Culture medium, culture method and preparation method of polysaccharide antigen suitable for Aspergillus

Abstract

The present application relates to a culture medium suitable for Aspergillus, a culture method and a method for preparing a polysaccharide antigen, the culture medium includes the following raw materials: glucose, glycerol, galactose, peptone, peanut sprout extract, germinated brown rice extract, microcrystalline cellulose, D-mannitol, calcium phosphate, zinc sulfate, sodium selenite, glutamine, hydroxyproline and tyrosine, and the remainder is water. The present application provides rich nutrition for the growth of Aspergillus through the coordination of carbon sources, nitrogen sources, plant sprout extracts and amino acids from multiple sources, which is conducive to increasing the yield of galactomannan; through the synergistic coordination of microcrystalline cellulose and calcium phosphate, the volume of the bacterial ball can be reduced, so that the inside and outside of the bacterial ball can obtain enough nutrition and grow stably, so that a galactomannan with a similar degree of polymerization and uniform structure and molecular weight can be obtained. The galactomannan obtained in the present application has a higher antigen immune activity, and the antibody obtained therefrom has the advantage of high titer.

Description

Technical Field

[0001] The present application relates to the field of biological fermentation technology, and more specifically, to a culture medium, a culture method and a preparation method of a polysaccharide antigen suitable for Aspergillus. Background Art

[0002] Aspergillus is a type of fungus, and its main species include Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, etc. Among them, Aspergillus fumigatus (Latin name Aspergillus fumigatus ) is an important fungus that often appears in large numbers in the middle and late stages of grain fever and mildew, promoting the rise of grain temperature and grain spoilage; it is also a pathogenic bacterium that can cause human pulmonary aspergillosis and other diseases, threatening people's health. Therefore, researchers are committed to developing various methods that can quickly and accurately detect Aspergillus fumigatus.

[0003] Studies have found that galactomannan (GM) is present at high levels in the serum of infected animal models and patients with invasive aspergillosis, so it can be used as a marker for diagnosing Aspergillus infection. Aspergillus can be detected by antibodies specific to the galactomannan antigen of Aspergillus fumigatus, with a blood sensitivity of over 80%.

[0004] In the related art, the in vitro culture of Aspergillus fumigatus galactomannan antigen generally involves culturing the Aspergillus strain at 22-30°C for a period of time using Sabouraud medium, then precipitating the polysaccharide in the sterilized fermentation broth with 80% ethanol, and extracting the Aspergillus galactomannan through a certain purification method.

[0005] However, the polymerization degree and composition of galactomannan obtained from Aspergillus fumigatus in liquid fermentation vary greatly, resulting in an inhomogeneous structure of galactomannan. When such galactomannan is used as an antigen for preparing antibodies, the immunogenicity is poor, resulting in a low titer of specific antibodies. Therefore, how to obtain galactomannan with uniform structure and stable molecular weight is an urgent problem to be solved. Summary of the invention

[0006] In order to solve the above technical problems, the present application provides a culture solution, a culture method and a polysaccharide antigen suitable for Aspergillus culture.

[0007] According to the first aspect of the present application, a culture solution suitable for culturing Aspergillus is provided, using the following technical solution:

[0008] A culture medium suitable for Aspergillus, the culture medium comprising a liquid fermentation medium, the liquid fermentation medium comprising the following raw materials:

[0009] Glucose 15000-20000 mg / L, glycerol 5000-10000 mg / L, galactose 2000-6000 mg / L, peptone 5000-15000 mg / L, peanut sprout extract 1000-3000 mg / L, germinated brown rice extract 1000-3000 mg / L, microcrystalline cellulose 5000-20000 mg / L, D-mannitol 300-500 mg / L, calcium phosphate 1000-2000 mg / L, zinc sulfate 10-20 mg / L, sodium selenite 10-20 mg / L, glutamine 10-30 mg / L, hydroxyproline 10-30 mg / L and tyrosine 10-30 mg / L, and the balance is water.

[0010] In some embodiments of the present application, the liquid fermentation medium is prepared by the following method:

[0011] According to the preset mass concentration, glucose, glycerol, galactose, peptone, peanut sprout extract, germinated brown rice extract, microcrystalline cellulose, D-mannitol, calcium phosphate, zinc sulfate and sodium selenite are taken, and water is added to make up to 500-800 mL; then steam sterilized at a temperature of 115-125° C. for 10-30 min to obtain a first mixture; after cooling the first mixture to room temperature, glutamine, hydroxyproline and tyrosine are added, and water is added to make up to 1000 mL to obtain a second mixture; the second mixture is filtered and sterilized with a 0.22-0.45 μm filter membrane to obtain the liquid fermentation medium.

[0012] In some embodiments of the present application, the culture medium includes a seed culture medium, and the seed culture medium includes the following raw materials:

[0013] Glucose 20000-30000 mg / L, glycerol 5000-10000 mg / L, peptone 3000-5000 mg / L, yeast extract 2000-4000 mg / L, microcrystalline cellulose 5000-20000 mg / L, peanut sprout extract 1000-3000 mg / L, germinated brown rice extract 1000-3000 mg / L, potassium dihydrogen phosphate 500-2000 mg / L and magnesium sulfate 200-800 mg / L, and the balance is water.

[0014] In some embodiments of the present application, the seed culture medium is prepared by the following method:

[0015] According to the preset mass concentration, glucose, glycerol, peptone, yeast extract, microcrystalline cellulose, peanut sprout extract, germinated brown rice extract, potassium dihydrogen phosphate and magnesium sulfate are taken, and water is added to make the volume to 1000 mL; then steam sterilized at a temperature of 115-125° C. for 10-30 minutes to obtain the seed culture medium.

