods. In S. B. Ross-Murphy (Ed.), Physical techniques for the study of food biopolymers (pp. 343–345). Glasgow: Blackie Academic and Professional. Rubinstein, M., & Colby, R. H. (2003). Polymer physics. Oxford, UK: Oxford University Press. Scheller, H. V., & Ulvskov, P. (2010). Hemicelluloses. Annual Review of Plant Biology, 61, 263–289.

227

Scherrenberg, R., Coussens, B., Van Vliet, P., Edouard, G., Brackman, J., et al. (1998). The molecular characteristics of poly(propyleneimine) dendrimers as studied with small-angle neutron scattering, viscosimetry, and molecular dynamics. Macromolecules, 31, 456–461. Siegmund, B., & Murkovic, M. (2004). Changes in chemical composition of pumpkin seeds during the roasting process for production of pumpkin seed oil (Part 2: Volatile compounds). Food Chemistry, 84, 367–374. Song, Y., Li, J., Hu, X. S., Ni, Y. Y., & Li, Q. H. (2011). Structural characterization of a polysaccharide isolated from Lady Godiva pumpkins (Cucurbita pepo lady godiva). Macromolecular Research, 19, 1172–1178. Song, Y., Yang, Y., Zhang, Y. Y., Duan, L. S., Zhou, C. L., Ni, Y. Y., et al. (2013). Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva). Carbohydrate Polymers, 98, 686–691. Sun, S. F. (1994). Physical chemistry of macromolecules: Basic principles and issues. New York, NY: John Wiley and Sons. Szopinski, D., Kulicke, W. M., & Luinstra, G. A. (2015). Structure–property relationships of carboxymethyl hydroxypropyl guar gum in water and a hyperentanglement parameter. Carbohydrate Polymers, 119, 159–166. Tayal, A., & Khan, S. A. (2000). Degradation of a water-soluble polymer: Molecular weight changes and chain scission characteristics. Macromolecules, 33, 9488–9493. Traiphol, R., Charoenthai, N., Srikhirin, T., Kerdcharoen, T., Osotchan, T., & Maturos, T. (2007). Chain organization and photophysics of conjugated polymer in poor solvents: Aggregates, agglomerates and collapsed coils. Polymers, 48, 813–826. Trappe, V., Bauer, J., Weissmüller, M., & Burchard, W. (1997). Angular dependence in static and dynamic light scattering from randomly branched systems. Macromolecules, 30, 2365–2372. Tuinier, R., Zoon, P., Cohen Stuart, M. A., Fleer, G. J., & de Kruif, C. (1999). Concentration and shear-rate dependence of the viscosity of an exocellular polysaccharide. Biopolymers, 50, 641–646. Zhang, M., Cheung, P. C. K., Zhang, L., Chiu, C. M., & Ooi, V. E. C. (2004). Carboxymethylated ␤-glucans from mushroom sclerotium of Pleurotus tuber-regium as novel water-soluble anti-tumor agent. Carbohydrate Polymers, 57, 319–325.