CN105169491A - Method for preparing fungus hyperbranched polysaccharide-xanthan gum hydrogel bracket - Google Patents

Abstract

The invention discloses a method for preparing a fungus hyperbranched polysaccharide-xanthan gum hydrogel bracket, and belongs to the technical field of natural macromolecular materials. The preparation method comprises the following steps: dissolving pleurotus tuber-regium hyperbranched polysaccharide and xanthan gum into a NaOH aqueous solution according to different mass ratios, generating crosslinking reaction by an obtained pleurotus tuber-regium hyperbranched polysaccharide and xanthan gum solution and a sodium trimetaphosphate aqueous solution under the temperature of 37 DEG C for 10 minutes to 48 hours, and obtaining the fungus hyperbranched polysaccharide-xanthan gum hydrogel bracket. The method disclosed by the invention is easy and convenient to operate; the adopted raw materials are abundant in source; an adopted crosslinking agent is water-soluble and nontoxic; furthermore, the prepared bracket material has medicament controlled release performance, and is high in mechanical property and biocompatibility. The fungus hyperbranched polysaccharide-xanthan gum hydrogel bracket can be used for preparing an artificial tissue bracket and a food or nutritional substance and medicament controlled release carrier.

Description

A method for preparing fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold

technical field

The invention relates to a method for preparing fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold. Belonging to the technical field of natural polymer materials, the fungal highly branched polysaccharide-xanthan gum hydrogel three-dimensional penetrating porous scaffold can be widely used in industries such as tissue engineering materials, biomedical materials, and drug controllable release.

Background technique

Polymer hydrogel has a certain degree of elasticity and a three-dimensional through-porous structure, which is suitable for simulating the extracellular matrix, providing a three-dimensional microenvironment suitable for cell growth and biophysical signals between cells and the extracellular matrix, and maintaining the normal phenotype and physiology of cells. Function. Polysaccharides are biological macromolecules that widely exist in nature. They participate in various life activities of cells and produce a variety of biological functions. They are closely related to various physiological functions that sustain life. In recent years, fungal polysaccharides have attracted increasing attention as an important source of bioactive polysaccharides. A large number of studies have shown that fungal polysaccharides have rich biological activities such as anti-cancer, liver protection, immunity, anti-coagulation, hypoglycemia, anti-virus and anti-oxidation, and have been widely used in the field of health food and new drug resources, thus becoming a very active research field.

Fungal polysaccharides are rich in sources, and the depth and breadth of its research and development need to be further strengthened. Tiger Milk Mushroom is an edible fungus that grows in tropical and subtropical regions. It has a delicious taste and rich nutritional value; it has certain medicinal effects on some diseases, such as asthma, smallpox and high blood pressure; it can promote fetal development and increase the survival rate. In recent years, it has been found that the polysaccharides and their derivatives extracted from tiger milk mushroom have anti-tumor activity and play an immunoregulatory role on the body. The polysaccharide extracted from tiger milk mushroom has a highly branched structure. The highly branched polysaccharide is in a spherical chain conformation, and there are a large number of functionalized hydroxyl groups on its periphery. They can undergo functional reactions or interact with surrounding substances. From the material point of view, these spherical polysaccharide molecules with a large number of sugar residues are conducive to the multi-bonding and clustering effects with other polysaccharides or proteins. However, the aqueous solution of highly branched polysaccharides from tiger milk mushroom has low viscosity and is not easy to form hydrogel. Polysaccharides can be physically cross-linked with other polysaccharides or other biomacromolecules through hydrogen bonds or electrostatic attraction between ions, or through direct chemical cross-linking, and cross-linking after chemical modification to form hydrogels.