[0016] In some embodiments of the present application, the culture medium includes an activated culture medium, and the activated culture medium includes the following raw materials:

[0017] Glucose 10000-15000 mg / L, yeast extract 1000-2000 mg / L, nitrate mother solution 30-40 mL / L, trace element mother solution 1-2 mL / L and agar 15000-200000 mg / L, the balance is water.

[0018] According to a second aspect of the present application, a method for culturing Aspergillus is provided, wherein the liquid fermentation medium is used to perform liquid fermentation on Aspergillus, comprising the following steps:

[0019] Inoculating Aspergillus into the liquid fermentation medium, and culturing with shaking at a temperature of 46-48° C. for 30-40 minutes to obtain a first culture solution;

[0020] Transferring the first culture solution to a shaking culture at a temperature of 20-25° C. for 10-12 hours to obtain a second culture solution;

[0021] The second culture solution was transferred to a shaking culture at a temperature of 30-35° C. for 48-72 hours, and then the culture was stopped when the pH of the system was 4.2-4.5.

[0022] In some embodiments of the present application, the inoculation amount of Aspergillus is 2-8%, and the speed of the shaking culture is 180-200 rpm.

[0023] In some embodiments of the present application, before inoculating Aspergillus into the liquid fermentation medium, the culture method further comprises using the above-mentioned activation medium to activate the Aspergillus and using the above-mentioned seed culture medium to perform seed culture on the Aspergillus, comprising the following steps:

[0024] Inoculating Aspergillus into the activation culture medium for activation culture, and ending the culture after the Aspergillus spores mature;

[0025] The Aspergillus spores are washed with sterile physiological saline and then a monospore suspension is prepared with the sterile physiological saline;

[0026] The monospore suspension is inoculated into the seed culture medium for seed culture to obtain Aspergillus seed culture solution.

[0027] In some embodiments of the present application, the conditions for the activation culture are: culturing at a temperature of 28-30°C for 3-5 days; the conditions for the seed culture are: culturing at a temperature of 28-30°C for 5-7 days.

[0028] According to the third aspect of the present application, a method for preparing a polysaccharide antigen is provided, comprising the following steps:

[0029] The Aspergillus niger is cultured by the above-mentioned culture method to obtain a bacterial liquid;

[0030] After sterilizing the bacterial solution, filtering to remove the bacterial bodies, and obtaining a filtrate;

[0031] The filtrate is centrifuged to obtain a crude polysaccharide extract;

[0032] The polysaccharide antigen is obtained by purifying the polysaccharide crude extract.

[0033] In summary, this application has the following beneficial effects:

[0034] The present application uses glucose, glycerol, galactose, microcrystalline cellulose and peptone as different carbon sources and nitrogen sources, and through the synergistic coordination of plant bud extracts, amino acids and inorganic salts, it can promote cell adhesion and cell growth in the case of removing animal tissue fluid and serum to increase the yield of galactomannan. Through the synergistic coordination of microcrystalline cellulose and calcium phosphate, the volume of the bacterial ball can be reduced, so that the inside and outside of the bacterial ball can grow stably, so that galactomannan with similar degree of polymerization and uniform structure and molecular weight can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0036] Figure 1 This is a liquid phase diagram of the purification and preparation of Aspergillus galactomannan in Example 1. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application. It should be noted that, in the absence of conflict, the embodiments in the present application and the features in the embodiments can be arbitrarily combined with each other.

[0038] During the liquid fermentation culture, Aspergillus fumigatus is very easy to form balls and aggregate, which makes the balls too large. The large balls will lead to the lack of nutrition inside the balls, resulting in the unstable growth state of Aspergillus fumigatus. The polymerization degree and composition of the galactomannan obtained vary greatly, resulting in the inhomogeneous structure of galactomannan, which often contains octamers, hexamers, etc. When such galactomannan is used as an antigen for preparing antibodies, the immunological expression is poor, which will lead to a low titer of specific antibodies.

[0039] Based on this, in an exemplary embodiment, the present application provides a culture medium suitable for Aspergillus, the culture medium includes a liquid fermentation medium, and the liquid fermentation medium includes the following raw materials:

[0040] Glucose 15000-20000 mg / L, glycerol 5000-10000 mg / L, galactose 2000-6000 mg / L, peptone 5000-15000 mg / L, peanut sprout extract 1000-3000 mg / L, microcrystalline cellulose 5000-20000 mg / L, D-mannitol 300-500 mg / L, calcium phosphate 1000-2000 mg / L, zinc sulfate 10-20 mg / L, sodium selenite 10-20 mg / L, glutamine 10-30 mg / L, hydroxyproline 10-30 mg / L and tyrosine 10-30 mg / L, and the balance is water.

[0041] In order to obtain galactomannan with uniform structure and stable molecular weight, the present application adjusts the formula of the liquid fermentation medium and adds microcrystalline cellulose on the basis of ordinary Sabouraud liquid medium. The main component of microcrystalline cellulose is a straight-chain polysaccharide substance bonded by β-1,4-glucosidic bonds, which can not only serve as a polysaccharide carbon source to provide nutrition for the growth of Aspergillus, but also has a synergistic effect with calcium phosphate, which can reduce the volume of the bacterial ball and enable the bacterial ball to grow stably inside and outside. In this way, galactomannan with similar degree of polymerization and uniform structure and molecular weight can be obtained.

[0042] Normally, animal tissue fluid or serum contains a variety of hormones, growth factors, adhesion factors and nutrients, which can promote cell growth and increase the production of their metabolites. However, due to the complex composition of animal tissue fluid and serum, the composition of animal tissue fluid and serum in different batches varies greatly. The culture medium with added animal tissue fluid and serum has large uncontrollable factors, which will affect the instability of the galactomannan molecular structure during Aspergillus culture.

