CN101297821A - Phellinus linteus mycelia active glucoprotein and use thereof and preparation - Google Patents

Abstract

The invention discloses a submerged fermentation phellinus linteus mycelium glycoprotein, a usage and a separation extraction preparation method thereof, the complex is the complex of heteropolysaccharide and protein, wherein, the content of the heteropolysaccharide is 15 to 20 percent, and the heteropolysaccharide is composed of three monosaccharides of glucose monosaccharide, xylose and mannose; the content of the protein is 80 to 85 percent, and the protein is composed of 18 amino acids of aspartic acid, glutamic acid, arginine and so on; and the weight-average molecular weight is 20 to 40KD. The glycoprotein complex uses bran extract liquid as a main ingredient for preparing a culture medium, the phellinus linteus mycelium is produced by the submerged fermentation of the phellinus linteus bacterial strain liquid, the homogenization, the cold-water extraction, the centrifugalization, the collection of supernatant liquid, the precipitation of ammonium sulfate, the dialysis and the DEAE-Sepharose Fast Flow column chromatography are carried out for system separating and purifying the glycoprotein complex. The anti-bacterial glycoprotein is used for preparing the anti-bacterial dugs which have inhibitory effects on escherichia coil and staphylococcus aureus. At the same time, the glycoprotein complex can be used for the separation and the purification of the glycoprotein from the mycelium obtained by the submerged fermentation of various medical and edible fungi.

Description

Phellinus mycelium active glycoprotein and its use and preparation method

Technical field:

The invention relates to a Phellinus submerged fermentation mycelia glycoprotein complex with antibacterial and other activities, the use of the complex and a preparation method for separation and purification thereof.

Background technique

It is impossible for any kind of organism to exist in isolation in nature. All kinds of organisms can survive because of their own defense mechanisms. Plants and microorganisms can synthesize some small molecular antibiotic substances, such as phenols, tannins and other small molecular substances. At the same time, it can also synthesize antibacterial macromolecules such as polysaccharides, proteins, and glycoprotein complexes. The role of antimicrobial protein is to resist the invasion of antigenic bacteria, improve the disease resistance of the organism itself, and maintain the continuation and normal activities of life activities. The research on antibacterial macromolecules has made great progress in recent years. With the deepening of separation and purification technology and the rapid development of biological mass spectrometry technology, people's research and understanding of antibacterial macromolecules continue to deepen, and the self-defense of various organisms in nature The system has a further understanding. Hundreds of antimicrobial proteins, antimicrobial peptides, and antimicrobial glycoprotein complexes have been discovered.

Large medicinal edible fungi are an important part of traditional Chinese herbal medicine. Because they are rich in biologically active substances, they have become an important natural resource for the development of new drugs and have attracted more and more attention.

Phellinus belongs to Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymenomycetes), Polyporales (Polyporales), Rust Poraceae (Hymenochaetacae), and the medicinal fungi of the genus Phellinus (Phellinus), mainly distributed in In China, Japan, South Korea, North Korea, Russia, Australia, North America, Central and South America and other places, its fruiting body is used as medicine, and it has the reputation of "forest gold". "Medicine Properties" records: Phellinus japonica tastes slightly bitter, cold in nature, can benefit the five internal organs, soften hard, detoxify; stop bleeding, invigorate blood circulation; Diarrhea and so on. At present, some studies mainly focus on the domestication of strains and artificial cultivation techniques to obtain fruiting bodies. However, due to the shortcomings of artificial solid cultivation techniques, such as difficult cultivation, long growth cycle, and easy to be restricted by seasons, etc., the mulberry produced from fruiting bodies Yellow active substances are expensive and difficult to popularize; and the preparation of mycelium by submerged fermentation can basically overcome the above shortcomings, and can also obtain a large amount of extracellular active substances. But at present, there are only a few documents that conduct systematic research on Phellinus submerged fermentation technology. Chinese patent CN1556212 introduces the process of liquid submerged fermentation to produce Phellinus water extract and polysaccharides. yellow polysaccharide. Patent CN1908181 introduced the solid fermentation of Phellinus 4.6302 to obtain mycelia and obtain polysaccharides with antitumor activity.

