Rheological and physicochemical properties of polysaccharides extracted from stems of Dendrobium officinale

Journal Pre-proof Rheological and physicochemical properties of polysaccharides extracted from stems of Dendrobium officinale

Bo Wang, Weimin Zhang, Xinpeng Bai, Congfa Li, Dong Xiang PII:

S0268-005X(19)32042-9

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105706

Reference:

FOOHYD 105706

To appear in:

Food Hydrocolloids

Received Date:

04 September 2019

Accepted Date:

22 January 2020

Please cite this article as: Bo Wang, Weimin Zhang, Xinpeng Bai, Congfa Li, Dong Xiang, Rheological and physicochemical properties of polysaccharides extracted from stems of Dendrobium officinale, Food Hydrocolloids (2020), https://doi.org/10.1016/j.foodhyd.2020.105706

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Journal Pre-proof Rheological and physicochemical properties of polysaccharides extracted from stems of Dendrobium officinale Bo Wang b,1，Weimin Zhang b,2, Xinpeng Bai a, 3, Congfa Li b,4, Dong Xiang a, b, * a

Engineering Research Center of Utilization of Tropical polysaccharide resources, Ministry of Education, No.58

Haikou 570228, China b College

of Food Science and Engineering, Hainan University, No.58 Haikou 570228, China

Abstract The physicochemical and rheological properties of polysaccharides extracted from stems of Dendrobium officinale (DOP) were investigated. Two samples were prepared: the first sample (HEPDOP) was prepared by pretreating D. officinale stems by hot water scalding, followed by water extraction and ethanol precipitation； and the second sample (EPDOP) was obtained by water extraction and ethanol precipitation without pretreatment. The contents of total carbohydrate and protein, mannanase activity, infrared spectra, molecular weight distribution, thermal properties, and hydrophobicity of the two samples were analyzed. The results showed that mannanase activity of HEPDOP (0.0247 U) was much lower than that of EPDOP (0.1158 U). HEPDOP had higher molecular weight and carbohydrate content, and lower polydispersity index (PDI) value and protein content compared to EPDOP. The results further showed that the decomposition temperature of HEPDOP was 311 °C, which is higher than that of EPDOP (306 °C); and its hydrophobicity was significantly lower than that of EPDOP. Interestingly, at the same concentration (2%(w/w)), HEPDOP had higher viscosity than EPDOP，and acidity, alkalinity, the addition of salt ions, and temperature was found to reduce their viscosity. Finally, we observed that increasing temperature caused the decrease of viscosity and modulus of both DOPs. Keywords: Dendrobium officinale; polysaccharides; Rheological properties; mannanase activity; hydrophobicity 1. Introduction Dendrobium officinale is a perennial herb mainly distributed in southern China; it is an edible plant with high medicinal value (Kaiwei., et al., 2016; Yu, Yang, Teixeira da Silva, Luo, & Duan, 2019). In medicine, it is often used for the treatment of high blood sugar, warm stomach, lung diseases, throat inflammation, etc., and fresh stem of D. officinale, which has health benefits, is often used as an ingredient in a soup tea (Chen, et al., 2015; Fan, Meng, Xiao, & Zhang, 2018; Meng, Fan, Li, & Zhang, 2017; Ye, et al., 2017; Yu, et al., 2019; Zhou, Xie, Lei, Huang, & Wei, 2018). Previous studies have shown that the main active ingredients of D. officinale include alkaloids, flavonoids, polyphenols, amino acids, polysaccharides, etc. (Chan, et al., 2018; Guo, et al., 2013; Ye, et al., 2017; Zhou, et al., 2018; Zhu, et al., 2017). Other studies have also demonstrated that polysaccharides extracted from D. officinale (DOP) have many biological activities and effects, such as ability to treat enteritis, immunomodulatory effects, antioxidant activities, etc. (Huifan, et al., 2018; Kaiwei., et al., 2016; Liang, et al., 2018; Luo, et al., 2016; Tao-Bin., et al., 2016). Thus far, most studies on DOP have emphasized on characterization of components obtained by column chromatographic purification (Kaiwei., et al., 2016; Luo, et al., 2016; Tao-Bin., et al., 2016; Wei, et al., 2016). Thus, studies on DOP obtained by alcohol precipitation are rare, among which is Fan et al. (Fan, et al., 2018), who have carried out a series of studies on six DOPs with different average molecular weights obtained using ethanol fractionation. Rheology is the study of flow and deformation of objects, especially for behavior during the transition between solid and fluid (Tabilo-Munizaga & Barbosa-Cánovas, 2005; Xu, Zhang, Liu, 1

