Preparation method of a novel bletilla striata polysaccharide and its composite hydrogel

Abstract

The present invention discloses a preparation method of a novel Bletilla striata polysaccharide and its composite hydrogel, belonging to the food field. The preparation method of the novel Bletilla striata polysaccharide includes: Step 1, pulverize dry Bletilla striata tubers, sieve them through a sieve to obtain Bletilla striata powder; Step 2, dissolve the Bletilla striata powder in deionized water, extract it 2-3 times at 80-90 °C; concentrate the extract, then add absolute ethanol to precipitate at 2-4 °C, let it stand and then centrifuge to collect the precipitate; Step 3, redissolve the precipitate in deionized water, perform deproteinization treatment, dialyze the obtained substance after deproteinization with a dialysis bag, collect the non-dialyzable solution and freeze-dry it to obtain crude polysaccharide; Step 4, purify it with DEAE-52 cellulose and a fully automatic gel purification system to obtain Bletilla striata polysaccharide, named BSP-182. The composite hydrogel is prepared by mixing BSP-182 and HA powder in deionized water. The present invention solves the problems of low gel strength and poor viscosity of Bletilla striata polysaccharide, and provides feasible guidance for the development and application of BSP food.

Description

Technical Field

[0001] The present invention relates to the field of food, and specifically to a preparation method of a novel Bletilla striata polysaccharide and its composite hydrogel. Background Art

[0002] Bletilla striata has a history of use for thousands of years and is an important traditional Chinese medicine resource listed in the "Chinese Pharmacopoeia (2020 Edition)" and "Shennong Ben Cao Jing". Bletilla striata is widely used as an ingredient in many preparations in traditional Chinese medicine preparations and has various biological activities. In recent years, studies have confirmed that polysaccharides are the most important biological active components in the tubers of Bletilla striata plants and have various biological activities such as immunomodulation, anti-inflammatory, anti-tumor, anti-fibrosis, hemostasis, and protection of gastrointestinal function. At the same time, due to its special physical and chemical properties, Bletilla striata polysaccharide (BSP) can also be used as an additive or ingredient in the research and development of drugs, health products, and the food industry.

[0003] BSP is a promising natural functional polysaccharide with a linear polymer having a molecular weight range of 100 - 300KDa, composed of mannose and glucose linked by β-glycosidic bonds. As a water-soluble plant β-glucan, BSP exhibits excellent hydrophilicity, swelling property, and permeability, and its structure contains numerous hydroxyl groups suitable for chemical modification. In addition, BSP exhibits good stability, biocompatibility, and tissue adhesion, indicating its potential use in the preparation of hydrogels.

[0004] Natural hydrogels derived from biopolymers, such as polysaccharides and proteins, are attracting increasing attention. These natural hydrogels can be potential substitutes for synthetic hydrogels due to their structural characteristics, excellent biocompatibility, low toxicity, and biodegradability. Hyaluronic acid (HA) is a natural acidic mucopolysaccharide composed of a disaccharide repeat sequence of N-acetylglucosamine and glucuronic acid, linked together by β-1,4 and β-1,3 glycosidic bonds. HA has excellent moisturizing, lubricating, and antioxidant properties and has a wide range of applications in the fields of pharmaceuticals, food, cosmetics, biomaterials, etc. HA also exhibits high hydrophilicity because its disaccharide units contain abundant carboxylic acid (COO-) and hydroxyl (OH-) groups, providing unique advantages in hydrogel formation and making it a promising new food raw material.

[0005] In food processing, gelation is an important functional property of polysaccharides, which enhances the texture of foods, retains moisture, improves food stability, extends shelf life, and plays a crucial role in the development of new food products. Research has shown that the interaction between polysaccharides can affect the gelation ability, gel strength, water-holding capacity and other properties of mixed gels. This synergistic effect can be achieved by changing the conformation or structure of polysaccharides and is affected by various factors such as ion concentration, pH, and chemical structure. Appropriate structural modification of polysaccharides under specific conditions can broaden their applications as polymeric materials. Typical examples include chitosan CS / HA, HA / alginate, and xanthan gum / glucomannan. There may be a synergistic effect between BSP and HA, both of which are biodegradable endogenous substances with potential carrier applications. The combination of these properties with the physical structure of the gel can lead to the formation of bioactive materials. This physical cross-linking method does not require strict conditions, is suitable for the preparation of food materials, enhances the mechanical properties of hydrogels, and avoids the potential toxicity of certain chemical cross-linking agents or initiators.

[0006] However, the poor solubility, low viscosity, and poor gelation ability of BSP in water limit its application in the food industry. Summary of the Invention

[0007] Object of the Invention: The object of the present invention is to provide a preparation method of a novel Bletilla striata polysaccharide and its composite hydrogel in view of the deficiencies of the prior art, to solve the problems of low gel strength and poor viscosity of Bletilla striata polysaccharide gels, and to provide feasible guidance for the development and application of BSP foods.

