Structural Characteristics, Rheological Properties, and Antioxidant Activity of Novel Polysaccharides from “Deer

School of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China Scientific Research Base of Edible Mushroom Processing Technology Integration of Ministry of Agriculture and Rural Affairs, Changchun 130118, China Key Laboratory of Technological Innovations for Grain Deep-Processing and High-Effeciency Utilization of By-Products of Jilin Province, Changchun 130118, China Engineering Research Center of Grain Deep-Processing and High-Effeciency Utilization of Jilin Province, Changchun 130118, China

and cholesterol, anti-inflammatory, antioxidation, antitumor, antivirus, immune regulation, etc. [5][6][7][8][9]. Polysaccharide is the main active component in Auricularia. However, compared with polysaccharides of Auricularia auricula and Auricularia cornea, which are widely cultivated in China, there are few studies on the edible value and processing properties of novel polysaccharide of deer tripe mushroom.
Rheology is the study of flow and deformation, especially the behaviour of transitions between solids and fluids [10]. Carrageenan and gellan are materials with unique rheological and gel properties, which have been widely used in the food industry. Pectin is primarily used in the food industry because of its gelling, thickening, and stability ingredient [11].
erefore, some studies of physicochemical and rheological properties are very important and need to be further successfully developed and understood for application. In recent years, the rheological properties of polysaccharides extracted from various natural plants including mushrooms have been studied extensively [12,13]. In addition, with the study of structure identification and physical properties of edible fungus polysaccharide, it has been widely used in the field of food. Some edible fungus polysaccharides dissolve in water to form gels, which can be widely used as thickening agent, gelling agent, stabilizer, emulsifier, and other applications in food [14]. Xu et al. isolated a polysaccharide fraction (FMPS) from floral mushrooms cultivated in Huangshan Mountain, and the rheological properties of FMPS in aqueous solutions were investigated, which exhibited weak gel behaviour with the change of concentration and temperature [12]. Bao et al. [15] studied the rheological properties of Auricularia auricula polysaccharide and found that Auricularia auricula polysaccharide has high viscosity and the ability to form a heat stable gel, which can be used as a new hydrogel in functional foods. Zhu et al. [16] extracted ß-glucan from Auricularia auricularia and prepared a novel ß-glucan-based hydrogel. However, whether there are related properties of Auricularia auricularia and its domesticated deer's ear has not been reported yet.
In this study, the deer tripe mushroom polysaccharide was extracted by hot water extraction method, and the structure characterization, rheological properties, and antioxidant activity of polysaccharide were studied, in order to provide a solid foundation for the in-depth research and industrial application of polysaccharide from the deer tripe mushroom. is research provides information of DTMP that may be useful for its application in the food industry.

Preparation of DTMP.
A 10.0 g sample was added to 1000 mL of distilled water and extracted under (kept at 90°C or 4 hours), repeated for 2 times, after which it was filtered and centrifuged (4000 g, 15 min, 4°C). e supernatant was concentrated to 1/3 of the original volume, and 4 volumes of 95% ethanol was then added, after which the mixture was stirred until precipitation completed at 4°C for 24 h. Protein was removed 6 times with the Sevag method [17]. en, the precipitate was obtained after centrifugation (4000 g, 20 min) and washed with 95% ethanol (3-4 times). e DTMP was obtained after freeze drying (ALPHA, 1-2 LDplus, Marin Christ Co., Osterode, Germany). e extraction rate of DTMP extracted using hot water was 12.5%.

FT-IR Analysis.
e dried polysaccharide powder (2.0 mg) was put in an agate mortar and dried KBr crystal (200 mg) was added, it was evenly ground, which was pressed into tablets. It was measured using an infrared spectrometer (Bruker Optics Co., Berlin, Germany) in the range of 4 000-500 cm −1 .

Nuclear Magnetic Resonance (NMR) Spectroscopy.
e samples were dissolved in 99.9% D 2 O, and the 1 H-NMR and 13 C NMR spectra of the samples were determined by Bruker 400 MHz nuclear magnetic resonance spectroscopy (Bruker, Fallanden, Switzerland).

