Zinc-rich phellinus igniarius polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses zinc-rich phellinus igniarius polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Adding SOD, CAT and a zinc-containing compound into the liquid culture medium to obtain a tolerance treatment culture medium; inoculating a phellinus igniarius strain and culturing to obtain a phellinus igniarius seed solution subjected to tolerance treatment; adding a zinc-containing compound and 6-benzyladenine into the liquid culture medium to obtain an enrichment culture medium; inoculating a phellinus igniarius seed solution subjected to tolerance treatment for culture, separating mycelia after culture is finished, washing, freeze-drying and crushing to obtain zinc-rich mycelium powder; the zinc-rich phellinus igniarius polysaccharide is obtained through hot water extraction and alcohol precipitation extraction. The method comprises the following steps: adding SOD, CAT and zinc sulfate into a liquid culture medium to carry out tolerance culture on a bacterial strain for the first time; carrying out enrichment culture by using a liquid culture medium added with zinc sulfate and 6-benzyladenine; the strain does not need to be subjected to tolerance domestication for many times, and the finally prepared zinc-rich phellinus linteus polysaccharide is high in zinc content, so that the antibacterial effect can be remarkably improved.

Description

Technical Field

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

[0002] Phellinus belongs to the phylum Basidiomycetes, class Agaricaceae, order Aphyllophorales, and genus Xylophora. It is currently internationally recognized as the first effective medicinal fungus in the field of biological cancer treatment. In recent years, as a new type of anti-cancer fungus, Phellinus has attracted the attention of many experts in the international pharmaceutical industry and health care products industry. Since fungal polysaccharides are one of the main active ingredients with biological activity in fungi, and some products derived from fungal polysaccharides have entered the clinic, the development and utilization of Phellinus polysaccharides have attracted the attention of many scholars. Phellinus polysaccharides have good physiological activities such as antioxidant, anti-tumor, immunomodulatory, hypoglycemic, anti-aging, and non-biological toxicity, and have become a hot spot in the field of fungal research for scholars at home and abroad. The extraction of Phellinus polysaccharides is the same as other polysaccharides, which are extracted from Phellinus by leaching methods, such as hot water leaching. Trace elements are essential nutrients for animals to maintain life and production. One of its notable characteristics is that the dosage is small but the effect is great. Although the content of trace elements in organisms is less than 0.01%, they participate in almost all physiological and biochemical processes of the body and are closely related to animal growth and health. They participate in the formation and activation of enzymes, vitamins and hormones in animals; they also participate in regulating material metabolism and determine the growth, development and reproductive function of the body, as well as the production efficiency and product quality of animals. Therefore, the supply and absorption of trace elements are crucial. Compared with inorganic trace elements, organic trace elements have a higher bioavailability. This is mainly because after the general trace element inorganic salts and organic salts are ingested by the body, they must first form chelates or complexes with amino acids or other substances with the help of coenzymes before they can be absorbed by the body. Inorganic trace elements are unstable and easy to combine, and the possibility of interaction is easier than other substances. In recent years, a large number of studies have shown that organic trace elements are superior to inorganic trace elements in improving 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, it has high biological conversion efficiency, simple process and high conversion rate.

[0003] If trace elements are combined with mulberry linterus polysaccharide and prepared by bioconversion, mulberry linterus polysaccharide containing organic trace elements can be obtained. For example, the patent with application number CN202010336748.4 discloses a selenium-rich mulberry linterus polysaccharide and its preparation method and application. By adding inorganic selenium to the mulberry linterus culture medium, the inorganic selenium is organicized by mulberry linterus mycelium, and the selenium-rich mulberry linterus polysaccharide is extracted, and the inorganic selenium is organically converted by microorganisms to obtain polysaccharides to promote wound healing. Zinc is an important trace element and has multiple effects and functions on human health, including: promoting immune system function, supporting growth and development, maintaining skin health, promoting taste and appetite, protecting eyesight, and anti-oxidation. Both zinc and polysaccharides have the effect of promoting wound healing. If zinc is loaded into mulberry linterus polysaccharide, a more effective wound healing drug can be developed. However, zinc also has a broad-spectrum antibacterial property. Zinc will inhibit the growth of mulberry linterus strains, and it is difficult to prepare zinc-rich mulberry linterus 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-enriching effect. Therefore, a method is needed to cultivate Phellinus linteus strains that can not only make them zinc-resistant but also obtain Phellinus linteus polysaccharides with high zinc content. Summary of the invention