[0008] In order to improve the performance of BSP and broaden its application in the food industry, the present invention first separates BSP by using water extraction and ethanol precipitation techniques. The structure of BSP is elucidated by molecular weight determination, monosaccharide composition analysis, methylation analysis, and nuclear magnetic resonance studies. In addition, by analyzing the structural changes and gel strength changes of hydrogels at different BSP / HA ratios, the synergistic effect between BSP and HA in mixed gels is studied. The aim is to provide a reference for the rational selection of BSP in food texture design or improvement, and to provide new perspectives and data support for the application of BSP / HA as potential food additives.

[0009] Technical Solution: A preparation method of a novel Bletilla striata polysaccharide, comprising the following steps:

[0010] Step 1: Crush the dried Bletilla striata tubers, sieve them through a sieve to obtain Bletilla striata powder;

[0011] Step 2: Dissolve the Bletilla striata powder in deionized water and extract it 2-3 times at 80-90 °C; concentrate the extract, then add anhydrous ethanol to precipitate at 2-4 °C, let it stand for at least 12 h, and then centrifuge to collect the precipitate;

[0012] Step 3: Redissolve the precipitate in deionized water and perform protein removal treatment by the Sevage method; dialyze the obtained material after protein removal against deionized water in a dialysis bag at 2-4 °C for 72 hours; collect the non-dialyzable solution and lyophilize to obtain crude polysaccharide;

[0013] Step 4: Finally, purify using DEAE-52 cellulose and an automatic gel purification system to obtain Bletilla striata polysaccharide with a higher purity, named BSP-182.

[0014] Further, in Step 1, the mesh number of the sieve is 10-12 meshes.

[0015] Further, in Step 2:

[0016] 1) Dissolve Bletilla striata powder in deionized water, and the mass-to-volume ratio of Bletilla striata powder to deionized water is (1-1.5) g:(10-12) mL;

[0017] 2) The extraction solution concentration method is: evaporate to dryness using a rotary evaporator;

[0018] 3) The volume ratio of the concentrated extraction solution to the added absolute ethanol is (1-1.5):(4-5).

[0019] Further, in Step 3:

[0020] 1) Redissolve the precipitate in deionized water, and the mass-to-volume ratio of the precipitate to deionized water is (1-1.5) g:(10-12) mL;

[0021] 2) The pore size of the dialysis bag is (1-1.2) kDa.

[0022] For the Bletilla striata polysaccharide prepared by the above method, the molecular weight of BSP-182 is 182.10 kDa; BSP-182 includes mannose and glucose, and the molar ratio of mannose to glucose is 8:2.

[0023] A preparation method of a composite hydrogel based on Bletilla striata polysaccharide, comprising the following steps:

[0024] S1: Dissolve BSP-182 and HA powder in deionized water respectively, and magnetically stir at 25-30 °C for 10-15 minutes to prepare a completely dissolved BSP-182 solution and HA solution;

[0025] S2: Mix the BSP-182 solution and the HA solution, and magnetically stir evenly at 25-30 °C for 10-15 minutes to obtain a mixture;

[0026] S3: Place the mixture at 25-30 °C for swelling and induce gel formation to form a composite hydrogel.

[0027] Further, in S1, the concentration of the BSP-182 solution is 0.5%, w / v; the concentration of the HA solution is 1%, w / v.

[0028] Further, in S2, the volume ratio of the BSP-182 solution to the HA solution is (3 - 7):(3 - 7).

[0029] Further, in S2, the volume ratio of the BSP-182 solution to the HA solution is 7:3.

[0030] Beneficial effects:

[0031] (1) In the present invention, a new molecular weight neutral polysaccharide (BSP-182) was extracted and purified from the tubers of Bletilla striata by DEAE-52 cellulose and a fully automatic gel purification system (BRT-GS); and the structure of BSP-182 was characterized. HPLC and monosaccharide analysis showed that the molecular weight of BSP-182 was 182.10 kDa, mainly composed of mannose and glucose with a molar ratio of 7.8:2.2. FTIR, methylation analysis and nuclear magnetic resonance spectra showed that BSP-182 mainly contained β-1,4-Glc and β-1,4-Man.

[0032] (2) The present invention studied the synergistic gelation effect of Bletilla striata polysaccharide (BSP-182) and hyaluronic acid (HA). BSP-182 and HA formed a gel through hydrogen bonds at 25°C. Compared with the pure BSP-182 solution, the synergistic gelation effect with HA could improve the rheological properties of BSP-182. When the temperature was equal to 40°C, the viscosity and thermoreversibility of the hydrogel were the best, and the viscoelasticity of the gel was the best when HA:BSP-182 = 7:3.