Rheological Properties of DTMP
2.6.1. Preparation of DTMP Solutions. In order to accelerate the dissolution of DTMP, DTMP was added to 100 mL of distilled water to prepare 0.1%, 0.5%, 1%, 1.5%, and 2% (w/v) DTMP solutions followed by stirring at 50°C for 1 h with a magnetic stirrer.

Steady Flow Behaviour.
e rheological properties of the DTMP solutions were determined using a DHR-1 Rheometer (TA Instruments, USA). A parallel plate rotor (diameter 50.0 mm) with a gap of 0.104 mm was installed on the rheometer. Static rheology was performed allowing 2 min before testing. All measurements were taken at 25 ± 0.1°C, and all experiments were carried out at least in triplicate.
According to the solution fluid type, the typical formula of the curtain law model was used to describe its rheology: where η s is the apparent viscosity (Pa·s), K is the consistency index (Pa·s n ), and n is the flow behaviour index. Using the solution system, rheological curve fitting was performed to obtain the rheological curve of the K value and n value.
(1) Determination of Flow Properties at Different Concentrations. DTMP solutions with different mass concentrations (0.1%, 0.5%, 1.0%, 1.5%, and 2.0% (w/v)) were accurately prepared and incubated for 20 min in a 70°C water bath with an DHR-1 Rheometer (TA) (diameter 40.0 mm). Using a spacing of 1 mm, shear rate of 0.1 -1000 s −1 , and measured temperature of 25°C, the viscosity of the solution was observed with the shear rate changes.
(2) Determination of Flow Properties at Different pH Values. Before the viscosity measurements, 1.0% of the pH of the DTMP solution was adjusted to around 2, 4, 6, 8, or 10 using 4 M NaOH or HCl. e temperature was also controlled at 25°C in a water bath, and the shear rate ranged from 0.1 to 1000 s −1 .
(3) Determination of Flow Properties at Different Temperatures. According to Determination of Flow Properties at Different Concentrations, 1.0% of the DTMP solution was loaded onto the rheometer substrate using parallel plates, and the sample was allowed to sit for 1 h to stabilise the temperature. e experimental temperatures were 25, 35, 45, 55, 65, and 75°C. e shear rate ranged from 0.1 to 100 s −1 .

Dynamic Viscoelasticity Tests.
Dynamic viscoelasticity measurements were used to determine the storage modulus (G′) and loss modulus (G″) of the DTMP solution (1.0-2.0% (w/v)). e linear viscoelastic region of the DTMP was determined by a strain sweep ranging from 0.1 to 1000% with 1 Hz of frequency at 25°C. G′ and G″ were measured at a frequency sweep of 0.1-100 Hz at 1.0% strain.
e maximum sustained force was the gel strength. Each sample was tested 3 times.

SEM Test.
e samples were frozen at −20°C, which were observed through a Hitachi S3000 N scanning electron microscope (Tokyo, Japan) at an accelerating voltage of 20 kV.

Statistical Analysis.
In this study, all experimental values were expressed as the mean of three trials ± the standard deviation. An analysis of variance (ANOVA) was performed using Origin 2018 and SPSS20.0.

