Oyster mushroom fermentation polysaccharide as well as preparation method and application thereof

Abstract

The invention provides oyster mushroom fermented polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of biopolymers. The method comprises the following steps: preparing a liquid culture medium from glucose, yeast extract powder, KH2PO4, MgSO4. 7H2O and vitamin B1 in proportion; inoculating the oyster mushroom seed solution into a liquid culture medium, and culturing under the conditions of proper rotating speed, pH and the like; mixing the culture solution with ethanol, and centrifuging to obtain a precipitate; and adding deionized water into the precipitate for redissolving, and freeze-drying to obtain the oyster mushroom liquid fermentation polysaccharide. The invention discloses physicochemical properties and in-vivo anti-hypoxia activity of pleurotus ostreatus liquid fermentation polysaccharide.

Description

Technical Field

[0001] The present invention relates to the technical field of biopolymers, and in particular to a fermented polysaccharide of Pleurotus ostreatus and a preparation method and application thereof. Background Art

[0002] Pleurotus ostreatus is one of the most cultivated edible mushrooms in the world, possessing significant edible, medicinal, and economic value. Polysaccharides are the primary biological basis for the biological activity of Pleurotus ostreatus, accounting for approximately 57.6% to 81.8% of its content. Pleurotus ostreatus polysaccharides exhibit antioxidant, anti-aging, and immunomodulatory properties, making them a valuable resource for the development of functional foods. Pleurotus ostreatus fermented polysaccharides are polysaccharides with diverse biological activities, produced and accumulated through the growth and metabolism of Pleurotus ostreatus (including its mycelium) under suitable culture conditions through a specific fermentation process.

[0003] Currently, the production and preparation of oyster mushroom polysaccharides primarily relies on extraction from oyster mushroom fruiting bodies. These fruiting bodies are primarily obtained through solid cultivation techniques. The fruiting body growth cycle is approximately 60 days, progressing through stages such as formation, growth, and development. This requires strict control of environmental factors such as humidity, temperature, and light, resulting in high production costs and a long growth cycle. Furthermore, the extraction steps for oyster mushroom polysaccharides are complex and the extraction yield is low. Large-scale industrial production is inefficient and expensive, hindering its practical application in the functional food sector. Therefore, developing a method for preparing oyster mushroom fermented polysaccharides with high production efficiency, stable product quality, and ease of automated control has become a pressing technical challenge facing the prior art. Summary of the Invention

[0004] Based on the above technical problems to be solved by the present invention, the present invention proposes a fermented polysaccharide of Pleurotus ostreatus and a preparation method and application thereof.

[0005] One of the purposes of the present invention is to provide a method for preparing fermented polysaccharides from Pleurotus ostreatus, the method comprising:

[0006] S1. Prepare PDA culture medium, culture Pleurotus ostreatus strains, and obtain activated Pleurotus ostreatus strains;

[0007] S2, transferring the activated Pleurotus ostreatus spawn to a seed culture medium to obtain Pleurotus ostreatus seed liquid;

[0008] S3, inoculating the oyster mushroom seed liquid into a liquid culture medium composed of glucose, yeast extract, KH2PO4, MgSO4·7H2O, and vitamin B1, wherein the mass fractions of each component are: 50-70 parts of glucose, 12-22 parts of yeast extract, 1.5-4.5 parts of KH2PO4, 0.5-1.5 parts of MgSO4·7H2O, and 0.2-2 parts of vitamin B1; the culture time is 4-6 days to obtain a oyster mushroom liquid fermentation liquid;

[0009] S4, mixing the oyster mushroom liquid fermentation liquid with 85% to 98% ethanol and subjecting it to alcohol precipitation, wherein the volume ratio of the fermentation liquid to the 85% to 98% ethanol is 1: (2 to 5), the precipitation temperature is 2 to 8° C., and the precipitation time is 48 to 96 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 3000 to 5000 rpm and the centrifugation time is 5 to 15 min, to obtain an alcohol precipitation product of the oyster mushroom liquid fermentation liquid;

[0010] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0011] Furthermore, the preparation of the activated Pleurotus ostreatus spawn includes:

[0012] S21. Take potatoes, add water to boil, and continue to cook for 15 to 30 minutes, separate the filter residue and the filtrate, add glucose and agar powder to the filtrate in order, each in an amount of 5% to 15% of the weight of the potato; add water to make the volume ratio of the potato mass to volume 150 to 250 g / L; sterilize under high pressure at 110 to 120° C. for 20 to 40 minutes to obtain a PDA solid culture medium;

[0013] S22, transferring the Pleurotus ostreatus strain to the PDA solid culture medium, and culturing in the dark at 22-30° C. for 4-8 days until the mycelium covers the surface of the culture medium, thereby obtaining the activated Pleurotus ostreatus strain.