Xanthan gum is a linear water-soluble natural polysaccharide composed of D-glucan, D-mannose, D-glucuronic acid, acetic acid and pyruvic acid linked by "pentasaccharide repeating units". Hydrogels prepared from xanthan gum are highly hydrophilic, non-toxic, degradable, and biocompatible. They are often used as superabsorbent resins, drug carriers, and microcapsules. They have a wide range of applications in the biomedical field. Application prospects. Xanthan gum biomacromolecules tend to form double helix structures through hydrogen bonds, and these double helix structures further form network-like physically cross-linked hydrogels through intermolecular forces, such as electrostatic forces, hydrogen bonds, and interchain entanglements. However, the xanthan gum physical hydrogel has the following problems: it is easily soluble in water, has poor water resistance and mechanical properties, and is easily damaged. The glucuronic acid and pyruvic acid groups on the side chain of xanthan gum and a large number of hydroxyl groups in the main molecular chain structure are beneficial to the chemical modification and modification of xanthan gum. Sodium trimetaphosphate is a food additive, which is water-soluble and non-toxic. Under physiological temperature (37°C) and weak alkaline conditions, it can be used as an esterification reagent to undergo a chemical cross-linking reaction with hydroxyl groups on polysaccharides to form insoluble Hydrogels with water and greatly improved mechanical properties. Xanthan gum hydrogel is rigid, brittle and brittle. By introducing another polymer into the xanthan gum molecular network to form an interpenetrating network, the mechanical properties of xanthan gum hydrogel can be further improved by means of forced mutual solubility and synergistic effect. . Alireza Shalviri et al. (Carbohydrate Polymers, 2010, 79:898-907) used sodium trimetaphosphate as a cross-linking agent to prepare starch/xanthan gum interpenetrating network hydrogel, which has good film-forming properties and can be used according to the charge on the drug. The gel membrane is selectively permeable and can be used as a variety of drug carriers. Based on the above analysis, combined with the advantages of highly branched polysaccharides of tiger milk mushroom and xanthan gum, there are great advantages in the preparation of composite polysaccharide biomedical hydrogel scaffold materials cross-linked by sodium trimetaphosphate. The hydrogel can not only provide a microenvironment for maintaining cell growth, but also has specific biological activities, so it is expected to be applied in the fields of biomedicine and tissue engineering. Fungal hyperbranched polysaccharide-xanthan gum hydrogel, as a good scaffold material, not only depends on its biocompatibility and biodegradability, but more importantly, its unique chemical structure and biological activity. Therefore, it is superior to synthetic polymer hydrogels or other polysaccharide hydrogels. Fungal hyperbranched polysaccharide-xanthan gum hydrogel has broad application prospects in the fields of tissue engineering, drug controlled release, food and nutrient carriers, etc.

Because of the great application value of natural polysaccharide hydrogel, its preparation, application and development have become one of the research hotspots at home and abroad. It is one of the important topics in the fields of biomaterial science and medicine to develop ideal artificial scaffold materials to replace organ transplantation to repair tissue defects or lesions. At present, sodium alginate and chitosan are mainly used as raw materials for the preparation of polysaccharide hydrogels. For example: the Chinese patent publication number is CN103087334A, the publication date is May 8, 2013, and the application name of the invention is "Preparation method of sodium alginate-artemisia gum composite hydrogel". This application discloses that the alginic acid-artemisia gum composite hydrogel is formed in situ by introducing microporous calcium carbonate into the ^{gluconolactone} solution by introducing Artemisia gum into the sodium alginate solution system. The properties of sodium alginate and artemisia gum are complementary, thereby enhancing the water absorption and mechanical properties of sodium alginate-based hydrogel. The disadvantage of this method is that the cross-linking agent used is Ca ²⁺ , which relies on electrostatic force to form a cross-linked network, and the obtained physical hydrogel is obtained. Therefore, in the process of controlled drug release, the cross-linking agent Ca ²⁺ in the hydrogel is easily replaced by other ions in the diffusion system, making the hydrogel soluble in water and other problems.

