CN112029669B - Cordyceps sinensis and nematode grass fermentation liquor, method, antioxidant and application - Google Patents

Cordyceps sinensis and nematode grass fermentation liquor, method, antioxidant and application Download PDF

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CN112029669B
CN112029669B CN202010968126.3A CN202010968126A CN112029669B CN 112029669 B CN112029669 B CN 112029669B CN 202010968126 A CN202010968126 A CN 202010968126A CN 112029669 B CN112029669 B CN 112029669B
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王志方
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Kebang Biotechnology Shanghai Co ltd
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Abstract

The invention relates to the technical field of cordyceps strains and microbial fermentation, in particular to cordyceps sinensis with the preservation number of CGMCC No. 13886. The preparation method of the cordyceps militaris fermentation liquor comprises the following steps: inoculating the nematodiasis into a sterilized shake flask liquid culture medium for culture, inoculating shake flask seed liquid into a sterilized primary seed fermentation culture medium for fermentation, inoculating primary seed fermentation liquid into a sterilized secondary seed fermentation culture medium for fermentation, and inoculating secondary seed fermentation liquid into a sterilized production-grade fermentation culture medium for fermentation to obtain nematodiasis fermentation liquid. The above fermented liquid is subjected to iron reduction antioxidation detection with activity of 437.95 μ M FeSO and DPPH free radical scavenging method4The DPPH radical clearance is 61.78%. The oxidation resistance of the cordyceps militaris fermentation liquor shows good high-temperature resistance.

Description

Cordyceps sinensis and nematode grass fermentation liquor, method, antioxidant and application
Technical Field
The invention relates to the technical field of cordyceps sinensis strains and microbial fermentation, in particular to cordyceps sinensis and nematodiella fermentation liquor, a method, an antioxidant and application.
Background
In recent years, the relationship between free radicals and various diseases has been attracting attention. A large number of researches show that the body can generate oxidative stress by bad stimulation to generate a large number of free radicals, and if the free radicals cannot be removed in time, biomolecules such as protein, DNA, lipid and the like can be damaged to cause peroxidation, unsaturated fatty acid in a cell membrane is oxidized to damage a cell structure; meanwhile, toxic substances such as hydroperoxide and Malondialdehyde (MDA) are generated, and the normal metabolic activity of the human body is interfered, so that corresponding diseases are caused, and the aging process is accelerated. Therefore, more and more attention is paid to the development of safe and effective products with antioxidant effect. Since the hot spot of finding effective antioxidants in the 80's of the last century, the most studied first-generation antioxidants are mainly vitamins, such as vitamin C, E; the second generation antioxidant comprises beta-carotene, coenzyme Q10, superoxide dismutase, etc.; the third generation antioxidant is widely present in grape seed, blueberry, green leaf plant or other Chinese medicinal extracts, such as anthocyanin, lycopene, resveratrol, tea polyphenol and other polyphenols.
Xinjiang Cordyceps (Cordyceps grsacilisis (Grev.) Dur et Mont) is a complex of Cordyceps fungi parasitizing on insect larvae of Altai bat moth (Hepialus altacicola W.), is a traditional Kazakh ethnic drug in Xinjiang, and has the main effects of nourishing marrow and essence, protecting lung and kidney, reducing phlegm and the like. By analyzing chemical components, the Xinjiang cordyceps sinensis is relatively similar to the cordyceps sinensis in chemical components, and the content of part of the components is similar to or even slightly higher than that of the cordyceps sinensis. In recent years, due to the rapid price rise of Xinjiang cordyceps, wild cordyceps is crazy to be dug, and natural resources are in imminent danger.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a cordyceps militaris strain, which is used for solving the endangered problem of natural resources of wild cordyceps militaris in the prior art; meanwhile, the invention also provides a cordyceps militaris fermentation broth and a preparation method of the cordyceps militaris fermentation broth; in addition, the invention also provides an antioxidant and application of the nematodiasis. The above herba Ceratophylli Thalictri Elati fermentation liquid adopts iron reduction antioxidation (FRAP) method and DPPH free radical scavenging method to detect antioxidation of the fermentation liquid, and the iron reduction antioxidation activity of the herba Ceratophylli Elongati fermentation liquid is 437.95 μ M FeSO4The DPPH free radical clearance rate of the nematodia fermentation liquor is 61.78%. The oxidation resistance of the cordyceps militaris fermentation liquor shows good high-temperature resistance.
In order to attain the above and other related objects,
the invention provides cordyceps sinensis (Ophiocerdyceps sp.), which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13886.
A fungus is separated from Xinjiang Cordyceps sinensis (Cordyceps grsacilisis (Grev.) Dur et Mont), is identified as a Cordyceps sinensis, is preserved in China general microbiological culture Collection center (China institute of sciences, national institute of microbiology, 3, North Cheng Xilu No.1 institute of China, Taiyang district, Beijing) in 2017 at 17 th month, and is named as: the nematophagous grass bacteria KB024 (Ophiocordiceps sp. KB024) with the preservation number of CGMCC No. 13886.