[0014] Furthermore, the preparation of the Pleurotus ostreatus seed liquid includes:

[0015] S31, preparing 15-25 g / L of glucose, 2.5-7.5 g / L of yeast extract powder, 0.5-1.5 g / L of MgSO4·7H2O, and 0.5-1.5 g / L of KH2PO4 to obtain the seed culture medium;

[0016] S32, inoculating the activated Pleurotus ostreatus strain into the seed culture medium, culturing at 22-30° C. for 0.5-2 days; and shaking culturing at 22-30° C. for 1-3 days to obtain the Pleurotus ostreatus seed liquid.

[0017] Furthermore, the mass proportions of the components of the liquid culture medium are: 55-65 parts of glucose, 15-19 parts of yeast extract powder, 2-4 parts of KH2PO4, 0.8-1.2 parts of MgSO4 and 0.15-1.5 parts of vitamin B1; the initial fermentation pH value of the liquid culture medium is 5.8-6.2, the inoculation amount is 5%-7%, and the rotation speed is 150-200 rpm.

[0018] Furthermore, the ethanol is 93% to 97% ethanol; the volume ratio of the oyster mushroom liquid fermentation liquid to the ethanol is 1:(1 to 4); and the precipitation temperature is 2 to 4°C.

[0019] The second purpose of the present invention is to provide a fermented polysaccharide of Pleurotus ostreatus, which is prepared according to the preparation method of the fermented polysaccharide of Pleurotus ostreatus.

[0020] Furthermore, the oyster mushroom fermentation polysaccharide is oyster mushroom liquid fermentation polysaccharide; the components of the oyster mushroom liquid fermentation polysaccharide include sugar, protein and water; the mass percentages of the sugar, protein and water are (50-60):(10-20):(4-8).

[0021] The third object of the present invention is to provide an application of fermented polysaccharide of Pleurotus ostreatus in the preparation of anti-hypoxia medicines / foods / health products.

[0022] Furthermore, the fermented polysaccharide of Pleurotus ostreatus can prolong the survival time of mice in normal pressure closed hypoxia.

[0023] Furthermore, the fermented polysaccharide of Pleurotus ostreatus can prolong the survival time of mice poisoned by sodium nitrite.

[0024] Compared with the prior art, the present invention provides a fermented polysaccharide of Pleurotus ostreatus and its preparation method and application, which has the following beneficial effects:

[0025] The present invention adopts liquid fermentation technology to prepare liquid fermentation polysaccharide of Pleurotus ostreatus. Compared with solid fermentation technology, the liquid fermentation technology proposed in the present invention has the advantages of high production efficiency, stable product quality, and easy automation control.

[0026] Furthermore, the fermentation broth of the present invention contains rich extracellular polysaccharides of Pleurotus ostreatus, which can be obtained through simple steps such as alcohol precipitation and redissolution, and is easy to prepare for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 An ion exchange chromatography elution curve of a liquid fermentation polysaccharide of Pleurotus ostreatus according to an embodiment of the present invention is shown;

[0028] Figure 2 The figure shows the appearance and properties of liquid fermented polysaccharide of Pleurotus ostreatus according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The following describes exemplary embodiments of the present application in more detail with reference to the accompanying drawings. Although exemplary embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0030] In the following examples, unless otherwise specified, the technical means adopted are conventional means well known to those skilled in the art, and the reagents and materials in the present invention are obtained from the market or other public channels.

[0031] The experimental materials and equipment involved in the present invention mainly include but are not limited to:

[0032] Reagents:

[0033] Yeast extract powder, analytical grade, Oxoid Company, UK; glucose, analytical grade, Sinopharm Chemical Reagent Co., Ltd.; magnesium sulfate heptahydrate, analytical grade, Tianjin North Tianyi Chemical Reagent Factory; potassium dihydrogen phosphate, analytical grade, Sinopharm Chemical Reagent Co., Ltd.; vitamin B1, analytical grade, Beijing Solebold Technology Co., Ltd.; anhydrous ethanol, 100%, Tianjin Yuanli Chemical Co., Ltd.

[0034] instrument:

[0035] Constant temperature shaking incubator, HYG, Shanghai Xinrui Automation Equipment Co., Ltd.; high-speed centrifuge, TGL16G, Shanghai Medical Analytical Instrument Factory; vacuum freeze dryer, ALPHA1-2 / LD, CHRIS, Germany.

[0036] The technical principles of this invention primarily involve inoculating oyster mushroom seeds into a liquid culture medium rich in carbon and nitrogen sources, various inorganic salts, and growth factors, and culturing under appropriate temperature, pH, and aeration conditions. As the seeds grow and multiply, they synthesize and secrete polysaccharides into the fermentation broth. The oyster mushrooms themselves also contain a certain amount of polysaccharides. The fermented oyster mushroom polysaccharides are complex macromolecular polymers composed of multiple monosaccharides linked by different types of glycosidic bonds, exhibiting a quaternary structure. The resulting fermented oyster mushroom polysaccharides exhibit biological activities such as immunomodulation, anti-tumor, antioxidant, and hypoglycemic properties, and are widely used in food, pharmaceuticals, and health supplements.

