Culture medium and application thereof

Abstract

The invention provides a culture medium. The culture medium comprises a culture medium main body and culture medium auxiliary materials, the culture medium main body comprises a solid culture base material, a paste culture base material and a liquid culture base material, and the mass ratio of the solid culture base material to the paste culture base material to the liquid culture base material is 1: 1: (3-4); the solid culture base material is a dried and cured pit mud compound, the pasty culture base material is a pasty mixture formed by mixing pit mud and water, and the liquid culture base material is a pit mud extracting solution; the culture medium auxiliary material is prepared from the following raw materials in parts by mass: 800 to 1500 g of distilled water, 10 to 30 g of glucose, 10 to 30 g of peptone, 2 to 10 g of sodium chloride, 5 to 10 g of disodium hydrogen phosphate, 0.3 to 1 g of beef extract and 10 to 35 g of agar; the mass ratio of the liquid culture base material to the culture medium auxiliary material is 1: (2-4). According to the method, the biological activity can be effectively improved, the strain breeding efficiency is improved, the protease generation efficiency and activity are improved, and finally the generation efficiency and quality of plant polypeptide are improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a culture medium and application thereof. Background Art

[0002] In the process of brewing liquor, sorghum, wheat or corn and other grains are generally fermented in a fermentation tank containing cellar mud to produce liquor with a certain aroma. The cellar mud is generally adhered to the wall of the fermentation tank. With the improvement of people's living standards, people have higher and higher requirements for the quality of liquor. As an important material in the grain fermentation process, the quality of the cellar mud has an important influence on the quality of liquor. However, the current application of cellar mud is only in winemaking, so it is necessary to study it and explore its application in other aspects to improve its economic value and use value. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a culture medium and application thereof, which has the advantages of good strain cultivation effect, good fermentation effect, effective utilization of cellar mud, and reusable culture medium.

[0004] The above technical objectives of the present invention are achieved through the following technical solutions:

[0005] A culture medium, comprising a culture medium main body and culture medium auxiliary materials; the culture medium main body comprises a solid culture medium material, a paste culture medium material and a liquid culture medium material, wherein the mass ratio of the solid culture medium material, the paste culture medium material and the liquid culture medium material is 1:1:(3-4); the solid culture medium material is a dried and solidified pit mud composite, the paste culture medium material is a paste mixture formed by mixing pit mud and water, and the liquid culture medium material is a pit mud extract; the culture medium auxiliary materials are composed of the following raw materials in mass fractions: 800-1500g of distilled water, 10-30g of glucose, 10-30g of peptone, 2-10g of sodium chloride, 5-10g of disodium hydrogen phosphate, 0.3-1g of beef extract and 10-35g of agar; and the mass ratio of the liquid culture medium material to the culture medium auxiliary materials is 1:(2-4).

[0006] Furthermore, the cellar mud compound is composed of the following raw materials in terms of mass: 50-100 g of cellar mud, 20-30 g of medical stone powder, 20-30 g of green shou stone powder, and 2-6 g of cellulose.

[0007] Furthermore, the culture medium auxiliary material is composed of the following raw materials by mass: 1000g of distilled water, 20g of glucose, 15g of peptone, 5g of sodium chloride, 8g of disodium hydrogen phosphate, 0.5g of beef extract, and 20g of agar.

[0008] Furthermore, the cellar mud composite is composed of the following raw materials in terms of mass: 80 g of cellar mud, 25 g of medical stone powder, 25 g of green shou stone powder, and 5 g of cellulose.

[0009] Furthermore, the cellar mud is selected from red mud in the cellar used for fermentation of Maotai-flavor liquor.

[0010] Furthermore, the mass ratio of the solid culture medium material, the paste culture medium material and the liquid culture medium material is 1:1:3, and the mass ratio of the liquid culture medium material to the culture medium auxiliary material is 1:3.