The biological characteristics of the grass nematodiasis are as follows: the nematophagous grass fungi grows on the potato glucose agar culture medium to form a colony, the substrate hypha at the bottom is tightly combined with the culture medium, and a large amount of white filament needle-shaped aerial hypha are emitted from the substrate hypha to form a white hemispherical colony.
The specific separation process of the nematophagous fungi comprises the following steps: cleaning fresh Xinjiang cordyceps sinensis, cleaning the Xinjiang cordyceps sinensis with clear water, wiping the surface of the Xinjiang cordyceps sinensis with 75% alcohol, cleaning the Xinjiang cordyceps sinensis with sterile water, repeating the cleaning for 3 times, cutting the Xinjiang cordyceps sinensis into slices, inoculating the slices on a PDA (PDA) slant culture medium, and culturing for 1-2 weeks in a thermostat at 25-28 ℃ to grow visible white and off-white short-melting mycelium. And (3) inoculating the grown mycelium on a new PDA slant culture medium, culturing at 22-25 ℃, and forming a circular or elliptical colony after 1-2 months. And continuously culturing for 1-2 weeks, wherein a large amount of white fine needle-shaped aerial hyphae are emitted from the hyphae in the substrate to form white hemispherical colonies.
Through tests, the liquid culture medium suitable for the growth of the grass nematodiasis is screened out, and specifically comprises the following components: 2-5 parts of glucose, 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour and 83-92 parts of water. The liquid culture medium is suitable for shake flask production culture.
The fermented filtrate of the nematodiferous grass fungi (Ophiocerdyceps sp) contains rich functional factors such as cordyceps polysaccharide, betaine, protocatechuic acid, inositol, carnitine and the like, and can replace wild cordyceps to be used in the technical fields of medicines, foods, cosmetics and the like, so that the problem that the natural resources of the wild cordyceps are endangered is solved.
In a second aspect of the invention, a cordyceps militaris fermentation broth is provided, wherein the cordyceps militaris fermentation broth is prepared by fermenting the cordyceps militaris.
The cordyceps sinensis fermentation liquor utilizes cordyceps sinensis to carry out liquid submerged fermentation, and the obtained fermentation filtrate contains rich functional factors such as cordyceps polysaccharide, betaine, protocatechuic acid, inositol, carnitine and the like, can effectively eliminate free radicals in vivo, improve sub-health state, enhance immunity, eliminate color spots and the like, and can effectively relieve symptoms such as headache, insomnia and the like caused by pressure; meanwhile, the product can also be used for maintaining beauty, keeping young, improving eyesight, reducing weight and prolonging life.
The cordycepin fermentation liquor shows good oxidation resistance after analysis, and the oxidation resistance of the cordycepin fermentation liquor basically keeps unchanged at high temperature, so that the cordycepin fermentation liquor can be used in the fields of human health-care food, cosmetic raw materials, livestock and poultry health-care feed additives and the like.
The third aspect of the invention provides a preparation method of the cordyceps militaris fermentation broth, which comprises the following steps:
step one, inoculating the grass nematodiasis into a sterilized shake flask liquid culture medium for culture to prepare shake flask seed liquid; wherein, the shake flask liquid culture medium comprises the following components: 2-5 parts of glucose, 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour and 83-92 parts of water;
step two, inoculating the shake flask seed liquid into a sterilized primary seed fermentation culture medium for fermentation to obtain primary seed fermentation liquid;
thirdly, inoculating the primary seed fermentation liquor into a sterilized secondary seed fermentation culture medium for fermentation to obtain secondary seed fermentation liquor;
step four, inoculating the secondary seed fermentation liquor into a sterilized production-grade fermentation culture medium for fermentation to obtain a nematodiasis fermentation liquor; wherein, the first-stage seed fermentation culture medium, the second-stage seed fermentation culture medium and the production-stage fermentation culture medium all comprise the following components: 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour, 0.08-0.12 part of edible oil and 88-94 parts of water.
The cordyceps sinensis fermentation liquor utilizes cordyceps sinensis to carry out liquid submerged fermentation, and the obtained fermentation filtrate contains rich functional factors such as cordyceps polysaccharide, betaine, protocatechuic acid, inositol, carnitine and the like, can effectively eliminate free radicals in vivo, improve sub-health state, enhance immunity, eliminate color spots and the like, and can effectively relieve symptoms such as headache, insomnia and the like caused by pressure; meanwhile, the product can also be used for maintaining beauty, keeping young, improving eyesight, reducing weight and prolonging life.
The above herba Ceratophylli Thalictri Elati fermentation liquid adopts iron reduction antioxidation (FRAP) method and DPPH free radical scavenging method to detect antioxidation of the fermentation liquid, and the iron reduction antioxidation activity of the herba Ceratophylli Elongati fermentation liquid is 437.95 μ M FeSO4The DPPH free radical clearance rate of the nematodia fermentation liquor is 61.78%.
After the cordyceps militaris fermentation liquor is subjected to water bath treatment at 40-100 ℃ for 30min, the oxidation resistance is not obviously reduced. After the cordyceps sinensis fermentation liquor is treated by water bath at 100 ℃ for 30min, the iron reduction antioxidant activity of the cordyceps sinensis fermentation liquor is 391.76 mu M FeSO4The DPPH free radical clearance rate of the nematodia fermentation liquor is 58.03%. Therefore, the oxidation resistance of the cordyceps militaris fermentation liquor shows good high-temperature resistance.