[0037] Based on the above principles, the present invention proposes a fermented polysaccharide of Pleurotus ostreatus and its preparation method and application, including:

[0038] S1. Inoculating a liquid culture medium with a seed liquid of Pleurotus ostreatus into the liquid culture medium is performed, wherein the culture medium is composed of glucose, yeast extract powder, KH2PO4, MgSO4·7H2O, and vitamin B1, wherein the weight proportions of the components of the culture medium are as follows: 50-70 parts of glucose, 12-22 parts of yeast extract powder, 1.5-4.5 parts of KH2PO4, 0.5-1.5 parts of MgSO4·7H2O, and 0.2-2 parts of vitamin B1; the initial pH value of the fermentation is 5.5-6.5, the inoculation amount is 4%-8%, the rotation speed is 150-200 rpm, and the culture time is 4-6 days to obtain a liquid fermentation liquid of Pleurotus ostreatus.

[0039] S2. Mixing the Pleurotus ostreatus liquid fermentation liquid with 85% to 98% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation liquid to the 85% to 98% ethanol is 1:(2 to 5), the precipitation temperature is 2 to 8° C., and the precipitation time is 48 to 96 hours; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 3000 to 5000 rpm and the centrifugation time is 5 to 15 minutes, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation liquid.

[0040] S3. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0041] The prepared liquid fermentation polysaccharide of Pleurotus ostreatus can significantly improve the body's tolerance to hypoxia.

[0042] Furthermore, compared with the existing liquid fermentation technology of Pleurotus ostreatus, the present invention innovates the composition of the culture medium, and the yield of the prepared liquid fermentation polysaccharide of Pleurotus ostreatus is significantly improved, reaching 2.63g / L.

[0043] Furthermore, the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention can prolong the survival time of mice in normal pressure closed hypoxia by 37.38% and the survival time of sodium nitrite poisoning by 27.00%, thereby improving the body's tolerance to hypoxia.

[0044] Therefore, the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention can be applied in the field of anti-hypoxia and can bring significant social and economic benefits.

[0045] Example 1

[0046] The invention provides a method for preparing liquid fermentation polysaccharide of Pleurotus ostreatus.

[0047] Mainly include:

[0048] (1) The preparation experiment of liquid fermentation polysaccharide of Pleurotus ostreatus includes the following steps:

[0049] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0050] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm3 Take 3-5 squares of different sizes and inoculate them into a 250mL triangular shake flask containing 100mL seed culture medium, and culture at a constant temperature of 26℃ for 24h; place it on a shaker at 160rpm and shake at 26℃ for 48h to obtain the oyster mushroom seed liquid.

[0051] S3. Inoculate the oyster mushroom seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, 8.5 g / L yeast extract powder, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; fermentation conditions are initial pH 6, inoculation size 6%, and rotation speed 180 rpm; and culture time is 5 days to obtain oyster mushroom liquid fermentation liquid.

[0052] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0053] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0054] (2) Another experiment for preparing liquid fermentation polysaccharide of Pleurotus ostreatus includes the following steps:

[0055] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0056] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm 3 Take 3-5 squares of different sizes and inoculate them into a 250mL triangular shake flask containing 100mL seed culture medium, and culture at a constant temperature of 26℃ for 24h; place it on a shaker at 160rpm and shake at 26℃ for 48h to obtain the oyster mushroom seed liquid.

[0057] S3. Inoculating the oyster mushroom seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, 8.5 g / L yeast extract, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; fermentation conditions are initial pH 6, inoculum size 6%, and rotation speeds of 120, 140, 160, 180, and 200 rpm respectively; and culture time is 5 days to obtain oyster mushroom liquid fermentation liquid.

[0058] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0059] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0060] (3) Another experiment for preparing liquid fermentation polysaccharide of Pleurotus ostreatus includes the following steps:

[0061] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0062] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm 3 Take 3 to 5 squares of different sizes and inoculate them into a 250 mL triangular shake flask containing 100 mL seed culture medium, and culture them at a constant temperature of 26°C for 24 h; place them on a shaker at 160 rpm and culture them at 26°C for 48 h to obtain the oyster mushroom seed liquid.

[0063] S3. Inoculating the Pleurotus ostreatus seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, 8.5 g / L yeast extract, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; the fermentation conditions are initial pH 6, inoculation amounts are 4%, 6%, 8%, 10%, and 12%, respectively, and the rotation speed is 160 rpm; the culture time is 5 days, and a Pleurotus ostreatus liquid fermentation liquid is obtained.

[0064] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0065] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0066] (IV) Another experiment for preparing liquid fermentation polysaccharide of Pleurotus ostreatus includes the following steps:

[0067] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0068] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm 3 Take 3-5 squares of different sizes and inoculate them into a 250mL triangular shake flask containing 100mL seed culture medium, and culture at a constant temperature of 26℃ for 24h; place it on a shaker at 160rpm and shake at 26℃ for 48h to obtain the oyster mushroom seed liquid.