[0043] Therefore, in order to avoid the influence of animal tissue fluid or serum on the molecular structure stability of galactomannan during Aspergillus culture, no animal tissue fluid or serum is added to the liquid fermentation medium of the present application. Instead, glucose, glycerol, galactose, microcrystalline cellulose, and peptone are used as different carbon sources and nitrogen sources. In addition, peanut sprout extract, germinated brown rice extract, amino acids, and inorganic salts are synergistically combined to promote cell adhesion and cell growth while removing animal tissue fluid and serum, thereby increasing the yield of galactomannan.

[0044] By using the liquid fermentation medium of the present application, the volume of the bacterial ball can be reduced, so that the bacteria can form a small and uniform bacterial ball, and the inside and outside of the bacterial ball can grow stably, thereby improving the stability of the structure and molecular weight of galactomannan. In this way, galactomannan with high purity, large yield, similar degree of polymerization, and uniform structure and molecular weight can be obtained.

[0045] In the present application, the peanut sprout extract is obtained by germinating peanuts, grinding the peanut sprouts, and extracting them with alcohol. Peanut sprouts are rich in vitamins, amino acids, and trace elements, which can provide nutrition for the growth of Aspergillus.

[0046] Exemplarily, the peanut sprout extract is prepared by the following method: After washing the peanuts, immerse them in water at a temperature of 70-80°C for 5-10 minutes; then soak the peanuts soaked for the first time in water at 20-30°C for 10-12 hours; then take out the peanuts soaked for the second time from the water, spread them flat in a container, and cover the peanuts soaked for the second time with moistened gauze, keep the gauze moist at a temperature of 20-30°C for 5-10 days, and obtain the peanut sprouts. Cut the peanut sprouts from the peanuts, grind the peanut sprouts, extract them with a volume fraction of 60-70% ethanol solution, filter to remove the residue, and concentrate and dry the filtrate to obtain the peanut sprout extract.

[0047] Germinated brown rice extract is obtained by grinding and alcohol extraction of the germinated brown rice. Since brown rice retains the rice bran and germ, after germination, the active enzymes are activated and released, so that the germinated brown rice is not only rich in vitamins and trace elements, but also contains growth factors such as glutathione and γ-aminobutyric acid, which can provide nutrition for the growth of Aspergillus.

[0048] Exemplarily, the germinated brown rice extract is prepared by the following method: after the brown rice is cleaned, it is immersed in water and soaked at a temperature of 70-80°C for 5-10 minutes; then the brown rice soaked for the first time is soaked in water at 20-30°C for 10-12 hours; then the brown rice soaked for the second time is taken out of the water, spread in a container, and the wet gauze is covered on the brown rice soaked for the second time, and the gauze is kept wet at a temperature of 20-30°C for 3-5 days to obtain the germinated brown rice. After the germinated brown rice is ground, it is extracted with a volume fraction of 60-70% ethanol solution, filtered to remove the filter residue, and the filtrate is concentrated and dried to obtain the germinated brown rice extract.

[0049] In an exemplary embodiment, the liquid fermentation medium is prepared by the following method:

[0050] According to the preset mass concentration, glucose, glycerol, galactose, peptone, peanut sprout extract, germinated brown rice extract, microcrystalline cellulose, D-mannitol, calcium phosphate, zinc sulfate and sodium selenite are taken, and water is added to make up to 500-800 mL; then steam sterilized at a temperature of 115-125° C. for 10-30 min to obtain a first mixture; after cooling the first mixture to room temperature, glutamine, hydroxyproline and tyrosine are added, and water is added to make up to 1000 mL to obtain a second mixture; the second mixture is filtered and sterilized with a 0.22-0.45 μm filter membrane to obtain a liquid fermentation medium.

[0051] The present application can remove harmful bacteria in liquid fermentation culture medium by high-temperature steam sterilization; in order to avoid the influence of high temperature on amino acids, after obtaining the first mixture, glutamine, hydroxyproline and tyrosine are added, and then filtered and sterilized through a 0.22-0.45μm filter membrane, which can retain the activity of effective ingredients in the culture medium while removing bacteria and spores.

[0052] Exemplarily, the liquid fermentation medium is prepared by the following method:

[0053] Take 15000 mg of glucose, 5000 mg of glycerol, 2000 mg of galactose, 5000 mg of peptone, 1000 mg of peanut sprout extract, 1000 mg of fermented brown rice extract, 5000 mg of microcrystalline cellulose, 300 mg of D-mannitol, 1000 mg of calcium phosphate, 10 mg of zinc sulfate, and 10 mg of sodium selenite, add water to make up to 800 mL; then steam sterilize at a temperature of 115°C for 30 min to obtain a first mixture; after cooling the first mixture to room temperature, add 10 mg of glutamine, 10 mg of hydroxyproline and 10 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0054] Exemplarily, the liquid fermentation medium is prepared by the following method:

[0055] Take 20000 mg of glucose, 10000 mg of glycerol, 6000 mg of galactose, 15000 mg of peptone, 3000 mg of peanut sprout extract, 3000 mg of germinated brown rice extract, 20000 mg of microcrystalline cellulose, 500 mg of D-mannitol, 2000 mg of calcium phosphate, 20 mg of zinc sulfate, and 20 mg of sodium selenite, add water to make up to 500 mL; then steam sterilize at a temperature of 125°C for 10 min to obtain a first mixture; after cooling the first mixture to room temperature, add 30 mg of glutamine, 30 mg of hydroxyproline and 30 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0056] In an exemplary embodiment, the culture medium includes a seed culture medium, and the seed culture medium includes the following raw materials:

[0057] Glucose 20000-30000 mg / L, glycerol 5000-10000 mg / L, peptone 3000-5000 mg / L, yeast extract 2000-4000 mg / L, microcrystalline cellulose 5000-20000 mg / L, peanut sprout extract 1000-3000 mg / L, germinated brown rice extract 1000-3000 mg / L, potassium dihydrogen phosphate 500-2000 mg / L and magnesium sulfate 200-800 mg / L, and the balance is water.