Based on the above, it is not difficult to find that most of the research on Phellinus Phellinus bioactive substances mainly focuses on the separation and purification or structural identification of active small molecular compounds or active polysaccharides in its fruiting bodies, but among them, the separation and purification of antibacterial active glycoproteins, identification of physical and chemical properties and Related biological functions have not been reported.

Contents of the invention

The object of the present invention is to provide a submerged fermented Phellinus Phellinus mycelium glycoprotein complex with antibacterial activity, its application and its extraction, separation and preparation method. The method uses bioengineering fermentation technology to cultivate Phellinus mycelium, extracts Phellinus mycelium glycoprotein with antibacterial activity, and obtains its basic physical and chemical properties. The method steps are clear and easy to operate. This complex can be used in the production of healthcare products or in the further development of new natural antibacterial drugs.

For realizing the above-mentioned purpose of the present invention:

(1) a Phellinus mycelia glycoprotein complex, characterized in that: the heteropolysaccharide content in the glycoprotein complex is 15-20%, and the protein content is 80-85%, wherein the heteropolysaccharide comprises glucose, wood Sugar and mannose are three kinds of monosaccharides, proteins include 18 kinds of amino acids including aspartic acid, glutamic acid and arginine; the weight-average molecular weight of the glycoprotein complex is: 20-40kD

(2) The antibacterial glycoprotein is used to prepare antibacterial drugs that have inhibitory effects on Escherichia coli and Staphylococcus aureus.

(3) A method for preparing the above-mentioned Phellinus mycelium glycoprotein complex with antibacterial activity:

The present invention adopts Phellinus AS3.637 (purchased from China Microorganism Strain Preservation Center), and preserves on the slope of PDA medium, activates the strain before use, cuts the slope of the strain into small pieces and inoculates it in the liquid seed culture medium at 25°C, After culturing at 160 rpm for 7 days, the seed liquid was collected, inoculated in a fermenter for fermentation for 9 to 14 days, and centrifuged at 8000 rpm to obtain Phellinus mycelium. The yield of the obtained mycelium was 80 g per liter of culture medium. Add 1 to 5 times the volume of water to the Phellinus mycelium, homogenate and break it, extract at 4-30°C, centrifuge, discard the precipitate, collect the extract, concentrate the extract and add ammonium sulfate to a concentration of 80-90% (m/V), let it stand for 24-72 hours, take the precipitate by high-speed centrifugation, add a small amount of water to redissolve the precipitate, put the precipitate reconstituted solution into a dialysis bag of 8,000-10,000 Daltons, dialyze with deionized water, collect the dialysate and then lower the temperature Concentrate, load the sample on the DEAE-Sepharose FastFlow chromatography column, and use distilled water and 0.1, 0.3, 0.5, 2.0mol/L NaCl solution as eluents for step-wise elution. The results are shown in Figure 1. Detected at OD280nm of ultraviolet light, there are four absorption peaks of A, B, C, and D, among which the B component with the highest content is selected to freeze-dry to obtain Phellinus mycelium glycoprotein complex.

Its physical and chemical properties can be determined by infrared, high performance liquid chromatography, amino acid analyzer, SDS-PAGE, etc. Determination test of various performances of Phellinus antibacterial glycoprotein complex prepared by the method of the present invention

(1) Determination of antibacterial activity

The antibacterial activity adopts the plate punching method. Add 10 ⁶ cfu·mL ^-1 of Escherichia coli and Staphylococcus aureus liquid cultured for 18 to 20 hours in a sterilized petri dish with a diameter of 9 cm, and make a bacterial suspension with sterile physiological saline Spread 1mL on the corresponding solid medium respectively, add each type of bacteria into a petri dish, then inject 20mL of nutrient agar medium that has been melted and cooled to 45-50°C, rotate and mix immediately, after the agar solidifies, use externally Punch 4 equidistant holes on the culture medium with a sterilized stainless steel hole puncher with a diameter of 6 mm, and then use a dropper to absorb the melted agar culture solution, and drop 1 drop into each hole to make up the holes to avoid leakage at the bottom. Use a microsampler to draw 50 μL of each sample into the wells, and place them in a 35°C incubator for 24 hours.