Contents of the invention

In view of the deficiencies in the above-mentioned technologies, the purpose of the present invention is to provide a simple and convenient process, less pollution, and the obtained product has good mechanical properties, controlled drug release, good biocompatibility, biodegradability and biological activity. Preparation method of hydrogel scaffold.

To achieve the above object, the technical solution provided by the invention is:

A method for preparing fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold, said preparation method comprising the following steps:

a The dried tiger milk mushroom sclerotia is crushed, followed by Soxhlet extraction with ethyl acetate and acetone to remove fat, and then the fat-removed tiger milk mushroom sclerotia is soaked in normal saline, wherein every 100g of tiger milk mushroom sclerotia Soak in 1L of normal saline, extract under high pressure at 120°C, centrifuge to obtain the extract, cool the extract and then centrifuge to collect the residue; the residue is centrifugally washed with deionized water and freeze-dried to obtain the highly branched polysaccharide of tiger milk mushroom.

b Dissolve the hyperbranched polysaccharide of tiger milk mushroom in NaOH aqueous solution with a pH value of 12-14, stir for 2 hours to prepare a solution of hyperbranched polysaccharide of tiger milk mushroom with a concentration of 2% w/v.

c. Add xanthan gum to the hyperbranched polysaccharide solution of tiger milk mushroom obtained in step b, and stir for 12 hours to obtain a uniformly mixed hyperbranched polysaccharide-xanthan gum solution. The concentration of xanthan gum is 0.5% to 5%. w/v, the mass ratio of hyperbranched polysaccharide to xanthan gum is 1:0.25～1:2.5.

d Dissolve sodium trimetaphosphate in deionized water and stir until completely dissolved to obtain an aqueous solution of sodium trimetaphosphate with a concentration of 75-262.5 mg/mL.

e is that the concentration of sodium trimetaphosphate aqueous solution of 75～262.5mg/mL obtained through step d is added in the hyperbranched polysaccharide-xanthan gum solution of tiger milk mushroom obtained through step c, wherein the hyperbranched polysaccharide of tiger milk mushroom - The volume ratio of the xanthan gum solution to the sodium trimetaphosphate aqueous solution is 25:8, stirring rapidly for 5 minutes, and cross-linking at 37° C. for 10 minutes to 48 hours to obtain the fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold.

Due to the adoption of the above technical scheme, the technical scheme of the present invention aims at the structural characteristics of the highly branched polysaccharides of tiger milk mushroom and xanthan gum, and adopts water-soluble, non-toxic sodium trimetaphosphate esterification and cross-linking to prepare drugs with controlled release and mechanical properties. Highly branched polysaccharide-xanthan gum hydrogel scaffold with good performance, drugs or cell growth factors are embedded in the hydrogel scaffold, and the mechanical properties of the hydrogel are adjusted by using the content ratio of highly branched polysaccharide and xanthan gum performance and pore size of the porous structure. The bioactivity of fungal hyperbranched polysaccharides facilitates cell adhesion and growth. The hydroxyl groups on the periphery of the hyperbranched polysaccharide in the hydrogel, the hydroxyl and carboxyl groups on the xanthan gum sugar ring, and the sodium metaphosphate group introduced by the cross-linking agent have certain interactions with drugs or cell growth factors. The pore size and the charge can regulate the release of drugs or cell growth factors at different rates, thereby simulating the controlled release behavior of growth factors in the extracellular matrix and controlling the induction of cell proliferation and differentiation. In addition, xanthan gum exists in the conformation of rigid aggregates formed by double helix chains, highly branched polysaccharides are evenly distributed in the pores of these double helix chains, and sodium trimetaphosphate is esterified along the helix chains. Cross-linking forms highly branched polysaccharides and xanthan gum hydrogel scaffolds. The presence of highly branched polysaccharides and the cross-linking reaction have no significant effect on the double helix structure of xanthan gum, and cross-linking has a good effect on enhancing the strength of the hydrogel. .

