CN108893503B

CN108893503B - Porous composite polysaccharide hydrogel and preparation method thereof

Info

Publication number: CN108893503B
Application number: CN201810550600.3A
Authority: CN
Inventors: 蒋建新; 吉骊; 琚斯怡; 雷福厚; 段久芳
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2021-09-14
Anticipated expiration: 2038-05-31
Also published as: CN108893503A

Abstract

The invention relates to a preparation method of porous composite polysaccharide hydrogel, which is mainly improved in that cassava residue saccharification liquid is used for replacing a conventional carbon source to culture microorganisms capable of producing bacterial cellulose. According to the method, the cassava residue saccharified liquid is used for replacing a conventional carbon source to carry out fermentation culture on the microorganisms producing the bacterial cellulose, so that the performance of the obtained gel can be improved, the cassava residue can be converted into a material with a high attachment value, and the reduction treatment of the solid waste cassava residue is realized. Furthermore, the invention adopts a biological 'in-situ synthesis' mode to form the composite hydrogel of hyaluronic acid and bacterial cellulose, so that the composite polysaccharide hydrogel with a more stable structure can be prepared.

Description

Porous composite polysaccharide hydrogel and preparation method thereof

Technical Field

The invention relates to a composite polysaccharide hydrogel and a preparation method thereof, belonging to the technical field of biomass chemistry and chemical engineering.

Background

Bacterial cellulose (BC cellulose for short) is a natural ultramicro fiber net which is synthesized by specific microorganisms and consists of ultramicro fibers, and has a unique three-dimensional nanofiber net structure, high water holding capacity and good mechanical strength and biocompatibility. The bacterial cellulose is a chain high molecular polymer formed by connecting beta-glucopyranose residues by beta-1, 4 glycosidic bonds, a large number of oxygen bonds exist in molecules and among molecules, and the bacterial cellulose contains a large number of hydroxyl groups and ether bonds and has a plurality of effective reactive active sites. Compared with natural plant cellulose, the cellulose has more excellent characteristics such as high purity, high crystallinity, large specific surface area, good hydrophilicity and biocompatibility, and is easy to degrade in the environment. At present, in developed countries, the bacterial cellulose industry has already formed a market with billions of dollars in adult value at first step, and relates to the industries of food, chemical industry, medicine, textile, paper making and the like, and the research of China in the industry field is still in the first stage. Under the conditions of continuous increase of world population and increasing shortage of resources, bacterial cellulose as an environment-friendly and renewable biological material has great commercial value and good development prospect.