The preparation method of the cordyceps militaris fermentation liquor is simple, the technical production cost is low, the cordyceps militaris fermentation liquor is not limited by environmental conditions, and the cordyceps militaris fermentation liquor is suitable for large-scale industrial production and popularization.
In one embodiment of the present invention, the shake flask liquid culture medium in the first step comprises the following components: 2-5 parts of glucose, 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour and 83-92 parts of water. Selecting food-grade glucose, high-quality corn flour, high-quality soybean and high-quality chickpea, grinding the materials, sieving the ground materials by a 100-mesh sieve, and preparing a shake flask liquid culture medium.
In an embodiment of the present invention, the specific conditions for the cultivation in the first step are as follows: culturing for 5-7 days under the conditions that the rotating speed of a shaking table is 100-150 r/min and the culturing temperature is 22-25 ℃.
In an embodiment of the present invention, the primary seed fermentation medium, the secondary seed fermentation medium, and the production-stage fermentation medium all comprise the following components: 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour, 0.08-0.12 part of edible oil and 88-94 parts of water. Selecting high-quality corn flour, high-quality soybeans and high-quality chickpeas, grinding the corn flour, the high-quality soybeans and the high-quality chickpeas, sieving the ground corn flour, the high-quality soybeans and the high-quality chickpeas with a 100-mesh sieve, and preparing a corresponding culture medium.
In an embodiment of the present invention, the specific conditions of the fermentation in the second step are as follows: culturing for 5-7 days under the conditions of ventilation volume of 4-6L/min, stirring speed of 90-110 r/min, culture temperature of 22-25 ℃ and culture pressure of 0.04-0.06 MPa;
the concrete conditions of the fermentation in the third step are as follows: culturing for 5-7 days under the conditions of ventilation volume of 40-50L/min, stirring speed of 70-90 r/min, culture temperature of 22-25 ℃ and culture pressure of 0.04-0.06 MPa;
the specific conditions of the fermentation in the fourth step are as follows: culturing for 5-7 days under the conditions of air flow of 80-100L/min, stirring speed of 60-80 r/min and culture temperature of 22-25 ℃.
In an embodiment of the present invention, the volume ratio of the shake flask seed liquid to the primary seed fermentation medium in the second step is (3-4): (96-97);
the volume ratio of the primary seed fermentation liquid to the secondary seed fermentation medium in the third step is (8-12): (88 to 92);
and the volume ratio of the secondary seed fermentation liquid to the production-grade fermentation medium in the fourth step is (8-12): (88-92).
In an embodiment of the present invention, the sterilization temperature of the shake flask liquid culture medium is 110-120 ℃, and the sterilization time is 20-40 min;
the sterilization temperature of the primary seed fermentation culture medium, the secondary seed fermentation culture medium and the production-grade fermentation culture medium is 115-125 ℃, and the sterilization time is 20-40 min.
In one embodiment of the present invention, the sterilization temperature of the shake flask liquid culture medium is 115 ℃, and the sterilization time is 30 min;
the sterilization temperature of the primary seed fermentation culture medium, the secondary seed fermentation culture medium and the production-level fermentation culture medium is 121 ℃, and the sterilization time is 30 min.
In an embodiment of the invention, the amount of dry bacteria in the primary seed fermentation broth is 29-31 g/L; the amount of dry bacteria in the secondary seed fermentation liquor is 34-36 g/L; the dry bacterium amount in the nematodia fermentation liquor is 39-41 g/L.
In a fourth aspect of the invention, an antioxidant is provided, which comprises the cordyceps militaris fermentation broth. The nematodiferous grass fermentation liquor is added into the antioxidant, so that the antioxidant can effectively remove free radicals in vivo, improve sub-health state, enhance immunity, eliminate color spots and the like, and can effectively relieve symptoms such as headache, insomnia and the like caused by pressure; meanwhile, the product can also be used for maintaining beauty, keeping young, improving eyesight, reducing weight and prolonging life.
In a fifth aspect of the invention, the application of cordyceps militaris in preparing antioxidant food or medicine is provided, wherein the preservation number of the cordyceps militaris (Ophiocordyceps sp.) is CGMCC No. 13886. The above-mentioned nematodiella (Ophiocerdyceps sp) can effectively remove free radical in vivo, improve sub-health state, enhance immunity and eliminate mottle, so that it has good antioxidant function.
As mentioned above, the fermentation broth of Cordyceps sinensis and nematode grass, the method, the antioxidant and the application of the invention have the following beneficial effects:
1. the cordyceps sinensis fermentation liquor utilizes cordyceps sinensis to carry out liquid submerged fermentation, and the obtained fermentation filtrate contains rich functional factors such as cordyceps polysaccharide, betaine, protocatechuic acid, inositol, carnitine and the like, can effectively eliminate free radicals in vivo, improve sub-health state, enhance immunity, eliminate color spots and the like, and can effectively relieve symptoms such as headache, insomnia and the like caused by pressure; meanwhile, the product can also be used for maintaining beauty, keeping young, improving eyesight, reducing weight and prolonging life.