Compared with the prior art, the method for preparing the highly branched polysaccharide-silk fibroin hydrogel scaffold of the present invention has the following advantages:

The preparation method of the present invention has the advantages of simple operation, low cost, and rich sources of raw materials. One of the raw materials used is the highly branched polysaccharide of tiger milk mushroom, which has biological activity, and can prepare hydrogel in situ under physiological conditions suitable for cell growth, thereby The highly branched polysaccharide-xanthan gum hydrogel scaffold obtained by the preparation method can control the slow release of drugs and improve drug efficacy when used as a drug carrier; when used as a tissue engineering scaffold material, it can be loaded with cell growth factors and simulate extracellular matrix to control cell growth The function of the slow release of factors can induce cell proliferation and differentiation into regenerative tissues, and the presence of fungal hyperbranched polysaccharides can endow the bioactivity of the hydrogel scaffold. Experiments show that the hyperbranched polysaccharide-xanthan gum hydrogel scaffold obtained by the method has a three-dimensional through-hole porous structure, and the model molecule bovine serum albumin has a large amount of embedding in the hydrogel, and has good controllable release behavior. Therefore, this method can be widely used in the preparation of artificial tissue scaffold materials, and also has broad application prospects in the fields of drug controlled release and food. It is worth noting that the hyperbranched polysaccharide-xanthan gum hydrogel of tiger milk mushroom is not only similar in structure to the extracellular matrix, but more importantly, it contains fungal hyperbranched polysaccharides, which have specific biological activities and serve as A highly branched polysaccharide whose structure is easy to flexibly adjust physical properties or carry various chemical signaling molecules to optimize cell survival and induce cell-specific differentiation behaviors. Therefore, embedding cell growth factors into hyperbranched polysaccharide-xanthan gum hydrogel of tiger milk mushroom and regulating its release behavior through the unique chemical structure of hyperbranched polysaccharides can better simulate its release in vivo and induce neogenesis Uniform formation of tissue. In addition, in the hydrogel scaffold, xanthan gum maintains a rigid double-helix aggregate structure, and the carboxyl group on the sugar ring can induce the adsorption and deposition of Ca ²⁺ and PO ₄ ^3- added in the cell culture solution, making the scaffold Mineralization is beneficial to the adhesion and proliferation of bone cells.

Description of drawings

FIG. 1 is a scanning electron microscope picture of the hyperbranched polysaccharide-xanthan gum hydrogel scaffold of Example 6.

Fig. 2 is the controlled release curve of bovine serum albumin from the hyperbranched polysaccharide-xanthan gum hydrogel scaffold of Examples 3, 4, 5, and 6.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

A method for preparing fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold, said preparation method comprising the following steps:

a. The dried sclerotia of tiger milk mushroom was crushed, and the fat was removed by Soxhlet extraction with ethyl acetate and acetone for 6 hours in sequence. Both ethyl acetate and acetone were chemically pure reagents. Then soak the sclerotium of tiger milk mushroom after fat removal in physiological saline at a temperature of 80°C for 2 hours, and centrifuge; the residue is soaked at a high pressure of 120°C for 30 minutes, wherein every 100g of tiger milk mushroom sclerotia is soaked in 1L of physiological saline, Centrifuge at 8000 rpm for 20 minutes to obtain the extract, cool the extract and then centrifuge and collect the residue; the residue is centrifugally washed with deionized water and freeze-dried to obtain the highly branched polysaccharide of tiger milk mushroom, which can also be dried by other methods to obtain tiger milk mushroom Highly branched polysaccharides.

b At room temperature, dissolve the hyperbranched polysaccharide of tiger milk mushroom in NaOH aqueous solution with a pH value of 12-14, and prepare a solution of hyperbranched polysaccharide of tiger milk mushroom with a concentration of 2% w/v after magnetic stirring for 2 hours, here The concentration of NaOH is 0.01-1mol/L, and 2% w/v represents the mass volume concentration, which means that 2 grams of solute are dissolved in 100 grams of solvent.