The cassava industry is one of important post industries in the western region, the planting area of the cassava in Guangxi is 30 ten thousand hm2, and the fresh cassava yield is more than 700 ten thousand t. The cassava dregs are solid waste materials produced by cassava starch, and the main components of the cassava dregs are carbohydrates, including starch, cellulose, hemicellulose, protein and a small amount of lignin. At present, only a small part of the cassava residues are used as feed, alcohol and single-cell protein are produced, and the disposal problem of the cassava residues becomes an important factor for restricting the development of the cassava industry. The solid waste cassava residue can be converted into biological energy by using a biotechnology strategy, so that the solid waste cassava residue is reduced, the problem of treatment of the cassava residue is solved, the aim of reducing environmental pollution is fulfilled, and a high-value biochemical product can be produced. The enzymolysis of the cassava residues by using amylase, cellulase and the like is one of effective ways for efficient biological utilization, so that on one hand, harmless treatment can be achieved, the environmental pollution is reduced, on the other hand, residual starch and cellulose can be effectively utilized, and the utilization rate of biomass resources is increased. At present, carbon sources of a biological bacterial cellulose culture medium are mainly commercial sugar raw materials such as glucose, sucrose and the like. Monosaccharide obtained after hydrolysis of fiber raw materials is used as a carbon source in a culture medium to realize green preparation and clean production of the materials, and agricultural and forestry waste is effectively applied to the aspect of biological medicine materials, so that the production and preparation of the biological materials have more selectivity. The saccharified liquid after the cassava residue is saccharified can also be used as an effective carbon source to replace glucose or sucrose in the culture medium. The most important two points in the hydrogel production process are cost control and yield improvement, if cassava dregs are used as a carbon source, the relationship among the enzyme dosage, substrate concentration and yield is well processed, the problem of industrial treatment of the cassava dregs is solved, a new choice is provided for the lack of bacterial cellulose raw materials, and the raw material cost can be effectively reduced. In addition, the cassava dregs have the advantage that no pretreatment is needed, which not only reduces equipment investment, but also reduces energy consumption. Although researchers do a lot of work on the preparation and production of the composite bacterial cellulose, the preparation and production of the composite bacterial cellulose are still in the basic research stage, and the problems of low product purity, high production cost, poor operability and the like exist in order to realize large-scale production. Therefore, research and development of a novel method for producing composite bacterial cellulose are needed to obtain a production means which is low in cost, high in purity, simple and convenient to operate and environment-friendly. And then, by testing the physical and chemical properties such as mechanical property and the like, a better composite process and raw material proportion are found, the large-scale production and application demonstration of the green biological process are promoted, the material consumption and energy consumption, the industrial solid waste discharge and the like are obviously reduced, and a biological process system with ecological safety, environmental friendliness, low carbon and circular development is established.

As a natural hydrogel material, the bacterial cellulose hydrogel also has obvious defects of poor wet strength and poor water holding capacity under stress, and the service performance of the material is influenced. Therefore, by using the in-situ compounding method, natural polymer materials such as alginic acid, hyaluronic acid and the like are added into the culture medium, and a bacterial cellulose compound is formed in the synthesis process of the natural polymer materials, so that the hydrogel material with excellent performance is obtained. Hyaluronic acid is another polyaminoglucose commonly found in most mammalian tissues, and its basic structure is a large polysaccharide composed of two disaccharide units, D-glucuronic acid and N-acetylglucosamine, which, unlike other mucopolysaccharides, is sulfur-free. Its hyaluronic molecule can carry over 500 times of water, and can be widely used in cosmetics and cosmetics for promoting wound healing. If the bacterial cellulose is used as a matrix, a hyperfine three-dimensional nano network structure of the bacterial cellulose is utilized, hyaluronic acid is selected as a good macromolecular substance to be compounded with the bacterial cellulose, the good biodegradability and biocompatibility of the hyaluronic acid are combined, a new composite material is synthesized through an in-situ synthesis route, polymer molecules which are easy to disperse and stable are introduced into the network structure, and the produced composite hydrogel material endows the bacterial cellulose with more excellent water holding performance and higher mechanical strength, is expected to be used as a novel tissue engineering scaffold and is applied to the medical fields of wound accessories and the like. The compounding process can improve the difficult problem that the hydrogel is difficult to permeate cells due to the compactness of the fiber network, and further can be used as a medical material with higher practicability. In the prior art, there are various methods for preparing hydrogel by compounding bacterial cellulose and other macromolecular substances, but the hydrogel is prepared by firstly obtaining bacterial cellulose through fermentation and then compounding the bacterial cellulose with other macromolecular substances, for example, the methods disclosed in patents CN104130424 and CN 103044694 a, and the hydrogel prepared by the method has limited strength and cannot meet the requirement of high strength.

Disclosure of Invention

The invention aims to provide a preparation method of porous composite polysaccharide hydrogel, which is mainly characterized in that a liquid culture medium taking cassava residue saccharification liquid as a carbon source is adopted to culture microorganisms capable of producing bacterial cellulose, and a bacterial cellulose membrane formed on the surface of the culture solution after the culture is finished is the porous composite polysaccharide hydrogel.