[0033] (3) The present invention provides feasible guidance for the development and application of BSP-182 foods. The HA / BSP-182 hydrogel can be used as a promising excipient in the food industry. Description of the drawings

[0034] Figure 1 It is the ion chromatogram of BSP-182 and the 16-sugar mixed standard, the determination diagram of the average molar mass of BSP-182, and the qualitative analysis diagram of the polysaccharide functional groups of BSP-182 in Example 1 of the present invention;

[0035] Figure 2 It is the 1D and 2D NMR spectra of BSP-182 in Example 1 of the present invention;

[0036] Figure 3 It is for the Figure 2 characteristic correlation map of the signals in the region between 58 - 65 ppm shown in B in Example 1 of the present invention established by DEPT spectral analysis of the signals in this region;

[0037] Figure 4 It is the molecular structure diagram of BSP-182 in Example 1 of the present invention;

[0038] Figure 5 It is the characterization diagram of the morphology and microstructure of BSP-182 by scanning electron microscope in Example 1 of the present invention;

[0039] Figure 6 It is the rheological characteristic diagram of the BSP-182 / HA mixture in Example 1 of the present invention;

[0040] Figure 7 It is to determine the linear viscoelastic region of the hybrid HA-BSP-182 system by strain sweep test in Example 2 of the present invention;

[0041] Figure 8 It is the scanning electron microscope images of the HA / BSP-182 mixture, HA and BSP-182 in Example 2 of the present invention;

[0042] Figure 9 It is the FTIR spectrum of the HA / BSP-182 mixture in Example 2 of the present invention. Detailed implementation manners

[0043] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the described embodiments.

[0044] Example 1: Extraction and purification of a novel Bletilla striata polysaccharide

[0045] 1. Material acquisition: Dry Bletilla striata tubers were collected from Bozhou City, Anhui Province, China. DEAE-52 cellulose was purchased from Solarbio Biochemical Technology Co., Ltd. (Beijing, China). Hyaluronic acid HA (purity >= 93%, molecular weight 200 - 400 kDa) was purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). All other chemical reagents and solvents were of analytical grade.

[0046] 2. Extraction and purification of BSP:

[0047] 1) The dry Bletilla striata tubers were crushed and passed through a 10-mesh (pore size 2 mm) sieve to obtain Bletilla striata powder.

[0048] 2) The Bletilla striata powder (200 g) was extracted with deionized water at a ratio of 1 g:10 mL at 90 °C for 2 - 3 times. The extract was concentrated (using a rotary evaporator until the water evaporated completely), and then four times the volume of absolute ethanol was added to precipitate at 4 °C. After standing for 12 h, the precipitate was collected by centrifugation (5000 r, 10 min).

[0049] 3) The precipitate was redissolved in deionized water and deproteinized by the Sevage method. The resulting material after deproteinization was dialyzed in a dialysis bag (1 kDa) at 4 °C for 72 hours; the non-dialyzable solution was collected and lyophilized to obtain crude polysaccharide.

[0050] 4) Finally, purification was carried out using DEAE-52 cellulose and an automatic gel purification system (BRT-GS) to obtain Bletilla striata polysaccharide with a relatively high purity, named BSP-182.

[0051] 3. Determination methods for the structural characterization of BSP-182:

[0052] 1) Monosaccharide composition

[0053] BSP-182 (5 mg) was placed in an ampoule containing 2 mL of trifluoroacetic acid (TFA, 3 M) and hydrolyzed at 120 °C for 3 h. The solution after TFA hydrolysis was aspirated and transferred to a test tube, then dried under nitrogen. 5 mL of water was added, vortexed and mixed evenly. 50 μL was aspirated and added to 950 μL of deionized water, centrifuged at 12000 rpm for 5 min, and the supernatant was taken. Finally, an ion chromatograph (ICS5000, USA), Dionex Carbopac TM PA20 chromatographic column (3 * 150 mm) and an electrochemical detector were used for analysis (Ye et al., 2021).

[0054] 2) Molecular weight determination

[0055] The sample (BSP-182 prepared in this example) and the standard product (polysaccharide with a known molecular weight) were accurately weighed. The sample was prepared into a 5 mg / mL solution, centrifuged at 12000 rpm for 10 min, the supernatant was filtered through a 0.22 μm microporous filter membrane, the sample was transferred to a 1.8 mL injection vial, and then a high performance liquid chromatograph (LC-10A, Shimadzu) was used to characterize the sample.

[0056] 3) Fourier transform infrared spectroscopy

[0057] 2 mg of BSP-182 and 200 mg of potassium bromide were mixed and pressed into tablets. The blank control was made by pressing potassium bromide powder into tablets. They were respectively placed in a Fourier transform infrared spectrometer FT-IR650 for scanning and recording.

