Zinc-rich phellinus igniarius polysaccharide and its preparation method and application

Abstract

The present invention discloses a zinc-rich mulberry linterinary polysaccharide and a preparation method and application thereof, and belongs to the field of biomedicine technology. SOD, CAT and a zinc-containing compound are added to a liquid culture medium to obtain a tolerance treatment culture medium; the mulberry linterinary strain is inoculated for cultivation to obtain a tolerance treatment mulberry linterinary seed liquid; the zinc-containing compound and 6-benzyladenine are added to the liquid culture medium to obtain an enrichment culture medium; the tolerance treatment mulberry linterinary seed liquid is inoculated for cultivation, and the mycelium is separated after the cultivation is completed, and the mycelium is washed, freeze-dried and crushed to obtain zinc-rich mycelium powder; zinc-rich mulberry linterinary polysaccharide is obtained by hot water extraction and alcohol precipitation extraction. The present invention performs a tolerance culture on the strain by adding SOD, CAT and zinc sulfate to the liquid culture medium; then enriching the culture with a liquid culture medium to which zinc sulfate and 6-benzyladenine are added; there is no need to repeatedly perform tolerance acclimation on the strain, and the zinc-rich mulberry linterinary polysaccharide finally prepared has a high zinc content, which can significantly improve the antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to zinc-rich phellinus igniarius polysaccharide and a preparation method and application thereof. Background Art

[0002] Phellinus igniarius, belonging to the phylum Basidiomycetes, class Agaricaceae, order Aphyllophorales, and genus Phellinus, is currently internationally recognized as the most effective medicinal fungus in the field of biological cancer treatment. In recent years, as a novel anticancer fungus, Phellinus igniarius has attracted considerable attention from experts in the international pharmaceutical and health product industries. Because fungal polysaccharides are one of the main biologically active ingredients in fungi, and some products derived from these polysaccharides have entered clinical use, the development and utilization of Phellinus igniarius polysaccharides has garnered considerable attention from researchers. Phellinus igniarius polysaccharides possess excellent antioxidant, anti-tumor, immunomodulatory, hypoglycemic, anti-aging, and biotoxic properties, making them a hot topic among researchers both domestically and internationally in the field of fungal research. Extraction of Phellinus igniarius polysaccharides, like other polysaccharides, is performed using extraction methods, such as hot water extraction.

[0003] Trace elements are essential nutrients for animal life and production. One of their notable characteristics is their high efficacy at low doses. Although they comprise less than 0.01% of an organism's content, trace elements participate in virtually all physiological and biochemical processes and are closely linked to animal growth and health. They contribute to the formation and activation of enzymes, vitamins, and hormones within the animal body; they also regulate metabolism and determine growth, development, and reproduction, as well as animal production efficiency and product quality. Therefore, the supply and absorption of trace elements are crucial. Organic trace elements have a higher bioavailability than inorganic trace elements. This is primarily because, after ingestion, inorganic and organic trace element salts must first form chelates or complexes with amino acids or other substances through the action of coenzymes before they can be absorbed. Inorganic trace elements are unstable and easily bind, making them more likely to interact with other substances. Numerous studies in recent years have demonstrated that organic trace elements are superior to inorganic trace elements in enhancing immune function, improving intestinal health, and reducing stress. It can enhance the body's ability to resist pain, maximize the body's immune response, promote cellular immunity and humoral immunity, and play an anti-stress and anti-disease role. Compared with artificially synthesized organic trace elements, biotransformation has high efficiency, simple process and high conversion rate.