c. At room temperature, add xanthan gum to the highly branched polysaccharide solution of tiger milk mushroom obtained through step b, and continue magnetic stirring for 12 hours to obtain a uniformly mixed tiger milk mushroom hyperbranched polysaccharide-xanthan gum solution, xanthan gum The concentration is 0.5%～5%w/v, and the mass ratio of the hyperbranched polysaccharide of tiger milk mushroom to xanthan gum is 1:0.25～1:2.5.

d Dissolve sodium trimetaphosphate in deionized water at room temperature, stir until completely dissolved, or shake well to obtain an aqueous solution of sodium trimetaphosphate with a concentration of 75-262.5 mg/mL.

e at room temperature, add the sodium trimetaphosphate aqueous solution with a concentration of 75 to 262.5 mg/mL obtained through step d into the highly branched polysaccharide-xanthan gum solution of tiger milk mushroom obtained through step c, wherein tiger milk mushroom has a high The volume ratio of the branched polysaccharide-xanthan gum solution to the sodium trimetaphosphate aqueous solution is 25:8, the molar ratio of the repeating sugar units of the sodium trimetaphosphate to the xanthan gum is 1.46-14.6, stir rapidly for 5 minutes, and here can be used Magnetic stirring or mechanical stirring can also be used, and the cross-linking reaction is carried out at 37°C for 10 minutes to 48 hours to obtain the fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold, and the rheological behavior of the hydrogel is studied by using a rheometer. Washed with ion water and freeze-dried to obtain a three-dimensional porous scaffold of highly branched polysaccharide-xanthan gum. The morphology of the scaffold after freeze-drying was observed by scanning electron microscopy, and the swelling rate of the scaffold material after drying and the effect on bovine serum Protein release behavior.

Example 1

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L of normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain the hyperbranched polysaccharide of tiger milk mushroom. Dissolve 0.5g of hyperbranched polysaccharides from tiger milk mushroom in 25mL of NaOH aqueous solution with pH=12, stir magnetically for 2 hours to prepare hyperbranched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, add 0.125g xanthan gum Into the above-mentioned tiger milk mushroom hyperbranched polysaccharide solution with a concentration of 2%w/v, continue magnetic stirring for 12h to obtain a homogeneously mixed tiger milk mushroom hyperbranched polysaccharide-xanthan gum solution, and 8mL75mg/mL triple bias Add sodium phosphate to the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at 37°C for 10 minutes to obtain fungal hyperbranched polysaccharide-xanthan gum Gel scaffold, using a rheometer to study the rheological behavior of the hydrogel scaffold, washing with deionized water and freeze-drying to obtain a three-dimensional porous scaffold of hyperbranched polysaccharide-xanthan gum, observing the shape of the scaffold after freeze-drying with a scanning electron microscope In phosphate buffered saline solution, the swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin were tested.

Example 2

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides from tiger milk mushroom in 25mL of NaOH aqueous solution with pH=13, stir magnetically for 2 hours to prepare hyperbranched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, add 0.25g xanthan gum Into the above-mentioned concentration of the tiger milk mushroom hyperbranched polysaccharide solution of 2%w/v, continue magnetic stirring for 12h to obtain a homogeneously mixed hyperbranched polysaccharide-xanthan gum solution, and 8mL112.5mg/mL trimetaphosphoric acid Add sodium to the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at 37°C for 1 hour to obtain a fungal hyperbranched polysaccharide-xanthan gum hydrogel Glue scaffold, use a rheometer to study the rheological behavior of the hydrogel scaffold, wash with deionized water and freeze-dry to obtain a three-dimensional porous scaffold with hyperbranched polysaccharide-xanthan gum, and observe the morphology of the scaffold after freeze-drying with a scanning electron microscope , in phosphate buffered saline to test the swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin.