[0058] 4) Methylation analysis and nuclear magnetic resonance

[0059] BSP-182 (3 mg) was dissolved in 0.9 mL of DMSO, and then 6 mg of sodium hydroxide was added and used with CH 3I was methylated. The methylated polysaccharide was taken, 2 mL of trifluoroacetic acid (TFA) was added, and hydrolysis was carried out for 90 min. It was evaporated to dryness with a rotary evaporator. It was reduced with 5 mg of sodium borohydride for 8 hours, and then 1 mL of acetic anhydride was added for acetylation reaction at 100 °C for 1 h (Peng et al., 2024). Finally, the acetylated product was dissolved in CH 2 Cl 2 and the acetylated product sample was determined by an Agilent GCMS 6890-5973 gas chromatography-mass spectrometry instrument;

[0060] BSP-182 (50 mg) was dissolved in D 2 O, and then freeze-dried. The 1D (1H and 13C NMR) and 2D NMR (correlation spectroscopy, COSY; total correlation spectroscopy, TOCSY; nuclear Overhauser effect spectroscopy, NOESY; heteronuclear single quantum coherence spectroscopy, HSQC; heteronuclear multiple bond spectroscopy, HMBC) of BSP were obtained at 25 °C using an NMR spectrometer (Brucker, Germany), and the relevant data were recorded.

[0061] 5) Scanning electron microscopy

[0062] The dried BSP-182 (5 mg) was adhered to a conductive carbon film containing a double-sided adhesive, placed in the sample chamber of an ion sputtering instrument, and sputtered with gold for about 40 s, and observed with a super-high-resolution scanning electron microscope (Nova NanoSEM 450) (FEI Company, USA).

[0063] 4. Structural characterization results of BSP-182:

[0064] 1) Monosaccharide composition and molecular weight determination

[0065] BSP was extracted from the tubers of Bletilla striata, and the yield was 20.3%. Figure 1 A and B in it depict the ion chromatograms of BSP-182 and the mixed standard 16 sugars. The main monosaccharides in BSP-182 are glucose (16.309 min) and mannose (19.425 min), and the molar ratio is 2.2:7.8 ( Figure 1 A in it). The ratio of mannose to glucose in BSP-182 varies due to different extraction and purification methods, thus affecting its physicochemical properties. BSP-182 showed a single, symmetric, and narrow peak on HPLC, with a retention time of 26.127 minutes, confirming its homogeneity ( Figure 1 C in it). The weight-average molecular weight (Mw) and number-average molar mass (Mn) of BSP-182 were measured to be 182.10 kDa and 169.94 kDa respectively ( Figure 1in C). These findings indicate that glucose and mannose are the dominant monosaccharides in BSP-182, consistent with previous studies that characterized BSP as a glucomannan polysaccharide.

[0066] 2) FTIR analysis

[0067] FTIR spectroscopy is helpful for the qualitative analysis of polysaccharide functional groups. The characteristic FTIR spectrum of BSP-182, as shown in Figure 1 D in -1 , has absorption bands at 3600 - 3200 cm -1 which are stretching vibration absorption peaks of -OH, and the absorption peaks in this region are characteristic peaks of sugars. Specifically as follows: 3375 cm -1 is the stretching vibration absorption peak of O-H, a characteristic peak of sugars. There is an absorption peak at 2887 cm -1 which may be attributed to C-H stretching vibration. There is an absorption peak at 1730 cm -1 which may be attributed to C=O stretching vibration. There is an absorption peak at 1637 cm -1 which may be attributed to N-H bending vibration. There is an absorption peak at 1377 cm -1 which may be attributed to C=O symmetric stretching vibration. There are absorption peaks at 1246 cm -1 and 1026 cm -1 which may be attributed to O-H bending vibration. There is an absorption peak at 809 cm

[0068] 3) Methylation analysis

[0069] Methylation analysis of BSP-182 revealed the main composition of mannose (Man) and glucose (Glc), with a ratio of approximately 8:2 (Table-1). This composition is consistent with the observed monosaccharide distribution. Notably, mannose as the terminal sugar accounts for 8.7% of the total, indicating a branching degree of approximately 1 / 11. This suggests a relatively high degree of branching in the polysaccharide. Primarily, the glycosidic bond is 1,4-linked, indicating that the backbone of the polysaccharide is mainly composed of 1,4-linked Man and Glc. Branching within the structure occurs at the 2,3,6 positions of Man residues and the 3,6 positions of Glc residues.

[0070] Table-1 Methylation analysis of BSP

[0071]

[0072] 4) Nuclear magnetic resonance analysis

[0073] The 1D and 2D NMR spectra of BSP-182 are as shown in Figure 2 .