[0004] 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 adds SOD, CAT and zinc-containing compounds to a liquid culture medium to perform tolerance culture on the strain; then enriches and cultures the strain with a liquid culture medium to which zinc-containing compounds and 6-benzyladenine are added; 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.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing zinc-rich phellin polysaccharide, comprising the following steps: (1) adding SOD (superoxide dismutase), CAT (catalase) and a zinc-containing compound to a liquid culture medium and mixing them evenly to obtain a tolerance treatment culture medium; inoculating the Phellinus igniarius strain into the tolerance treatment culture medium for culturing to obtain a tolerance treatment Phellinus igniarius seed liquid; (2) adding a zinc-containing compound and 6-benzyladenine (6-BA) to a liquid culture medium and mixing them evenly to obtain an enriched culture medium; inoculating the tolerant Phellinus linterus seed liquid obtained in step (1) into the enriched culture medium for culturing, separating mycelium after the culturing, washing, freeze-drying, and crushing to obtain zinc-enriched mycelium powder; (3) Zinc-rich mulberry linterus polysaccharide was extracted from zinc-rich mycelium powder by hot water extraction and alcohol precipitation.

[0006] 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 linterus strain in the tolerance treatment medium is 0.1-0.5 g / L; the Phellinus linterus strain is Fasciola baumii, and its preservation number is CFCC7230.

[0007] The preservation number of F. baumii is CFCC7230, and it is preserved in the China Forestry Microbial Culture Collection Management Center.

[0008] 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.

[0009] Preferably, in step (1), the culturing is carried out in a constant temperature shaker; the rotation speed of the constant temperature shaker is 120-180 rpm; the culturing temperature is 27-30° C., and the culturing time is 6-12 days.

[0010] Preferably, in step (2), the liquid culture medium is PAD liquid culture medium; and the zinc-containing compound is zinc sulfate.

[0011] 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.

[0012] Preferably, in step (2), the inoculation amount of the tolerant treated Phellinus linterus 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.

[0013] The second aspect of the present invention provides the application of the above preparation method in increasing the zinc content of zinc-rich Phellinus linterus polysaccharide.

[0014] The third aspect of the present invention provides zinc-rich Phellinus linteus polysaccharide obtained by the above preparation method. The zinc content in the zinc-rich Phellinus linteus polysaccharide is greater than 6 g / kg.

[0015] The fourth aspect of the present invention provides the use of zinc-rich mulberry linterus polysaccharide in the preparation of antibacterial drugs.

[0016] Preferably, the dosage form of the drug is liquid, paste, gel, dressing or spray; the concentration of zinc-rich mulberry linterus polysaccharide in the drug is 100-600 μg / mL.

[0017] The fifth aspect of the present invention provides the use of SOD, CAT and 6-BA in increasing the polysaccharide content of Phellinus linterus mycelium and the zinc content of zinc-rich Phellinus linterus polysaccharide.

[0018] Beneficial effects of the present invention: (1) The present invention successfully obtained a method for culturing zinc-rich mulberry linterus polysaccharides by tolerating and enriching mulberry linterus mycelium. The mulberry linterus strain was cultured in a tolerating medium with high concentrations of zinc to which superoxide dismutase (SOD) and catalase (CAT) were added, thereby achieving rapid tolerance of the mulberry linterus strain, and the tolerance treatment was completed in only one generation. In addition, in the process of enriching the culture medium, in addition to adding high concentrations of zinc, 6-benzyladenine (6-BA) was also added to increase the relative yield of mulberry linterus mycelium and the enrichment of zinc.