It has been determined that Phellinus antibacterial glycoprotein can significantly inhibit Staphylococcus aureus and Escherichia coli, and the results are shown in Figure 2 and Figure 3.

(2) Determination of biochemical characteristics

①Thermal stability

Phellinus antibacterial glycoprotein is treated in a boiling water bath, with less loss of antibacterial activity and good thermal stability;

② pH stability

When treated with different pH solutions, the antibacterial activity is stable in the range of pH 2-13;

③Isoelectric point

The PI value of Phellinus antibacterial glycoprotein was measured by isoelectric focusing electrophoresis: acidic protein with PI value of 5.0-7.0.

④Molecular weight

Using gel filtration chromatography and SDS-PAGE vertical plate power method, the molecular weight of the antibacterial glycoprotein was measured to be 3-5kD; the results are shown in Figure 4.

All Phellinus Phellinus AS3.637 of the present invention was purchased from China Microorganism Culture Collection Center. The liquid submerged fermentation medium is mainly made of the extract of agricultural and by-product bran, and other ingredients include corn flour and bean cake powder. The composition of the medium is as follows (/L):

Bran Extract 50g

Corn flour 30g

Glucose 10g

Bean cake powder 10g

Yeast paste 10g

corn steep liquor 10g

FeSO4 1g

KH ₂ PO ₄ 1g

the rest is water

pH Natural

Compared with other existing glycoprotein complex extraction methods, the invention has high technological content and obvious technical advantages, and can obtain active components with a single component.

The Phellinus mycelia glycoprotein complex is expected to be an ideal and promising antibacterial drug or drug precursor substance. The preparation method of the complex adopts the low-cost bran extract as the main raw material, uses the bioengineering fermentation technology to cultivate the Phellinus mycelia, and separates and purifies the glycoprotein with antibacterial activity from it. The production process is simple and easy. The production cycle is short, the production cost is low, and the glycoprotein yield is high, so it has broad application prospects and potential economic benefits.

Description of drawings

Fig. 1 is the DEAE-Sepharose Fast Flow chromatogram of Phellinus antibacterial glycoprotein of the present invention

Figure 2 is the inhibition result of Phellinus antibacterial glycoprotein on Staphylococcus aureus

Figure 3 is the inhibition result of Phellinus antibacterial glycoprotein Escherichia coli

Fig. 4 is the SDS-PAGE electrophoresis figure of Phellinus antibacterial glycoprotein of the present invention

Detailed ways

The preparation method of Phellinus antimicrobial protein and the steps of biological activity are further described below in conjunction with specific examples to the technical scheme of the present invention:

Example 1

Phellinus AS3.637 was purchased from China Microorganism Culture Collection Center. The strains were first cultured on slant medium. The test tubes and tampons used for culture need to be sterilized at 121°C for 30 minutes, then put into the culture medium and sterilized at 121°C for 30 minutes, and placed on an inclined plane to cool to solidify into a slope. Then the ultra-clean bench was inoculated. The slant culture was carried out at a constant temperature of about 25°C for a period of 7 days. Cut the slant bacteria into small pieces and inoculate them into the seed culture medium. After the medium is divided, put a cotton plug on the Erlenmeyer flask to prevent external microorganisms from entering the medium and cause pollution, and ensure good ventilation performance. . The composition of the seed culture solution is: 20g of glucose, 5g of yeast extract, 10g of corn steep liquor, 50g of bran, 0.5g of MgSO ₄ 7H ₂ O, 1g of KH ₂ PO ₄ , the rest is water, and the pH is natural; at 25°C and 160rpm After cultivating for 7 days, the seed liquid was collected and inoculated in a 20L fermenter to obtain a fermentation culture liquid. The liquid medium consisted of: 50 g of bran, 30 g of corn flour, 10 g of glucose, 10 g of bean cake powder, 10 g of yeast extract, 10 g of corn steep liquor, FeSO ₄ 1g, KH ₂ PO ₄ 1g, the rest is water, the pH is natural; ferment at 25°C for 9 days. The obtained mycelium is brownish yellow in color and has a strong fragrance.