[0074] In 1 the anomalous region (4.3 - 5.5 ppm) of the \(^1H\) NMR spectrum, multiple sets of isomeric hydrogen signals can be observed ( Figure 2 in A). The key peaks at 5.43, 5.33, 5.11, 5.03, 4.87, and 4.45 ppm indicate the presence of various sugar residues in the polysaccharide, mainly in the β configuration. The 1 interval of 3.0 - 4.2 ppm in the \(^1H\) NMR spectrum usually shows proton signals from non - isomeric hydrogens outside the residual sugar ring, exhibiting a diverse and complex pattern consistent with the general characteristics of the polysaccharide hydrogen spectrum.

[0075] In the \(^{13}C\) NMR spectrum ( Figure 2 in B), no significant signal corresponding to the carbonyl carbon of uronic acid was detected in the range of 170 - 210 ppm, preliminarily confirming the neutrality of BSP - 182. In the anomalous region (90 - 110 ppm) of the carbon spectrum, two main anomalous carbon signals were observed at 102.50 and 100.13 ppm. The positions of these peaks indicate that most of the residual sugars in BSP - 182 mainly adopt the β - configuration; multiple typical residual sugar carbon peaks were detected in the range of 55 - 85 ppm. The signal between 58 - 65 ppm corresponds to the outer - ring C6 methylene - \(CH_2 -\) carbon of pyranose - type residual sugars, confirming their presence in the monosaccharide composition. DEPT spectral analysis further supports this confirmation, which established the characteristic correlations of the signals in this region ( Figure 3 ). In addition, outside the anomalous carbons of the residual sugars within the sugar ring, signals of C2 - C5 carbons were observed in the range of 65 - 85 ppm, overlapping with the typical signals of the polysaccharide carbon spectrum.

[0076] The structure of BSP - 182 was analyzed using 2D nuclear magnetic resonance spectra, especially the HSQC spectrum ( Figure 2 in C) that reflects hydrogen - carbon correlations. In the anomalous region, multiple sets of H / C correlation signals were identified, mainly six sets at 5.44 / 71.42, 5.12 / 93.81, 4.91 / 97.72, 4.87 / 99.05, 4.69 / 100.11, 4.46 / 102.50 ppm. The signals at other positions showed lower responses, indicating lower abundances of the corresponding residual sugars. By combining the methylation composition ratios with the relative intensities of the anomalous hydrogen signals in the \(^1H\) spectrum and HSQC data, the preliminary assignments include: 4.69 / 100.11 ppm corresponding to H1 / C1 of β - 1,4 - Man residues, 4.46 / 102.50 ppm corresponding to H1 / C1 of β - 1,4 - Glc residues, and 5.12 / 93.81 ppm corresponding to H1 / C1 of α - t - Man residues.

[0077] We continued by analyzing multiple 2D NMR spectra (Figure 2 The hydrocarbon signal positions of the three main residues in the polysaccharide structure were mapped by D) in []. For example, the abnormal hydrogen at 4.69 ppm was identified as H1 of the β-1,4-Man residue. The adjacent proton correlations in the COSY and NOESY spectra revealed a correlated signal with H1 at 4.06 ppm, thus establishing the position of H2 at 4.06 ppm. Further analysis using the HSQC spectrum confirmed the H2 / C2 signal at 4.06 / 69.90 ppm, precisely locating the carbon signal at position 2 of this residue at 69.90 ppm. Using the COSY and NOESY data, the correlated signal at 4.06 / 3.74 ppm was located, verifying the position of H3 at 3.74 ppm. Subsequent integration with the HSQC spectrum led to the identification of the 13C spectral position of C3 at 3.75 / 71.42 ppm. Applying this methodology, the positions of H4 / C4, H5 / C5, and H6 / C6 were determined to be 3.75 / 76.42 ppm, 3.50 / 74.90 ppm, and 3.69 / 60.33 ppm, respectively. Using the same method, we further determined the potential assignments of the positions of the three main residues (Table 2).

[0078] Table 2. Main residue sugar data of BSP-182

[0079]

[0080] The HMBC spectrum is particularly valuable for identifying hydrogen-carbon correlations separated by 2-3 chemical bonds, which is crucial for analyzing BSP-182 to determine glycosidic linkages ([]) Figure 2 in E). In samples with a high content of β-1,4-Man residues, the HMBC spectrum revealed unique correlations for the abnormally positioned hydrogens: 4.69 / 70.02, 4.69 / 76.42. These correlations confirmed its association with C2 in the same residue and C4, respectively, thus establishing the glycosidic linkage as a 1→4 bond between β-1,4-Man residues.