[0004] If trace elements are combined with mulberry linterinary polysaccharide and prepared through bioconversion, mulberry linterinary polysaccharide containing organic trace elements can be obtained. For example, patent application number CN202010336748.4 discloses a selenium-rich mulberry linterinary polysaccharide and its preparation method and application. By adding inorganic selenium to the mulberry linterinary culture medium, the inorganic selenium is organicized using mulberry linterinary mycelium, and selenium-rich mulberry linterinary polysaccharide is extracted. The inorganic selenium is then organically converted using microorganisms to obtain polysaccharide that promotes wound healing. Zinc is an important trace element with multiple effects and benefits on human health, including: promoting immune system function, supporting growth and development, maintaining skin health, promoting taste and appetite, protecting eyesight, and having antioxidant effects. Both zinc and polysaccharides have the effect of promoting wound healing. If zinc is loaded into mulberry linterinary polysaccharide, more effective wound healing drugs can be developed. However, zinc also has broad-spectrum antibacterial properties and inhibits the growth of mulberry linterinary strains, making it difficult to prepare zinc-rich mulberry linterinary polysaccharide using the method of CN202010336748.4. However, the method of domesticating Phellinus linteus strains with zinc resistance is not only time-consuming but also has an insignificant zinc-enriched effect. Therefore, a method is needed to cultivate Phellinus linteus strains that can not only make them zinc-resistant but also produce Phellinus linteus polysaccharides with high zinc content. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a zinc-rich mulberry linterus polysaccharide and its preparation method and application. The present invention performs tolerance culture on the strain by adding SOD, CAT and a zinc-containing compound to a liquid culture medium; then enriches the strain with a liquid culture medium supplemented with a zinc-containing compound and 6-benzyladenine; and finally obtains the zinc-rich mulberry linterus polysaccharide by water extraction and alcohol precipitation. The method of the present invention does not require multiple tolerance acclimation of the strain, and the zinc-rich mulberry linterus polysaccharide finally prepared has a high zinc content, which can significantly improve the antibacterial effect.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing zinc-rich phellinus linteus polysaccharide, comprising the following steps:

[0008] (1) SOD (superoxide dismutase), CAT (catalase) and a zinc-containing compound are added to a liquid culture medium and mixed evenly to obtain a tolerance treatment medium; the Phellinus igniarius strain is inoculated into the tolerance treatment medium for culturing to obtain a tolerance treatment Phellinus igniarius seed liquid;

[0009] (2) adding a zinc-containing compound and 6-benzyladenine (6-BA) to a liquid culture medium and mixing them evenly to obtain an enrichment culture medium; inoculating the tolerant treated Phellinus igniarius seed liquid obtained in step (1) into the enrichment culture medium for culturing; separating the mycelium after the culturing, washing, freeze-drying, and crushing to obtain zinc-enriched mycelium powder;

[0010] (3) Zinc-rich mulberry igniarius polysaccharide was extracted from zinc-rich mycelium powder using hot water extraction and alcohol precipitation methods.

[0011] Preferably, in step (1), the liquid culture medium is prepared from potato extract, glucose, KH2PO4, vitamin B1 and ultrapure water; the zinc-containing compound is zinc sulfate; the inoculation amount of the Phellinus linteus strain in the tolerance treatment medium is 0.1-0.5 g / L; the Phellinus linteus strain is Facies baumii, and its preservation number is CFCC 7230.

[0012] The preservation number of F. baumii is CFCC 7230, and it is preserved in the China Forestry Microbial Culture Collection Center.

[0013] Preferably, the concentration of SOD in the liquid culture medium is 0.5 g / L; the concentration of CAT in the liquid culture medium is 1.0 g / L; and the concentration of zinc ions in the liquid culture medium is 0.1-1.0 g / L.

[0014] Preferably, in step (1), the culture is carried out in a constant temperature shaker; the rotation speed of the constant temperature shaker is 120-180 rpm; the culture temperature is 27-30°C, and the culture time is 6-12 days.

[0015] Preferably, in step (2), the liquid culture medium is PDA liquid culture medium; and the zinc-containing compound is zinc sulfate.

[0016] Preferably, the concentration of zinc ions in the liquid culture medium is 0.1-1.0 g / L; the concentration of 6-benzyladenine in the liquid culture medium is 2 g / L.

[0017] Preferably, in step (2), the inoculation amount of the tolerance-treated Phellinus igniarius seed solution in the enrichment culture medium is 0.5-1 mL / L; and the culture is carried out at 27-30° C. for 6-10 days.

[0018] The second aspect of the present invention provides the application of the above preparation method in increasing the zinc content of zinc-rich Phellinus igniarius polysaccharide.

[0019] The third aspect of the present invention provides zinc-rich Phellinus linteus polysaccharide obtained by the above preparation method, wherein the zinc content of the zinc-rich Phellinus linteus polysaccharide is greater than 6 g / kg.

[0020] The fourth aspect of the present invention provides the use of zinc-rich mulberry linterus polysaccharide in the preparation of antibacterial drugs.

[0021] Preferably, the dosage form of the drug is liquid, paste, gel, dressing or spray; the concentration of zinc-rich phellinus igniarius polysaccharide in the drug is 100-600 μg / mL.