Example 3

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides from tiger milk mushroom in 25mL NaOH aqueous solution with pH=14, stir magnetically for 2 hours to prepare hyperbranched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, add 0.5g xanthan gum In the above-mentioned highly branched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, continue magnetic stirring for 12 hours to obtain a uniformly mixed hyperbranched polysaccharide-xanthan gum solution, add 8 mL of 150 mg/mL sodium trimetaphosphate In the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at a temperature of 37° C. for 10 minutes to obtain a fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold , using a rheometer to study the rheological behavior of the hydrogel scaffold, washed with deionized water and freeze-dried to obtain a three-dimensional through-hole porous scaffold of hyperbranched polysaccharide-xanthan gum, and observed the morphology of the scaffold after freeze-drying with a scanning electron microscope. The swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin were tested in phosphate buffered saline solution.

Example 4

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides of tiger milk mushroom in 25mL of NaOH aqueous solution with pH=13, stir magnetically for 2 hours to prepare a solution of hyperbranched polysaccharides of tiger milk mushroom with a concentration of 2% w/v, add 0.75g of xanthan gum Into the above-mentioned concentration of the tiger milk mushroom hyperbranched polysaccharide solution of 2%w/v, continue magnetic stirring for 12h, obtain the homogeneously mixed hyperbranched polysaccharide-xanthan gum solution, mix 8mL187.5mg/mL trimetaphosphoric acid Add sodium to the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at 37°C for 24 hours to obtain a fungal hyperbranched polysaccharide-xanthan gum hydrogel Glue scaffold, use a rheometer to study the rheological behavior of the hydrogel scaffold, wash with deionized water and freeze-dry to obtain a three-dimensional porous scaffold with hyperbranched polysaccharide-xanthan gum, and observe the morphology of the scaffold after freeze-drying with a scanning electron microscope , in phosphate buffered saline to test the swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin.

Example 5

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides of tiger milk mushroom in 25mL of NaOH aqueous solution with pH=13, stir magnetically for 2 hours to prepare a solution of hyperbranched polysaccharides of tiger milk mushroom with a concentration of 2% w/v, add 1.0g of xanthan gum In the above-mentioned highly branched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, continue magnetic stirring for 12 hours to obtain a uniformly mixed hyperbranched polysaccharide-xanthan gum solution, add 8 mL of 225 mg/mL sodium trimetaphosphate In the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pre-gel solution, and cross-link the pre-gel solution at a temperature of 37°C for 48 hours to obtain a fungal hyper-branched polysaccharide-xanthan gum hydrogel scaffold , using a rheometer to study the rheological behavior of the hydrogel scaffold, washed with deionized water and freeze-dried to obtain a three-dimensional through-hole porous scaffold of hyperbranched polysaccharide-xanthan gum, and observed the morphology of the scaffold after freeze-drying with a scanning electron microscope. The swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin were tested in phosphate buffered saline solution.

Example 6

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides from tiger milk mushroom in 25mL of NaOH aqueous solution with pH=13, stir magnetically for 2 hours to prepare hyperbranched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, add 1.25g xanthan gum Into the above-mentioned concentration of the tiger milk mushroom hyperbranched polysaccharide solution of 2%w/v, continue magnetic stirring for 12h to obtain a homogeneously mixed hyperbranched polysaccharide-xanthan gum solution, and 8mL262.5mg/mL trimetaphosphoric acid Add sodium to the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at 37°C for 3 hours to obtain a fungal hyperbranched polysaccharide-xanthan gum hydrogel Glue scaffold, use a rheometer to study the rheological behavior of the hydrogel scaffold, wash with deionized water and freeze-dry to obtain a three-dimensional porous scaffold with hyperbranched polysaccharide-xanthan gum, and observe the morphology of the scaffold after freeze-drying with a scanning electron microscope , in phosphate buffered saline to test the swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin. (this embodiment is the best embodiment)