Journal Pre-proof Sun, & Wang, 2016). Examples of materials that have unique rheological and gel properties are xanthan gum and gellan gum, which are widely used in the food industry (Cho & Yoo, 2015; Danalache, Mata, Moldão-Martins, & Alves, 2015). In recent years, many researches have focused on studying rheological properties of polysaccharides extracted from various natural plants, including floral mushroom cultivation (Xu, et al., 2016), soy hull (Shengnan, et al., 2019), chia seed (Timilsena, Adhikari, Kasapis, & Adhikari, 2015), litchi pulp (HuangFei., et al., 2018) and onion (ZhuDan-Ye., et al., 2018); but the rheological properties of DOP remain to be uncovered. Enzymatic hydrolysis is a method commonly used in degradation of polysaccharide to reduce its molecular weight (Diao, et al., 2017; Mudgil, Barak, & Khatkar, 2012), and many natural polysaccharide-degrading enzymes in plants, such as cellulase, amylase, pectinase, etc., have been identified and used in such process (Agatonovic-Kustrin & Morton, 2018; Brown, Wiersma, & Olson, 2018; Jamsazzadeh Kermani, et al., 2015; Lohani, Trivedi, & Nath, 2004; Manrique & Lajolo, 2004; Tatsumi, Konishi, & Tsujiyama, 2016; Tavares, De Souza, & Buckeridge, 2015). The viscosity of a water-soluble polysaccharide solution is closely linked to its molecular weight, which also affects its rheological properties (Trujillo-Cayado, Alfaro, Munoz, Raymundo, & Sousa, 2016; Vardhanabhuti & Ikeda, 2006; Zhang, Chen, Ma, & Zhang, 2013). Many studies have reported the relationship between the enzymatic hydrolysis of polysaccharides and their rheological properties; for example, depolymerization of a polysaccharide aqueous solution by enzymatic hydrolysis has been demonstrated to decrease its viscosity (Diao, et al., 2017; Hanne R. Sørensen, 2006; Mudgil, et al., 2012; Poonsrisawat, et al., 2016; Zhang, et al., 2010). However, the effect of natural polysaccharide-degrading enzymes in plant on the rheological properties of the extracted polysaccharides has not been reported. In this study, we extracted polysaccharides from D. officinale stems (DOP). Two samples were prepared: the first sample (HEPDOP) was prepared by pretreating D. officinale stems by hot water scalding, followed by ethanol precipitation; and the second sample (EPDOP) was prepared only by ethanol precipitation without pretreatment. The two types of polysaccharides obtained were then subjected to chemical composition and mannanase activity analyses. The infrared spectra, molecular weight, thermal properties, hydrophobicity and rheological properties of the two types of samples were also analyzed. This research provides information of DOP that may be useful for its application in the food industry. 2. Materials and methods 2.1 Materials and reagents Dendrobium officinale stem used in this study was provided by Bao Yuan Tang Company (Hainan, China). The D. officinale stem was grown in Qiong Zhong County, Hainan Province and was harvested after one year. Dextran with different molecular weights was purchased from Shanghai Aladdin Biochemical Technology Co. Ltd (Shanghai, China). All other reagents used were of analytical grade. 2.2 Extraction of DOP 2.2.1 Water extraction method Fifty grams of fresh D. officinale stem was cut into small pieces (sizes of about 2-3 cm) and then placed in a crusher, into which 1000 ml of deionized water was added. The stem was crushed for 3 min and then filtered to obtain filtrate. The filtrate was centrifuged (10000 g, 30 min), and the supernatant was collected. The supernatant was precipitated with 4 volumes of 95% ethanol for 24 h. After centrifugation (10000 2