[0069] S3. Inoculate the oyster mushroom seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, 8.5 g / L yeast extract, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; the fermentation conditions are as follows: initial pH of 4, 5, 6, and 7, inoculation size of 6%, rotation speed of 160 rpm; and culture time of 5 days to obtain oyster mushroom liquid fermentation liquid.

[0070] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0071] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0072] (V) Another experiment for preparing liquid fermentation polysaccharide of Pleurotus ostreatus includes the following steps:

[0073] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0074] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm 3 Take 3-5 squares of different sizes and inoculate them into a 250mL triangular shake flask containing 100mL seed culture medium, and culture at a constant temperature of 26℃ for 24h; place it on a shaker at 160rpm and shake at 26℃ for 48h to obtain the oyster mushroom seed liquid.

[0075] S3. Inoculate the oyster mushroom seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, yeast extract powder (2.5, 5.0, and 7.5 g / L, respectively), 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; fermentation conditions are initial pH 6, inoculation size 6%, and rotation speed 180 rpm; and culture time is 5 days to obtain oyster mushroom liquid fermentation liquid.

[0076] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0077] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0078] result:

[0079] See also Figure 2The prepared liquid fermentation polysaccharide from Pleurotus ostreatus is a yellow-brown powder. Using the preparation conditions described in Example 1 (I), the yield of liquid fermentation polysaccharide from Pleurotus ostreatus can reach 2.63 g / L. Compared with existing technologies, this invention can significantly increase the yield of liquid fermentation polysaccharide from Pleurotus ostreatus, reduce industrial production costs, and improve production efficiency.

[0080] According to the preparation conditions described in Example 1 (II), see Table 1. Liquid fermentation polysaccharide of Pleurotus ostreatus can be prepared under different rotation speed conditions, but the yields vary, as shown in Table 1:

[0081] Table 1 Effect of different rotation speeds on the yield of polysaccharides from liquid fermentation of Pleurotus ostreatus

[0082]

[0083]

[0084] According to the preparation conditions described in Example 1 (III), see Table 2. Liquid fermentation polysaccharide of Pleurotus ostreatus can be prepared under different inoculation conditions, but the yields vary, as shown in Table 2:

[0085] Table 2 Effects of different inoculation amounts on the yield of polysaccharides from liquid fermentation of Pleurotus ostreatus

[0086]

[0087] According to the preparation conditions described in Example 1 (IV), see Table 3. Liquid fermentation polysaccharide of Pleurotus ostreatus can be prepared under different initial pH conditions, but the yields vary, as shown in Table 3:

[0088] Table 3 Effects of different inoculation amounts on the yield of polysaccharides from liquid fermentation of Pleurotus ostreatus

[0089]

[0090] According to the preparation conditions described in Example 1 (V), see Table 4. Liquid fermentation polysaccharide of Pleurotus ostreatus can be prepared under different yeast extract powder addition conditions, but the yields vary, as shown in Table 4:

[0091] Table 4 Effects of different inoculation amounts on polysaccharide yields from liquid fermentation of Pleurotus ostreatus

[0092]

[0093] In the preparation process of liquid fermentation polysaccharides from Pleurotus ostreatus according to the present invention, the choice of nitrogen source has a significant effect on the yield of polysaccharides. According to the steps described in Example 1 (I), the nitrogen source was replaced with yeast extract powder, peptone, beef extract, corn steep liquor, ammonium chloride, and ammonium sulfate, respectively. With other conditions remaining unchanged, the polysaccharide yield changed significantly. Please refer to Table 5:

[0094] Table 5 Effects of different nitrogen sources on polysaccharide yields from liquid fermentation of Pleurotus ostreatus

[0095]

[0096]

[0097] The present invention provides a method for preparing liquid-fermented polysaccharides from Pleurotus ostreatus, addressing the long production cycle and high costs of Pleurotus ostreatus polysaccharides, which hinder their large-scale application in functional foods. This method boasts a short production cycle and a simple preparation method, effectively improving the production efficiency of Pleurotus ostreatus polysaccharides and promoting their practical application in functional foods.

[0098] Example 2

[0099] The present invention proposes the use of liquid fermentation polysaccharide of Pleurotus ostreatus in the preparation of anti-hypoxia medicines / foods / health products.

[0100] Mainly include:

[0101] The preparation of liquid fermentation polysaccharide of Pleurotus ostreatus comprises the following steps:

[0102] (1) A method for preparing liquid fermentation polysaccharide of Pleurotus ostreatus, comprising the following steps:

[0103] S1. Take 200g of fresh peeled potatoes, add appropriate amount of distilled water, boil and continue to cook for 20min, separate the residue and filtrate, add 20g of glucose and agar powder to the filtrate in sequence; add distilled water to make the volume 1L, stir evenly, divide and package, and then sterilize at 115℃ for 30min to obtain PDA solid culture medium; cut the slant surface of Pleurotus ostreatus into pieces of about 1cm 3 The cubes were transferred to PDA solid culture medium and cultured at 26℃ in the dark for 6 days until the mycelium covered the surface of the flat culture medium to obtain activated Pleurotus ostreatus strains.