[0058] The seed culture medium is a culture medium used for expanding the culture of Aspergillus. In this embodiment, carbon sources, nitrogen sources and inorganic salts from multiple sources are used to supply the necessary nutrients required for spore germination and growth, and promote the massive reproduction of the fungus, making the fungus become thick and have strong vitality, so as to obtain Aspergillus seeds with strong vitality.

[0059] In an exemplary embodiment, the seed culture medium is prepared by the following method:

[0060] According to the preset mass concentration, glucose, glycerol, peptone, yeast extract, microcrystalline cellulose, peanut sprout extract, germinated brown rice extract, potassium dihydrogen phosphate and magnesium sulfate are taken, and water is added to make the volume to 1000 mL; then steam sterilize at a temperature of 115-125° C. for 10-30 minutes to obtain a seed culture medium.

[0061] Exemplarily, the seed culture medium is prepared by the following method:

[0062] Take 20000 mg of glucose, 5000 mg of glycerol, 3000 mg of peptone, 2000 mg of yeast extract, 5000 mg of microcrystalline cellulose, 1000 mg of peanut sprout extract, 1000 mg of germinated brown rice extract, 500 mg of potassium dihydrogen phosphate and 200 mg of magnesium sulfate, add water to make the volume to 1000 mL; then steam sterilize at 115°C for 30 min to obtain a seed culture medium.

[0063] Exemplarily, the seed culture medium is prepared by the following method:

[0064] 30000 mg of glucose, 10000 mg of glycerol, 5000 mg of peptone, 4000 mg of yeast extract, 20000 mg of microcrystalline cellulose, 3000 mg of peanut sprout extract, 3000 mg of germinated brown rice extract, 2000 mg of potassium dihydrogen phosphate and 800 mg of magnesium sulfate were added with water to make the volume to 1000 mL; then, the mixture was steam sterilized at 125°C for 10 min to obtain a seed culture medium.

[0065] In an exemplary embodiment, the culture medium includes an activated culture medium, and the activated culture medium includes the following raw materials:

[0066] Glucose 10000-15000 mg / L, yeast extract 1000-2000 mg / L, nitrate mother solution 30-40 mL / L, trace element mother solution 1-2 mL / L and agar 15000-200000 mg / L, the balance is water.

[0067] In this embodiment, the activation culture medium can activate the Aspergillus species in the preserved state and restore their activity.

[0068] Exemplarily, the activation medium is prepared by the following method:

[0069] Take 10000 mg of glucose, 1000 mg of yeast extract, 30 mL of nitrate stock solution, 1 mL of trace element stock solution and 15000 mg of agar, add 500 mL of water and stir evenly, then add water to make up to 1000 mL; then sterilize at 121°C for 15 min to obtain an activated culture medium.

[0070] Exemplarily, the activation medium is prepared by the following method:

[0071] Take 15000 mg of glucose, 2000 mg of yeast extract, 40 mL of nitrate stock solution, 2 mL of trace element stock solution, and 200000 mg of agar, add 500 mL of water and stir evenly, then add water to make up to 1000 mL; then sterilize at 121°C for 15 min to obtain an activated culture medium.

[0072] Among them, the raw materials of nitrate mother liquor include: NaNO3 120 g / L, KCl 10.4 g / L, MgSO4•7H2O 10.4 g / L, K2HPO4•3H2O 30.4 g / L, and the balance is distilled water.

[0073] The raw materials of trace element mother solution include: ZnSO4•7H2O 0.22 g / L, H3BO3 0.11 g / L, MnCl2•4H2O 0.05 g / L, FeSO4•7H2O 0.016 g / L, CoCl2•5H2O 0.016 g / L, CuSO4•5H2O 0.016 g / L, (NH4)6Mo7O 24 •4H2O 0.011 g / L, Na2EDTA 0.5 g / L, the balance is distilled water.

[0074] In an exemplary embodiment, the present application provides a method for culturing Aspergillus, comprising the following steps:

[0075] Inoculating Aspergillus into a liquid fermentation medium, and culturing with shaking at a temperature of 46-48° C. for 30-40 minutes to obtain a first culture solution;

[0076] The first culture solution was transferred to a shaking culture at a temperature of 20-25° C. for 10-12 hours to obtain a second culture solution;

[0077] The second culture solution was transferred to a shaking culture at a temperature of 30-35°C for 48-72 hours, and then the culture was stopped when the pH of the system was 4.2-4.5.

[0078] In this embodiment, by optimizing the culture conditions, the Aspergillus spores are first cultured at a temperature of 46-48° C. for a short time, and then cultured at a temperature of 20-25° C. The Aspergillus spores cultured under this temperature difference can form a ball morphology with small balls and many hyphae branches. This small and uniform ball can allow the inside and outside of the ball to absorb sufficient nutrients, thereby increasing the yield of galactomannan in the culture solution.

[0079] Exemplarily, a method for culturing Aspergillus comprises the following steps:

[0080] Inoculating Aspergillus into a liquid fermentation medium, and culturing with shaking at a temperature of 46° C. for 30 minutes to obtain a first culture solution;

[0081] The first culture solution was transferred to a shaking culture at 20° C. for 10 h to obtain a second culture solution;

[0082] The second culture solution was transferred to shaking culture at 30°C for 48 hours, and then the culture was stopped when the pH of the system reached 4.2.