Journal Pre-proof g, 20 min), the precipitate was freeze-dried to obtain DOP sample named as EPDOP. 2.2.2 Water extraction with hot water scalding pretreatment method Fifty grams of fresh D. officinale stem was cut into small pieces (sizes of about 2-3 cm) and then soaked in boiling water for 5 min. Subsequently, it was removed from boiling water and surface water droplets were removed. After that, it was subjected to the steps described in 2.2.1; the DOP sample named as HEPDOP was obtained. 2.3 Chemical composition analysis of DOP The total carbohydrate content of DOPs was determined by the phenol sulfuric acid method, the polysaccharide is hydrolyzed to monosaccharide by the action of sulfuric acid, and rapidly dehydrated to form an aldehyde derivative, which is then reacted with phenol to form an orangeyellow compound (Li, Liao, Thakur, Zhang, & Wei, 2018). In short, a 0.1 mg/ml solution was prepared from the freeze-dried sample, and its absorbance at 490 nm was measured. The standard curve was prepared using glucose with different concentrations (glucose concentration is the abscissa and the absorbance is the ordinate). The linear regression equation of the standard curve was y = 4.944x + 0.0424，R² = 0.9983. The protein content of DOPs was determined by the G-250 method (Zhang, Li, Xiong, Jiang, & Lai, 2013). The standard curve was plotted using BSA with different concentrations, in which BSA concentration is the abscissa and the absorbance is the ordinate. The linear regression equation of the standard curve was y = 4.0905x + 0.1662，R² = 0.9982. 2.4 Mannanase activity assay Mannanase activity was determined in the D. officinale filtrate using locust bean gum as the substrate, and the amount of reducing sugar released from the locust bean gum was determined by 3, 5-dinitrosalicylic acid (DNS) method (Ahirwar, et al., 2016; MILLER, 1959; Pongsapipatana, et al., 2016; C. Wang, et al., 2016). Briefly, 1 ml of D. officinale filtrate was reacted with 5 ml of locust bean gum solution (0.5% (w/v)) at room temperature for 30 min. After 2.5 ml of DNS solution was added, the mixture was heated in boiling water for 5 min and was then cooled down to room temperature. One unit of enzyme activity is defined as the amount of reducing sugar released from the substrate in one minute. 2.5 FT-IR analysis The infrared spectra of both DOP samples were recorded on an infrared spectrometer (Bruker Optics Co, Berlin, Germany) at the frequency range of 4000 cm -1 to 400 cm -1 (Li, Xiang, Wang, & Gong, 2019). Prior to the measurement, each freeze-dried DOP sample was mixed with KBr and compressed into tablets, and the ratio of sample to KBr was 1:80. 2.6 Determination of molecular weights of DOP The molecular weight of each DOP was determined by high performance gel permeation chromatography (HPGPC; Waters e2695, MA, USA). The HPGPC system was equipped with a series of a Waters Ultrahydro-gelTM500 column (7.8mm × 300mm) and a Waters UltrahydrogelTM120 column (7.8mm × 300 mm), and a Waters 2414 refractive index detector (Waters, MA, USA) (Luo, et al., 2016). The operating conditions were as follows: mobile phase, 0.05% NaN3; flow rate, 0.6 ml/min; column temperature, 40 ° C; and injection volume, 10 μl. The samples were prepared by dissolving 25 mg of freeze-dried DOPs in 10 ml of deionized water. The samples were passed through a 0.22-μm filter before injection. The standard curve was prepared using dextran with different molecular weights. 2.7 Measurement of thermal properties of DOP 3

Journal Pre-proof The thermal gravimetric analysis (TGA) of the samples was carried out using a Q600 thermogravimetric analyzer (TA Instruments, USA), operated at a heating rate of 10 °C/min and a temperature range of 30-900 °C; nitrogen was used as carrier gas. The differential scanning calorimetric (DSC) analysis of the samples was conducted using a Q100 differential calorimeter (TA Instruments, USA). Nitrogen was used the carrier gas, and the instrument was operated at a heating rate of 10 °C/min and at a temperature range of 30-200 °C (Fan, et al., 2018; Zhang, et al., 2016). 2.8 Determination of hydrophobicity of DOP The hydrophobicity of DOP was determined by fluorescence spectra using an F7000 fluorescence spectrometer (Hitachi, Japan), and pyrene was used as a probe. DOP sample solutions at different concentrations were prepared and were then mixed with pyrene (final concentration of pyrene was 4×10-6mol/L) (al., 1998; Hiromi Morimoto, 2003). To obtain the excitation spectra, the samples were scanned at an emission wavelength of 390 nm from 200 nm to 370 nm (al., 1998). To obtain the emission spectra, the samples were scanned at an excitation wavelength of 335 nm from 350 nm to 500 nm. The excitation and emission slit widths were set at 5 nm and 2.5 nm, respectively. The scanning speed was 1200 nm/min, and each sample was scanned 3 times. 2.9 The rheological properties of DOP The rheological properties of the samples were measured using a Discovery Hybrid rheometer2 (DHR-2; TA instruments, New Castle DE, USA). The 60-mm parallel plate was used and the measurement gap was 1mm. 2.9.1 Steady-state flow scanning of DOP To obtain steady-state flow curves, two types of 0.4-2.0%(w/w) DOP aqueous solutions were prepared (Chen, Zhang, Sun, & Wei, 2014), the measurement was carried out in flow sweep mode. The temperature was set at 25 °C, and the scans were conducted at a shear rate range of 0.01 to 1,000 s-1. 2.9.2 Effect of various salts on apparent viscosity NaCl or CaCl2 was added to DOP aqueous solution (1%w/w)) to final ion concentrations of 0, 0.1, 0.3 and 0.5 mol/L (Li, et al., 2018). After that, the samples were subjected to the measurements described in 2.9.1. 2.9.3 Effect of pH on apparent viscosity The pH of DOP aqueous solution (1% (w/w)) was adjusted to pH 3.0, 5.0, 6.5, 8.0, and 10.0. Subsequently, the solution was subjected to dialysis (through a membrane with MWCO of 3500 Da) for 24 h to remove residual ions. After that, the samples were subjected to the test described in 2.9.1. 2.9.4 Effect of temperature on apparent viscosity Two types of DOP aqueous solutions with various concentrations between 0.4% (w/w) and 2.0% (w/w) were prepared. The measurement was carried out in flow temperature ramp mode at a temperature range of 25°C - 85°C. The heating rate was 3°C/min, and the shear rate was 10.0 s-1. 2.9.5 Oscillatory measurements Two types of DOP aqueous solutions at various concentrations (0.4-2.0% (w/w)) were prepared. The measurement was conducted in oscillation amplitude mode at a constant temperature of 25 °C, an angular frequency of 10.0 rad/s, and a strain range of 0.1% - 1000%. After the linear viscoelastic region of DOPs was obtained, the measurement was carried out in oscillation frequency mode, in which the strain was fixed at 1.0% and the frequency sweep was in a range of 250 - 0.1 4