According to the research of the invention, the cassava residue saccharified liquid is used for replacing a common carbon source such as glucose in a microorganism culture medium, and compared with the method for producing hydrogel by fermenting with glucose as the carbon source, the indexes of the obtained hydrogel, such as tensile strength, sol multiple and the like, are obviously improved.

Preferably, the concentration of monosaccharide in the cassava saccharification liquid is 260-265 g/L. It was found that the higher the monosaccharide concentration, the higher the yield of the product.

Preferably, the cassava saccharification liquid is prepared by sequentially carrying out enzymolysis on cassava residues by high-temperature amylase and saccharifying enzyme. The high-temperature amylase can quickly liquefy the raw materials, and the saccharifying enzyme can further degrade the liquefied materials into monosaccharide.

Preferably, the cassava residue saccharification liquid is prepared by the following method: adding the cassava residues into water at the temperature of 45-55 ℃ according to the mass-volume ratio of 20%, preserving heat for 0.5-1 h, adding commercial high-temperature resistant amylase according to 20U/g of starch, and stirring and liquefying at the temperature of 85-90 ℃ for 2-2.5 h; then adjusting the pH value of the system to 3.8-4.5 by using 10% dilute sulfuric acid, adding glucoamylase into starch according to 150U/g for saccharification for 1-1.5 h at 60 ℃, and then performing vacuum concentration to enable the concentration of monosaccharide to be 260-265 g/L.

Preferably, the content of starch in the cassava residue is 45-50%, and the content of cellulose in the cassava residue is 22-27%.

Preferably, the improvement of the present application further comprises the operation of directly adding hyaluronic acid to the culture medium, comprising the following steps: culturing a microorganism capable of producing bacterial cellulose by using a liquid culture medium added with hyaluronic acid and taking cassava residue saccharification liquid as a carbon source, wherein a film formed on the surface of the culture solution after the culture is finished is the porous composite polysaccharide hydrogel.

By adding hyaluronic acid, the hyaluronic acid and the bacterial cellulose are coupled and crosslinked, so that the strength and other indexes of the obtained gel can be further improved.

Preferably, the microorganism capable of producing bacterial cellulose is one or more of acetobacter xylinum, rhizobium, sarcina, pseudomonas, achromobacter, alcaligenes, aerobacter or azotobacter.

Further preferably, the microorganism capable of producing bacterial cellulose is acetobacter xylinum.

Preferably, the addition amount of the hyaluronic acid in the liquid culture medium is 7.5-15 g/L.

As a preferred procedure, hyaluronic acid is formulated into an aqueous solution prior to addition.

As a preferred mode of operation, the method of the invention comprises the steps of:

1) sequentially carrying out enzymolysis on the cassava residues by using high-temperature amylase and saccharifying enzyme to obtain cassava residue saccharifying liquid, and adjusting the concentration of monosaccharide in the cassava residue saccharifying liquid to be 260-265 g/L;

2) the method comprises the steps of statically culturing acetobacter xylinum at 30 ℃ for 10-12 days by using a liquid culture medium which is added with hyaluronic acid with the concentration of 7.5-15 g/L and takes a cassava residue saccharification liquid as a carbon source, and forming a film on the surface of the culture solution after the culture is finished, namely the porous composite polysaccharide hydrogel.

Preferably, the liquid culture medium comprises 7.5-15 g/L of hyaluronic acid, 18-22 g/L of cassava dreg saccharification liquid, 4.5-5.5 g/L of peptone, 4.5-5.5 g/L of yeast extract, 2.5-3 g/L of disodium hydrogen phosphate and 1.1-1.2 g/L of citric acid monohydrate; the initial pH was 5.5.

Preferably, the liquid culture medium comprises 10-15 g/L of hyaluronic acid, 18-22 g/L of cassava residue saccharification liquid, 4.5-5.5 g/L of peptone, 4.5-5.5 g/L of yeast extract, 2.5-3 g/L of disodium hydrogen phosphate and 1.1-1.2 g/L of citric acid monohydrate; the initial pH was 5.5.