Example 2

100g of Phellinus mycelium was added to 1 times the volume of water, and the homogenate was broken, extracted at 4°C, centrifuged, discarded insoluble matter, and the extract was collected. After the extract was concentrated, ammonium sulfate was added to a concentration of 80% (m/V). Stand still for 24 hours, high-speed centrifuge to take the precipitate, add a small amount of water to redissolve the above precipitate, put the precipitate reconstituted solution into an 8000 Dalton dialysis bag, dialyze with deionized water, collect the dialysate, concentrate, and load the sample on DEAE-Sepharose Fast Flow The chromatographic column uses distilled water and NaCl as eluents for stepwise elution, collects the glycoprotein complex components eluted by the NaCl solution, and freezes and dries each collected solution.

Take each tube and measure the UV absorption at 280nm, and get 4 peaks. After the antibacterial activity test, B and C are the active ingredients, which have antibacterial effect. The Phellinus mycelia glycoprotein complex was collected after freeze-drying, and the sample was brown powder. Its physical and chemical properties were determined by infrared, high performance liquid chromatography, amino acid analyzer, and SDS-PAGE. The polysaccharide in the glycoprotein complex is a heteropolysaccharide, the polysaccharide content is 15%, and it is composed of three kinds of monosaccharides: glucose, xylose and mannose; the protein content is 85%, including aspartic acid, glutamic acid and arginine 18 kinds of amino acids including acid; the weight average molecular weight of the glycoprotein complex is: 20kD;

Example 3

After adding 3 times the volume of water to 100g Phellinus mycelium, the homogenate was broken, leached at 15°C, centrifuged, discarded the insoluble matter, collected the extract, concentrated the extract and added ammonium sulfate to a concentration of 85% (m/V), Stand still for 36 hours, high-speed centrifuge to take the precipitate, add a small amount of water to redissolve the precipitate, put the precipitate reconstituted solution into an 8000 Dalton dialysis bag, dialyze with deionized water, collect the dialysate, concentrate, and load the sample on the DEAE-Sepharose Fast Flow layer The column was analyzed, and distilled water and NaCl were used as eluents for stepwise elution, and the glycoprotein complex components eluted by the NaCl solution were collected, and each collected solution was freeze-dried.

Take each tube and measure the UV absorption at 280nm, and get 4 peaks. After the antibacterial activity test, B and C are the active ingredients, which have antibacterial effect. The Phellinus mycelia glycoprotein complex was collected after freeze-drying, and the sample was brown powder. Its physical and chemical properties were determined by infrared, high performance liquid chromatography, amino acid analyzer, and SDS-PAGE. The polysaccharide in the glycoprotein complex is a heteropolysaccharide, the polysaccharide content is 18%, and it is composed of three kinds of monosaccharides: glucose, xylose and mannose; the protein content is 82%, including aspartic acid, glutamic acid and arginine 18 kinds of amino acids including acid; the weight average molecular weight of the glycoprotein complex is: 25kD;

Example 4

Add 5 times the volume of water to 100g of Phellinus mycelium, then homogenate and break it, extract at 15°C, centrifuge, discard the insoluble matter, collect the extract, add ammonium sulfate to the concentration of 90% (m/V) after the extract is concentrated, Stand still for 72 hours, high-speed centrifuge to take the precipitate, add a small amount of water to redissolve the precipitate, put the precipitate reconstituted solution into an 8000 Dalton dialysis bag, dialyze with deionized water, collect the dialysate, concentrate, and load the sample on the DEAE-Sepharose Fast Flow layer The column was analyzed, and distilled water and NaCl were used as eluents for stepwise elution, and the glycoprotein complex components eluted by the NaCl solution were collected, and each collected solution was freeze-dried.