Journal Pre-proof rad/s. The values of frequency-dependent G' and G'' were recorded. 2.9.6 Effect of temperature on modulus In this measurement, the oscillation temperature ramp mode was adopted. The measurement was carried out at a temperature range of 15 °C - 85 °C, a heating rate and a cooling rate of 3 °C/min, and a fixed strain of 1.0%. The scan was initiated immediately after the temperature reached the set values, as well as when it began to drop. During the experiment, a circle of silicone oil was applied as a seal around the parallel plates to prevent the sample from volatilizing. 2.10 Statistical analysis All data were statistically analyzed using SPSS-Statistics V17 software, in which analysis of variance, followed by Duncan multiple comparisons (Tabilo-Munizaga, et al.) were performed. The difference at P < 0.01 is considered significant.

3. Results and discussion 3.1 Mannanase activity and chemical composition of DOP As shown in Table 1, HEPDOP contained higher total carbohydrate content and lower protein content compared with EPDOP. This difference might be due to the fact that most proteins were denatured after hot water scalding treatment and were not extracted during the ethanol precipitation step. In addition, mannanase activity of HEPDOP was significantly lower than that of EPDOP, suggesting that pretreatment by hot water scalding may inactivate mannanase in D. officinale stem. Table 1 Mannanase activity and contents of total carbohydrate and protein in DOP samples.

Mannanase activity (U)

Total carbohydrate (%)

Protein (%)

EPDOP

0.1158±0.0002 a

70.17%±0.31b

3.25±0.04a

HEPDOP

0.0247±0.0004 b

78.20%±0.40a

0.04±0.01b

Data are mean ± SD (n=3). a, b Data are significantly different (p<0.01).

3.2 FT-IR spectra of DOPs As shown in Fig. 1, EPDOP and HEPDOP exhibited similar FT-IR spectra, indicating that their structures are similar or are not significantly different. The FT-IR spectra showed a strong and broad absorption peak at 3418 cm-1 is the characteristic peak of O-H groups (Kaiwei., et al., 2016). Peaks at 2931 cm−1 and 2890 cm−1 are due to the stretching and bending vibrations of methyl and methylene C-H bonds; the peaks are the characteristic peaks of polysaccharides (Tao-Bin., et al., 2016). A high-intensity peak at 1738 cm−1 can be assigned to the valence vibration of C=O in acetyl or carboxylic acid ester. The presence of this peak indicates that DOP may contain uronic acid (He, et al., 2018). Furthermore, peaks at around 1379 cm−1 and 1250 cm−1 are due to symmetric bending vibration of C-H in methyl groups and vibration of C-O in O-acetyl groups, respectively (Wei, et al., 2016). Finally, peaks at 876 cm−1 indicates the presence of β-D-mannopyranose (Wei, et al., 2016).

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Fig. 1 FT-IR spectra of EODOP and HEPDOP obtained from different preparation methods.