[0022] The fifth aspect of the present invention provides the use of SOD, CAT and 6-BA in increasing the polysaccharide content of Phellinus linteus mycelium and the zinc content of zinc-rich Phellinus linteus polysaccharide.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention successfully obtained a method for culturing zinc-rich Phellinus igniarius polysaccharides by treating the mycelium with tolerance and enrichment. The Phellinus igniarius strain was cultured in a tolerance medium with the addition of high-concentration zinc, superoxide dismutase (SOD) and catalase (CAT), to achieve rapid tolerance of the strain, with tolerance treatment completed in just one generation. In addition, during the enrichment culture process, in addition to adding high-concentration zinc to the culture medium, 6-benzyladenine (6-BA) was also added, which increased the relative yield of the mycelium and the enrichment of zinc.

[0025] (2) The concentration of trace elements in the culture medium of the present invention is controllable, and the optimized formula (such as the addition of potato extract, glucose, vitamin B1 and other ingredients) provides a sufficient nutritional environment for the stable growth and efficient enrichment of mycelium. The preparation process includes strict condition control (such as temperature, time, material-liquid ratio, alcohol precipitation ratio, etc.) to ensure the purity of polysaccharide extraction and significantly improve the biological activity of mulberry ignia polysaccharide. Using natural mulberry ignia polysaccharide as the matrix and combining the efficient enrichment design of trace elements, compared with other chemically synthesized products, it has both biocompatibility and low toxicity. The zinc-rich mulberry ignia polysaccharide prepared by the present invention has good antibacterial effect, and the antibacterial effect is significantly improved compared with the use of mulberry ignia polysaccharide alone and zinc alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Results of Fourier transform infrared spectrum of zinc-rich phellinus linteus polysaccharide;

[0027] Figure 2 : The mycelia of Phellinus linteus cultured in Example 1 and Comparative Examples 1 to 5;

[0028] Figure 3 :The results of the effects of superoxide dismutase (SOD), catalase (CAT) and 6-benzyladenine (6-BA) addition on the growth of Phellinus igniarius;

[0029] Figure 4 : The results of the determination of the cytotoxicity of zinc-rich phellinus igniarius polysaccharide;

[0030] Figure 5 : (a) Representative bright-field images of the inhibition zones of E. coli and two strains of MRSA after 16 h of treatment with different patches: (b) Diameters of the inhibition zones against E. coli and two strains of MRSA, each point represents the mean ± SD (n = 3), ***P < 0.001. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0032] As introduced in the background technology section, adding selenium to the culture medium for fermenting and preparing mulberry igniarius can obtain selenium-rich polysaccharides, but zinc has an antibacterial effect. If zinc-rich polysaccharides are to be prepared, continuous zinc tolerance acclimatization is required, and the zinc content of mulberry igniarius polysaccharides is not high.

[0033] Based on this, the purpose of the present invention is to provide a zinc-rich phellinus polysaccharide and its preparation method and application. The present invention adds SOD, CAT and high-concentration zinc sulfate to the liquid culture medium to perform a tolerance culture on the strain, and then adds 6-BA and zinc sulfate to perform enrichment culture on the strain; the mycelium obtained by the two cultures contains more polysaccharides, and the polysaccharides contain more zinc.