Preferably, the prepared porous composite polysaccharide hydrogel is separated from the culture solution, granulated and dried by a fluidized bed at 90-110 ℃, and finally the porous composite polysaccharide hydrogel product is obtained.

Another objective of the invention is to protect the porous composite polysaccharide hydrogel prepared by the method of the invention.

The final purpose of the invention is to protect the application of the porous composite polysaccharide hydrogel in the preparation of biomedical materials, preferably in the preparation of wound accessories.

The invention has the following beneficial effects:

(1) the method degrades the starch in the cassava dregs through enzymolysis, can directly replace a carbon source in a culture medium, can improve the performance of the obtained gel, can convert the cassava dregs into chemicals or materials with high attached values, realizes the reduction treatment of the solid waste cassava dregs, has large resource amount and low price of the cassava dregs, and can reduce the biotransformation cost by converting the cassava dregs through an enzymolysis method.

(2) In the culture process, the method directly utilizes the carbon source of the cassava residue saccharification liquid to ferment and culture the acetobacter xylinum without any pretreatment, and is a green culture mode with low energy consumption and high yield.

(3) The method for preparing the bacterial cellulose hydrogel by replacing a pure glucose product with the saccharification liquid provides a new development idea for a wood fiber raw material sugar platform, and effectively applies agricultural and forestry wastes to the aspect of biological medical materials, so that the production and preparation of the biological materials have more selectivity.

(4) According to the invention, the composite hydrogel of hyaluronic acid and bacterial cellulose is formed in a biological 'in-situ synthesis' manner, and the hyaluronic acid in the culture medium is combined with the formed bacterial cellulose along with the growth of acetobacter xylinum, so that the composite polysaccharide hydrogel with a stable structure is obtained.

(5) The addition of the hyaluronic acid also endows the bacterial cellulose hydrogel with higher wet strength and certain toughness, improves the poor water holding capacity of the hydrogel when stressed, and improves the service performance of the material.

(6) The composite polysaccharide hydrogel prepared by the invention has rich and uniform pore-forming, and a good network structure can well solve the problem that the hydrogel is difficult to permeate cells due to the compactness of a fiber network, so that the hydrogel can be used as a novel tissue engineering scaffold and a medical material with higher practicability.

(7) The invention adopts granulation and fluidized bed to dry hydrogel, greatly improves the drying rate, and overcomes the defects of long drying time, high energy consumption and the like of hydrogel products.

Drawings

FIG. 1 is a scanning electron microscope image of the porous composite polysaccharide hydrogel prepared in example 4;

FIG. 2 is a scanning electron microscope image of a porous composite polysaccharide hydrogel prepared in a comparative example;

FIG. 3 is a flowchart illustrating the operation of examples 2 to 4.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The cassava saccharification liquid referred to in the following examples was prepared by the following method:

adding the cassava residues into water at 50 ℃ according to a ratio of 20% (w/v), preserving heat for 0.5h, adding commercial high-temperature resistant amylase according to a ratio of 20U/g of starch, and stirring and liquefying at 85-90 ℃ for 2 h; then adjusting the pH of the system to 3.8-4.5 by using 10% dilute sulfuric acid, adding saccharifying enzyme according to 150U/g starch, and saccharifying for 1h at 60 ℃. The resulting saccharified solution was concentrated under vacuum until the monosaccharide concentration in the concentrated solution was 263.67 g/L.

Example 1

This example relates to a method for preparing a polysaccharide hydrogel by fermentation culture of acetobacter xylinum alone, comprising the steps of:

replacing glucose in the culture medium with cassava residue saccharification liquid with monosaccharide concentration of 263.67g/L, wherein the acetobacter xylinum liquid culture medium comprises the following components: 20g/L of cassava residue saccharification liquid, 5g/L of peptone, 5g/L of yeast extract, 2.7g/L of disodium hydrogen phosphate and 1.15g/L of citric acid monohydrate, and adjusting the initial pH to 5.5. Inoculating activated acetobacter xylinum seed solution with volume fraction of 10%, and standing and culturing at 30 deg.C for 10 days to obtain bacterial cellulose polysaccharide hydrogel as a membrane formed on the surface of the culture solution.