[0083] Exemplarily, a method for culturing Aspergillus comprises the following steps:

[0084] Inoculating Aspergillus into a liquid fermentation medium, and culturing with shaking at a temperature of 48° C. for 40 minutes to obtain a first culture solution;

[0085] The first culture solution was transferred to a shaking culture at 25° C. for 12 h to obtain a second culture solution;

[0086] The second culture solution was transferred to a shaking culture at 35°C for 72 hours, and then the culture was stopped when the pH of the system reached 4.5.

[0087] In an exemplary embodiment, the inoculation amount of Aspergillus is 2-8%, and the shaking culture speed is 180-200 rpm.

[0088] Exemplarily, the inoculation amount of Aspergillus is 2%, and the speed of shaking culture is 180 rpm.

[0089] Exemplarily, the inoculation amount of Aspergillus is 8%, and the speed of shaking culture is 200 rpm.

[0090] In an exemplary embodiment, the present application provides a method for culturing Aspergillus, comprising the following steps:

[0091] Inoculating Aspergillus on an activation culture medium for activation culture, and terminating the culture after the Aspergillus spores mature;

[0092] After washing the Aspergillus spores with sterile saline, the Aspergillus spores are prepared into a single spore suspension with sterile saline;

[0093] inoculating the monospore suspension into a seed culture medium for seed culture to obtain an Aspergillus seed culture solution;

[0094] Inoculating the Aspergillus seed culture solution into the liquid fermentation medium, and shaking culture at a temperature of 46-48° C. for 30-40 minutes to obtain a first culture solution;

[0095] The first culture solution was transferred to a shaking culture at a temperature of 20-25° C. for 10-12 hours to obtain a second culture solution;

[0096] The second culture solution was transferred to a shaking culture at a temperature of 30-35°C for 48-72 hours, and then the culture was stopped when the pH of the system was 4.2-4.5.

[0097] In an exemplary embodiment, the conditions for activation culture are: culture at a temperature of 28-30° C. for 3-5 days; and the conditions for seed culture are: culture at a temperature of 28-30° C. for 5-7 days.

[0098] Illustratively, the conditions for activation culture are: culture at a temperature of 28° C. for 5 days; and the conditions for seed culture are: culture at a temperature of 28° C. for 7 days.

[0099] Illustratively, the conditions for activation culture are: culture at a temperature of 30° C. for 3 days; and the conditions for seed culture are: culture at a temperature of 30° C. for 5 days.

[0100] In an exemplary embodiment, the present application provides a method for preparing a polysaccharide antigen, comprising the following steps:

[0101] Inoculating Aspergillus into an activation culture medium for activation culture, and terminating the culture after the Aspergillus spores mature;

[0102] After washing the Aspergillus spores with sterile saline, the Aspergillus spores are prepared into a single spore suspension with sterile saline;

[0103] inoculating the monospore suspension into a seed culture medium for seed culture to obtain an Aspergillus seed culture solution;

[0104] Inoculating the Aspergillus seed culture solution into the liquid fermentation medium, and shaking culture at a temperature of 46-48° C. for 30-40 minutes to obtain a first culture solution;

[0105] The first culture solution was transferred to a shaking culture at a temperature of 20-25° C. for 10-12 hours to obtain a second culture solution;

[0106] The second culture solution is transferred to a shaking culture at a temperature of 30-35°C for 48-72 hours, and when the pH of the system is 4.2-4.5, the culture is stopped to obtain a bacterial solution;

[0107] After sterilizing the bacterial solution, the bacterial bodies are removed by filtration to obtain a filtrate;

[0108] The filtrate is centrifuged to obtain a crude polysaccharide extract;

[0109] The polysaccharide crude extract is purified to obtain the polysaccharide antigen.

[0110] Galactomannan exists in the cell wall of Aspergillus and is released into the extracellular fluid during the growth of Aspergillus. Therefore, galactomannan can be extracted from the cell wall of Aspergillus or from its culture fluid. Since the molecular weight of polysaccharides extracted from the cell wall is not uniform and the structure is relatively complex, compared with the cell wall polysaccharides, the polysaccharides extracted from the extracellular fluid have a higher purity and a uniform molecular weight. Therefore, in order to obtain galactomannan with uniform structure and stable molecular weight, this embodiment extracts galactomannan from the extracellular fluid, that is, after sterilizing the bacterial solution obtained by Aspergillus culture, the bacterial body is filtered to remove the bacterial body to avoid the galactomannan present in the cell wall of Aspergillus from entering the filtrate when the bacterial body is ruptured, thereby obtaining galactomannan with uniform structure, stable molecular weight and high purity.

[0111] In addition, since the present application adjusts the culture medium and culture method of Aspergillus, the fungus can be formed into small and uniform fungus balls, which can improve oxygen mass transfer to improve the growth stability of Aspergillus, thereby increasing the yield of galactomannan in the extracellular fluid.

[0112] In order to more clearly explain the technical solution of the present application, the present application lists specific examples of culture fluid for culturing Aspergillus, and the beneficial effects of selecting the above-mentioned ranges of the content of each component will be explained by giving specific experimental data through specific examples.

[0113] It should be noted that, unless otherwise specified, the raw materials of the following examples can be obtained from commercial sources. Aspergillus fumigatus ), the strain is ATCC13073.

[0114] Embodiment 1: A method for preparing galactomannan comprises the following steps:

[0115] 1. Preparation of activation medium:

[0116] Take 12000 mg of glucose, 1500 mg of yeast extract, 35 mL of nitrate stock solution, 1.5 mL of trace element stock solution and 18000 mg of agar, add 500 mL of water and stir evenly, then add water to make up to 1000 mL; then sterilize at 121°C for 15 min to obtain an activated culture medium.