[0034] Superoxide dismutase and catalase are culture additives primarily used in plant tissue culture and cell culture. In plant tissue culture, SOD catalyzes the conversion of superoxide anions into hydrogen peroxide, while CAT decomposes hydrogen peroxide into water and oxygen, thereby scavenging ROS, mitigating oxidative damage, and improving the survival and differentiation rates of explants. In animal cell culture, ROS are generated during cellular metabolism. High concentrations of ROS can damage cells and even lead to cell death. As antioxidant enzymes, SOD and CAT can promptly scavenge ROS generated by cells, protecting them from oxidative stress and maintaining normal physiological functions and activity. When culturing tumor or normal cell lines, the addition of SOD and CAT can improve cell survival and proliferation. The Phellinus igniarius strains used in this invention are fungi, which differ in many physical and chemical properties from plant and animal cells. For example, fungi have cell walls, while plant and animal cells only have cell membranes. Furthermore, fungi can produce mycelium, which is not a product of fungal proliferation but rather a product of the fungus itself. The present invention has found that the synergistic effect of exogenous addition of SOD and CAT can enhance the heavy metal stress response mechanism of Phellinus igniarius strains. When there is a high concentration of metal ions in the culture medium, a large amount of superoxide anion free radicals are produced in the fungus. SOD is a superoxide dismutase that can catalyze the superoxide anion free radical (O2 -) is an enzyme that disproportionates H2O2 and O2. Since H2O2 has a bactericidal effect, the present invention also adds CAT, a catalase that catalyzes the decomposition of H2O2 into water and oxygen. Therefore, the addition of SOD and CAT can increase the dissolved oxygen content in the liquid culture medium, which can accelerate bacterial proliferation and enter the logarithmic growth phase. However, if too much SOD is added, a large amount of H2O2 is produced, which can directly kill the fungi, so the amount of SOD and CAT added needs to be controlled. In addition, SOD and CAT may not only increase the oxygen content in the culture medium, but also increase the strain's tolerance to zinc ions through other effects. This is because the antibacterial principle of zinc ions mainly has two aspects: first, it binds to the bacterial membrane, thereby destroying the function of the bacterial membrane and killing the bacteria; second, the interaction between zinc ions and the negative ions of bacterial cells causes abnormalities in enzymes and other protein mechanisms within the bacterial cells. In addition, high concentrations of zinc ions can also cause DNA chain breaks in fungi, triggering cell necrosis or apoptosis. It can be seen that effectively eliminating various reactive oxygen groups is not enough to prevent the antibacterial effects of zinc ions. The combined dual-enzyme treatment significantly alleviated oxidative stress damage. This enhanced enzymatic defense system enables the mycelium to establish a stable zinc-tolerant system in a single generation. Furthermore, in an environment with high zinc ion concentrations, this system activates the secretion of extracellular polysaccharides (EPS) by the mycelium, which wrap the mycelial surface to form a "biofilm." The three-dimensional network structure formed by the polysaccharides increases the surface roughness of the mycelium and, by preferentially adsorbing zinc ions, reduces the toxic effects of direct contact with the cell membrane. In short, SOD and CAT synergistically protect the strain through multiple actions to improve zinc ion tolerance, eliminating the need for multiple domestication cycles and requiring only a single tolerance culture, significantly saving culture costs.

[0035] 6-benzyladenine is added to the enrichment culture of the present invention. 6-BA is a cytokinin that causes plant growth and development by stimulating cell division and inhibits respiratory kinase. The culture medium with the addition of 6-BA can promote cell division. The present invention has found through research that the addition of 6-BA to the enrichment culture medium can further increase the polysaccharide content in the mulberry ignia mycelium and the zinc content in the zinc-rich mulberry ignia polysaccharide.

[0036] The invention realizes efficient and rapid enrichment of zinc in the mulberry ignia mycelium by adding SOD, CAT and 6-BA. The zinc-enriched mulberry ignia polysaccharide in the mycelium is convenient and simple to prepare and has significant antibacterial activity.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0038] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0039] Example 1

[0040] (1) Preparation of Phellinus igniarius mycelium

[0041] Weigh 200g of potatoes and cut them into 1cm x 1cm x 1cm pieces. Add 500mL of ultrapure water to a pot and place the cut potato pieces in the pot. Boil for approximately 20 minutes. Remove from heat when the potato pieces are formed but crumble easily with a light pressure of chopsticks. After cooling, filter twice through eight layers of gauze to remove the residue to obtain a potato extract. Add 20g of glucose, 1.5g of potassium phosphate (KH2PO4), and 0.05g of vitamin B1 to a beaker and dissolve them in 300mL of ultrapure water. Pour the previously obtained potato extract into the beaker and mix. In a volumetric flask, dilute to 1L with ultrapure water and sterilize. Add 0.5g of lyophilized SOD powder, 1.0g of lyophilized CAT powder, and 1.23g of zinc sulfate to the sterilized culture medium to prepare a tolerance treatment medium with a zinc ion concentration of 500mg / L.

[0042] Using a sterile inoculating needle, pick a piece of golden mycelium from the Phellinus linteus strain (F. baumii) and streak it onto a PDA solid culture plate. Incubate upright in a fungal incubator at a constant temperature of 28°C for four days. After four days, golden spores will appear on the plate. Using a sterile inoculating loop, pick a piece of Phellinus linteus mycelium and place it into a tolerant liquid culture medium. Incubate in a shaker at 28°C, 150 rpm, for seven days.

[0043] Place 1.23g of zinc sulfate in a centrifuge tube, dissolve it in sterile water, and filter it through a 0.22μm filter to obtain a zinc sulfate solution. Add the zinc sulfate solution to the sterilized PDA liquid medium to a zinc sulfate concentration of 1.23g / L (zinc ion concentration of 500mg / L). Then add 6-BA to a 6-BA concentration of 2g / L to obtain an enrichment medium. During inoculation, break up the bacteria in the tolerance medium, then pipette the seed solution and inoculate it into the enrichment medium. Add 500μL of seed solution to each bottle (1L) of enrichment medium. Culture at 28°C for 8 days to obtain Phellinus igniarius mycelium.