[0019] (2) The concentration of trace elements in the culture medium of the present invention is adjustable, and the formula optimization (such as adding 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 linterus polysaccharide. Taking natural mulberry linterus 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 linterus polysaccharide prepared by the present invention has good antibacterial effect, and the antibacterial effect is significantly improved compared with the use of mulberry linterus polysaccharide alone and zinc alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Results of Fourier transform infrared spectrum of zinc-rich phellinus linterus polysaccharide; Figure 2 : The mycelia of Phellinus linteus cultured in Example 1 and Comparative Examples 1 to 5; 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; Figure 4 : The results of measuring the cytotoxicity of zinc-rich phellin polysaccharide; Figure 5 : (a) Representative bright field images of the inhibition zones of Escherichia coli and two strains of MRSA after 16 h of treatment with different patches: (b) The diameters of the inhibition zones of Escherichia coli and two strains of MRSA, each point represents the mean ± SD (n = 3), ***P < 0.001. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0022] As introduced in the background technology section, adding selenium to the culture medium for fermenting mulberry linterus can produce 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 linterus polysaccharides is not high.

[0023] Based on this, the purpose of the present invention is to provide a zinc-rich mulberry linterus 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 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.

[0024] Superoxide dismutase and catalase are used as culture additives, mainly for plant tissue culture and cell culture. During plant tissue culture, SOD can catalyze the conversion of superoxide anions into hydrogen peroxide, and CAT can decompose hydrogen peroxide into water and oxygen, thereby removing ROS, reducing oxidative damage, and improving the survival rate and differentiation rate of explants; during animal cell culture, ROS are produced during cell metabolism, and high concentrations of ROS can damage cells and even cause cell death. As antioxidant enzymes, SOD and CAT can promptly remove ROS produced by cells, protect cells from oxidative stress, and maintain the normal physiological function and activity of cells. When culturing tumor cell lines or normal cell lines, adding SOD and CAT can improve the survival rate and proliferation ability of cells. The Phellinus igniarius strain in the present invention is a fungus, and there are many differences in the physical and chemical properties between fungi and animal and plant cells. For example, fungi have cell walls, while animal and plant cells only have cell membranes; for example, fungi can produce mycelium, which is not a product of fungal proliferation, but is produced by 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 superoxide anion free radicals (O2 -) is an enzyme that dismutates H2O2 and O2. H2O2 has a bactericidal effect, so the present invention also adds CAT, which is a catalase that can catalyze the decomposition of H2O2 into water and oxygen. Therefore, adding SOD and CAT can increase the dissolved oxygen content in the liquid culture medium, and increasing the dissolved oxygen content can accelerate the proliferation of the bacteria to enter the logarithmic growth phase. However, if SOD is added too much, a large amount of H2O2 will be produced, which will directly kill the fungi, so it is also necessary to control the addition amount of SOD and CAT. In addition, SOD and CAT in the culture medium may also improve the tolerance of the strain to zinc ions through other effects in addition to increasing the oxygen content. Because the antibacterial principle of zinc ions mainly has two aspects: one is to combine with the bacterial membrane, thereby destroying the function of the bacterial membrane and killing the bacteria, and the other is to interact with the negative ions of the bacterial cells through the interaction of zinc ions and bacterial cells, resulting in abnormal enzymes and other protein mechanisms in the bacterial cells. In addition, high concentrations of zinc ions can also cause DNA chain breaks in fungi, causing cell necrosis or apoptosis. It can be seen that it is not possible to prevent the antibacterial effect of zinc ions by effectively removing various active oxygen groups. The combined treatment of double enzymes significantly alleviated the oxidative stress damage. The strengthening of this enzymatic defense system enables the mycelium to establish a stable zinc-tolerant system in one generation. On the other hand, in a high-concentration zinc ion environment, this system participates in activating the extracellular polysaccharide (EPS) secreted by the mycelium to wrap the mycelium surface and form a "biofilm". The three-dimensional network structure formed by the polysaccharide can increase the surface roughness of the mycelium and reduce the toxic effect of direct contact with the cell membrane by preferentially adsorbing zinc ions. In short, SOD and CAT synergistically protect the strain to improve zinc ion tolerance through multiple actions. There is no need for multiple domestication, and only one tolerance culture is required, which greatly saves the culture cost.