[0011] Further, the following steps are included:

[0012] S1, preparing solid culture medium: first drying the cellar mud, then weighing a certain amount of dry cellar mud, medical stone powder, green stone powder, cellulose and water, mixing and kneading, and then forming into sheets and drying and shaping;

[0013] S2, preparing a paste culture medium material: first drying the cellar mud, then weighing the cellar mud and distilled water in a certain proportion, mixing and stirring, and finally forming a paste culture medium material;

[0014] S3, preparing liquid culture medium material: first drying the cellar mud, then weighing the cellar mud and distilled water in a certain proportion, mixing and stirring, filtering to obtain a cellar mud extract, which is the liquid culture medium material;

[0015] S4, preparing culture medium auxiliary materials: weighing a certain amount of glucose, peptone, sodium chloride, disodium hydrogen phosphate, beef extract, agar and water, and stirring them evenly to form culture medium auxiliary materials;

[0016] S5, preparing a strain culture medium: adding a strain culture medium to a culture container, first placing a sheet of solid culture medium material at the bottom of the container, applying a paste culture medium material on the solid culture medium material, and then mixing and stirring a liquid culture medium material and a culture medium auxiliary material, and then pouring the mixture into the culture container; finally, a strain culture medium is formed with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of a liquid culture medium material and a culture medium auxiliary material on the surface;

[0017] S6, strain cultivation using strain culture medium: inoculate the strain seed liquid into the strain culture medium described in S5, set the culture temperature to 37-40°C, and culture on a shaking incubator at 250-300 r / min for 20-30 h.

[0018] S7, fermentation to prepare plant polypeptides: drying the plant meal and then crushing it, and then applying it to the liquid mixture of the liquid culture medium material and the culture medium auxiliary material soaked in S6, applying a low-intensity magnetic field during the fermentation process, setting the fermentation temperature to 40-42°C, and the fermentation time to 36-48h; after the fermentation is completed, cleaning the plant meal in the culture medium, extracting the fermentation liquid in the culture medium, centrifuging the fermentation liquid at 5500r / min for 30min, and then taking the upper clear liquid, which is the crude plant polypeptide extract; the strain culture medium is kept for use next time after cleaning.

[0019] Furthermore, in step S6, the culture temperature was set to 37°C, and the culture was performed on a shaking incubator at 270 r / min for 24 h.

[0020] Furthermore, in step S7, the fermentation temperature is set to 40°C and the fermentation time is set to 48 hours.

[0021] The present invention has the following beneficial effects:

[0022] 1. In the scheme of the present invention, the cellar mud in the fermentation cellar of Maotai-flavor liquor is used as part of the culture medium, which is beneficial to improving the growth efficiency of the strain and can improve the activity of the enzyme and the enzymolysis effect.

[0023] 2. In the scheme of the present invention, the culture medium is set as a multi-layer structure, and pit mud, medical stone and green shou stone are added to the solid culture medium at the bottom, which can effectively increase biological activity, improve strain propagation efficiency, and improve protease production efficiency and activity. After the fermentation is completed, after the upper layer of the culture medium is taken out, the middle layer and the lower layer can still be used as implantation of microorganisms, so that the microorganisms are retained for subsequent fermentation. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with embodiments.

[0025] Example 1

[0026] A culture medium comprises a solid culture medium, a paste culture medium, a liquid culture medium and culture medium supplementary materials.

[0027] in:

[0028] The solid culture medium, i.e., the cellar mud complex, is composed of the following raw materials in terms of mass: 5000 g of cellar mud, 2000 g of medical stone powder, 2000 g of green shou stone powder, and 200 g of cellulose.

[0029] The paste culture medium is a paste mixture with a water content of 30% formed by mixing pit mud and water.

[0030] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0031] The culture medium auxiliary materials are composed of the following raw materials by mass: 8000g of distilled water, 100g of glucose, 100g of peptone, 20g of sodium chloride, 50g of disodium hydrogen phosphate, 3g of beef extract, and 100g of agar.

[0032] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above is selected as 1:1:3:6 for subsequent experiments.