3.3 Molecular weight of DOP Molecular weight is an important parameter used in characterization of macromolecular polymers, including polysaccharides (Ma, et al., 2018). Fig. 2 shows the gel purification chromatogram (GPC) gel chromatogram of the DOP samples: while EPDOP had two peaks, HEPDOP had only one peak. As shown in Table 1, HEPDOP had larger average molecular weight (484 kDa). Polydispersity index (PDI), which is the width of the molecular weight distribution, of the two samples were in a range of 1.04 to 1.14, indicating that both samples have low heterogeneity in terms of both size and weight (Fan, et al., 2018). Mannan is a hemicellulose polysaccharide widely found in plant cell walls (Yuan, et al., 2007). Endo-β-mannanase in plants can degrade mannan into disaccharide, trisaccharide, tetrasaccharide and other mannose oligosaccharides (Xianfeng & Ping, 2000 ). Previous studies have shown that endo-β-mannanase is found in many types of plants, such as lilium bulbs (THOMAS WOZNIEWSKI, 1992), konjac tubers (Noboru SUGIYAMA, 1973), Sisymbrium officinale seeds (Carrillo-Barral, Matilla, Rodriguez-Gacio, & Iglesias-Fernandez, 2018), Coffea arabica L. seeds(Ferreira, et al., 2018; Marraccini, et al., 2014), carrot seeds (Homrichhausen, Hewitt, & Nonogaki, 2007), lettuce seeds(Toorop, Bewley, Abrams, & Hilhorst, 1999), barley (Hordeum vulgare L.) (Hrmova, Burton, Biely, Lahnstein, & Fincher, 2006), soybean (X. Chen, et al., 2018; Yan, Zhang, Guo, & Wang, 2012), tomato seeds (Peter E. Toorop 1, 1996), tomato fruit (Wang, Li, Zhang, Xu, & Bewley, 2009), Arabidopsis and poplar (Populus trichocarpa) (Yuan, et al., 2007). Because the content of mannose in D. officinale is above 80% (Fan, et al., 2018; Kaiwei., et al., 2016; Tao-Bin., et al., 2016; Wei, et al., 2016), we speculate that endo-β-mannanase may be present in the stem of D. officinale, and EPDOP may be partially hydrolyzed by mannanase causing the two peaks in molecular weight distributions in the GPC profile. In addition, the second peak of the GPC profile of EPDOP had a minimum PDI (1.04), which could be the 3.25% protein. It can be seen that the peak's relatively high area (for a 3% concentration) in line with a more compact protein structure as opposed to a loose carbohydrate. The former increases the refractive index difference between water and the folded protein interior, enhancing the RI signal. On the other hand, mannanase in HEPDOP may be inactivated during hot 6

Journal Pre-proof water scalding pretreatment; therefore, HEPDOP had higher molecular weight than EPDOP. This finding indicates that different pretreatment methods can result in DOPs with different molecular weights.

(A)

(B)

Fig. 2 (A) Standard curve of Mw. (B) GPC profiles of DOP obtained from different preparation methods. Table 2 Molecular weights of DOP.

EPDOP

RT (min)

Mw (kDa)

Mn (kDa)

PDI

peak1

18.31

411

360

1.14

peak2

21.86

99

95

1.04

18.10

484

436

1.11

HEPDOP

3.4 Thermal properties of DOP Fig. 3 shows the TGA and DSC curves of DOPs. The first weight loss observed in both samples, at which the total water content was about 11%, are due to water evaporation (Postulkova, et al., 2017), and EPDOP and HEPDOP had the thermal decomposition temperature of 306°C and 311°C, respectively. Additionally, no remaining residues observed after the thermal decomposition, indicating that the samples were completely decomposed. According to the DSC curves, the ΔH values of the two samples were significantly different. The ΔH value of HEPDOP was 401.4 J/g, which is much higher than that of EPDOP (336.4 J/g). This indicates that HEPDOP has higher thermal stability than EPDOP, which may be due to the difference between the molecular weights of both samples. Previous studies have shown that mannose and galactose contents and molecular weights of polysaccharides affect theirΔH value (Han, et al., 2016; Jia, et al., 2015).

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Fig. 3 TGA and DSC curves of the DOPs.

3.5 Hydrophobicity of DOP As illustrated in Fig. 4B, increasing concentration of DOP sample led to the increase of fluorescence intensity of pyrene probe, which is similar to the observation reported in previous studies (Amiji, 1995; Kazuhiro Nishikawa, 1998). The ratio between the fluorescence intensity of peak 1 (373 nm) and that of peak 3 (384 nm) or E1/E3 ratio can indicate the polarity of the microenvironment in which pyrene is located (Vieira, Moscardini, Tiera, & Tiera, 2003). The lower the E1 / E3 ratio, the stronger the hydrophobicity of the sample. At the same concentration (0.3 g/L), the E1/E3 ratios of the two samples were different, which may be due to their different protein contents. In other words, the E1/E3 ratio of EPDOP was 1.218, which is lower than that of HEPDOP (1.344), indicating that EPDOP is more hydrophobic than HEPDOP. As mentioned in 3.1, EPDOP contains more protein (3.25%), and due to the existence of hydrophobic aminoacids, the contribution of protein in hydrophobility on a per weight basis is expected to be much higher than that of the sugars.