Steady Shear Flow Behaviour
(1) Effect of Concentration on Viscosity. Based on the curves of apparent viscosity and shear rate of different concentrations of DTMP shown in Figure 1, the different concentrations of polysaccharide solution showed typical non-Newtonian behaviour and an obvious shear-thinning area. is phenomenon has been described previously in the literature [15,23]. e shear thinning behaviour of the DTMP solution may be due to the fact that the macromolecules are oriented along the streamline and the open entangled structure cannot be recovered in time. As predicted, the steady shear viscosity decreased as the shear rate increased. When the concentration of DTMP increased, the shear thinning phenomenon was still observed. As expected, as the polysaccharide concentration increased, the viscosity of all polysaccharide solutions increased at certain shear rates.
As shown in Table 1, the static rheological curve could be described by the power law model. Moreover, the K values increased as the concentration increased, whereas the opposite trend was observed with the n values, which decreased from 0.325 to 0.164. As shown in Table 1, the power law model was observed to be favourably applicable for the DTMP polysaccharides (R 2 > 0.99), and n was less than 1, which indicated the pseudoplasticity of non-Newtonian fluid. As the concentration of the solution increased, shear thinning was indicated, and the viscosity of the solution system increased as the DTMP solution concentration increased. Lower values of n were observed at higher DTMP concentrations, indicating typical pseudoplastic flow behaviours. K is a systematic indicator of a sticky nature. As the DTMP concentration increased from 0.5% to 2.0% (mg/mL), K increased from 1.8321 to 7.2946. In this region, it is generally the DTMP particle-particle interactions that influence the flow behaviour of DTMP solution [24,25]. As the polymer concentrations increased, individual particles began to overlap to form intermolecular linkages or "connecting regions" that limited the movement and stretching of the polymer chains in aqueous solution. erefore, the viscosity increased significantly as the polysaccharide concentration increased [26]. e results showed that the higher the concentration was, the stronger the shear-thinning, and at a high concentration, DTMP solution formed a pseudoplastic fluid. Polysaccharides extracted from other mushrooms, such as Ganoderma lucidum polysaccharides [27], also exhibit shear thinning behaviour.
(2) Effect of pH on Viscosity. Figure 2 presents the curves of apparent viscosity and shear rate of a DTMP solution (1.0%) under different pH conditions. e results indicated that the rheological behaviour of the DTMP solution (10 mg/mL) changed between acidic conditions (pH 2-6) and alkaline conditions (pH 6-10). e shear-thinning effect was seen over the whole range of pH values (Figure 2). e solutions exhibited a higher viscosity at pH � 10 and a lower apparent viscosity at pH � 2 and pH � 4. Strong acids may cause the hydrolysis of polysaccharides, thereby reducing the viscosity.
Conformational changes are structural changes caused by fluctuations in ion interactions or base compositions that are attributed to changes in pH [28]. However, this trend is different from that of Auricularia polysaccharides, in which the viscosity decreased when the pH became acid [15].
(3) Effect of Temperature on Viscosity. Temperature is an important factor affecting the rheological properties of fluids. Figure 3 shows the apparent viscosity curve of DTMP solution (1.0%) at 25-75°C. As can be seen from Figure 3, the viscosity of the solution increased with the increase of temperature. A possible reason for this trend may be that at higher temperatures, the molecular motion becomes more intense, and the molecular forces gradually stronger, which increases the molecular flow of the solution system and results in higher viscosity. However, the pseudoplastic fluid behaviour remains even at the highest temperature. Figure 4 shows the G′ and G″ data of DTMP. e DTMP is affected by both viscosity and elasticity, which can be represented using G′ and G″. G′ (storage modulus) represents the elastic component, and G″ (loss modulus) represents the sticky component. e dynamic rheological properties of DTMP in the linear viscoelastic region were measured to ensure that the polysaccharides had not been destroyed. G′ and G″ of DTMP were essentially constant with stresses from 0.1 to 1.0%, but when strain was >1.0%, G′ and G″ were reduced (Figure 4(a)). us, 1.0% strain was selected as the dynamic viscoelasticity measurement of DTMP. e dynamic viscoelasticity measurement must appear in the linear viscoelastic region to ensure the polysaccharide structure [29]. Regardless of what happens to the concentration of DTMP, G′ and G″ of both groups were enhanced with increased frequency (Figure 4(b)). In addition, G′ was always higher than G″; the DTMP exhibited a gel-like behaviour.

FT-IR Spectroscopy Analysis.
FT-IR analysis of DTMP was carried out by KBr tablet pressing method, and the infrared spectrum in the range of 4 000-500 cm −1 is shown in Figure 5. A wide peak was present at 3251.46 cm −1 , which was caused by the stretching vibration between the polysaccharide characteristic -OH and the O-H bond of water. And the absorption peak near 2918.17 cm −1 was observed C-H stretching vibration and variable angle vibration [25,31]. ere was a strong absorption peak at 1715.73 and 1600.56 cm −1 , which was caused by the C�O asymmetric stretching vibration, indicating that there might exist an aldehyde group -CHO. Generally, the sugar molecule-bound water was found as an absorption peak in this region [32]. e absorption peak at 1488.47 cm −1 was the variable angle vibration of C-H, and the above four are the characteristic absorption peaks of polysaccharide [33]. 1241.92 cm −1 and 1028.55 cm −1 characteristic absorption peaks indicated that DTMP contained pyran-type glycosylic bonds, and α-(1-6) glycosylic bonds might be existed. FT-IR results showed that DTMP might be pyran type and exhibited α-configuration.