2. The above herba Ceratophylli Thalictri Elati fermentation liquid adopts iron reduction antioxidation (FRAP) method and DPPH free radical scavenging method to detect antioxidation of the fermentation liquid, and the iron reduction antioxidation activity of the herba Ceratophylli Elongati fermentation liquid is 437.95 μ M FeSO4The DPPH free radical clearance rate of the nematodia fermentation liquor is 61.78%.
3. After the cordyceps militaris fermentation liquor is subjected to water bath treatment at 40-100 ℃ for 30min, the oxidation resistance is not obviously reduced. After the cordyceps sinensis fermentation liquor is treated by water bath at 100 ℃ for 30min, the iron reduction antioxidant activity of the cordyceps sinensis fermentation liquor is 391.76 mu M FeSO4The DPPH free radical clearance rate of the nematodia fermentation liquor is 58.03%. Therefore, the oxidation resistance of the cordyceps sinensis fermentation liquor shows good high-temperature resistance。
4. The preparation method of the cordyceps militaris fermentation liquor is simple, the technical production cost is low, the cordyceps militaris fermentation liquor is not limited by environmental conditions, and the cordyceps militaris fermentation liquor is suitable for large-scale industrial production and popularization.
Drawings
FIG. 1 shows a standard curve for iron reduction oxidation resistance (FRAP).
FIG. 2 shows the microscopic morphology of mycelia and spores of the grass nematodiasis KB024 (Ophiocericeps sp.
FIG. 3 shows the 18S phylogenetic tree of the grass nematode bacterium KB024 (Ophiocordiceps sp. KB024).
FIG. 4 shows the ITS phylogenetic tree of C.elegans KB024 (Ophiocordiceps sp. KB024).
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
The Ophiocordyceps sp, as referred to herein, has been deposited at the International depositary organization for microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC) preservation, address: the microbial research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation date of 2017, 04.17.2017 and a preservation number of CGMCC No. 13886.
A fungus is separated from Xinjiang Cordyceps sinensis (Cordyceps grsacilis (Grev.) Dur et Mont), is identified as a cordycepin fungus, and is classified and named as: the nematophagous grass bacteria KB024 (Ophiocordiceps sp. KB024) with the preservation number of CGMCC No. 13886.
Xinjiang Linnaeus is collected from the land managed by the Aleptai white Harpagin field of Xinjiang, a Linnaeus strain (Ophiocordyceps sp.) KB024 is obtained by separating the Linnaeus from the sclerotium, and the Linnaeus strain belongs to the Linnaeus genus by morphological and molecular biological identification. Deposited at the Budapest treaty on the International deposit of microorganisms: china general microbiological culture Collection center (CGMCC) preservation, address: the microbial research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation date of 2017, 04.17.2017 and a preservation number of CGMCC No. 13886.
The morphological and molecular biological identification of the grass nematodiasis:
culturing nematodiella KB024 (Ophiococcus sp.KB024) on PDA culture medium at 22-25 deg.C for 2 weeks to form creeping growth colony, which has a layer of white villous hypha on the surface, culturing for one month to form semispherical colony on the creeping colony, wherein the colony is composed of spiny spore silk, and yellow brown pigment is secreted into the culture medium. Observing under microscope, spore silk diameter is 1 μm, spore stalks are paired on spore silk every 15-20 μm, spores are radially and uniformly arranged on top of spore stalks, the spores are in water drop shape, top has fine-thread protrusion, and spore size is about 1 μm, as shown in FIG. 2.
18S rRNA and ITS sequences were PCR amplified by extracting the total DNA of M.elegans KB024 (Ophiocordyces sp. KB024), the sequencing results were retrieved from GenBank database using Blast software in NCBI (national Center for Biotechnology information), the 18S rRNA gene sequences and ITS sequences of related strains with higher similarity were aligned by sequence alignment using CLUSTAL X, and phylogenetic tree construction and homology comparison using the adjacency to Saitou and Nei (Neighbor Joining) using MEGA6.0 software. The 18S rRNA gene sequence of the M.elegans (Ophiocordyceps sp.) KB024 was determined to be 1208bp, the ITS sequence was 550bp, and the strains with the highest similarity between the 18S and ITS sequences were all Ophiocordyceps gracilis. The purified strain, M.nematocimum (Ophiocordiceps sp.) KB024 was identified as M.nematocimum (Ophiocordiceps sp.) by the above results and the results of homology analysis and phylogenetic analysis of 18S rRNA and ITS sequences.
The specific separation process of the nematophagous fungi comprises the following steps: cleaning fresh Xinjiang cordyceps sinensis, cleaning the Xinjiang cordyceps sinensis with clear water, wiping the surface of the Xinjiang cordyceps sinensis with 75% alcohol, cleaning the Xinjiang cordyceps sinensis with sterile water, repeating the cleaning for 3 times, cutting the Xinjiang cordyceps sinensis into slices, inoculating the slices on a PDA (PDA) slant culture medium, and culturing for 1-2 weeks in a thermostat at 25-28 ℃ to grow visible white and off-white short-melting mycelium. And (3) inoculating the grown mycelium on a new PDA slant culture medium, culturing at 22-25 ℃, and forming a circular or elliptical colony after 1-2 months. And continuously culturing for 1-2 weeks, wherein a large amount of white fine needle-shaped aerial hyphae are emitted from the hyphae in the substrate to form white hemispherical colonies.