[0104] S2, prepare seed culture medium with 20g / L glucose, 5g / L yeast extract powder, 1g / L MgSO4·7H2O, and 1g / L KH2PO4, and cut the activated oyster mushroom into pieces of about 1cm 3 Take 3 to 5 squares of different sizes and inoculate them into a 250 mL triangular shake flask containing 100 mL seed culture medium, and culture them at a constant temperature of 26°C for 24 h; place them on a shaker at 160 rpm and culture them at 26°C for 48 h to obtain the oyster mushroom seed liquid.

[0105] S3. Inoculate the oyster mushroom seed liquid into a liquid culture medium, wherein the culture medium ratio is 30 g / L glucose, 8.5 g / L yeast extract powder, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 0.1 g / L vitamin B1; fermentation conditions are initial pH 6, inoculation size 6%, and rotation speed 180 rpm; and culture time is 5 days to obtain oyster mushroom liquid fermentation liquid.

[0106] S4. Mixing the Pleurotus ostreatus liquid fermentation broth with 95% ethanol and subjecting the mixture to alcohol precipitation, wherein the volume ratio of the fermentation broth to the 95% ethanol is 1:3, the precipitation temperature is 4° C., and the precipitation time is 72 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 4000 rpm and the centrifugation time is 10 min, to obtain an alcohol precipitation product of the Pleurotus ostreatus liquid fermentation broth.

[0107] S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

[0108] (2) Qualitative and quantitative analysis of the prepared liquid fermentation polysaccharides of Pleurotus ostreatus

[0109] Firstly, the total sugar content in the prepared liquid fermentation polysaccharide of Pleurotus ostreatus was detected by phenol-sulfuric acid method.

[0110] Prepare a precise 100 μg / mL anhydrous glucose standard solution. Pipette 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μL of the glucose standard solution into a test tube. Make up to 2 mL with deionized water, add 1 mL of 5% phenol solution, mix thoroughly, and then add 5 mL of concentrated sulfuric acid. Let stand at room temperature for 20 minutes. Measure the absorbance at 490 nm using a UV spectrophotometer. Prepare a 1 mg / mL sample solution and dilute appropriately. Determine the total sugar content in the sample as described above. Detect protein content in the sample using the Coomassie Brilliant Blue method. A 100 μg / mL bovine serum albumin standard solution was accurately prepared. 100, 200, 400, 600, 800, and 1000 μL of the standard solution were pipetted into 10 mL EP tubes. The solution was made up to 1 mL with ultrapure water. 5 mL of Coomassie Brilliant Blue G-250 solution was added and allowed to react at room temperature for 5 minutes in the dark. The absorbance at 595 nm was measured using a UV spectrophotometer. A 1 mg / mL sample solution was prepared and diluted appropriately to determine the protein content. The moisture content of the samples was determined using the direct drying method. The samples were weighed and dried in an oven at 100°C. After cooling, they were weighed again. The drying, cooling, and weighing steps were repeated until the weight remained constant. The moisture content was calculated based on the difference in sample weight before and after drying. The composition of the polysaccharide obtained from the liquid fermentation of Pleurotus ostreatus is shown in Table 7.

[0111] Secondly, the prepared liquid fermentation polysaccharide of Pleurotus ostreatus is purified, and the molecular weight of the purified component is detected.

[0112] After deproteinization using the Sevage method and decolorization by adsorption using AB-8 macroporous resin, the polysaccharides from the liquid fermentation of Pleurotus ostreatus were purified using ion exchange column chromatography. DEAE-650M ion exchange resin was used, and elution was performed using deionized water, 0.2 mol / L, and 0.5 mol / L NaCl solutions, respectively, at a flow rate of 5 mL / min. 8 mL was collected from each tube. The polysaccharide content of each eluate was determined, and an elution curve was plotted. Fractions were collected according to the elution curve and dialyzed using a 2000 Da dialysis bag to a conductivity below 100 S / m. The dialyzed fractions were freeze-dried to obtain purified polysaccharides.

[0113] The molecular weight of purified polysaccharide fractions from liquid fermentation of Pleurotus ostreatus was determined using dextran gel chromatography. A standard curve was constructed based on the peak elution times of known dextran standards of varying molecular weights. Samples of the purified fractions were dissolved in deionized water to a concentration of 1 mg / mL and filtered through a 0.22 μm filter. The experiment was performed under the same chromatographic conditions as the standard. The peak elution times were recorded and substituted into the standard curve equation to calculate the molecular weight.

[0114] (3) applying the prepared liquid fermentation polysaccharide of Pleurotus ostreatus to the preparation of anti-hypoxia medicines / foods / health products.

[0115] First, in order to prove the application potential of the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention in anti-hypoxia food, the anti-hypoxia activity of the prepared liquid fermentation polysaccharide of Pleurotus ostreatus was verified by a normal pressure closed hypoxia experiment.