[0033] The method for preparing plant polypeptides using the strain culture medium prepared in Example 1 comprises the following steps:

[0034] S1, preparing solid culture medium: first drying the cellar mud, then weighing 5000g of dried cellar mud, 2000g of medical stone powder, 2000g of green stone powder, 200g of cellulose and 5000g of water, mixing and kneading, and then forming into a sheet with a length, width and height of 4cm×2cm×0.5cm respectively, and drying and shaping, the drying temperature is 45°C, and set aside;

[0035] S2, preparing a paste culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water at a mass ratio of 7:3, mixing and stirring, and finally forming a paste culture medium material for standby use;

[0036] S3, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering to obtain a cellar mud extract as the liquid culture medium material, and setting aside;

[0037] S4, prepare culture medium auxiliary materials: weigh 100 g glucose, 100 g peptone, 20 g sodium chloride, 50 g disodium hydrogen phosphate, 3 g beef extract, 100 g agar and 8000 g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0038] S5, prepare strain culture medium: select the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:1:3:6. Add the strain culture medium to the culture container, specifically: first place the stacked sheets of solid culture medium material at the bottom of the container, apply the paste culture medium material on the solid culture medium material, and then mix and stir the liquid culture medium material and the culture medium auxiliary material and pour them into the culture container; finally, a strain culture medium is formed with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of liquid culture medium material and culture medium auxiliary material on the surface;

[0039] S6, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S5. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0040] S7, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S6, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0041] Example 2

[0042] A culture medium comprises a solid culture medium, a paste culture medium, a liquid culture medium and culture medium supplementary materials.

[0043] in:

[0044] The solid culture medium, i.e., the cellar mud complex, is composed of the following raw materials in terms of mass: 10,000 g of cellar mud, 3,000 g of medical stone powder, 3,000 g of green shou stone powder, and 600 g of cellulose.

[0045] The paste culture medium is a paste mixture with a water content of 30% formed by mixing pit mud and water.

[0046] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0047] The culture medium auxiliary materials are composed of the following raw materials by mass: 15000g of distilled water, 300g of glucose, 300g of peptone, 100g of sodium chloride, 100g of disodium hydrogen phosphate, 10g of beef extract, and 350g of agar.

[0048] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above is selected as 1:1:3:6 for subsequent experiments.

[0049] The method for preparing plant polypeptides using the strain culture medium prepared in Example 2 comprises the following steps:

[0050] S1, preparing solid culture medium material: first drying the cellar mud, then weighing 10000g of dried cellar mud, 3000g of medical stone powder, 3000g of green stone powder, 600g of cellulose and 10000g of water, mixing and kneading, and then forming into a sheet with a length, width and height of 4cm×2cm×0.5cm respectively, and drying and shaping, the drying temperature is 45°C, and set aside;

[0051] S2, preparing a paste culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water at a mass ratio of 7:3, mixing and stirring, and finally forming a paste culture medium material for standby use;

[0052] S3, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering to obtain a cellar mud extract as the liquid culture medium material, and setting aside;

[0053] S4, prepare culture medium auxiliary materials: weigh 300 g glucose, 300 g peptone, 100 g sodium chloride, 100 g disodium hydrogen phosphate, 10 g beef extract, 350 g agar and 15000 g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0054] S5, prepare strain culture medium: select the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:1:3:6. Add the strain culture medium to the culture container, specifically: first place the stacked sheets of solid culture medium material at the bottom of the container, apply the paste culture medium material on the solid culture medium material, and then mix and stir the liquid culture medium material and the culture medium auxiliary material and pour them into the culture container; finally, a strain culture medium is formed with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of liquid culture medium material and culture medium auxiliary material on the surface;

[0055] S6, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S5. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0056] S7, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S6, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0057] Example 3

[0058] A culture medium comprises a solid culture medium, a paste culture medium, a liquid culture medium and culture medium supplementary materials.

[0059] in:

[0060] The solid culture medium, i.e., the cellar mud complex, is composed of the following raw materials in terms of mass: 8000 g of cellar mud, 2500 g of medical stone powder, 2500 g of green shou stone powder, and 500 g of cellulose.

[0061] The paste culture medium is a paste mixture with a water content of 30% formed by mixing pit mud and water.

[0062] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0063] The culture medium auxiliary materials are composed of the following raw materials by mass: 10000g of distilled water, 200g of glucose, 150g of peptone, 50g of sodium chloride, 80g of disodium hydrogen phosphate, 5g of beef extract, and 200g of agar.

[0064] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above is selected as 1:1:3:9 for subsequent experiments.