Example 1
A preparation method of cordyceps militaris fermentation liquor comprises the following steps:
step one, culture medium raw materials: selecting food-grade glucose, high-quality corn flour, high-quality soybean and chickpea, grinding the materials into powder, and sieving the powder with a 100-mesh sieve.
Step two, preparing a shake flask liquid culture medium: adding glucose 3%, corn flour 2%, semen glycines powder 3%, and semen Ciceris Arietini powder 3% into 100ml water, adding 500ml above culture medium into 1L glass triangular flask, and sterilizing at 115 deg.C for 30 min.
Step three, preparing shake flask seed liquid: taking a slant of Cordyceps militaris, scraping off a fungus block with a diameter of 0.5cm from the slant under aseptic condition, inoculating into a shake flask, placing on a shaking table, and culturing at a rotation speed of 120rpm and 22-25 deg.C for 5-7 days.
Step four, preparing first-stage seed fermentation liquor: according to the mass volume ratio, 3 percent of corn flour, 4 percent of soybean meal, 5 percent of chickpea powder and 0.1 percent of edible oil are respectively added into 100ml of tap water to prepare a first-grade seed fermentation culture medium; preparing 18L of the culture medium in a 25L seed fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; taking a bottle of shake flask seeds in the third step under aseptic condition, inoculating into a 25L fermentation tank, and inoculating according to the inoculation amount of 4%; wherein the fermentation process comprises the following steps: the ventilation volume is 5L/min, the stirring speed is 100r/min, the culture temperature is 22-25 ℃, the tank pressure is 0.06MPa, the culture is carried out for 5-7 days, and the fermentation process is finished until the dry bacterial mass in the primary seed fermentation liquid reaches 30 g/L.
Step five, preparing secondary seed fermentation liquor: preparing 180L of secondary seed fermentation culture medium according to the culture medium formula in the fourth step in a secondary liquid seed tank (250L fermentation tank), sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; under the aseptic condition, transferring the primary seed fermentation liquor prepared in the fourth step into the fermentation liquor for culturing, and inoculating according to the inoculation amount of 10%; wherein the fermentation process comprises the following steps: the ventilation volume is 50L/min, the stirring speed is 80r/min, the culture temperature is 22-25 ℃, the culture pressure is 0.06MPa, the culture is carried out for 5-7 days, and the fermentation is finished when the dry bacterial mass in the secondary seed fermentation liquid reaches 35 g/L.
Step six, the nematodia fermentation liquor: preparing a production-grade fermentation culture medium in a large-scale fermentation tank (1000L fermentation tank) according to the formula in the fourth step, wherein the total volume of the culture medium is 70% of that of the large-scale fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; transferring the secondary seed fermentation liquor prepared in the fifth step to the fermentation liquor under the aseptic condition for culture, and inoculating according to the inoculation amount of 10%; wherein the fermentation process comprises the following steps: the ventilation capacity is 100L/min, the stirring speed is 80r/min, the culture time is 5-7 days, and the fermentation is finished when the dry bacterium amount in the fermentation liquor reaches 40 g/L.
Step seven, preparing fermentation filtrate: and (4) centrifuging the fermentation liquor obtained in the step six under the aseptic condition, centrifuging for 5min at 4000r/min, and taking supernate, namely the fermentation filtrate.
Example 2
A preparation method of cordyceps militaris fermentation liquor comprises the following steps:
step one, culture medium raw materials: selecting food-grade glucose, high-quality corn flour, high-quality soybean and chickpea, grinding the materials into powder, and sieving the powder with a 100-mesh sieve.
Step two, preparing a shake flask liquid culture medium: adding glucose 3%, corn flour 2%, semen glycines powder 3%, and semen Ciceris Arietini powder 3% into 100ml water, adding 500ml above culture medium into 1L glass triangular flask, and sterilizing at 115 deg.C for 30 min.
Step three, preparing shake flask seed liquid: taking a slant of Cordyceps militaris, scraping off a fungus block with a diameter of 0.5cm from the slant under aseptic condition, inoculating into a shake flask, placing on a shaking table, and culturing at a rotation speed of 120rpm and 22-25 deg.C for 5-7 days.
Step four, preparing first-stage seed fermentation liquor: according to the mass volume ratio, 2 percent of corn flour, 3 percent of soybean meal, 4 percent of chickpea powder and 0.1 percent of edible oil are respectively added into 100ml of tap water to prepare a first-grade seed fermentation culture medium; preparing 18L of the culture medium in a 25L seed fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; taking a bottle of shake flask seeds in the third step under aseptic condition, inoculating into a 25L fermentation tank, and inoculating according to the inoculation amount of 3%; wherein the fermentation process comprises the following steps: the ventilation volume is 5L/min, the stirring speed is 100r/min, the culture temperature is 22-25 ℃, the tank pressure is 0.4MPa, the culture is carried out for 5-7 days, and the fermentation process is finished until the dry bacterial mass in the primary seed fermentation liquid reaches 30 g/L.