And (3) separating the prepared polysaccharide hydrogel from the culture solution, granulating, and drying by a fluidized bed at 90-110 ℃ to obtain the polysaccharide hydrogel product.

The solid yield of the hydrogel (the solid yield of the hydrogel is calculated by the method of the dry basis weight of the hydrogel/the dry basis weight of the raw material cassava residue) is 22.48 percent, the crystallinity is 28.1 degrees, the swelling multiple after the swelling equilibrium is 678.4, the tensile strength is 0.28N, and a plurality of bundles of dense and staggered filaments are observed under a scanning electron microscope.

Example 2

The embodiment relates to a method for preparing polysaccharide hydrogel by coupling bacterial cellulose and hyaluronic acid, wherein cassava residue saccharification liquid is used for replacing glucose in a culture medium, and hyaluronic acid is added into the culture medium, and the method comprises the following steps:

preparing a 50g/L aqueous solution of hyaluronic acid, and heating at 60 ℃ for 1h before adding the aqueous solution into a culture medium;

replacing glucose in the culture medium with cassava residue saccharification liquid with monosaccharide concentration of 263.67g/L, wherein the acetobacter xylinum liquid culture medium comprises the following components: 20g/L of cassava residue saccharification liquid, 5g/L of peptone, 5g/L of yeast extract, 2.7g/L of disodium hydrogen phosphate, 1.15g/L of citric acid monohydrate and 7.5g/L of hyaluronic acid, and the initial pH is adjusted to be 5.5. Inoculating 10% volume fraction of activated cassava bacillus seed liquid, and standing and culturing at 30 ℃ for 10 days to form a film on the surface of the culture solution, namely the polysaccharide hydrogel.

And separating the prepared porous composite polysaccharide hydrogel from the culture solution, granulating, and drying by a fluidized bed at 90-110 ℃ to obtain the composite polysaccharide hydrogel product.

The polysaccharide hydrogel solid yield (the solid yield of the polysaccharide hydrogel is calculated by the method of polysaccharide hydrogel dry basis mass/raw material cassava residue dry basis mass) is 23.57%, the crystallinity is 27.4 degrees, the swelling multiple after swelling balance is 742.6, the tensile strength is 0.62N, and the observation of a scanning electron microscope shows that thinner filaments begin to form a net shape. The operation flow chart of the present embodiment is shown in fig. 3.

Example 3

replacing glucose in the culture medium with cassava residue saccharification liquid with monosaccharide concentration of 263.67g/L, wherein the acetobacter xylinum liquid culture medium comprises the following components: 20g/L of cassava residue saccharification liquid, 5g/L of peptone, 5g/L of yeast extract, 2.7g/L of disodium hydrogen phosphate, 1.15g/L of citric acid monohydrate and 10g/L of hyaluronic acid, and the initial pH is adjusted to be 5.5. Inoculating 10% volume fraction of activated cassava bacillus seed liquid, and standing and culturing at 30 ℃ for 10 days to form a film on the surface of the culture solution, namely the polysaccharide hydrogel.

The polysaccharide hydrogel solid yield is 27.79%, the crystallinity is 27.0 degrees, the swelling multiple after swelling balance is 872.6, the tensile strength is 0.75N, and the filaments are stronger under the observation of a scanning electron microscope, have obvious net structures and certain porosity. The operation flow chart of the present embodiment is shown in fig. 3.