[0116] Male Kunming mice (SPF, 6-8 weeks old, 18-20 g) were randomly divided into five groups of 10 mice each after one week of adaptive feeding. The groups were divided into the following groups: solvent control (NC), Rhodiola rosea capsule positive control (Rhoc), low-dose Pleurotus ostreatus polysaccharide (EPS-L), medium-dose Pleurotus ostreatus polysaccharide (EPS-M), and high-dose Pleurotus ostreatus polysaccharide (EPS-H). Mice were treated with oral gavage for 14 consecutive days according to the protocol in Table 6.

[0117] Table 6 Experimental animal grouping design

[0118]

[0119] One hour after the last oral gavage, the mice were placed individually in 250 mL ground-mouth bottles containing 5 g of soda lime (one mouse per bottle), and the bottle mouths were carefully sealed with vaseline. The survival time of the mice from entering the bottle to complete cessation of breathing was recorded and recorded as the normal pressure closed hypoxia survival time.

[0120] Secondly, in order to prove the application potential of the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention in anti-hypoxia food, the anti-hypoxia activity of the prepared liquid fermentation polysaccharide of Pleurotus ostreatus was verified by a sodium nitrite poisoning experiment.

[0121] Animal pretreatment was the same as in the anti-hypoxic activity validation experiment. One hour after the final oral gavage, each group of mice was intraperitoneally injected with 240 mg / kg body weight of sodium nitrite solution. The time from the end of injection to respiratory arrest was recorded and used as the survival time after sodium nitrite poisoning.

[0122] result:

[0123] Table 7 shows the composition of the liquid fermented polysaccharide from Pleurotus ostreatus prepared in the present invention. The total sugar content accounts for the highest proportion, at 55.53±0.09%, while the protein content and moisture content are 14.50±0.90% and 5.18±0.08%, respectively. Compared with polysaccharides from Pleurotus ostreatus fruiting bodies and fermented polysaccharides prepared in existing studies, the composition shows significant differences, indicating that the liquid fermented polysaccharide from Pleurotus ostreatus prepared in the present invention is a novel polysaccharide.

[0124] Table 7 Composition of polysaccharides from liquid fermentation of Pleurotus ostreatus

[0125]

[0126] See also Figure 1 , after ion exchange chromatography, the liquid fermentation polysaccharide of Pleurotus ostreatus was separated into two components, named EPS-0 and EPS-1, respectively, of which EPS-0 is a neutral polysaccharide and EPS-1 is an acidic polysaccharide. The two active components contained in the EPS prepared by the present invention are neutral polysaccharides and acidic polysaccharides. Studies have shown that neutral polysaccharides can improve the body's anaerobic metabolic function and enhance the body's hypoxia tolerance by activating the HIF-1α signaling pathway and upregulating the activity of glycolytic enzymes. Acidic polysaccharides usually contain more uronic acid and can alleviate hypoxia-induced body damage by reducing inflammatory responses. The EPS prepared by the present invention contains neutral polysaccharides and acidic polysaccharide components, respectively, which may promote the body's hypoxia tolerance through various pathways, alleviate hypoxia-induced body damage, and exert anti-hypoxia function.

[0127] Please refer to Table 8. The molecular weights of EPS-0 and EPS-1 are similar, with retention times of 15.876 min and 15.264 min, respectively, and molecular weights of 35.47 kDa and 51.83 kDa, respectively. Molecular weight is an important structural basis for the biological activity of natural polysaccharides. Low molecular weight polysaccharides (<10 kDa) have high solubility and are easily absorbed and utilized by the body through biological barriers. They have high bioavailability, but may not be able to form specific active conformations due to their overly simple structures, resulting in reduced biological activity. High molecular weight polysaccharides (>100 kDa) have low solubility and are prone to forming aggregates or even gels, resulting in reduced bioavailability and loss of biological activity in the body. Therefore, polysaccharides with a molecular weight of 10 to 100 kDa are generally beneficial for the body to absorb and efficiently exert biological activity. The molecular weights of the two active components of the EPS prepared by the present invention are 35.47 kDa and 51.83 kDa, respectively, which may be beneficial for EPS to be absorbed by the body through biological barriers and effectively exert its anti-hypoxia function.

[0128] Table 8 Molecular weight of purified polysaccharide fractions from liquid fermentation of Pleurotus ostreatus

[0129]

[0130] The effects of liquid fermentation polysaccharides of Pleurotus ostreatus on the survival time of mice subjected to normal pressure and airtight hypoxia are shown in Table 9. The effect of liquid fermentation polysaccharides of Pleurotus ostreatus on prolonging the survival time of mice subjected to normal pressure and airtight hypoxia is dose-dependent. The medium dose and high dose can prolong the survival time of mice by 22.10% and 37.38%, respectively (P<0.05). Under hypoxic conditions, a decrease in oxygen partial pressure leads to the obstruction of mitochondrial oxidative phosphorylation, a decrease in ATP synthesis, a compensatory enhancement of anaerobic glycolysis, and the accumulation of lactic acid, which can eventually lead to energy depletion, acidosis, and multiple organ failure. The survival time of normal pressure and airtight hypoxia comprehensively reflects the tolerance of key organs such as the heart, brain, and lungs to hypoxia, and is a core indicator for evaluating the anti-hypoxic activity of functional factors. The liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention can significantly prolong the survival time of mice subjected to normal pressure and airtight hypoxia, improve the tolerance of mice to hypoxic environments, and has obvious anti-hypoxic activity.