Step five, preparing secondary seed fermentation liquor: preparing 180L of secondary seed fermentation culture medium according to the culture medium formula in the fourth step in a secondary liquid seed tank (250L fermentation tank), sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; under the aseptic condition, transferring the primary seed fermentation liquor prepared in the fourth step into the fermentation liquor for culturing, and inoculating according to the inoculation amount of 9%; wherein the fermentation process comprises the following steps: the ventilation volume is 50L/min, the stirring speed is 80r/min, the culture temperature is 22-25 ℃, the culture pressure is 0.04MPa, the culture is carried out for 5-7 days, and the fermentation is finished when the dry bacterial mass in the secondary seed fermentation liquid reaches 35 g/L.
Step six, the nematodia fermentation liquor: preparing a production-grade fermentation culture medium in a large-scale fermentation tank (1000L fermentation tank) according to the formula in the fourth step, wherein the total volume of the culture medium is 70% of that of the large-scale fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; transferring the secondary seed fermentation liquor prepared in the fifth step to the fermentation liquor under the aseptic condition for culture, and inoculating according to the inoculation amount of 10%; wherein the fermentation process comprises the following steps: the ventilation capacity is 100L/min, the stirring speed is 80r/min, the culture time is 5-7 days, and the fermentation is finished when the dry bacterium amount in the fermentation liquor reaches 40 g/L.
Step seven, preparing fermentation filtrate: and (4) centrifuging the fermentation liquor obtained in the step six under the aseptic condition, centrifuging for 5min at 4000r/min, and taking supernate, namely the fermentation filtrate.
Example 3
A preparation method of cordyceps militaris fermentation liquor comprises the following steps:
step one, culture medium raw materials: selecting food-grade glucose, high-quality corn flour, high-quality soybean and chickpea, grinding the materials into powder, and sieving the powder with a 100-mesh sieve.
Step two, preparing a shake flask liquid culture medium: adding glucose 3%, corn flour 2%, semen glycines powder 3%, and semen Ciceris Arietini powder 3% into 100ml water, adding 500ml above culture medium into 1L glass triangular flask, and sterilizing at 115 deg.C for 30 min.
Step three, preparing shake flask seed liquid: taking a slant of Cordyceps militaris, scraping off a fungus block with a diameter of 0.5cm from the slant under aseptic condition, inoculating into a shake flask, placing on a shaking table, and culturing at a rotation speed of 120rpm and 22-25 deg.C for 5-7 days.
Step four, preparing first-stage seed fermentation liquor: according to the mass volume ratio, 1 percent of corn flour, 2 percent of soybean meal, 3 percent of chickpea powder and 0.1 percent of edible oil are respectively added into 100ml of tap water to prepare a first-grade seed fermentation culture medium; preparing 18L of the culture medium in a 25L seed fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; taking a bottle of shake flask seeds in the third step under aseptic condition, inoculating into a 25L fermentation tank, and inoculating according to the inoculation amount of 4%; wherein the fermentation process comprises the following steps: the ventilation volume is 5L/min, the stirring speed is 100r/min, the culture temperature is 22-25 ℃, the tank pressure is 0.06MPa, the culture is carried out for 5-7 days, and the fermentation process is finished until the dry bacterial mass in the primary seed fermentation liquid reaches 30 g/L.
Step five, preparing secondary seed fermentation liquor: preparing 180L of secondary seed fermentation culture medium according to the culture medium formula in the fourth step in a secondary liquid seed tank (250L fermentation tank), sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; under the aseptic condition, transferring the primary seed fermentation liquor prepared in the fourth step into the fermentation liquor for culturing, and inoculating according to 12% of inoculation amount; wherein the fermentation process comprises the following steps: the ventilation volume is 50L/min, the stirring speed is 80r/min, the culture temperature is 22-25 ℃, the culture pressure is 0.06MPa, the culture is carried out for 5-7 days, and the fermentation is finished when the dry bacterial mass in the secondary seed fermentation liquid reaches 35 g/L.
Step six, the nematodia fermentation liquor: preparing a production-grade fermentation culture medium in a large-scale fermentation tank (1000L fermentation tank) according to the formula in the fourth step, wherein the total volume of the culture medium is 70% of that of the large-scale fermentation tank, sterilizing at 121 ℃ for 30min, and naturally cooling to room temperature; transferring the secondary seed fermentation liquor prepared in the fifth step to the fermentation liquor under the aseptic condition for culture, and inoculating according to the inoculation amount of 10%; wherein the fermentation process comprises the following steps: the ventilation capacity is 100L/min, the stirring speed is 80r/min, the culture time is 5-7 days, and the fermentation is finished when the dry bacterium amount in the fermentation liquor reaches 40 g/L.
Step seven, preparing fermentation filtrate: and (4) centrifuging the fermentation liquor obtained in the step six under the aseptic condition, centrifuging for 5min at 4000r/min, and taking supernate, namely the fermentation filtrate.