[0081] The NOESY spectrum helps to identify proton-correlated signals of spatially proximal polysaccharide residues and also significantly affects the analysis of glycosidic bond linkages between sugar residues ([]) Figure 2In F). In the β-1,4-Man residues with a high compositional percentage, the NOESY spectrum revealed abnormal hydrogen correlation signals at 4.69 / 3.49, 4.69 / 3.74, and 4.69 / 4.06. These signals corresponded to the correlations between abnormal H1 and H5, H3, H2 within the same ring, and H4 of the same residue, respectively, confirming the glycosidic bond of the 1→4 bond in β-1,4-Man. Similarly, in the β-1,4-Glc residues, the correlations at 4.46 / 3.61 and 4.46 / 3.73 were identified, indicating the residue itself and the 1→4 glycosidic bond between β-1,4-Glc and β-1,4-Man, respectively. In addition, the signal at 5.12 / 3.75 in the α-t-Man residue indicated a 1→4 glycosidic bond with β-1,4-Man.

[0082] The main structure of BSP-182 can be depicted based on the monosaccharide composition ratio and the results of methylation analysis, using the structures of three primary sugar residues and considering the types of other remaining residues ( Figure 4 ). ("X" represents a set of sugar residues present in a smaller amount: →3,4)-Glcp-(1→, →3,4)-Manp-(1→, →2,4)-Manp-(1→, →4,6)-Manp-(1→, →4,6)-Glcp-(1→).

[0083] 5) Scanning electron microscopy analysis

[0084] Scanning electron microscopy characterized the morphology and microstructure of BSP, and the results are as Figure 5 shown.

[0085] The morphology and microstructure of BSP-182 were characterized using a scanning electron microscope (SEM). At a magnification of 200X, the sample presented as a thin layer, showing a thin, incomplete, irregularly shaped structure with irregular stacking and obvious gaps ( Figure 5 in A). These morphological characteristics of BSP-182 are common and may be due to the strong attraction between functional groups on the polysaccharide surface, resulting in the aggregation of polysaccharide chains. When magnified to 500X, the edges of the thin layer were clear and the fracture surface was obvious, characterized by a smooth surface. At higher magnifications of 1000X and 2000X, filamentous branches were observed at the layer edges, showing an obvious branched structure with neat and smooth edges ( Figure 5 in B).

[0086] Example 2: Co-gelation of BSP and HA

[0087] 1. Preparation of the colloid

[0088] 1) Dissolve BSP and HA powders separately in deionized water and stir magnetically at 25 °C for 10 minutes to prepare a completely dissolved BSP solution (0.5%, w / v) and HA solution (1%, w / v).

[0089] 2) Then, magnetically stir the BSP solution and HA solution evenly and mix them for 10 minutes at 25 °C to prepare different volume mixing ratios (HA:BSP, 7:3, 5:5, and 3:7).

[0090] 3) Then place the mixture at room temperature (25 °C) to fully swell to induce gel formation. The prepared hydrogels are equilibrated at room temperature for 12 hours before testing. Equilibrating the prepared hydrogels at room temperature for 10 - 12 hours before testing can help eliminate the surface moisture that may be introduced during the preparation process and make the moisture distribution inside and outside the hydrogels reach stability. This can reduce the size and mass fluctuations caused by moisture changes and ensure the reliability and consistency during testing.

[0091] 2. Testing methods

[0092] 1) Rheological detection

[0093] Apply a rheometer Thermo Fisher HAAKE MARS 60 (Karlsruhe, Germany) equipped with a cone plate (60 mm, diameter 1°) to study the rheological properties of the HA / BSP mixture, including apparent viscosity, viscoelasticity, and temperature-varying properties. The apparent viscosity is measured as a function of the shear rate (within 0.01 - 1000 s -1 at 25 °C). To obtain sol characterization, initially perform a strain sweep procedure in the strain range of 0.1–1000% at 25.0 °C to define the linear viscoelastic region (LVR) of all mixed gels. Perform a dynamic oscillatory frequency sweep test (0.1–100 rad / s) at 500% strain (within the LVR) to study the mechanical response of the sol.

[0094] First, scan the sol with a temperature sweep procedure from 80 °C to 5.0 °C at a cooling rate of 1 °C / min with a strain force of 100.0% and a frequency of 6.28 rad / s, and then heat and scan the sol from 5 °C to 80 °C at the same heating rate.

[0095] 2) Scanning electron microscopy

[0096] The gels with different BSP and HA ratios prepared above were equilibrated at room temperature for 12 hours. All the gels were quickly frozen by immersion in liquid nitrogen, and then the frozen gels were placed in a vacuum freeze dryer for freeze-drying. The freeze-dried gels were manually crushed, a small amount of the sample was directly stuck onto the conductive adhesive, and gold was sputtered onto the sample for 45 s using a Quorum SC7620 sputter coater at a current of 10 mA; subsequently, the morphology of the sample was photographed using a scanning electron microscope (SEM, TESCAN MIRA LMS), and the acceleration voltage was 3 kV during the morphology photography.