[0117] Among them, the raw materials of nitrate mother liquor include: NaNO3 120 g / L, KCl 10.4 g / L, MgSO4•7H2O 10.4 g / L, K2HPO4•3H2O 30.4 g / L, and the balance is distilled water.

[0118] The raw materials of trace element mother solution include: ZnSO4•7H2O 0.22 g / L, H3BO3 0.11 g / L, MnCl2•4H2O0.05 g / L, FeSO4•7H2O 0.016 g / L, CoCl2•5H2O 0.016 g / L, CuSO4•5H2O 0.016 g / L, (NH4)6Mo7O 24 •4H2O 0.011 g / L, Na2EDTA 0.5 g / L, the balance is distilled water.

[0119] 2. Preparation of seed culture medium:

[0120] Take 25000 mg of glucose, 8000 mg of glycerol, 4000 mg of peptone, 3000 mg of yeast extract, 10000 mg of microcrystalline cellulose, 2000 mg of peanut sprout extract, 2000 mg of germinated brown rice extract, 1000 mg of potassium dihydrogen phosphate and 500 mg of magnesium sulfate, add water to make the volume to 1000 mL; then steam sterilize at 121°C for 15 min to obtain a seed culture medium.

[0121] The peanut sprout extract is prepared by the following method: washing the peanuts, immersing them in water, and soaking them at 75°C for 8 minutes; then soaking the peanuts soaked for the first time in water at 25°C for 11 hours; then taking the peanuts soaked for the second time out of the water, laying them flat in a container, and covering the peanuts soaked for the second time with moistened gauze, keeping the gauze moist at 25°C for 8 days, and obtaining the peanut sprouts. The peanut sprouts are cut off from the peanuts, ground, extracted with a 65% ethanol solution, filtered to remove the residue, and the filtrate is concentrated and dried to 60% of the original volume to obtain the peanut sprout extract.

[0122] The germinated brown rice extract was prepared by the following method: the brown rice was washed and immersed in water at 75°C for 8 minutes; the brown rice soaked for the first time was then soaked in water at 25°C for 11 hours; the brown rice soaked for the second time was taken out of the water and spread in a container, and the brown rice soaked for the second time was covered with wet gauze, and the gauze was kept wet at 25°C for 4 days to obtain the germinated brown rice. The germinated brown rice was ground and extracted with a 65% ethanol solution by volume, and the residue was removed by filtration, and the filtrate was concentrated and dried to 60% of the original volume to obtain the germinated brown rice extract.

[0123] 3. Preparation of liquid fermentation medium:

[0124] Take 18000 mg of glucose, 8000 mg of glycerol, 4000 mg of galactose, 10000 mg of peptone, 2000 mg of peanut sprout extract, 2000 mg of germinated brown rice extract, 10000 mg of microcrystalline cellulose, 400 mg of D-mannitol, 1500 mg of calcium phosphate, 15 mg of zinc sulfate and 15 mg of sodium selenite, add water to make up to 500 mL; then steam sterilize at a temperature of 121°C for 15 min to obtain a first mixture; after cooling the first mixture to room temperature, add 20 mg of glutamine, 20 mg of hydroxyproline and 20 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0125] 4. Preparation of bacterial solution:

[0126] (1) Inoculate Aspergillus into activation culture medium for activation culture at 28°C for 5 days. When the Aspergillus spores mature, terminate the culture. Wash the Aspergillus spores with sterile saline and mix with sterile saline to make 10 6 / mL of single spore suspension;

[0127] (2) inoculating the monospore suspension into a seed culture medium for seed culture, and culturing at 30° C. for 6 days to obtain an Aspergillus seed culture solution;

[0128] (3) Inoculate the Aspergillus seed culture medium at an inoculum rate of 6% into the liquid fermentation medium, and culture the culture medium at a temperature of 48°C and a rotation speed of 180 rpm for 35 min to obtain a first culture medium; transfer the first culture medium to a temperature of 22°C and a rotation speed of 180 rpm for 11 h to obtain a second culture medium; transfer the second culture medium to a temperature of 32°C and a rotation speed of 180 rpm for 72 h, and stop the culture when the pH of the system is 4.4 to obtain a bacterial liquid.

[0129] 5. Preparation of galactomannan:

[0130] (1) The bacterial solution was sterilized by wet heat sterilization at 121°C for 40 minutes, and the bacterial cells were removed by filtration using a 0.22 μm filter membrane to obtain a filtrate; the filtrate was transferred to a centrifuge tube, and anhydrous ethanol 4 times the volume of the filtrate was added, and the mixture was allowed to stand overnight at 4°C; then the mixture was centrifuged at 15000 g for 15 minutes to obtain a primary precipitate; the primary precipitate was dissolved in deionized water, and the above centrifugation operation was repeated to obtain a secondary precipitate; the secondary precipitate was dissolved in deionized water, and activated carbon was added under stirring, and the mixture was decolorized at room temperature for 8 hours, and then a 0.22 μm filter membrane was used to discard the supernatant to obtain a tertiary precipitate, which was transferred to a 10KD centrifugal ultrafiltration tube, and after discarding the supernatant, the mixture was centrifuged at 5000 g for 10 minutes to obtain a crude polysaccharide extract;

[0131] (2) The crude polysaccharide extract was treated with anhydrous hydrazine and sodium nitrite, and then treated with a Mono Q anion exchange column, a ConA-Sepharose 4B affinity chromatography column, and a gel chromatography column to purify galactomannan. Figure 1 The liquid phase diagram for the purification of Aspergillus galactomannan. The purification method in this embodiment is a prior art and will not be described in detail here.