[0025] 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 added with 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 linterus mycelium and the zinc content in the zinc-rich mulberry linterus polysaccharide.

[0026] The invention realizes efficient and rapid enrichment of zinc in Phellinus igniarius mycelium by adding SOD, CAT and 6-BA. The preparation of the zinc-enriched Phellinus igniarius polysaccharide in the mycelium is convenient and simple, and has significant antibacterial activity.

[0027] 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 in conjunction with specific embodiments.

[0028] 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.

[0029] Example 1 (1) Preparation of Phellinus igniarius mycelium Weigh 200g of potatoes, cut into small pieces of 1cm×1cm×1cm, add 500mL of ultrapure water to the pot, put the cut potato pieces into the pot, and boil for about 20 minutes. When the potato pieces are formed but crushed by light pressure with chopsticks, turn off the heat, wait for cooling, filter twice with eight layers of gauze, remove the residue to obtain potato extract. Add 20g of glucose, 1.5g of KH2PO4, and 0.05g of vitamin B1 to a beaker, dissolve with 300mL of ultrapure water; pour in the potato extract obtained above and mix, dilute to 1L in a volumetric flask with ultrapure water, and sterilize. Add 0.5g of SOD freeze-dried powder, 1.0g of CAT freeze-dried powder, and 1.23g of zinc sulfate to the sterilized culture medium to prepare a tolerant treatment medium with a zinc ion concentration of 500mg / L.

[0030] Use a sterile inoculation needle to pick out a piece of golden mycelium from the Phellinus igniarius strain (Baumyophila) and streak it on a PDA solid culture medium plate. Cultivate it upright in a fungal incubator for 4 days at a constant temperature of 28°C. After four days, golden spores will grow on the plate. Use a sterile inoculation loop to pick up the mycelium of Phellinus igniarius and place it in a tolerant liquid culture medium. Cultivate it in a constant temperature shaker at 28°C and 150rpm for 7 days.

[0031] Put 1.23g zinc sulfate in a centrifuge tube, add sterile water to dissolve, and filter with a 0.22μm filter membrane to obtain a zinc sulfate solution. Add zinc sulfate solution to the sterilized PDA liquid culture medium to make the zinc sulfate concentration in the PDA liquid culture medium 1.23g / L (zinc ion concentration is 500mg / L), and then add 6-BA to make the 6-BA concentration in the PDA liquid culture medium 2g / L to obtain an enriched culture medium. When inoculating, break the bacteria in the tolerant culture medium in the tolerant culture medium, then use a pipette to absorb the seed liquid and inoculate it into the enriched culture medium. Each bottle (1L) of enriched culture medium is inoculated with 500μL seed liquid. Culture at 28℃ for 8 days to obtain the mycelium of Phellinus igniarius.

[0032] (2) Preparation of zinc-rich mulberry linterus polysaccharide: The mulberry linterus mycelium prepared in step (1) was collected by filtering through eight layers of gauze, and the mycelium was rinsed three times with double distilled water to remove free unconsumed sugars in the culture medium. The collected mycelium was freeze-dried at -80°C under a vacuum environment for 3 days, and the mycelium powder was obtained by grinding in a mortar.

[0033] Mycelial powder was weighed and mixed with double distilled water in a ratio of 1:50 (w / v), and stirred at 95°C for 3h using a temperature-controlled magnetic stirrer. The mixture was centrifuged, and the insoluble residue after centrifugation was treated twice as above. The three supernatants were mixed, and most of the water was removed by rotary evaporator at 55°C to prepare 300 mL of concentrated extract. Three volumes of anhydrous ethanol (900 mL) were added to the concentrated extract, and the extract was precipitated with alcohol at 4°C for 12h. The obtained precipitate was centrifuged at 8000rpm at 4°C for 5 min, the supernatant was discarded, 100 mL of anhydrous ethanol was added to the precipitate, the precipitate was vortexed to break up the precipitate, centrifuged at 8000rpm at 4°C for 5 min, the supernatant was discarded, and the above steps were repeated three times. The obtained alcohol-precipitated crude polysaccharide was redissolved with double distilled water, placed in a 4°C refrigerator overnight, centrifuged at 8000rpm at 4°C for 5 min, the precipitate was discarded, and the sugar solution was filtered with filter paper and filter membrane in turn until it was clear. This step was repeated three times.