[0097] 3) Fourier transform infrared spectroscopy

[0098] The gels with different BSP and HA ratios prepared above were freeze-dried, and the FTIR spectra of the freeze-dried samples were obtained using a Fourier transform infrared spectrometer (Thermo Scientific Nicolet iS20, USA). In a dry environment, about 10 mg of the sample and an appropriate amount of dry potassium bromide powder were added to a mortar and ground thoroughly several times, and then pressed into a tablet (a transparent thin film) on a tablet press. The test wavenumber range was 400 - 4000 cm -1 , with a resolution of 4 cm -1 , and the infrared spectra of the samples were collected.

[0099] 3. Test results

[0100] 1) Rheology of the BSP / HA mixture

[0101] The rheological properties of the HA / BSP-182 mixture indicated that the apparent viscosity of all HA / BSP-182 mixtures varied with the shear rate ( Figure 6 A in it). The BSP-182 solution exhibited Newtonian behavior at high shear rates, while mixing with HA increased the viscosity of the solution, showing significant shear thinning behavior. These results were highly consistent with the rheological properties of other composite gels of BSP. Shear thinning is a typical behavior of polysaccharide hydrogels with high molecular weight and polymer structure, which is helpful for material preparation, shaping, and structure improvement, especially in cosmetic applications, oral liquids, and food additives, where finer textures are required. The shear thinning behavior may be related to several factors, including polymer structure, additives, external conditions, and polymer concentration. In addition, when the HA / BSP-182 ratio was 7:3, the mixture solution exhibited the highest viscosity.

[0102] As Figure 6As shown in B of [reference], the storage modulus (G') and loss modulus (G") increase as the frequency rises from 1 rad / s to 100 rad / s. During the entire frequency sweep test, the material behaves like a viscoelastic liquid below the crossover frequency (G">G'), and the crossover occurs at a very high frequency. This behavior may be due to the increasing tendency of entangled polymers to collide at higher frequencies, leading to a faster increase in the storage modulus. Additionally, the modulus values are highest when the HA / BSP-182 ratio is 7:3, indicating better viscoelastic properties of the hydrogel at this ratio.

[0103] The strain sweep test determines the linear viscoelastic region (LVR) of the hybrid HA-BSP-182 system ( Figure 7 ). The results show that the samples exhibit a wide LVR over all strain ranges. Moreover, all ratios of HA / BSP-182 show no yield point and flow without additional stress, facilitating easy liquid flow. When the strain amplitude is less than 10%, both G' and G" of the mixtures of HA and BSP-182 with different mass ratios are in a plateau state. However, when the strain exceeds 10%, molecular entanglements may be disrupted. For the ratio of HA to BSP-182 of 7:3, the hydrogel exhibits the highest G" and G' values, indicating the strongest resistance to mechanical deformation.

[0104] During the cooling process from 80 °C to 5 °C, both G" and G' increase due to the decrease in molecular mobility, where G" remains higher than G' ( Figure 6 in C of [reference]). The HA / BSP-182 mixture always remains in a colloidal state. When the temperature drops below 40 °C, the rapid increase in the viscous modulus indicates a high viscosity at this time. When heated, the viscous modulus of the hydrogel decreases with increasing temperature and levels off at 40 °C ( Figure 6 in D of [reference]). This demonstrates the good thermoreversibility of G' and G" in the hydrogel. Throughout the heating and cooling processes, the modulus values of the HA / BSP-182 = 7:3 mixture always remain the highest.

[0105] These results indicate that compared with pure BSP solution, the HA / BSP mixed hydrocolloid forms a stronger sol through synergistic action, suggesting that HA may be an effective coagulant for BSP.

[0106] 2) Scanning electron microscopy

[0107] SEM is used to visualize the hydrogel network structure. BSP-182 exhibits a flaky and smooth structure, while HA exhibits a fibrous structure with entangled molecular chains ( Figure 8 ).

[0108] In contrast, the mixture of HA and BSP-182 exhibited a more distinct honeycomb structure. This may be attributed to the strong synergistic effect between HA and BSP-182 in the co-hydrocolloid, which led to a decrease in the pore network size in the mixed colloid. The outer surfaces of all samples appeared rough, with distinct grooves and pores, which helped to maintain the spatial structure and made the penetration and retention of moisture possible. Notably, a more regular and denser network was observed at the ratio of HA:BSP-182 = 7:3, which was characterized by more and smaller pores. This ratio corresponded to the maximum value of hydrogen bond connection.

[0109] 3)FTIR

[0110] The interaction between HA and BSP-182 was confirmed by FTIR, as Figure 9 shown. The infrared characteristics of the BSP-182 sample were similar to those of HA, such as the absorption peaks at 3389 cm -1 , 2892 cm -1 , 1643 cm -1 and 1033 cm -1 originated from the O-H, C-H, C=O and C-O structures respectively, or because both belonged to the polysaccharide structure. Especially at 1732 cm -1 , its unique ester group C=O stretching vibration absorption peak appeared, and the absorption peak at 895 cm -1 was related to the β-glycosidic bond, indicating that the glycosidic bond in this component was mainly in the β-configuration.