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Fig. 4 Fluorescence spectra of different DOP aqueous solutions. (A) Excitation spectra of 0.05%(w/w) DOP aqueous solutions, λem = 390 nm.（B）Emission spectra of DOP aqueous solutions of varying concentrations, λex = 335 nm. (C) E1(373.2nm)/E3(384nm) ratio of 0.3%(w/w) DOP aqueous solutions. a, b indicates significant difference (p<0.01). Pyrene at a concentration of 4×10-6 mol/L was used as a probe.

3.6 Steady-state flow curves of DOP Flow curves of the two types of DOP aqueous solutions were similar. At all concentrations tested, the viscosity decreased with increasing shear rate. At the same shear rate, the viscosity increased with increasing concentration. This indicates that the DOP aqueous solution exhibits shear thinning behavior (Xu, et al., 2016), which is caused by orientations of its molecular components (Vardhanabhuti, et al., 2006). When the shear rate is increased, the entanglement between the molecular chains is disrupted, causing the orientation of the molecular chains to become more random. As a result, the interactions between adjacent chains are reduced, thus the viscosity is decreased (Qiao, et al., 2016; Xu, et al., 2016; Yaich, et al., 2014). At the same shear rate, comparison of DOPs at the same concentration (2.0% (w/w)) showed that HEPDOP had higher viscosity than EPDOP. This is likely due to different molecular weights of the two samples, and another reason may be that HEPDOP has a higher carbohydrate content. Previous studies have shown that polysaccharides with lower Mw has lower number of molecular interactions, which causes the viscosity to decrease (Zhang, et al., 2013).

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Fig. 5 Viscosity-shear rate profiles of DOP aqueous solutions with different concentrations and viscosity of two types of DOP aqueous solutions at 2% (w/w) concentration.

3.7 Effect of NaCl or CaCl2 on viscosity The apparent Zeta potential of DOPs and the effects of NaCl or CaCl2 on the viscosity of DOP samples are shown in Fig. 6. Both EPDOP and HEPDOP had negative charges and HEPDOP had the larger amount of negative charge. The addition of Na+ or Ca2+ into DOP aqueous solutions causes their viscosity to decrease, and the decrease was more prominent at higher salt ion concentrations. Compared with that of HEPDOP, the viscosity of EPDOP aqueous solution decreased more significantly, which might be due to the fact that EPDOP has less negative charge. This result is slightly different from the observation by Shi et al. Shi et al. found that the addition of Na+ increased the viscosity of peony seed dreg polysaccharides (PSDPs), the addition of Ca2+ reduced the viscosity of PSDPs, and the viscosity of the fractions obtained by different extraction methods were slightly different (Shi, et al., 2016). Similarly to those observed by Shi et al., the results observed by Zhu et al. showed that the viscosity of ALCP (HBSS) extracted by hot buffer increased when Na+ concentration was low, but decreased when Na+ concentration was high (ZhuDan-Ye., et al., 2018). They also found that the viscosity of ALCP (CASS) obtained by alkali extraction reduced when Ca2+ was added. The effect of salt ions on the viscosity of DOP may be due to the salting out effect: the presence of positive ions reduces the repulsive force between molecules causing them to expand, thereby can decrease the viscosity (Li, et al., 2019; Li, et al., 2018). Furthermore, the addition of Ca2+ promotes the formation of a dense polysaccharide conformation, increasing the interactions between polysaccharide chains, thus causing the viscosity to reduce (HuangFei., et al., 2018; Li, et al., 2019).

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Fig. 6 Apparent Zeta potential of DOPs (1% (w/w)) and Viscosity-shear rate profiles of two 1%(w/w) DOP aqueous solutions containing different amounts of NaCl or CaCl2.

3.8 Effect of different pH on viscosity As depicted in Fig. 7, the DOP samples had shear thinning behavior at all pH values; however, comparing the two flow curves showed that EPDOP was more sensitive to pH than HEPDOP, which may be due to the fact that EPDOP contains more protein, and the sensitivity of the protein to pH is well known. The change in pH is likely to result in a weakening of the interaction between the protein and the polysaccharide, resulting in a decrease in viscosity. Under acidic pH, the viscosity of both DOPs aqueous solution decreased due to the destructive effect of strong acid and acid hydrolysis (Huang, et al., 2016). The viscosity of both samples also decreased at alkaline pH, which may be due to that alkaline pH causes hydroxyl groups in DOP molecular chains to become ionized, thereby diminishing the intermolecular hydrogen bonds (Wu, Ding, Jia, & He, 2015). 11

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Fig. 7 Viscosity-shear rate profiles of two 1%(w/w) DOP aqueous solutions at different pH.

3.9 The linear viscoelastic region of DOP The linear viscoelastic region of the DOP samples was determined by strain scanning. The results illustrated in Fig. 8 showed that the storage modulus G' and the loss modulus G'' of the DOP samples remained intact over a wide range of strain. Thus, subsequent oscillation measurements were carried out only in the linear viscoelastic region to ensure that the DOPs remain intact: the measurement was performed at 1% strain.