The fermentation filtrates obtained in the above examples 1 to 3 were subjected to oxidation resistance measurement including iron reduction oxidation resistance (FRAP) measurement, DPPH radical clearance measurement and fermentation filtrate oxidation resistance measurement, and the test examples specifically included the following steps:
test example 1
And (3) measuring the iron reduction oxidation resistance (FRAP) and the high-temperature resistance of the fermentation filtrate: the measurement of iron reduction Antioxidant Power (FRAP) is firstly used for measuring the Reducing Power of serum and then used for analyzing Antioxidant substances of plant origin. Antioxidant reduction of Fe in low pH solutions3+TPTZ (2, 4, 6-trypticyl-s-triaine, tripyridyltriazine) complex, forming blue Fe2+TPTZ solution, with a maximum absorbance at 593nm, the increase in absorbance being directly proportional to the antioxidant capacity.
The test method of iron reduction oxidation resistance (FRAP) is as follows:
1) preparing an FRAP reagent: acetic acid buffer (300mmol/L, pH3.6), 10mmol/L TPTZ (prepared with 40mmol/L HCl), and 20mol/L FeCl3The volume ratio of 10: 1: 1 and mixing.
2) Drawing a standard curve: preparing 0-500 mu mol/L FeSO4Mixing 0.5ml of the solution with 2ml of LFRAP reagent, placing in water bath at 37 ℃ for 20min, taking out, cooling to room temperature, measuring the light absorption value at 593nm, and making a standard curve (shown in figure 1); wherein the abscissa is FeSO4The ordinate of the concentration of (c) is the corresponding absorbance, and the regression equation of the standard curve is that y is 0.0014x +0.0082 (R)2＝0.9951)。
3) And (3) measuring iron reduction antioxidation of the fermentation filtrate: the fermentation filtrates (0.5mL) obtained in example 1, example 2 and example 3 were mixed with 2mL of LFRAP reagent, placed in a 37 ℃ water bath for 20min, taken out and cooled to room temperature, and the absorbance at 593nm was measured. The ferrous sulfate equivalent concentration of the sample to be measured was calculated from the standard curve, and the results are shown in table 1.
4) And (3) measuring the iron reduction oxidation resistance and high temperature resistance of the fermentation filtrate: the fermentation filtrates obtained in example 1, example 2 and example 3 were treated in a water bath at 40 to 100 ℃ for 30min, and then measured by the method of step 3. The ferrous sulfate equivalent concentration of the sample to be measured was calculated from the standard curve, and the results are shown in table 1.
Table 1
Figure BDA0002683088860000101
The above herba Ceratophylli Nigrae fermentation liquid adopts iron reduction antioxidation (FRAP) method to detect the antioxidation of the fermentation liquid, and the iron reduction antioxidation activity of the herba Ceratophylli Nigrae fermentation liquid is 437.95 μ M FeSO4After the cordyceps militaris fermentation liquor is subjected to water bath treatment at 40-100 ℃ for 30min, the oxidation resistance is not remarkably reduced. After the cordyceps sinensis fermentation liquor is treated by water bath at 100 ℃ for 30min, the iron reduction antioxidant activity of the cordyceps sinensis fermentation liquor is 391.76 mu M FeSO4Therefore, the oxidation resistance of the cordyceps militaris fermentation liquor shows good high-temperature resistance.
Test example 2
And (3) measuring the DPPH free radical scavenging capacity and the high temperature resistance of the fermentation filtrate: the DPPH free radical is an artificially synthesized, stable and organic free radical, the structure of the DPPH free radical contains 3 benzene rings, and 1N atom has a lone pair of electrons. The methanol or ethanol solution of DPPH free radicals is dark purple red, and has a maximum absorption peak in the range of 515-520nm, after the DPPH free radicals react with the antioxidant, lone pair electrons are paired, the dark purple DPPH free radicals are reduced into a yellow DPPH-H non-free radical form, the absorption value at 517nm is reduced, the reduction degree and the received electrons (the activity of the antioxidant for removing the free radicals) are in a quantitative relation, the reaction process is easily monitored by a spectrophotometer, and the analysis is simple, sensitive and rapid, and the application is most extensive.
The method for testing the DPPH free radical scavenging capacity specifically comprises the following steps:
1) preparation of a DPPH test solution: dissolving DPPH 1mg in about 20mL of methanol, sonicating for 5min, and shaking thoroughly to homogenize the upper and lower portions. Taking 1mL of the DPPH solution, measuring the absorbance A at 519nm0The value is obtained. The DPPH solution is preferably stored protected from light and is used up within 3.5 hours.
2) And (3) measuring DPPH free radical scavenging capacity of the fermentation filtrate: 1mL of each of the fermentation filtrates obtained in example 1, example 2 and example 3 was added to 2mL of DPPH solution, mixed well, allowed to stand at room temperature for 30min, and the absorbance A at 519nm was measured.
3) And (3) high-temperature resistance measurement of DPPH free radical scavenging capacity of fermentation filtrate: the fermentation filtrates obtained in example 1, example 2 and example 3 were treated in a water bath at 40 to 100 ℃ for 30min, and measured by the method in step 2.
4) Clearance calculation formula: DPPH clearance (%) - (a)0-a)/a0 x 100, the results are given in table 2.