[0111] When the infrared of the sample after crosslinking BSP-182 with HA showed the spectral characteristics of HA, the mixed hydrocolloid HA:BSP = 7:3 showed the C=O stretching vibration absorption peak of BSP at 1726 cm -1 and 1412 cm -1 . The C=O peak of the other two ratios of mixed hydrocolloids gradually weakened with the decrease of the BSP ratio. In addition, the peak shape of the O-H stretching vibration absorption peak also changed with the decrease of the BSP ratio. The intensity of the hydroxyl peak of the mixed hydrocolloid was higher than that of the pure BSP solution, indicating that BSP had been coated by HA and formed complex hydrogen bond interactions through structures such as hydroxyl groups.

[0112] In summary, HPLC and monosaccharide analysis showed that the molecular weight of BSP-182 was 182.10 kDa, which was a novel neutral polysaccharide with a molecular weight, mainly composed of mannose and glucose with a molar ratio of 8:2. FTIR, methylation analysis and nuclear magnetic resonance spectra showed that BSP mainly contained β-1,4-Glc and β-1,4-Man.

[0113] BSP-182 and HA form a gel through hydrogen bonding at 25 °C. Compared with the pure BSP-182 solution, the synergistic gelation with HA can improve the rheological properties of BSP-182. When the temperature is equal to 40 °C, the viscosity and thermoreversibility of the hydrogel are the best. When HA:BSP-182 = 7:3, the viscoelasticity of the gel is the best. This study provides feasible guidance for the development and application of BSP-182 foods. The HA / BSP-182 hydrogel can be used as a promising excipient in the food industry.

[0114] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing a composite hydrogel based on Bletilla striata polysaccharide, characterized in that: The steps include: S1. Dissolve BSP-182 and hyaluronic acid powder in deionized water respectively, and stir magnetically at 25-30° C. for 10-15 minutes to prepare a completely dissolved BSP-182 solution and a hyaluronic acid solution; S2, mixing the BSP-182 solution and the hyaluronic acid solution, and uniformly mixing them under magnetic stirring at 25-30° C. for 10-15 minutes to obtain a mixture; S3, placing the mixture at 25-30°C to swell and induce gelation to form a composite hydrogel; The preparation method of the Bletilla striata polysaccharide BSP-182 comprises the following steps: Step 1: crush the dried tubers of Bletilla striata, sieve through a mesh, and obtain Bletilla striata powder; Step 2: Dissolve the Bletilla striata powder in deionized water and extract at 90°C for 2-3 times; concentrate the extract, then add anhydrous ethanol to precipitate at 2-4°C, let stand for at least 12 hours, and then collect the precipitate by centrifugation; Step 3: Re-dissolve the precipitate in deionized water and deproteinize it by Sevage method; dialyze the deproteinized substance at 2-4°C for 72 hours using a dialysis bag; The non-dialyzable solution was collected and freeze-dried to obtain crude polysaccharide; Step 4: Purify with DEAE-52 cellulose and fully automatic gel purification system to obtain Bletilla striata polysaccharide, named BSP-182; The molecular weight of the BSP-182 is 182.10 kDa; the BSP-182 includes mannose and glucose, and the molar ratio of mannose to glucose is 8:2; the molecular weight of the hyaluronic acid is 200-400 kDa; the concentration of the BSP-182 solution is 0.5%, w / v; the concentration of the hyaluronic acid solution is 1%, w / v; in S2, the volume ratio of the hyaluronic acid solution to the BSP-182 solution is 7:

3.

2. The method for preparing a composite hydrogel based on Bletilla striata polysaccharide according to claim 1, characterized in that: In the step 1, the mesh size of the sieve is 10-12 meshes.

3. The method for preparing a composite hydrogel based on Bletilla striata polysaccharide according to claim 1, characterized in that: In the step 2: 1) Dissolve Bletilla striata powder in deionized water, the mass volume ratio of Bletilla striata powder to deionized water is (1-1.5) g: (10-12) mL; 2) The extract concentration method is: evaporating until there is no water in the rotary evaporator; 3) The volume ratio of the concentrated extract to the added anhydrous ethanol is (1-1.5): (4-5).

4. The method for preparing a composite hydrogel based on Bletilla striata polysaccharide according to claim 1, characterized in that: In the step three: 1) The precipitate is redissolved in deionized water, and the mass volume ratio of the precipitate to deionized water is (1-1.5) g: (10-12) mL; 2) The pore size of the dialysis bag is (1-1.2) kDa.

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