Fig. 8 Linear viscoelastic regions of DOPs determined by strain scanning in oscillation amplitude mode at a frequency of 10.0 rad/s.

3.10 Frequency sweep of DOP The storage modulus (G’) and the loss modulus (G’’) represent solid-like and liquid-like properties of viscoelastic materials, respectively (Lin, et al., 2018). Analysis of the data in Fig.9 showed that both G' and G'' of DOP samples at all concentrations increased with the increase of angular frequency, which is similar to observations by previous studies (Chaux-Gutiérrez, PérezMonterroza, & Mauro, 2019; Liu, et al., 2019). The intercept of G' and G'' was also observed at high angular frequency. At angular frequencies lower than that of the intercept, G'' was higher than G', and the DOP solution exhibited fluid-like behavior. On the other hand, at angular frequencies higher than that of the intercept, G' was higher than G''，and the DOP solution exhibited solid-like behavior. Based on this observation, it appears that the entanglement of the molecular chain was temporary: at low frequencies, long-term oscillation causes the entanglement to become unraveled; by contrast, at high frequencies, short-term oscillation did not cause disentanglement, but rather appears to promote the formation of a temporary network structure between the molecules (Du, Li, Chen, & Li, 2012; Huang, et al., 2016; Jin, et al., 2014). Furthermore, the viscoelastic behavior of the DOP 12

Journal Pre-proof samples appears to be concentration-dependent: the intercept of G' and G'' was at a lower frequency value as the DOP concentration was increased. Interestingly, at low concentration (1.2% (w/w)), the intercept of G' and G'' was only observed in the HEPDOP sample. This may be due to different viscosity and molecular chain entanglement between the two DOP samples (Wu, et al., 2015).

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Fig. 9 Frequency-dependent G’ and G’’ of two types of DOP aqueous solutions at various concentrations (from 0.8% (w/w) to 2% (w/w)).

3.11 Effect of temperature on rheological properties of DOP 3.11.1 Effect on viscosity As shown in Fig. 10(A), at temperatures between 25 °C and 85 °C, the viscosity of DOPs decreased with increasing temperature. Similar results have been reported by previous studies, in which increasing temperature was found to accelerate the thermal motion of the molecular chain, weakening the interaction between them; as a result, the viscosity was decreased (Lin, et al., 2018; Nwokocha & Williams, 2016; Wu, et al., 2015). 3.11.2 Effect on viscoelastic behavior Fig. 10(B) shows the effect of temperature on the viscoelastic behavior of DOP samples, each at a concentration of 2% (w/w). According to the data, both the G' and G'' decreased with increasing temperature. The decrease of G' is likely due to the increase in molecular thermal motion caused by increased temperature, which in turn leads to the increase of free volume of the molecules and the weakening of hydrogen bonds between them (Seo, Kang, Lee, Lee, & Chang, 2018). In addition, all samples exhibited thermal hysteresis: their heating and the cooling curves did not completely coincide, and the hysteresis areas were different. However, HEPDOP had a smaller hysteresis area compared with EPDOP. It is possible that there is a denser network consisting of more interacting molecular chains in the HEPDOP solution; thus, it can retain more water (Seo, et al., 2018; Yaich, et al., 2014). The G'' of EPDOP solution was also higher than the G' over the temperature range tested, indicating that EPDOP is more viscosity advantage. For the HEPDOP solution, the value of G' was initially higher than that of G'' until the temperature reached about 20 °C, after which the G' was rapidly declined, while G'' became dominated. This indicates that the gel transition temperature of HEPDOP is about 20 °C. (A)

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(B)

Fig. 10 (A) Viscosity-temperature profiles of DOP aqueous solutions at different concentrations. (B) Temperature dependence of G' and G'' for two types of DOP aqueous solutions at various concentrations from 0.8% (w/w) to 2% (w/w).

4. Conclusion The structure, and the physicochemical, thermal and rheological properties of two types of DOP (HEPDOP and EPDOP) were studied. Compared to EPDOP, HEPDOP had higher carbohydrate content and molecular weight, as well as lower protein content, and more importantly, mannanase activity in HEPDOP was significantly reduced. This result confirmed our hypothesis that hot water scald pretreatment could effectively reduce the activity of mannanase in the Dendrobium officinale stem. Furthermore, HEPDOP had a lower hydrophobicity, which is consistent with the higher protein content of EPDOP. The higher viscosity of HEPDOP is attributed to the higher carbohydrate content and larger average molecular weight of HEPDOP. The above of experimental results proved that the chemical composition and physical and chemical properties of Dendrobium polysaccharide had changed significantly after hot water scald pretreatment. However, the pretreatment did not change the structure of Dendrobium polysaccharide, and the infrared spectroscopy results could illustrate this point. Besides, both DOPs degraded at temperatures of above 300 °C, but the thermal stability of HEPDOP was slightly higher than that of EPDOP. The aqueous solutions of the two DOPs exhibited typical shear thinning behavior, and temperature, salt ions, acidity and alkalinity were found to have significant effects on their viscosity. In addition, EPDOP was more sensitive to pH and salt ions, which may be attributed to the fact that EPDOP contained more protein and the amount of surface negative charge of EPDOP was fewer. Compared 15