Example 4

preparing an aqueous solution of which the hyaluronic acid content is 50g/L, and heating the aqueous solution at 60 ℃ for 1 hour before preparing a culture medium;

replacing glucose in the culture medium with cassava residue saccharification liquid with monosaccharide concentration of 263.67g/L, wherein the acetobacter xylinum liquid culture medium comprises the following components: 20g/L of cassava residue saccharification liquid, 5g/L of peptone, 5g/L of yeast extract, 2.7g/L of disodium hydrogen phosphate, 1.15g/L of citric acid monohydrate and 15g/L of hyaluronic acid, and the initial pH is adjusted to be 5.5. Inoculating 10% volume fraction of activated cassava bacillus seed liquid, and standing and culturing at 30 ℃ for 10 days to form a film on the surface of the culture solution, namely the polysaccharide hydrogel.

The polysaccharide hydrogel solid yield was 28.14%, the crystallinity was 26.8 °, the swelling factor after swelling equilibrium was 944.1, and the tensile strength was 0.93N. The net structure stacked layer by layer is observed under a scanning electron microscope, and the net structure is uniform in shape and uniform in distribution, as shown in fig. 1. The operation flow chart of the present embodiment is shown in fig. 3.

Comparative example

This comparative example was used for the fermentative culture of Acetobacter xylinum using grape as a carbon source under the same conditions. The components of the culture medium: 20g/L glucose, 5g/L peptone, 5g/L yeast extract, 2.7g/L disodium hydrogen phosphate, 1.15g/L citric acid monohydrate, initial pH 5.5, culture temperature 30 ℃, after 10 days of culture, polysaccharide hydrogel.

The polysaccharide hydrogel has a solid yield of 21.24%, a crystallinity of 28.3 °, a swelling multiple of 464.6 after swelling equilibrium, and a tensile strength of 0.15N, and multiple strands of filaments were observed under a scanning electron microscope, as shown in fig. 2. The same magnification of scanning electron microscope is shown in FIG. 1 and FIG. 2, and it is clear that the degree of crosslinking of the gel obtained in example 4 is significantly better than that of the comparative example.

According to the data, the indexes such as tensile strength of the obtained hydrogel can be obviously improved after the cassava residue hydrolysate is used as the carbon source to replace glucose, and after hyaluronic acid is added, all the indexes are obviously improved.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A preparation method of a porous composite polysaccharide hydrogel is characterized in that a liquid culture medium added with hyaluronic acid and taking cassava residue saccharification liquid as a carbon source is adopted to culture microorganisms capable of producing bacterial cellulose, and a bacterial cellulose membrane formed on the surface of the culture solution after the culture is finished is the porous composite polysaccharide hydrogel;

the concentration of monosaccharide in the cassava residue saccharification liquid is 260-265 g/L;

the addition amount of the cassava residue saccharification liquid in the liquid culture medium is 18-22 g/L; the addition amount of the hyaluronic acid in the liquid culture medium is 7.5-15 g/L;

the cassava residue saccharification liquid is prepared by sequentially carrying out enzymolysis on cassava residues by high-temperature amylase and saccharifying enzyme;

the microorganism capable of producing bacterial cellulose is acetobacter xylinum.

2. The method of claim 1, comprising the steps of:

2) the method comprises the steps of statically culturing acetobacter xylinum for 10-12 days at the temperature of 30 ℃ by using a liquid culture medium which is added with hyaluronic acid with the concentration of 7.5-15 g/L and takes a cassava residue saccharification liquid as a carbon source, and forming a film on the surface of the culture solution after the culture is finished, namely the porous composite polysaccharide hydrogel.

3. The method according to claim 1 or 2, wherein the composition of the liquid medium is hyaluronic acid 7.5-15 g/L, cassava dreg saccharification liquid 18-22 g/L, peptone 4.5-5.5 g/L, yeast extract 4.5-5.5 g/L, disodium hydrogen phosphate 2.5-3 g/L, citric acid monohydrate 1.1-1.2 g/L; the initial pH was 5.5.