[0034] 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: the upper, middle and lower layers. The upper layer was the sugar solution, the middle layer was the milky white deformed protein, and the lower layer was the 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 middle milky white deformed protein layer no longer appears after sedimentation. Carefully separate and collect the sugar solution, and purify it using a DEAE-52 cellulose column (3cm×25cm). Through ion adsorption, the positively charged substances are adsorbed on the fiber column, while the neutral polysaccharides can be eluted smoothly. The sugar-containing eluate was collected, dialyzed for desalination for 2 days, and freeze-dried to obtain zinc-rich mulberry linterus polysaccharide. The infrared spectrum of zinc-rich mulberry linterus polysaccharide is shown in Figure 1 .

[0035] Comparative Example 1: Preparation of zinc-rich phellin polysaccharide The difference from Example 1 is that: SOD freeze-dried powder, CAT freeze-dried powder and 6-BA are not added; due to the high zinc ion concentration, the growth of Phellinus linteus mycelium is inhibited, a small amount of Phellinus linteus mycelium is obtained, and zinc-rich Phellinus linteus polysaccharide cannot be extracted; Phellinus linteus mycelium is obtained by expanding the culture by 10 times. Finally, zinc-rich Phellinus linteus polysaccharide is extracted.

[0036] Comparative Example 2: Preparation of zinc-rich phellin polysaccharide The difference from Example 1 is that: SOD freeze-dried powder and 6-BA are not added; a small amount of Phellinus linterus mycelium is obtained, and zinc-rich Phellinus linterus polysaccharide is finally extracted.

[0037] Comparative Example 3: Preparation of zinc-rich phellin polysaccharide 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.

[0038] Comparative Example 4: Preparation of zinc-rich phellin polysaccharide 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, and the mycelium of Phellinus linteus is obtained. Finally, zinc-rich Phellinus linteus polysaccharide is extracted.

[0039] Comparative Example 5: Preparation of zinc-rich phellin polysaccharide The difference from Example 1 is that the concentration of SOD is 1 g / L, the concentration of CAT is 2 g / L, and the mycelium of Phellinus linteus is obtained. Finally, zinc-rich Phellinus linteus polysaccharide is extracted.

[0040] The mycelia of Phellinus linteus cultured in Example 1 and Comparative Examples 1 to 5 were Figure 2 .

[0041] Test Example 1: Composition Determination (1) The mycelia of Phellinus linteus 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: Biomass (g / L) = [total weight (g) - dish weight (g)] / culture medium volume (L).

[0042] 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 mycelium can be obtained by using the method of Example 1.

[0043] (2) The reducing sugar content in the zinc-rich mulberry linterus polysaccharides prepared in Example 1 and Comparative Examples 1 to 5 was determined by the phenol-sulfate method. The protein content was determined by a total protein quantitative test kit (BCA method, purchased from Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 1.

[0044] Table 1 Reducing sugar and protein content in zinc-rich phellin polysaccharide According to Table 1, it can be seen that the content of reducing sugar in the zinc-rich Phellinus linteus polysaccharides prepared in Example 1 and Comparative Examples 1 to 5 is substantially the same, with a difference of about ±1%. Since the method of extracting zinc-rich Phellinus linteus polysaccharides from Phellinus linteus mycelium is the same in Example 1 and Comparative Examples 1 to 5, it is shown that the purity of the zinc-rich Phellinus linteus polysaccharides extracted in Comparative Examples 1 to 5 and Example 1 is substantially the same. Therefore, the comparison of the content of zinc-rich Phellinus linteus polysaccharides extracted in Comparative Examples 1 to 5 and Example 1 and the zinc content in the polysaccharide is comparable.