[0044] (2) Preparation of zinc-rich mulberry linterus polysaccharide: The mulberry linterus mycelia prepared in step (1) were collected by filtering through eight layers of gauze, and the mycelia were rinsed three times with double-distilled water to remove the free unconsumed sugars in the culture medium. The collected mycelia were freeze-dried at -80°C under vacuum for 3 days, and the mycelia powder was obtained by grinding in a mortar.

[0045] Mycelial powder was weighed and mixed with double-distilled water at a ratio of 1:50 (w / v). The mixture was stirred at 95°C for 3 h using a temperature-controlled magnetic stirrer. The mixture was centrifuged, and the insoluble residue was processed twice more as described above. The three supernatants were combined and the majority of the water was removed using a rotary evaporator at 55°C to prepare a 300 mL concentrated extract. Three volumes of anhydrous ethanol (900 mL) were added to the concentrated extract, and the extract was precipitated at 4°C for 12 h. The resulting precipitate was centrifuged at 8000 rpm for 5 min at 4°C in a refrigerated centrifuge. The supernatant was discarded, and 100 mL of anhydrous ethanol was added to the precipitate. The precipitate was vortexed to disperse, and the extract was centrifuged at 8000 rpm for 5 min at 4°C. The supernatant was discarded. This step was repeated three times. The resulting alcohol-precipitated crude polysaccharide was re-dissolved in double-distilled water, refrigerated at 4°C overnight, and centrifuged at 8000 rpm for 5 min at 4°C. The precipitate was discarded, and the sugar solution was filtered through filter paper and then a membrane filter until clear. This step was repeated three times.

[0046] The obtained clarified sugar solution was mixed in the ratio of sugar solution: chloroform: n-butanol (v / v / v) = 16:4:1 and shaken thoroughly. After natural sedimentation, the entire liquid was divided into three layers: upper, middle and lower. The upper layer was sugar solution, the middle layer was milky white deformed protein, and the lower layer was organic reagents chloroform and n-butanol. Carefully separate the sugar solution with a pipette and repeat the above steps again in the above proportion until the milky white deformed protein layer in the middle no longer appears after sedimentation. Carefully separate and collect the sugar solution and purify it with a DEAE-52 cellulose column (3cm×25cm). By ion adsorption, the positively charged substance is adsorbed on the fiber column, while the neutral polysaccharide can be eluted smoothly. Collect the sugar-containing eluate, dialyze and desalt for 2 days, and freeze-dry to obtain zinc-rich phellinus polysaccharide. The infrared spectrum of zinc-rich phellinus polysaccharide is shown in Figure 1 .

[0047] Comparative Example 1: Preparation of zinc-rich phellinus igniarius polysaccharide

[0048] The difference from Example 1 is that: SOD freeze-dried powder, CAT freeze-dried powder and 6-BA were not added; due to the high zinc ion concentration, the growth of Phellinus linteus mycelium was inhibited, resulting in a small amount of Phellinus linteus mycelium and failure to extract zinc-rich Phellinus linteus polysaccharide; Phellinus linteus mycelium was obtained by expanding the culture by 10 times, and finally zinc-rich Phellinus linteus polysaccharide was extracted.

[0049] Comparative Example 2: Preparation of zinc-rich phellinus igniarius polysaccharide

[0050] The difference from Example 1 is that: SOD freeze-dried powder and 6-BA were not added; a small amount of Phellinus linteus mycelium was obtained, and zinc-rich Phellinus linteus polysaccharide was finally extracted.

[0051] Comparative Example 3: Preparation of zinc-rich phellinus igniarius polysaccharide

[0052] The difference from Example 1 is that: 6-BA is not added; the mycelium of Phellinus linteus is obtained; and zinc-rich Phellinus linteus polysaccharide is finally extracted.

[0053] Comparative Example 4: Preparation of zinc-rich phellinus igniarius polysaccharide

[0054] The difference from Example 1 is that the concentration of SOD is 0.1 g / L, the concentration of CAT is 0.2 g / L, the mycelium of Phellinus linteus is obtained, and zinc-rich Phellinus linteus polysaccharide is finally extracted.