Example 7

Crush 500g of dried tiger milk mushroom sclerotia, perform Soxhlet extraction with ethyl acetate and acetone for 6 hours to remove fat, then soak the fat-free tiger milk mushroom sclerotia in 5L of normal saline at 80°C for 2 hours, centrifuge , the residue was soaked in 5L normal saline at a high pressure of 120°C for 30 minutes, centrifuged to obtain an extract, the extract was cooled and then centrifuged to collect the residue, the residue was centrifugally washed with deionized water and freeze-dried to obtain highly branched tiger milk mushroom polysaccharide. Dissolve 0.5g of hyperbranched polysaccharides from tiger milk mushroom in 25mL of NaOH aqueous solution with pH=13, stir magnetically for 2 hours to prepare hyperbranched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, add 1.25g xanthan gum In the above-mentioned highly branched polysaccharide solution of tiger milk mushroom with a concentration of 2% w/v, continue magnetic stirring for 12 hours to obtain a uniformly mixed highly branched polysaccharide-xanthan gum solution, add 8mL75mg/mL sodium trimetaphosphate In the above hyperbranched polysaccharide-xanthan gum solution, stir rapidly for 5 minutes to obtain a pregel solution, and cross-link the pregel solution at 37°C for 3 hours to obtain a fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold , using a rheometer to study the rheological behavior of the hydrogel scaffold, washed with deionized water and freeze-dried to obtain a three-dimensional through-hole porous scaffold of hyperbranched polysaccharide-xanthan gum, and observed the morphology of the scaffold after freeze-drying with a scanning electron microscope. The swelling rate of the scaffold material after drying and the release behavior to bovine serum albumin were tested in phosphate buffered saline solution.

The performance of the fungal hyperbranched polysaccharide-xanthan gum hydrogel scaffold of Examples 3-7 is shown in Table 1

Table I

Claims (1)

1. prepare a method for fungus highly-branched polysaccharide-xanthan gum hydrogel scaffold, it is characterized in that: described preparation method comprises the following steps:

The Sclerotium of Pleurotus tuber regium of drying is pulverized by a, carry out surname extraction with ethyl acetate, acetone successively and remove fat, then the Sclerotium of Pleurotus tuber regium after degrease is immersed in normal saline, wherein every 100g Sclerotium of Pleurotus tuber regium 1L normal saline soaks, extract at high pressure 120 DEG C of temperature, centrifugal extracting solution, centrifugal again and collect residue after cooling extracting solution, residue deionized water eccentric cleaning and lyophilization obtains Pleurotus tuber-regium highly-branched polysaccharide;

It is in the NaOH aqueous solution of 12 ~ 14 that Pleurotus tuber-regium highly-branched polysaccharide is dissolved in pH value by b, is prepared into the Pleurotus tuber-regium highly-branched polysaccharide solution that concentration is 2%w/v after stirring 2h;

Xanthan gum joins in the Pleurotus tuber-regium highly-branched polysaccharide solution obtained through b step by c, stir 12h, obtain the highly-branched polysaccharide-xanthan gum solution of mix homogeneously, the concentration of xanthan gum is 0.5% ~ 5%w/v, and the mass ratio of highly-branched polysaccharide and xanthan gum is 1:0.25 ~ 1:2.5;

Sodium trimetaphosphate dissolves in deionized water by d, is stirred to and dissolves completely, obtain the sodium trimetaphosphate aqueous solution that concentration is 75 ~ 262.5mg/mL;

The concentration obtained through Step d is that the sodium trimetaphosphate aqueous solution of 75 ~ 262.5mg/mL is added in the Pleurotus tuber-regium highly-branched polysaccharide-xanthan gum solution obtained through step c by e, wherein the volume ratio of Pleurotus tuber-regium highly-branched polysaccharide-xanthan gum solution and sodium trimetaphosphate aqueous solution is 25:8, rapid stirring 5min, cross-linking reaction 10min ~ 48h at 37 DEG C of temperature, obtains fungus highly-branched polysaccharide-xanthan gum hydrogel scaffold.