[0045] 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.

[0046] Take an equal amount of zinc-rich mulberry linterus polysaccharide 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: 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, and after the reaction is completed, cover and seal it, put it into a microwave digestion instrument for digestion, and after the temperature is cooled to below 50°C, take out the digestion tank and put it into a fume hood, open the digestion tank, rinse it with ultrapure water, transfer it to a 25mL volumetric flask, rinse it at least 3 to 4 times, dilute it with ultrapure water to the scale, and test it. The blank control is the above solvent without adding zinc-rich Phellinus linteus polysaccharide.

[0047] The zinc content is calculated according to the formula: ; Where: W——Zinc content in the sample, in mg / kg; C——Zinc concentration in the sample solution, in ug / L; C0——Zinc concentration in blank control, unit: ug / L; V——fixed volume, unit: mL; N——dilution multiple; m – sampling volume of the test specimen, in g. The polysaccharide content obtained from Phellinus igniarius mycelium and the zinc content in the polysaccharide are shown in Table 2.

[0048] Table 2 Polysaccharide content and zinc content in polysaccharides Obtaining more Phellinus linteus mycelia does not mean that 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 method of Example 1 obtains the highest polysaccharide content from the Phellinus linteus mycelia, and the zinc enrichment in the polysaccharide is the highest. Compared with Comparative Example 1, after adding SOD and CAT in Comparative Example 3, the polysaccharide content increased by nearly one-fold, and the polysaccharide zinc content increased 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, only 6-BA is added to the enrichment medium, because the zinc ion concentration is too high, the thalline cannot tolerate it, and the result is similar to Comparative Example 1, and only a small amount of Phellinus linteus mycelia can be obtained. Therefore, 6-BA needs to synergize with SOD and CAT to improve the polysaccharide content and the content of zinc in the polysaccharide.

[0049] Comparative Example 6: Preparation of Phellinus igniarius polysaccharide (1) Use a homogenizer to break up the mycelium of Phellinus linteus, and inoculate 500 μL of liquid PDA medium (PDA liquid medium / L: 200 g potato, 20 g glucose, 1.5 g potassium dihydrogen phosphate, 1 g magnesium sulfate heptahydrate) per bottle. Ferment and culture in a constant temperature shaker at 150 rpm and 28°C for 8 days. After fermentation, separate the mycelium, wash, freeze-dry, weigh, and crush to obtain mycelium powder.

[0050] (2) Phellinus linteus polysaccharides were extracted from the mycelium prepared in step (1) according to the method of Example 1.

[0051] Test Example 2: Cytotoxicity Test The human epidermal immortalized cell line (HaCaT) was used to conduct a cytotoxicity test on the zinc-rich mulberry linterus polysaccharide prepared in Example 1. 2.5-5×10 4 Cells were added to 3, 5, 10, 50, 100, 500, and 1000 μg / ml zinc-rich phellin polysaccharide solution prepared with DMEM and incubated for 20 h. MTT was then added and incubated for 4 h. DMSO was used for color development and the photometric analysis was performed at 490 nm to calculate the cytotoxicity. The results are shown in Figure 4 shown.

[0052] Cell survival rate (%) = (OD drug group-OD blank group) / (OD control group-OD blank group) × 100%; Blank group—containing culture medium and CCK-8 solution, but no cells or drugs; Control group—containing cells, culture medium, and CCK-8 solution without drugs; Drug-added group—contains cells, culture medium, CCK-8 solution and drug solution.

[0053] according to Figure 4 It can be seen that when the zinc-rich mulberry linterus polysaccharide prepared in Example 1 is 1000 μg / mL, the cell survival rate is still around 90%.

[0054] Test Example 3: Antibacterial Test Freshly cultured Escherichia coli (E. coli, deposit number ATCC23519, deposited in China Medical Microbiology Collection Center) and two strains of methicillin-resistant Staphylococcus aureus (MRSA, deposit numbers: ATCC43300 and ATCC33591, deposited in China Medical Microbiology Collection Center) were picked (18-24 hours) and diluted with sterile saline to 1.5×10 8 CFU / mL, and let it stand for 15 minutes after mixing to prevent bacterial precipitation from affecting the coating uniformity.