[0065] The method for preparing plant polypeptides using the strain culture medium prepared in Example 3 comprises the following steps:

[0066] S1, preparing solid culture medium: first drying the cellar mud, then weighing 8000g of dried cellar mud, 2500g of medical stone powder, 2500g of green stone powder, 500g of cellulose and 8000g of water, mixing and kneading, and then forming into a sheet with a length, width and height of 4cm×2cm×0.5cm respectively, and drying and shaping, the drying temperature is 45°C, and set aside;

[0067] S2, preparing a paste culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water at a mass ratio of 7:3, mixing and stirring, and finally forming a paste culture medium material for standby use;

[0068] S3, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering to obtain a cellar mud extract as the liquid culture medium material, and setting aside;

[0069] S4, prepare culture medium auxiliary materials: weigh 200g glucose, 150g peptone, 50g sodium chloride, 80g disodium hydrogen phosphate, 5g beef extract, 200g agar and 10000g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0070] S5, prepare strain culture medium: select the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:1:3:9. Add the strain culture medium into the culture container, specifically: first place the stacked sheets of solid culture medium material at the bottom of the container, apply the paste culture medium material on the solid culture medium material, and then mix and stir the liquid culture medium material and the culture medium auxiliary material and pour them into the culture container; finally, a strain culture medium is formed with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of liquid culture medium material and culture medium auxiliary material on the surface;

[0071] S6, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S5. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0072] S7, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S6, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0073] Example 4

[0074] A culture medium comprises a solid culture medium, a paste culture medium, a liquid culture medium and culture medium supplementary materials.

[0075] in:

[0076] The solid culture medium, i.e., the cellar mud complex, is composed of the following raw materials in terms of mass: 8000 g of cellar mud, 2500 g of medical stone powder, 2500 g of green shou stone powder, and 500 g of cellulose.

[0077] The paste culture medium is a paste mixture with a water content of 30% formed by mixing pit mud and water.

[0078] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0079] The culture medium auxiliary materials are composed of the following raw materials by mass: 10000g of distilled water, 200g of glucose, 150g of peptone, 50g of sodium chloride, 80g of disodium hydrogen phosphate, 5g of beef extract, and 200g of agar.

[0080] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above is selected as 1:1:3:12 for subsequent experiments.

[0081] The method for preparing plant polypeptides using the strain culture medium prepared in Example 4 comprises the following steps:

[0082] S1, preparing solid culture medium: first drying the cellar mud, then weighing 8000g of dried cellar mud, 2500g of medical stone powder, 2500g of green stone powder, 500g of cellulose and 8000g of water, mixing and kneading, and then forming into a sheet with a length, width and height of 4cm×2cm×0.5cm respectively, and drying and shaping, the drying temperature is 45°C, and set aside;

[0083] S2, preparing a paste culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water at a mass ratio of 7:3, mixing and stirring, and finally forming a paste culture medium material for standby use;

[0084] S3, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering to obtain a cellar mud extract as the liquid culture medium material, and setting aside;

[0085] S4, prepare culture medium auxiliary materials: weigh 200 g glucose, 150 g peptone, 50 g sodium chloride, 80 g disodium hydrogen phosphate, 5 g beef extract, 200 g agar and 10000 g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0086] S5, prepare strain culture medium: select the mass ratio of the solid culture medium material, paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:1:3:12. Add the strain culture medium to the culture container, specifically: first place the stacked sheets of solid culture medium material at the bottom of the container, apply the paste culture medium material on the solid culture medium material, and then mix and stir the liquid culture medium material and the culture medium auxiliary material and pour them into the culture container; finally, a strain culture medium is formed with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of liquid culture medium material and culture medium auxiliary material on the surface;

[0087] S6, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S5. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0088] S7, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S6, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0089] Comparative Example 1

[0090] A culture medium comprises a paste culture medium, a liquid culture medium and culture medium auxiliary materials.

[0091] in:

[0092] The paste culture medium is a paste mixture with a water content of 30% formed by mixing pit mud and water.