[0055] Comparative Example 5: Preparation of zinc-rich phellinus igniarius polysaccharide

[0056] The difference from Example 1 is that the concentration of SOD is 1 g / L, the concentration of CAT is 2 g / L, the mycelium of Phellinus linteus is obtained, and zinc-rich Phellinus linteus polysaccharide is finally extracted.

[0057] The mycelia of Phellinus linteus cultured in Example 1 and Comparative Examples 1 to 5 were Figure 2 .

[0058] Test Example 1: Composition Determination

[0059] (1) The mycelia of Phellinus igniarius obtained in Example 1 and Comparative Examples 1 to 5 were freeze-dried on a plate and weighed, and the biomass was calculated according to the following formula:

[0060] Biomass (g / L) = [total weight (g) - dish weight (g)] / culture medium volume (L).

[0061] The results are as follows Figure 3 As shown, the biomass obtained in Example 1 is higher than the biomass obtained in Comparative Examples 1 to 5, that is, more Phellinus igniarius mycelia can be obtained by adopting the method of Example 1.

[0062] (2) The reducing sugar content of the zinc-rich mulberry linterus polysaccharides prepared in Example 1 and Comparative Examples 1 to 5 was determined using the phenol-sulfate method. The protein content was determined using a total protein quantitative test kit (BCA method, purchased from Nanjing Jiancheng Bioengineering Research Institute). The results are shown in Table 1.

[0063] Table 1 Reducing sugar and protein content in zinc-rich phellinus igniarius polysaccharide

[0064]

[0065] According to Table 1, it can be seen that the content of reducing sugar in the zinc-rich mulberry linterinary polysaccharides prepared in Example 1 and Comparative Examples 1 to 5 is substantially the same, with a difference of about ±1%. Since the method for extracting zinc-rich mulberry linterinary polysaccharides from mulberry linterinary mycelium in Example 1 and Comparative Examples 1 to 5 is the same, it is shown that the purity of the zinc-rich mulberry linterinary polysaccharides extracted in Comparative Examples 1 to 5 and Example 1 is substantially the same. Therefore, the comparison of the content of zinc-rich mulberry linterinary polysaccharides extracted in Comparative Examples 1 to 5 and Example 1 and the zinc content in the polysaccharide is comparable.

[0066] An equal amount of the mulberry linterus mycelia cultured in Example 1 or Comparative Examples 1 to 5 was taken to extract zinc-rich mulberry linterus polysaccharide according to the method of Example 1 and Comparative Examples 1 to 5, and the content of zinc-rich mulberry linterus polysaccharide obtained from the mulberry linterus mycelia was calculated.

[0067] Take equal amounts of zinc-rich phellinus igniarius polysaccharides obtained in Example 1 and Comparative Examples 1 to 5 and perform zinc element detection according to "GB 5009.268-2016 National Food Safety Standard Determination of Multiple Elements in Food", and the specific steps are as follows:

[0068] Weigh an appropriate amount of zinc-rich phellinus linteus polysaccharide obtained in Example 1 and Comparative Examples 1 to 5 into a polytetrafluoroethylene digestion tank and add 5 mL of nitric acid. Let it stand for a while. After the reaction is complete, seal the lid and place it in a microwave digester for digestion. After the temperature cools to below 50°C, take out the digestion tank and place it in a fume hood. Open the digestion tank, rinse with ultrapure water, transfer it to a 25 mL volumetric flask, rinse at least 3 to 4 times, dilute with ultrapure water to the scale, and test. The blank control is the above solvent without the addition of zinc-rich phellinus linteus polysaccharide.

[0069] The zinc content is calculated according to the formula: ;

[0070] Where:

[0071] W——Zinc content in the sample, in mg / kg;

[0072] C——Zinc concentration in the sample solution, unit: ug / L;

[0073] C0——Zinc concentration in blank control, unit: ug / L;

[0074] V——constant volume, unit: mL;

[0075] N——dilution multiple;

[0076] m——the sampling amount of the test sample, in g.

[0077] The polysaccharide content obtained from the mycelium of Phellinus igniarius and the zinc content in the polysaccharide are shown in Table 2.