[0131] Table 9 Survival time of mice under normal pressure and closed hypoxia

[0132]

[0133] Note: * indicates significant difference compared with the blank control group (P<0.05).

[0134] The effects of liquid fermentation polysaccharide of Pleurotus ostreatus on the survival time of mice poisoned by sodium nitrite are shown in Table 10. The effect of liquid fermentation polysaccharide of Pleurotus ostreatus on prolonging the survival time of mice poisoned by sodium nitrite is dose-dependent, and can prolong the survival time of mice by up to 27.00% (P<0.05). After sodium nitrite enters the body, it oxidizes hemoglobin into methemoglobin. Methemoglobin cannot transport oxygen, resulting in insufficient oxygen supply to the various tissues and organs of the body, thereby causing hypoxia in the body. Anti-hypoxia functional components can maintain the homeostasis of the body's internal environment under hypoxia by improving anaerobic metabolic capacity, accelerating erythropoiesis, and other means, thereby prolonging the survival time of sodium nitrite poisoning. The liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention can significantly improve the survival time of mice poisoned by sodium nitrite, and has obvious anti-hypoxia activity.

[0135] It is worth emphasizing that according to the provisions of the "Health Food Function Test and Evaluation Method (2023 Edition)", the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention showed positive results in the normal pressure hypoxia tolerance experiment and sodium nitrite poisoning survival experiment in mice, proving that it has anti-hypoxia function.

[0136] Table 10 Survival time of mice poisoned by sodium nitrite

[0137]

[0138] Note: * indicates significant difference compared with the blank control group (P<0.05).

[0139] Compared with the existing technology, the comparative analysis of the biological activities of Pleurotus ostreatus polysaccharide and Pleurotus ostreatus fermented polysaccharide is shown in Table 11.

[0140] According to the "Health Food Functional Test and Evaluation Method (2023 Edition)", the evaluation criteria for anti-hypoxia health foods are that two of the following tests are positive in the mouse normal pressure hypoxia tolerance test, the sodium nitrite poisoning survival test, and the acute cerebral ischemic hypoxia test to determine whether the functional factor has an anti-hypoxia function. The liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention showed positive results in both the normal pressure hypoxia tolerance test and the sodium nitrite poisoning survival test in mice, and the extension of the survival time of mice was dose-dependent, demonstrating that the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention has an anti-hypoxia function.

[0141] Existing research on oyster mushroom polysaccharides or fermented oyster mushroom polysaccharides mainly focuses on functional activities such as antioxidant, anti-aging, and immunomodulatory. According to the mechanism of functional factor activity verification, antioxidant functional factors mainly alleviate the body's oxidative stress state by scavenging free radicals, enhancing antioxidant enzyme activity, and reducing lipid peroxide content; anti-aging functional factors mainly delay the body's aging by inhibiting the formation of advanced glycation end products, increasing telomerase activity, and reducing inflammation levels; immunomodulatory functional factors mainly enhance the body's immunity by increasing thymus and spleen indexes, improving spleen lymphocyte proliferation and transformation capacity, and increasing serum antibody levels. The above activity studies cannot indicate the impact of functional factors on the body's anaerobic metabolic capacity and erythropoiesis capacity, nor can they speculate on their impact on the body's survival time in normal pressure closed hypoxia, sodium nitrite poisoning, and acute cerebral ischemia and hypoxia, nor can they speculate on their anti-hypoxia function.

[0142] In addition, compared with the anti-hypoxia activity of natural product polysaccharides in other existing technologies, as shown in Table 12 (showing the highest extension rate), the liquid fermentation polysaccharide of Pleurotus ostreatus prepared by the present invention significantly improves the extension rate of the survival time of mice in normal pressure closed hypoxia, and has a more outstanding anti-hypoxia effect.

[0143] Table 11: Reported biological activities of Pleurotus ostreatus polysaccharides or Pleurotus ostreatus fermented polysaccharides

[0144]

[0145]

[0146] Table 12 Comparison of the anti-hypoxia effects of liquid fermentation polysaccharides of Pleurotus ostreatus and common natural product polysaccharides

[0147]

[0148] It should be noted that the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article, or apparatus that includes a series of elements includes not only those elements but also other elements not explicitly listed, or includes elements inherent to such process, method, article, or apparatus. In the absence of further limitations, the elements defined by the phrase "comprises..." do not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the elements.