[0093] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0094] The culture medium auxiliary materials are composed of the following raw materials by mass: 10000g of distilled water, 200g of glucose, 150g of peptone, 50g of sodium chloride, 80g of disodium hydrogen phosphate, 5g of beef extract, and 200g of agar.

[0095] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the above prepared paste culture medium material, liquid culture medium material, and culture medium auxiliary material is selected as 1:3:9 for subsequent experiments.

[0096] The method for preparing plant polypeptides using the strain culture medium prepared in this comparative example 1 comprises the following steps:

[0097] S1, preparing a paste culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water at a mass ratio of 7:3, mixing and stirring, and finally forming a paste culture medium material for standby use;

[0098] S2, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering and obtaining a cellar mud extract as the liquid culture medium material, and setting aside;

[0099] S3, prepare culture medium auxiliary materials: weigh 200g glucose, 150g peptone, 50g sodium chloride, 80g disodium hydrogen phosphate, 5g beef extract, 200g agar and 10000g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0100] S4, prepare strain culture medium: select the mass ratio of the paste culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:3:9. Add strain culture medium into the culture container, specifically: first lay the paste culture medium material at the bottom of the container, then mix and stir the liquid culture medium material and culture medium auxiliary material, and pour them into the culture container; finally, a strain culture medium with the paste culture medium material at the bottom and the liquid culture medium material and culture medium auxiliary material as the surface is formed;

[0101] S5, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S4. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0102] S6, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S5, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0103] Comparative Example 2

[0104] A culture medium comprises a solid culture medium, a liquid culture medium and culture medium supplementary materials.

[0105] in:

[0106] The solid culture medium, i.e., the cellar mud complex, is composed of the following raw materials in terms of mass: 8000 g of cellar mud, 2500 g of medical stone powder, 2500 g of green shou stone powder, and 500 g of cellulose.

[0107] The liquid culture medium is a cellar mud extract, and in this embodiment, the cellar mud from a Luzhou-flavor liquor fermentation cellar for more than 10 years is selected.

[0108] The culture medium auxiliary materials are composed of the following raw materials by mass: 10000g of distilled water, 200g of glucose, 150g of peptone, 50g of sodium chloride, 80g of disodium hydrogen phosphate, 5g of beef extract, and 200g of agar.

[0109] In this embodiment, the above culture medium is prepared and set aside for use, and finally the mass ratio of the solid culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above is selected to be 1:3:9 for subsequent experiments.

[0110] The method for preparing plant polypeptides using the strain culture medium prepared in this comparative example 2 comprises the following steps:

[0111] S1, preparing solid culture medium: first drying the cellar mud, then weighing 8000g of dried cellar mud, 2500g of medical stone powder, 2500g of green stone powder, 500g of cellulose and 8000g of water, mixing and kneading, and then forming into a sheet with a length, width and height of 4cm×2cm×0.5cm respectively, and drying and shaping, the drying temperature is 45°C, and set aside;

[0112] S2, preparing liquid culture medium material: first drying the cellar mud at a drying temperature of 45° C., then weighing the cellar mud and distilled water in a ratio of 1:5, mixing and stirring, filtering and obtaining a cellar mud extract as the liquid culture medium material, and setting aside;

[0113] S3, prepare culture medium auxiliary materials: weigh 200g glucose, 150g peptone, 50g sodium chloride, 80g disodium hydrogen phosphate, 5g beef extract, 200g agar and 10000g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0114] S4, prepare strain culture medium: select the mass ratio of the solid culture medium material, liquid culture medium material, and culture medium auxiliary material prepared above to be 1:3:9. Add strain culture medium into the culture container, specifically: first place the stacked sheets of solid culture medium material at the bottom of the container, then mix and stir the liquid culture medium material and culture medium auxiliary material and pour them into the culture container; finally, a strain culture medium with solid culture medium material at the bottom and a liquid mixture of liquid culture medium material and culture medium auxiliary material on the surface is formed;

[0115] S5, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S4. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0116] S6, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the liquid mixture of the liquid culture medium and the culture medium auxiliary material in S5, and a low-intensity magnetic field is applied during the fermentation process. The fermentation temperature is set to 40°C and the fermentation time is 48h. After the fermentation is completed, the plant meal in the culture medium is cleaned, and the fermentation liquid in the culture medium is extracted. The fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0117] Comparative Example 3

[0118] A culture medium consisting only of culture medium supplements.