Journal Pre-proof with that of EPDOP, the intercept of the G' and the G'' values of HEPDOP occurred at a lower concentration. Interestingly, the gel transition temperature was observed only in HEPDOP solution. This phenomenon can be related to the length and number of molecular chains, and the difference in the degree of molecular chain entanglement of EPDOP and HEPDOP leads to this result. This research provides information of Dendrobium officinale polysaccharide obtained by water extraction and ethanol precipitation, and inactivated the mannanase in Dendrobium officinale by pretreatment. This provides a new way for the industrial application of Dendrobium officinale. Currently, detailed analyses of DOP structure by methods such as nuclear magnetic, methylation and periodic acid oxidation, are being conducted in our laboratory. Acknowledgements This work was financially supported by the Provincial Key Research and Development Project, Hainan province, China (ZDYF2018071). References Agatonovic-Kustrin, S., & Morton, D. W. (2018). HPTLC - Bioautographic methods for selective detection of the antioxidant and alpha-amylase inhibitory activity in plant extracts. MethodsX, 5, 797-802. Ahirwar, S., Soni, H., Rawat, H. K., Ganaie, M. A., Pranaw, K., & Kango, N. (2016). Production optimization and functional characterization of thermostable β-mannanase from Malbranchea cinnamomea NFCCI 3724 and its applicability in mannotetraose (M4) generation. Journal of the Taiwan Institute of Chemical Engineers, 63, 344-353. al., A. F. e. (1998). Detection of Intramolecular Associations in Hydrophobically Modified Pectin Derivatives Using Fluorescent Probes. Langmuir. Amiji, M. M. (1995). Pyrene fluorescence study of chitosan self-association in aqueous solution. Carbohydrate Polymers. Brown, L. K., Wiersma, A. T., & Olson, E. L. (2018). Preharvest sprouting and α-amylase activity in soft winter wheat. Journal of Cereal Science, 79, 311-318. Carrillo-Barral, N., Matilla, A. J., Rodriguez-Gacio, M. D. C., & Iglesias-Fernandez, R. (2018). Mannans and endo-beta-mannanase transcripts are located in different seed compartments during Brassicaceae germination. Planta, 247(3), 649-661. Chan, C. F., Wu, C. T., Huang, W. Y., Lin, W. S., Wu, H. W., Huang, T. K., Chang, M. Y., & Lin, Y. S. (2018). Antioxidation and Melanogenesis Inhibition of Various Dendrobium tosaense Extracts. Molecules, 23(7). Chaux-Gutiérrez, A. M., Pérez-Monterroza, E. J., & Mauro, M. A. (2019). Rheological and structural characterization of gels from albumin and low methoxyl amidated pectin mixtures. Food Hydrocolloids, 92, 60-68. Chen, N.-D., Chen, N.-F., Li, J., Cao, C.-Y., Wang, J.-M., & Huang, H.-P. (2015). Similarity Evaluation of Different Origins and Species of Dendrobiums by GC-MS and FTIR Analysis of Polysaccharides. International Journal of Analytical Chemistry, 2015, 1-8. Chen, X., Jin, C., Wang, P., Chen, K., Zhang, X., Li, J., Gong, C., & Wang, A. (2018). Genome-wide analysis and endo-β-mannanase gene family expression profiling of tomato and soybean. Nordic Journal of Botany, 36(6). Chen, Y., Zhang, J. G., Sun, H. J., & Wei, Z. J. (2014). Pectin from Abelmoschus esculentus: optimization of extraction and rheological properties. Int J Biol Macromol, 70, 498-505. Cho, H. M., & Yoo, B. (2015). Rheological characteristics of cold thickened beverages containing 16

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Journal Pre-proof Conflict of interest statement We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Journal Pre-proof Author Statement All authors have read the revised manuscript and approved to submit it to your journal.

Journal Pre-proof Highlights: 1. HEPDOP had higher carbohydrate content and lower protein content compared to EPDOP. 2. Pretreatment by hot water scalding could inactivate mannanase in D. officinale stems. 3. HEPDOP had lower hydrophobicity and higher viscosity compared to EPDOP. 4. The intercept of the G' and the G'' values of HEPDOP occurred at low concentrations.