[0132] Example 2: The main difference between this example and Example 1 is that the liquid fermentation medium is prepared by the following method:

[0133] Take 15000 mg of glucose, 5000 mg of glycerol, 2000 mg of galactose, 5000 mg of peptone, 1000 mg of peanut sprout extract, 1000 mg of fermented brown rice extract, 5000 mg of microcrystalline cellulose, 300 mg of D-mannitol, 1000 mg of calcium phosphate, 10 mg of zinc sulfate and 10 mg of sodium selenite, add water to make up to 800 mL; then steam sterilize at a temperature of 115°C for 30 min to obtain a first mixture; after cooling the first mixture to room temperature, add 10 mg of glutamine, 10 mg of hydroxyproline and 10 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0134] Example 3: The main difference between this example and Example 1 is that the liquid fermentation medium is prepared by the following method:

[0135] Take 20000 mg of glucose, 10000 mg of glycerol, 6000 mg of galactose, 15000 mg of peptone, 3000 mg of peanut sprout extract, 3000 mg of germinated brown rice extract, 20000 mg of microcrystalline cellulose, 500 mg of D-mannitol, 2000 mg of calcium phosphate, 20 mg of zinc sulfate and 20 mg of sodium selenite, add water to make up to 500 mL; then steam sterilize at a temperature of 125°C for 10 min to obtain a first mixture; after cooling the first mixture to room temperature, add 30 mg of glutamine, 30 mg of hydroxyproline and 30 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0136] Example 4: The main difference between this example and Example 1 is that the seed culture medium is prepared by the following method: take 47000 mg of glucose, 4000 mg of peptone, 3000 mg of yeast extract, 1000 mg of potassium dihydrogen phosphate and 500 mg of magnesium sulfate, add water to make the volume to 1000 mL; then steam sterilize at 121°C for 15 min to obtain the seed culture medium.

[0137] Example 5: The main difference between this example and Example 1 is that the method for preparing the bacterial solution includes the following steps:

[0138] (1) Inoculate Aspergillus into activation culture medium for activation culture at 28°C for 5 days. When the Aspergillus spores mature, terminate the culture. Wash the Aspergillus spores with sterile saline and mix with sterile saline to make 10 6 / mL of single spore suspension;

[0139] (2) inoculating the monospore suspension into a seed culture medium for seed culture, and culturing at 30° C. for 6 days to obtain an Aspergillus seed culture solution;

[0140] (3) The Aspergillus seed culture fluid was inoculated into the liquid fermentation medium at an inoculum rate of 6%, and the culture was shaken at 180 rpm at a temperature of 32°C for 72 h. When the pH of the system reached 4.4, the culture was stopped to obtain a bacterial liquid.

[0141] Comparative Example 1: The main difference between this example and Example 1 is that the liquid fermentation medium is prepared by the following method:

[0142] Take 40000 mg of glucose, 10000 mg of peptone, 15 mg of zinc sulfate and 15 mg of sodium selenite, add water to make up to 500 mL; then steam sterilize at 121°C for 15 min to obtain a first mixture; after cooling the first mixture to room temperature, add 20 mg of glutamine, 20 mg of hydroxyproline and 20 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0143] Comparative Example 2: The main difference between this example and Example 1 is that the liquid fermentation medium is prepared by the following method:

[0144] Take 32000 mg of glucose, 8000 mg of glycerol, 4000 mg of galactose, 10000 mg of peptone, 400 mg of D-mannitol, 1500 mg of calcium phosphate, 15 mg of zinc sulfate and 15 mg of sodium selenite, add water to make up to 500 mL; then steam sterilize at 121°C for 15 min to obtain a first mixture; after cooling the first mixture to room temperature, add 20 mg of glutamine, 20 mg of hydroxyproline and 20 mg of tyrosine, stir evenly, add water to make up to 1000 mL to obtain a second mixture; filter the second mixture with a 0.45 μm filter membrane for sterilization to obtain a liquid fermentation medium.

[0145] Performance Testing

[0146] The polysaccharide antigens prepared in the examples and comparative examples were used as samples. The performance of the samples was tested according to the following method, and the test results are recorded in Table 1.

[0147] 1. Bacterial morphology: On the third day of the fermentation culture of Aspergillus fumigatus, the bacterial morphology of Aspergillus fumigatus was observed and the diameter of the bacterial ball was measured.

[0148] 2. Production of galactomannan: The polysaccharide content of the samples was determined by the Dubois-sulfuric acid phenol method. The volume of the bacterial liquid after Aspergillus fumigatus fermentation was 1000 mL.

[0149] 3. Purity of galactomannan: The samples were subjected to HPLC analysis using high performance gel chromatography.

[0150] Table 1

[0151]

[0152] Combining the data in Table 1 and Example 1, it can be seen that the bacteria in Example 1 are dispersed hyphae and small balls, the average diameter of the balls is less than 1 mm, and the obtained galactomannan is 3.541 g / L, and the purity is 96.2%, indicating that the galactomannan prepared by the method of Example 1 of the present application has the advantages of high yield and high purity. The molecular weight Mw of the sample determined by high-performance gel chromatography analysis is 20498 Da; the sample is subjected to HPLC detection by high-performance gel chromatography analysis, and the sample peak is single, sharp and narrow, and there is no obvious miscellaneous peak, indicating that the galactomannan obtained in Example 1 of the present application is uniform in size and high in purity.

[0153] Combining the data in Table 1, Examples 1-5 and Comparative Examples 1-2, it can be seen that when Aspergillus is cultured, changes in the formula of the liquid fermentation medium, the formula of the seed medium and the Aspergillus culture conditions will have different degrees of influence on the growth of Aspergillus, thereby affecting the uniformity, purity and yield of the galactomannan structure.

[0154] In summary, the present application adjusts the culture medium and culture method of Aspergillus so that the fungus can form small and uniform fungal balls, improve oxygen mass transfer, and thus improve the growth stability of Aspergillus, thereby increasing the yield of galactomannan in the extracellular fluid.