[0119] in:

[0120] The culture medium auxiliary materials are composed of the following raw materials by mass: 10000g of distilled water, 200g of glucose, 150g of peptone, 50g of sodium chloride, 80g of disodium hydrogen phosphate, 5g of beef extract, and 200g of agar.

[0121] The method for preparing plant polypeptides using the strain culture medium prepared in Example 3 comprises the following steps:

[0122] S1, prepare culture medium auxiliary materials: weigh 200g glucose, 150g peptone, 50g sodium chloride, 80g disodium hydrogen phosphate, 5g beef extract, 200g agar and 10000g water, stir evenly to form culture medium auxiliary materials, and set aside;

[0123] S2, preparing strain culture medium: adding strain culture medium into the culture container;

[0124] S3, strain cultivation using strain culture medium: inoculate strain seed liquid into the strain culture medium described in S2. In this embodiment, the inoculated strain is Bacillus subtilis. According to the inoculation density of 0.1 biomass, the culture temperature is set to 40°C, and the culture is carried out for 30 hours on a shaking incubator at 300 rpm.

[0125] S4, fermentation to prepare plant polypeptides: take plant meal, dry it and then crush it. In this embodiment, soybean meal is selected, and the mass of the selected soybean meal is equal to the mass of the liquid culture medium. Soybean meal is laid in the culture medium auxiliary material soaked in S3, a low-intensity magnetic field is applied during the fermentation process, the fermentation temperature is set to 40°C, and the fermentation time is 48h; after the fermentation is completed, the plant meal in the culture medium is cleaned, the fermentation liquid in the culture medium is extracted, the fermentation liquid is centrifuged at 5500r / min for 30min, and then the supernatant is taken, which is the crude plant polypeptide extract.

[0126] The differences between the above Examples 1-4 and Comparative Examples 1-3 are as follows:

[0127]

[0128] The difference between Example 1 and Example 2 is that the component ratios of the solid culture medium material and the liquid culture medium material are different.

[0129] By conducting a mouse feeding experiment with the crude plant polypeptide extracts in the above Examples 1-4 and Comparative Examples 1-3, an equal amount of crude polypeptide extracts prepared by the above method were added to the drinking water of seven groups of mice. After a period of feeding comparison, it was found that the crude plant polypeptide extracts in Examples 1-4 had a good effect on biological growth, and the group feeding growth effect in Example 3 was the best. It is proved that by adding pit mud to the culture medium and setting it as a multi-layer structure, the biological activity can be effectively increased, the bacterial reproduction efficiency can be improved, the protease generation efficiency and activity can be improved, and finally the generation efficiency of plant polypeptides can be improved, which can better improve the body's immunity and promote growth and development. And because the culture medium of this embodiment has a solid culture medium material and a paste culture medium material, the two layers of culture medium materials can be reused many times, and the effect will increase with the increase in the number of uses.

[0130] In addition, the culture medium proposed in this scheme also has a purification function.

[0131] When the culture medium in this scheme is applied in coatings, the specific setting is: the culture medium in this application is filtered, washed with clean water, and then dried, and then subjected to a high temperature treatment of 800°C, and crushed and ground into powder, and added to the coating product, which can effectively purify the air;

[0132] When the culture medium in this scheme is used in a filter, the specific configuration is as follows: the culture medium in this application is filtered and washed with clean water and then dried, and then subjected to a high temperature treatment of 800°C, and crushed and ground into powder, and when added to filter cotton or filter plate products, it can effectively purify the air or water quality;

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A culture medium, characterized in that: The culture medium comprises a culture medium main body and culture medium auxiliary materials; the culture medium main body comprises a solid culture medium material, a paste culture medium material and a liquid culture medium material, wherein the mass ratio of the solid culture medium material, the paste culture medium material and the liquid culture medium material is 1:1:(3-4); the solid culture medium material is a dried and solidified cellar mud complex, the paste culture medium material is a paste mixture formed by mixing cellar mud and water, and the liquid culture medium material is a cellar mud extract; the culture medium auxiliary materials are composed of the following raw materials in mass fractions: 800-1500g of distilled water, 10-30g of glucose, 10-30g of peptone, 2-10g of sodium chloride, 5-10g of disodium hydrogen phosphate, 0.3-1g of beef extract and 10-35g of agar; and the mass ratio of the liquid culture medium material to the culture medium auxiliary materials is 1:(2-4).