[0078] Table 2 Polysaccharide content and zinc content in polysaccharides

[0079]

[0080] Obtaining more Phellinus linteus mycelia does not mean obtaining more Phellinus linteus polysaccharides. If the polysaccharide content contained in the Phellinus linteus mycelia is high, more Phellinus linteus polysaccharides can still be obtained. According to Table 2, it can be seen that the polysaccharide content obtained from the Phellinus linteus mycelia by the method of Example 1 is the highest, and the zinc element enrichment amount in the polysaccharide is the highest. Compared with Comparative Example 1, after Comparative Example 3 adds SOD and CAT, the polysaccharide content increases by nearly one-fold, and the polysaccharide zinc element content increases by 790 times, indicating that SOD and CAT can not only improve the tolerance of the Phellinus linteus mycelia, but also improve the content of polysaccharides and zinc. Compared with Comparative Example 3, Example 1 also adds 6-BA, and the content of polysaccharides and zinc is further improved, indicating that 6-BA has a good enrichment effect. But if SOD and CAT are not added, and 6-BA is only added to the enrichment medium, because the zinc ion concentration is too high, the thalline cannot tolerate it, and its result is similar to Comparative Example 1, and can only obtain a small amount of Phellinus linteus mycelia. Therefore, 6-BA needs to synergize with SOD and CAT to improve the polysaccharide content and the content of zinc in the polysaccharide.

[0081] Comparative Example 6: Preparation of Phellinus igniarius polysaccharide

[0082] (1) Phellinus linteus mycelia were broken up using a homogenizer and inoculated into liquid PDA medium (PDA liquid medium / L: 200 g potato, 20 g glucose, 1.5 g potassium dihydrogen phosphate, 1 g magnesium sulfate heptahydrate) at a volume of 500 μL per bottle. Fermentation was carried out in a constant temperature shaker at 150 rpm and 28°C for 8 days. After fermentation, the mycelia were isolated, washed, freeze-dried, weighed, and pulverized to obtain mycelial powder.

[0083] (2) Phellinus igniarius polysaccharide was extracted from the mycelium prepared in step (1) according to the method of Example 1.

[0084] Test Example 2: Cytotoxicity Test

[0085] The cytotoxicity test of zinc-rich phellinus linteus polysaccharide prepared in Example 1 was conducted using human epidermal immortalized cell line (HaCaT). 4 Cells were added to the culture medium at 3, 5, 10, 50, 100, 500, and 1000 μg / ml of zinc-rich phellinus linteus polysaccharide solution prepared with DMEM and incubated for 20 h. MTT was then added and incubated for 4 h. DMSO was then used for color development. The color was read at 490 nm for photometric analysis and the cytotoxicity was calculated. The results are shown in the table below. Figure 4 shown.

[0086] Cell survival rate (%) = (OD of drug-treated group - OD of blank group) / (OD of control group - OD of blank group) × 100%;

[0087] Blank group—contains culture medium and CCK-8 solution, but no cells or drugs;

[0088] Control group—containing cells, culture medium, and CCK-8 solution without drugs;

[0089] Drug-added group—contains cells, culture medium, CCK-8 solution, and drug solution.

[0090] according to Figure 4 It can be seen that when the zinc-rich phellinus igniarius polysaccharide prepared in Example 1 is at 1000 μg / mL, the cell survival rate is still around 90%.

[0091] Test Example 3: Antibacterial Test

[0092] Freshly cultured Escherichia coli (E. coli, deposit number ATCC23519, deposited in China Center for Medical Microbiology Collection) and two strains of methicillin-resistant Staphylococcus aureus (MRSA, deposit numbers: ATCC43300 and ATCC33591, deposited in China Center for Medical Microbiology Collection) were picked (18-24 hours) and diluted with sterile saline to 1.5 × 10 8 CFU / mL, mix well and let it stand for 15 minutes to avoid bacterial precipitation affecting the coating uniformity.

[0093] Dip the bacterial suspension in a sterile cotton swab and evenly apply it to the surface of the LB solid plate. Repeat 3 times to ensure the formation of a uniform bacterial lawn. Let it stand for 5 minutes to allow the bacterial liquid to absorb and avoid slipping when the paper is attached. A control group, a positive control group, the mulberry linterinary polysaccharide group prepared in Comparative Example 6, and the zinc-rich mulberry linterinary polysaccharide group prepared in Example 1 were set. Wherein normal saline was used as the control group, and a zinc sulfate solution with a concentration of 100 μg / mL was configured with normal saline and zinc sulfate solid as a positive control. The mulberry linterinary polysaccharide extracted from Comparative Example 6 and normal saline were configured to a concentration of 100 μg / mL mulberry linterinary polysaccharide solution as the mulberry linterinary polysaccharide group. The mulberry linterinary polysaccharide prepared in Example 1 and normal saline were configured to a concentration of 100 μg / mL zinc-rich mulberry linterinary polysaccharide solution as the zinc-rich mulberry linterinary polysaccharide group. Sterile drug-sensitive paper was added to a mixture of normal saline, 100 μg / mL zinc sulfate solution, 100 μg / mL mulberry linterinary polysaccharide solution, and 100 μg / mL zinc-rich mulberry linterinary polysaccharide solution and infiltrated for 15 min. Use sterile tweezers to remove a drug-sensitive paper strip and place it on the surface of the plate. Lightly press the strip with the tip of the tweezers to flatten it. The spacing between each strip should be no less than 24 mm, and the center of the strip should be no less than 15 mm from the edge of the plate. Incubate at 35 ± 2°C for 16 hours, with the plate upright to prevent condensation from affecting the diffusion of the strip. After incubating the plate with the strip for 16 hours, measure the diameter of the inhibition zone with a vernier caliper.