Monosaccharide Composition
Analysis. DTMP was degraded by TFA and then derivatized with pyridine and acetic anhydride for GC-MS analysis. Figure 6 shows the monosaccharide composition of strandards and DTMP. As could   Journal of Food Quality be seen from the figure, DTMP was mainly composed of mannose, fructose, glucose, and galacturonic acid, of which galacturonic acid was the main component. By calculation, the molar ratio of mannose : fructose : glucose : galacuronic acid in DTMP was 0.8 : 14.8 : 1.0 : 26.32.

1 H NMR and l3 C NMIR Spectroscopy.
In general, there are several signals in the proton region of 1 H-NMR (δ4. 3-5.9), indicating the presence of several sugar residues. In addition, 1 H-NMR signals of the sugar residues in α-configuration were greater than δ � 5.0, and that of the sugar residues in ß-configuration was less than δ5.0. e 1 H-NMR spectrum of the DTMP is shown in Figure 7(a); signal (δ5.20) appeared in the heterocephalic proton region, which indicated that DTMP contained a glycoside bond and existed in a configuration pyranose. H 2 -H 6 signals of sugar residues appeared in the range of δ3.0-4.2, with serious overlap and difficult to be attributed. In addition, there is a weak signal in the range δ 3.2-3.5, indicating the presence of less -OCH 3 . In addition, there is a relatively obvious -CH signal in the range of δ1.0-1.3, which is classified as fructose signal in methylhexose according to the monosaccharide composition of DTMP [34].
Generally speaking, the proton signal of heterocentric carbon is in the range of δ 90-112, while the proton signal of most a configurational sugar residues is in the range of δ 90-102. e proton signals of heteroheader carbon of b-configuration sugar residues are located between δ102 and 112 [35]. e magnetic resonance carbon spectrum of DTMP is shown in Figure 7, and there are three heteroheader carbon signals between δ 90 and 112 (92.05, 93.19, and 95.86), indicating that there are three monosaccharide residues in DTMP. In addition, since the C3 or C5 proton signal of pyronose is less than 80, the combination of monosaccharide composition and end-group carbon proton signal 93.19. In the 13 C NMR spectra, the peaks at C-1 (93.19 ppm), C-2 (75.91 ppm), C-3 (74.09 ppm), C-4 (72.48 ppm), C-5 (71.11 ppm), and C-6 (60.48 ppm) contributed to the backbone chain of DTMP. 1 H NMR and 13 C NMR spectra showed that DTMP was a polysaccharide with α-1,4-glucoside bond. e NMR indicated that the conformation and linkages of DTMP were consistent with the results of GC-MS and FT-IR.

SEM Analysis.
As can be seen from Figure 8, the microstructure of DTMP gels with different concentrations is significantly different. When the concentration is low (2.0%-4.0%), the sample presents a flow transition state, and the resulting gel has a uniform microstructure and pore arrangement (Figures 8(a) and 8(b)), which is mainly the result of intermolecular cross-linking of TFP under hydration.
With the increase of sample concentration (Figures 8(c)-8(e)), the gel structure changed significantly. e pores became larger, the pore arrangement was irregular, and the three-dimensional honeycomb structure gradually weakened. When the concentration increases to 10% (Figure 8(e)), the gel state presents solid state, and the microstructure of the gel becomes very dense, presenting  continuous filamentous and flake bonded aggregates. is is consistent with the results of texture analysis and gel strength analysis.

Strength and Structure Properties of DTMP Gel.
As shown in Figure 9, 5 different concentrations of deer polysaccharide gels were placed in test tubes and inverted at      increased 1.39% to 14.57%, respectively. ese results indicated that there was a synergistic effect between DTMP and water in the system. is synergistic effect was enhanced by the addition of DTMP, which produced a filling effect in the gel. Depending on the hardness and elasticity values, DTMP can be developed into some products, such as frozen and fudge. is means that DTMP could be used as a substitute for some gels in food.