Take each tube and measure the UV absorption at 280nm, and get 4 peaks. After the antibacterial activity test, B and C are the active ingredients, which have antibacterial effect. The Phellinus mycelia glycoprotein complex was collected after freeze-drying, and the sample was brown powder. Its physical and chemical properties were determined by infrared, high performance liquid chromatography, amino acid analyzer, and SDS-PAGE. The polysaccharide in the glycoprotein complex is a heteropolysaccharide, the polysaccharide content is 20%, and it is composed of three kinds of monosaccharides: glucose, xylose and mannose; the protein content is 80%, including aspartic acid, glutamic acid and 18 kinds of amino acids including arginine; the weight average molecular weight of the glycoprotein complex is: 40kD;

Example 5

The antimicrobial glycoprotein antibacterial spectrum was determined by plate punching method, and the pathogenic bacteria were Escherichia coli and Staphylococcus aureus. The above pathogenic bacteria were cultured in beef extract peptone medium (beef extract: 3g; peptone: 10g; NaCl: 5g; agar: 15g; water: 1000mL). After the fresh colony grows, place a sterilized stainless steel puncher on the diagonal, and use a micro-sampler to absorb the separated antibacterial glycoprotein and add it to the hole. Each batch was treated 3 times, and the bacteria were grown for a period of time to observe the antibacterial activity of antibacterial glycoproteins against different pathogenic bacteria. It was found that antibacterial glycoproteins had strong antibacterial and antibacterial activities against Staphylococcus aureus and Escherichia coli.

The invention uses submerged fermented Phellinus mycelium as raw material, separates and purifies the glycoprotein in the mycelium, maintains the activity of the glycoprotein, and is verified to have antibacterial activity of the protein. The invention can be generally used for the separation and purification of glycoproteins in various fermented mycelia.

Claims (4)

1. phellinus igniarius mycelium sugar-protein compound, it is characterized in that: heteropolysaccharide content is 15～20% in the described sugar-protein compound, protein content is 80～85%, wherein heteropolysaccharide comprises glucose, 3 kinds of monosaccharide of xylose and mannose, protein comprises 18 seed amino acids of aspartic acid, glutamic acid and arginine; Described sugar-protein compound weight average molecular weight is: 20～40kD.

2. the purposes of the described phellinus igniarius mycelium sugar-protein compound of claim 1, it is characterized in that: described antibiotic glycoprotein is used to prepare to escherichia coli and the inhibited anti-microbial type medicine of staphylococcus aureus.

3. the preparation method of the described phellinus igniarius mycelium sugar-protein compound of claim 1 is characterized in that following steps:

(1) adopt the Phellinus igniarius (L. ex Fr.) Quel. strain after conventional liquid submerged fermentation is cultivated, to obtain phellinus igniarius mycelium;

(2) with homogenate fragmentation behind the water of 1～5 times of volume of this phellinus igniarius mycelium adding, 4～30 ℃ of lixiviates, centrifugal, abandon insoluble matter, collect extracting solution; Adding ammonium sulfate to concentration behind the cryoconcentration extracting solution is 80～90%m/V, leaves standstill 24～72h, and high speed centrifugation is got precipitation;

(3) add low amounts of water and redissolve above-mentioned precipitation, will precipitate and redissolve liquid and insert 8000～10000 daltonian bag filters, deionized water is dialysed;

(4) collect above-mentioned dialysis solution, behind the cryoconcentration, be splined on DEAE-Sepharose Fast Flow chromatographic column, use distilled water and 0.1mol/L successively, 0.3mol/L, 0.5mol/L, 2mol/L NaCl are that eluant carries out stage eluting;

(5) detect at ultraviolet light OD280nm place, in A, B, four absworption peaks of C, D, select the highest B component lyophilization of content, promptly obtain the phellinus igniarius mycelium sugar-protein compound.

4. mycelium preparation method according to claim 3 is characterized in that: in described every liter of seed culture medium: glucose 20g, yeast extract 5g, Semen Maydis pulp 10g, wheat bran 50g, MgSO ₄7H ₂O 0.5g, KH ₂PO ₄1g, all the other are water, the pH nature; Consisting of of every liter of fermentation broth: wheat bran 50g, Semen Maydis powder 30g, glucose 10g, soybean cake powder 10g, yeast extract 10g, Semen Maydis pulp 10g, FeSO ₄1g, KH ₂PO ₄1g, all the other are water, the pH nature.

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