[0149] The above are merely embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for preparing fermented polysaccharide from Pleurotus ostreatus, characterized in that: The method comprises: S1. Prepare PDA culture medium, culture Pleurotus ostreatus strains, and obtain activated Pleurotus ostreatus strains; S2, transferring the activated Pleurotus ostreatus spawn to a seed culture medium to obtain Pleurotus ostreatus seed liquid; S3, inoculating the oyster mushroom seed liquid into a liquid culture medium composed of glucose, yeast extract, KH2PO4, MgSO4·7H2O, and vitamin B1, wherein the mass fractions of each component are: 50-70 parts of glucose, 12-22 parts of yeast extract, 1.5-4.5 parts of KH2PO4, 0.5-1.5 parts of MgSO4·7H2O, and 0.2-2 parts of vitamin B1; the culture time is 4-6 days to obtain a oyster mushroom liquid fermentation liquid; S4, mixing the oyster mushroom liquid fermentation liquid with 85% to 98% ethanol and subjecting it to alcohol precipitation, wherein the volume ratio of the fermentation liquid to the 85% to 98% ethanol is 1: (2 to 5), the precipitation temperature is 2 to 8° C., and the precipitation time is 48 to 96 h; obtaining a precipitate by centrifugation, wherein the centrifugal speed is 3000 to 5000 rpm and the centrifugation time is 5 to 15 min, to obtain an alcohol precipitation product of the oyster mushroom liquid fermentation liquid; S5. Adding deionized water to re-dissolve the alcohol precipitation product of the liquid fermentation liquid of Pleurotus ostreatus, and freeze-drying to obtain liquid fermentation polysaccharide of Pleurotus ostreatus.

2. The method for preparing the fermented polysaccharide of Pleurotus ostreatus according to claim 1, wherein: The preparation of the activated oyster mushroom strain comprises: S21. Take potatoes, add water to boil, and continue to cook for 15 to 30 minutes, separate the filter residue and the filtrate, add glucose and agar powder to the filtrate in order, each in an amount of 5% to 15% of the weight of the potato; add water to make the volume ratio of the potato mass to volume 150 to 250 g / L; sterilize under high pressure at 110 to 120° C. for 20 to 40 minutes to obtain a PDA solid culture medium; S22, transferring the Pleurotus ostreatus strain to the PDA solid culture medium, and culturing in the dark at 22-30° C. for 4-8 days until the mycelium covers the surface of the culture medium, thereby obtaining the activated Pleurotus ostreatus strain.

3. The method for preparing the fermented polysaccharide of Pleurotus ostreatus according to claim 1, wherein: The preparation of the oyster mushroom seed liquid comprises: S31, preparing 15-25 g / L of glucose, 2.5-7.5 g / L of yeast extract powder, 0.5-1.5 g / L of MgSO4·7H2O, and 0.5-1.5 g / L of KH2PO4 to obtain the seed culture medium; S32, inoculating the activated Pleurotus ostreatus strain into the seed culture medium, culturing at 22-30° C. for 0.5-2 days; and shaking culturing at 22-30° C. for 1-3 days to obtain the Pleurotus ostreatus seed liquid.

4. The method for preparing the fermented polysaccharide of Pleurotus ostreatus according to claim 1, wherein The mass proportions of the components of the liquid culture medium are: 55-65 parts of glucose, 15-19 parts of yeast extract powder, 2-4 parts of KH2PO4, 0.8-1.2 parts of MgSO4·7H2O and 0.15-1.5 parts of vitamin B1; the initial fermentation pH value of the liquid culture medium is 4-7, the inoculation amount is 4%-12%, and the rotation speed is 120-200 rpm.

5. The method for preparing fermented polysaccharide of Pleurotus ostreatus according to claim 1, characterized in that: The ethanol is 93% to 97% ethanol; the volume ratio of the oyster mushroom liquid fermentation liquid to the ethanol is 1:(1 to 4); and the precipitation temperature is 2 to 4°C.

6. A fermented polysaccharide of Pleurotus ostreatus, characterized in that: The Pleurotus ostreatus fermented polysaccharide is prepared according to the preparation method of Pleurotus ostreatus fermented polysaccharide according to any one of claims 1 to 5.

7. The fermented polysaccharide of Pleurotus ostreatus according to claim 6, characterized in that The oyster mushroom fermentation polysaccharide is oyster mushroom liquid fermentation polysaccharide; the components of the oyster mushroom liquid fermentation polysaccharide include sugar, protein and water; the mass percentages of the sugar, protein and water are (50-60): (10-20): (4-8).

8. Use of the fermented Pleurotus ostreatus polysaccharide prepared by the preparation method according to any one of claims 1 to 5 or the fermented Pleurotus ostreatus polysaccharide according to claim 6 in the preparation of anti-hypoxia medicines / foods / health products.

9. The use according to claim 8, characterized in that The fermented polysaccharide of Pleurotus ostreatus can prolong the survival time of mice in normal pressure closed hypoxia.

10. The use according to claim 8, characterized in that The fermented polysaccharide of Pleurotus ostreatus can prolong the survival time of mice poisoned by sodium nitrite.

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