Antioxidant Activity Analysis
3.7.1. DPPH Radical Scavenging Activity. DPPH free radical is a stable lipid free radical. When the free radical scavenger presents, purple gradually fades to a stable yellow, and the degree of depigment is quantitatively related to the number of electrons received [36]. e ability to scavenge DPPH free radicals of vitamin C (VC) and DTMP is shown in Figure 10.  Each value represents the mean ± standard deviation (n � 3). ere were significant differences in the values of different letters in the same column (P < 0.05).
When the concentration of VC from 1.0 to 3.0 mg/mL, free radical scavenging rate flattened at (80.25-90.32%). As shown in Figure 9, with the concentration of 0.5-3.0 mg/mL, the scavenging ability of the samples to DPPH free radicals increased, but relatively weak relative to VC. When the concentration reached 3.0 mg/mL, the DPPH free radical rate of DTMP was 67.53 ± 0.47%. In addition, the half maximal inhibitory concentration (IC 50 ) of DTMP was 2.09 mg/mL, respectively, indicating that the antioxidant effect of DTMP was relatively good.

ABTS Radical Scavenging
Activity. ABTS method is widely used for the evaluation of antioxidant capacity of bioactive substances in vitro due to its simple operation and good repeatability [37]. Figure 11 shows the ABTS radical scavenging ability of DTMP. When the VC concentration reached 2.0 mg/mL, the clearance rate reached 95.85% and then flattened. As shown in Figure 11, as the concentration ranged from 0.5 to 3.0 mg/mL, the scavenging ability of the samples about ABTS radical was increased, but not as good as VC. In addition, the IC 50 values of DTMP were 3.81 mg/ mL, indicating that the antioxidant effect of DTMP had a certain antioxidant effect.

OH Scavenging Activity. Hydroxyl free radical (-OH)
is the most reactive free radical in ROS, which may directly or indirectly cause tissue damage, induce a variety of human diseases including cancer, and promote aging [38]. As shown in Figure 12, when the DTMP concentrations of 0.5-3.0 mg/ mL, the scavenging ability of all samples to hydroxyl free radicals increased with the increase of DTMP concentration, but all of them were lower than VC. In addition, the IC 50 of DTMP was 3.49 mg/mL, indicating that the -OH free scavenging effect of DTMP was weaker than DPPH free radical scavenging ability.

O 2− Scavenging Activity.
e O 2− is a kind of ROS anionic free radical in the human body, which leads to lipid peroxidation in vivo and accelerates body aging. Figure 13 shows the O 2− scavenging ability of DTMP and VC. At concentrations from 0.5 to 3.0 mg/mL, DTMP showed a dependence on concentrations. At 3.0 mg/mL, DTMP were 52.76 ± 0.48%, both were lower than for VC. e ability of polysaccharides scavenge superoxide anionradicals is associated with the quantity of active phenolic and alcohol hydroxyl groups in the molecule [39]. In addition, the IC 50 value of DTMP was 3.07 mg/mL, respectively, indicating that the -OH free scavenging effect of DTMP was just below DPPH free radical scavenging ability.
e rheological results showed the following: DTMP solution showed a shearthinning (pseudoplastic) behaviour, and its pseudoplasticity was more obvious at a concentration of 2%. e power law model was used to evaluate the viscosity curves of DTMP, and its viscosity and consistency indices both increased as the concentration increased, whereas both indices decreased as the concentration decreased. e viscosity of the polysaccharide solution changed as the pH and temperature changed: the polysaccharide solutions had a higher viscosity at pH � 10 and 35°C. DTEP had good thermal stability. Viscoelastic analyses showed gel-like behaviour, with G′ always higher than G″. e gel strength and rupture strength enhanced with the increase of concentration 2%-10% (W/V). Additionally, the antioxidant experiments of the crude polysaccharide showed that the crude polysaccharide had good antioxidant activity, and there was an obvious dose-effect relationship between its activity and concentration in the low concentration range.
is research provides a solid foundation for the in-depth research and industrial application of polysaccharide from the deer tripe mushroom.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors declare that there are no conflicts of interest. Journal of Food Quality 11