[0094] The results are as follows Figure 5As shown in the figure, although zinc sulfate has an antibacterial effect, at a concentration of 100 μg / mL, zinc sulfate has no or only a weak antibacterial effect, and its inhibition zone is extremely unclear. Although mulberry linterus has an antibacterial effect, it relies on the phenols and flavonoids contained in mulberry linterus, and mulberry linterus polysaccharide has no antibacterial effect. Under the same concentration conditions, zinc-rich mulberry linterus polysaccharide has a significant antibacterial effect. This shows that the zinc-rich mulberry linterus polysaccharide prepared by the method of the present invention has a significant antibacterial effect.

[0095] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing zinc-rich phellinus linteus polysaccharide, characterized in that: The following steps are involved: (1) SOD, CAT and a zinc-containing compound are added to a liquid culture medium and mixed evenly to obtain a tolerant treatment medium; the Phellinus linteus strain cultured on a PDA solid is inoculated into the tolerant treatment medium for culture to obtain a tolerant treatment Phellinus linteus seed solution; the liquid culture medium is prepared from potato extract, glucose, KH2PO4, vitamin B1 and ultrapure water; the Phellinus linteus strain is Fasciola baumii, and its preservation number is CFCC 7230; the concentration of SOD in the liquid culture medium is 0.1-1 g / L; the concentration of CAT in the liquid culture medium is 0.2-2.0 g / L; the concentration of zinc ions in the liquid culture medium is 0.1-1.0 g / L; (2) adding a zinc-containing compound and 6-benzyladenine to a liquid culture medium and mixing them evenly to obtain an enrichment culture medium; inoculating the tolerant treated Phellinus igniarius seed liquid obtained in step (1) into the enrichment culture medium for culturing; separating the mycelium after the culturing, washing, freeze-drying, and pulverizing to obtain zinc-enriched mycelium powder; the concentration of 6-benzyladenine in the liquid culture medium is 2 g / L; (3) Zinc-rich mulberry igniarius polysaccharide was extracted from zinc-rich mycelium powder using hot water extraction and alcohol precipitation methods.

2. The preparation method according to claim 1, characterized in that In step (1), the zinc-containing compound is zinc sulfate; and the inoculation amount of the Phellinus igniarius strain in the tolerance treatment medium is 0.1-0.5 g / mL.

3. The preparation method according to claim 1, characterized in that In step (1), the culture is carried out in a constant temperature shaker; the rotation speed of the constant temperature shaker is 120-180 rpm; the culture temperature is 27-30° C., and the culture time is 6-12 days.

4. The preparation method according to claim 1, characterized in that In step (2), the liquid culture medium is PDA liquid culture medium; and the zinc-containing compound is zinc sulfate.

5. The preparation method according to claim 1, characterized in that In step (2), the inoculation amount of the tolerance-treated Phellinus igniarius seed solution in the enrichment medium is 0.5-1 mL / L; and the culture is carried out at 27-30° C. for 6-10 days.

6. Use of the preparation method according to any one of claims 1 to 5 in increasing the zinc content of zinc-rich phellinaceous polysaccharide.

7. Zinc-rich phellinaceous polysaccharide obtained by the preparation method according to any one of claims 1 to 5.

8. Use of the zinc-rich phellinus igniarius polysaccharide according to claim 7 in the preparation of antibacterial drugs.

9. The use according to claim 8, characterized in that The dosage form of the drug is liquid, paste, gel, dressing or spray; the concentration of zinc-rich phellinus igniarius polysaccharide in the drug is 100-600 μg / mL.

Images