[0155] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for culturing Aspergillus, characterized in that: The steps include: Inoculating Aspergillus into a liquid fermentation medium, and culturing with shaking at a temperature of 46-48° C. for 30-40 minutes to obtain a first culture solution; Transferring the first culture solution to a shaking culture at a temperature of 20-25° C. for 10-12 hours to obtain a second culture solution; The second culture solution is transferred to a shaking culture at a temperature of 30-35° C. for 48-72 hours, and the culture is stopped when the pH of the system is 4.2-4.5; The liquid fermentation medium is composed of the following raw materials: Glucose 15000-20000 mg / L, glycerol 5000-10000 mg / L, galactose 2000-6000 mg / L, peptone 5000-15000 mg / L, peanut sprout extract 1000-3000 mg / L, germinated brown rice extract 1000-3000 mg / L, microcrystalline cellulose 5000-20000 mg / L, D-mannitol 300-500 mg / L, calcium phosphate 1000-2000 mg / L, zinc sulfate 10-20 mg / L, sodium selenite 10-20 mg / L, glutamine 10-30 mg / L, hydroxyproline 10-30 mg / L and tyrosine 10-30 mg / L, the balance is water; The peanut sprout extract is obtained by germinating peanuts to obtain peanut sprouts, grinding the peanut sprouts, and extracting with an ethanol solution; The germinated brown rice extract is obtained by germinating brown rice to obtain germinated brown rice, and then grinding the germinated brown rice and extracting it with an ethanol solution; Before inoculating Aspergillus into the liquid fermentation medium, the culture method further comprises using an activation medium to activate the Aspergillus and using a seed culture medium to perform seed culture on the Aspergillus, comprising the following steps: Inoculating Aspergillus into the activation culture medium for activation culture, and ending the culture after the Aspergillus spores mature; The Aspergillus spores are washed with sterile saline and then a monospore suspension is prepared with the sterile saline; inoculating the monospore suspension into the seed culture medium for seed culture to obtain Aspergillus seed culture solution; The seed culture medium is composed of the following raw materials: Glucose 20000-30000 mg / L, glycerol 5000-10000 mg / L, peptone 3000-5000 mg / L, yeast extract 2000-4000 mg / L, microcrystalline cellulose 5000-20000 mg / L, peanut sprout extract 1000-3000 mg / L, germinated brown rice extract 1000-3000 mg / L, potassium dihydrogen phosphate 500-2000 mg / L and magnesium sulfate 200-800 mg / L, the balance is water; The Aspergillus is Aspergillus fumigatus.

2. The method for culturing Aspergillus according to claim 1, characterized in that The liquid fermentation medium is prepared by the following method: According to the preset mass concentration, glucose, glycerol, galactose, peptone, peanut sprout extract, germinated brown rice extract, microcrystalline cellulose, D-mannitol, calcium phosphate, zinc sulfate and sodium selenite are taken, and water is added to make up to 500-800 mL; then steam sterilized at a temperature of 115-125° C. for 10-30 min to obtain a first mixture; after cooling the first mixture to room temperature, glutamine, hydroxyproline and tyrosine are added, and water is added to make up to 1000 mL to obtain a second mixture; the second mixture is filtered and sterilized with a 0.22-0.45 μm filter membrane to obtain the liquid fermentation medium.

3. The method for cultivating Aspergillus according to claim 1, characterized in that The seed culture medium is prepared by the following method: According to the preset mass concentration, glucose, glycerol, peptone, yeast extract, microcrystalline cellulose, peanut sprout extract, germinated brown rice extract, potassium dihydrogen phosphate and magnesium sulfate are taken, and water is added to make the volume to 1000 mL; then steam sterilized at a temperature of 115-125° C. for 10-30 minutes to obtain the seed culture medium.

4. The method for culturing Aspergillus according to claim 1, characterized in that The activation culture medium is composed of the following raw materials: Glucose 10000-15000 mg / L, yeast extract 1000-2000 mg / L, nitrate mother solution 30-40 mL / L, trace element mother solution 1-2 mL / L and agar 15000-200000 mg / L, the balance is water; The nitrate mother solution is composed of the following raw materials: NaNO3 120 g / L, KCl 10.4 g / L, MgSO4•7H2O 10.4 g / L, K2HPO4•3H2O 30.4 g / L, and the balance is distilled water; The trace element mother solution is composed of the following raw materials: ZnSO4•7H2O 0.22 g / L, H3BO3 0.11 g / L, MnCl2•4H2O 0.05 g / L, FeSO4•7H2O 0.016 g / L, CoCl2•5H2O 0.016 g / L, CuSO4•5H2O 0.016 g / L, (NH4)6Mo7O 24 •4H2O 0.011 g / L, Na2EDTA 0.5 g / L, the balance is distilled water.

5. The method for culturing Aspergillus according to claim 1, characterized in that: The inoculation amount of Aspergillus is 2-8%, and the speed of the shaking culture is 180-200 rpm.

6. The method for culturing Aspergillus according to claim 1, characterized in that: The conditions for the activation culture are: culturing at a temperature of 28-30°C for 3-5 days; the conditions for the seed culture are: culturing at a temperature of 28-30°C for 5-7 days.

7. A method for preparing a polysaccharide antigen, characterized in that: The steps include: Cultivating Aspergillus niger by the culturing method according to any one of claims 1 to 6 to obtain a bacterial liquid; After sterilizing the bacterial solution, filtering to remove bacterial bodies to obtain a filtrate; The filtrate is precipitated and centrifuged to obtain a crude polysaccharide extract; Purifying the polysaccharide crude extract to obtain the polysaccharide antigen; The polysaccharide antigen is galactomannan.

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