Table 2
Figure BDA0002683088860000111
The oxidation resistance of the cordyceps sinensis fermentation liquor is detected by adopting a DPPH free radical scavenging method, and the DPPH free radical scavenging rate of the cordyceps sinensis fermentation liquor is 61.78%. After the cordyceps militaris fermentation liquor is subjected to water bath treatment at 40-100 ℃ for 30min, the oxidation resistance is not obviously reduced. The DPPH free radical clearance rate of the cordyceps militaris fermentation liquor is 58.03 percent after the cordyceps militaris fermentation liquor is treated by water bath at the temperature of 100 ℃ for 30 min. Therefore, the oxidation resistance of the cordyceps militaris fermentation liquor shows good high-temperature resistance.
In conclusion, the nematodiferous grass fermentation liquor disclosed by the invention utilizes nematodiferous grass bacteria to carry out liquid submerged fermentation, and the obtained fermentation filtrate contains rich functional factors such as cordyceps polysaccharide, betaine, protocatechuic acid, inositol, carnitine and the like, so that the nematodiferous grass fermentation liquor can effectively eliminate in-vivo free radicals, improve the sub-health state, enhance the immunity, eliminate color spots and the like, and can effectively relieve symptoms such as headache, insomnia and the like caused by pressure; meanwhile, the product can also be used for maintaining beauty, keeping young, improving eyesight, reducing weight and prolonging life. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (7)

1. A preparation method of cordyceps militaris fermentation liquor is characterized by comprising the following steps:
firstly, inoculating nematodiferous grass fungi (Ophiocerdyceps sp) into a sterilized shake flask liquid culture medium for culture to prepare shake flask seed liquid; wherein, the shake flask liquid culture medium comprises the following components: 2-5 parts of glucose, 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour and 83-92 parts of water;
step two, inoculating the shake flask seed liquid into a sterilized primary seed fermentation culture medium for fermentation to obtain primary seed fermentation liquid;
thirdly, inoculating the primary seed fermentation liquor into a sterilized secondary seed fermentation culture medium for fermentation to obtain secondary seed fermentation liquor;
step four, inoculating the secondary seed fermentation liquor into a sterilized production-grade fermentation culture medium for fermentation to obtain a nematodiasis fermentation liquor; the first-stage seed fermentation culture medium, the second-stage seed fermentation culture medium and the production-stage fermentation culture medium comprise the following components in parts by weight: 1-3 parts of corn flour, 2-4 parts of soybean flour, 3-5 parts of chickpea flour, 0.08-0.12 part of edible oil and 88-94 parts of water;
the preservation number of the nematodiella (Ophiocerdyceps sp.) is CGMCC No. 13886.
2. The method for preparing cordyceps militaris fermentation broth, according to claim 1, is characterized in that: the specific conditions for culturing in the first step are as follows: culturing for 5-7 days under the conditions that the rotating speed of a shaking table is 100-150 r/min and the culturing temperature is 22-25 ℃.
3. The method for preparing cordyceps militaris fermentation broth, according to claim 1, is characterized in that: the specific conditions of the fermentation in the second step are as follows: culturing for 5-7 days under the conditions of ventilation volume of 4-6L/min, stirring speed of 90-110 r/min, culture temperature of 22-25 ℃ and culture pressure of 0.04-0.06 MPa;
the concrete conditions of the fermentation in the third step are as follows: culturing for 5-7 days under the conditions of ventilation volume of 40-50L/min, stirring speed of 70-90 r/min, culture temperature of 22-25 ℃ and culture pressure of 0.04-0.06 MPa;
the specific conditions of the fermentation in the fourth step are as follows: culturing for 5-7 days under the conditions of air flow of 80-100L/min, stirring speed of 60-80 r/min and culture temperature of 22-25 ℃.
4. The method for preparing cordyceps militaris fermentation broth according to claim 1 or 3, wherein the method comprises the following steps: and in the second step, the volume ratio of the shake flask seed liquid to the primary seed fermentation medium is (3-4): (96-97);
the volume ratio of the primary seed fermentation liquid to the secondary seed fermentation medium in the third step is (8-12): (88 to 92);
and the volume ratio of the secondary seed fermentation liquid to the production-grade fermentation medium in the fourth step is (8-12): (88-92).
5. The method for preparing cordyceps militaris fermentation liquor according to any one of claims 1 to 3, which is characterized by comprising the following steps of: the sterilization temperature of the shake flask liquid culture medium is 110-120 ℃, and the sterilization time is 20-40 min;
the sterilization temperature of the primary seed fermentation culture medium, the secondary seed fermentation culture medium and the production-grade fermentation culture medium is 115-125 ℃, and the sterilization time is 20-40 min.
6. The method for preparing cordyceps militaris fermentation broth, according to claim 1, is characterized in that: the amount of dry bacteria in the primary seed fermentation liquid is 29-31 g/L; the amount of dry bacteria in the secondary seed fermentation liquor is 34-36 g/L; the dry bacterium amount in the nematodia fermentation liquor is 39-41 g/L.
7. An antioxidant, characterized by: the antioxidant comprises the nematodiferous grass fermentation liquor as claimed in any one of claims 1 to 6.
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