2. A culture medium according to claim 1, characterized in that: The pit mud compound is composed of the following raw materials in terms of mass: 50-100g of pit mud, 20-30g of medical stone powder, 20-30g of green shou stone powder, and 2-6g of cellulose.

3. A culture medium according to claim 1, characterized in that: The culture medium auxiliary material is composed of the following raw materials by mass: 1000 g of distilled water, 20 g of glucose, 15 g of peptone, 5 g of sodium chloride, 8 g of disodium hydrogen phosphate, 0.5 g of beef extract, and 20 g of agar.

4. A culture medium according to claim 2, characterized in that: The pit mud compound is composed of the following raw materials in terms of mass: 80 g pit mud, 25 g medical stone powder, 25 g green shou stone powder, and 5 g cellulose.

5. A culture medium according to claim 1, characterized in that: The cellar mud is selected from red mud in the cellar used for fermenting Maotai-flavor liquor.

6. A culture medium according to claim 1, characterized in that: The mass ratio of solid culture medium material, paste culture medium material and liquid culture medium material is 1:1:3, and the mass ratio of liquid culture medium material to culture medium auxiliary material is 1:

3.

7. Use of the culture medium according to claim 1 in preparing plant polypeptides, characterized in that: The preparation method comprises the following steps: S1, preparing solid culture medium: first drying the cellar mud, then weighing a certain amount of dry cellar mud, medical stone powder, green stone powder, cellulose and water, mixing and kneading, and then forming into sheets and drying and shaping; S2, preparing a paste culture medium material: first drying the cellar mud, then weighing the cellar mud and distilled water in a certain proportion, mixing and stirring, and finally forming a paste culture medium material; S3, preparing liquid culture medium material: first drying the cellar mud, then weighing the cellar mud and distilled water in a certain proportion, mixing and stirring, filtering to obtain a cellar mud extract, which is the liquid culture medium material; S4, preparing culture medium auxiliary materials: weighing a certain amount of glucose, peptone, sodium chloride, disodium hydrogen phosphate, beef extract, agar and water, and stirring them evenly to form culture medium auxiliary materials; S5, preparing a culture medium: adding a culture medium to a culture container, first placing a sheet of solid culture medium material at the bottom of the container, applying a paste culture medium material on the solid culture medium material, and then mixing and stirring a liquid culture medium material and a culture medium auxiliary material, and then pouring the mixture into the culture container; finally forming a strain culture medium with a solid culture medium material at the bottom, a paste culture medium material in the middle layer, and a liquid mixture of a liquid culture medium material and a culture medium auxiliary material on the surface; S6, strain cultivation using strain culture medium: inoculate the strain seed liquid into the strain culture medium described in S5, set the culture temperature to 37-40°C, and culture on a shaking incubator at 250-300 r / min for 20-30 h. S7, fermentation to prepare plant polypeptides: drying the plant meal and then crushing it, and then applying it to the liquid mixture of the liquid culture medium material and the culture medium auxiliary material soaked in S6, applying a low-intensity magnetic field during the fermentation process, setting the fermentation temperature to 40-42°C, and the fermentation time to 36-48h; after the fermentation is completed, cleaning the plant meal in the culture medium, extracting the fermentation liquid in the culture medium, centrifuging the fermentation liquid at 5500r / min for 30min, and then taking the upper clear liquid, which is the crude plant polypeptide extract; the strain culture medium is kept for next use after cleaning.

8. Use of a culture medium according to claim 7 in preparing plant polypeptides, characterized in that: In step S6, the culture temperature was set to 37°C, and the culture was performed on a shaking incubator at 270 r / min for 24 h.

9. Use of a culture medium according to claim 7 in preparing plant polypeptides, characterized in that: In step S7, the fermentation temperature is set to 40°C and the fermentation time is set to 48 hours.

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