[0055] Dip the bacterial suspension with a sterile cotton swab and evenly apply it on the surface of the LB solid plate, repeat 3 times to ensure the formation of a uniform bacterial moss, let it stand for 5 minutes to absorb the bacterial liquid, and avoid sliding when the paper is attached. A control group, a positive control group, a mulberry linterinary polysaccharide group prepared in comparative example 6, and a zinc-rich mulberry linterinary polysaccharide group prepared in Example 1 were set. Among them, physiological saline was used as the control group, and a zinc sulfate solution with a concentration of 100 μg / mL was configured with physiological saline and zinc sulfate solid as a positive control. The mulberry linterinary polysaccharide extracted from comparative example 6 and physiological saline were configured with a concentration of 100 μg / mL mulberry linterinary polysaccharide solution as the mulberry linterinary polysaccharide group, and the mulberry linterinary polysaccharide prepared in Example 1 and physiological saline were configured with a concentration of 100 μg / mL zinc-rich mulberry linterinary polysaccharide solution as the zinc-rich mulberry linterinary group. Sterile drug-sensitive paper sheets were added to physiological 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 minutes. Use sterile tweezers to take the drug-sensitive paper, stick it on the surface of the plate, and gently press the paper with the tip of the tweezers to make it flat. The distance between each paper piece should not be less than 24mm, and the center of the paper piece should not be less than 15mm from the edge of the plate. Incubate at 35±2℃ for 16h, and place the plate upright to prevent condensation from affecting the diffusion of the paper. After the plate with the paper piece is incubated for 16h, use a vernier caliper to measure the diameter of the inhibition zone.

[0056] The results are as follows Figure 5 As 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 antibacterial zone is extremely unclear; although Phellinus linteus has an antibacterial effect, it relies on the phenols and flavonoids contained in Phellinus linteus, and Phellinus linteus polysaccharide has no antibacterial effect. Under the same concentration conditions, zinc-rich Phellinus linteus polysaccharide has a significant antibacterial effect. This shows that the zinc-rich Phellinus linteus polysaccharide prepared by the method of the present invention has a significant antibacterial effect.

[0057] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope 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 tolerance treatment medium; the Phellinus linterus strain cultured on a PDA solid is inoculated into the tolerance treatment medium for culturing to obtain a tolerance treatment Phellinus linterus seed liquid; (2) adding a zinc-containing compound and 6-benzyladenine to a liquid culture medium and mixing them evenly to obtain an enriched culture medium; inoculating the tolerant Phellinus linterus seed liquid obtained in step (1) into the enriched culture medium for culturing, separating mycelium after the culturing, washing, freeze-drying, and crushing to obtain zinc-enriched mycelium powder; (3) Zinc-rich mulberry linterus polysaccharide was extracted from zinc-rich mycelium powder by hot water extraction and alcohol precipitation.

2. The preparation method according to claim 1, characterized in that: 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 linterus strain in the tolerance treatment medium is 0.1-0.5 g / mL; the Phellinus linterus strain is Fasciola baumii, and its preservation number is CFCC7230.

3. The preparation method according to claim 2, characterized in that: 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.

4. 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.

5. The preparation method according to claim 1, characterized in that: In step (2), the liquid culture medium is PAD liquid culture medium; the zinc-containing compound is zinc sulfate; the concentration of zinc ions in the liquid culture medium is 0.1-1.0 g / L; and the concentration of 6-benzyladenine in the liquid culture medium is 2 g / L.

6. The preparation method according to claim 1, characterized in that: In step (2), the inoculation amount of the tolerance-treated Phellinus linterus 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.

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

8. Zinc-rich phellin polysaccharide obtained by the preparation method according to any one of claims 1 to 6.

9. Use of the zinc-rich mulberry linterus polysaccharide according to claim 8 in the preparation of antibacterial drugs.

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

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