CN106544241B - Preparation method of attractive cordyceps militaris wine - Google Patents
Preparation method of attractive cordyceps militaris wine Download PDFInfo
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Abstract
The invention relates to a preparation method of beautiful cordyceps wine, which comprises the following steps: step 1, pressing cordyceps sinensis strains on the surface of a culture medium by using a porous cover plate in a solid culture process; step 2, when the sporophores grow to a proper height from the small holes, separating the sporophores at the contact part of the cover plate and the culture medium from the culture medium; step 3, placing the cover plate and the sporophore bundles growing in the cover plate into a container together; step 4, injecting wine into the container; and step 5, storing. The preparation method of the invention not only ensures uniform growth of the sporophore bundles, but also can remove the wine turbidity phenomenon caused by long-term soaking of the culture medium in the wine, and ensures the quality of the cordyceps wine.
Description
Technical Field
The invention relates to a preparation method of cordyceps wine, and in particular relates to a method for culturing cordyceps by using a cover plate with holes and then preparing cordyceps wine.
Background
Cordyceps militaris (Cordyceps militaris) is a model species of Ascomycotina, Clavicipitales, Cordyceps, and has a scientific name of Cordyceps militaris (Vuill.) Fr. Modern science proves that the cordyceps militaris not only has special nutritional value, but also has obvious medicinal value. Wherein the extract mainly contains cordycepic acid, cordycepin, adenosine, and Cordyceps polysaccharide. Has effects in tonifying lung yin and kidney yang, and can be used for treating kidney deficiency, sexual impotence, spermatorrhea, soreness of waist and knees, and asthenia after illness. The traditional Chinese medicine considers that the traditional Chinese medicine has the effects of strengthening body resistance and consolidating constitution, has obvious curative effects on senile chronic bronchitis and pulmonary heart disease, can improve the detoxifying capability of the liver, has the effect of protecting the liver, and improves the antiviral and anti-radiation capability of the body. The traditional Chinese medicine considers that the cordyceps sinensis enters the lung and kidney two channels, can tonify lung yin and kidney yang, is mainly used for treating kidney deficiency, impotence and spermatorrhea, soreness and pain of waist and knees, weakness after illness, chronic cough and weakness, cough with phlegm and blood, spontaneous perspiration and night sweat and the like, and is the only traditional Chinese medicine capable of balancing and adjusting yin and yang simultaneously.
The cordyceps sinensis wine is a commonly adopted nutrition absorption method, and the brewing process is as follows: culturing Cordyceps in wine bottle, adding Chinese liquor, sealing, and standing for appropriate days. By adopting the brewing method, the cordyceps sinensis has long culture period, uneven growth vigor, uneven height, uneven density and poor shape and appearance.
Disclosure of Invention
The invention provides a preparation method of cordyceps wine, aiming at the problems in the prior art, in the solid cordyceps culture process, a cover plate with small holes is pressed on a culture medium, and sporophores grow out from the small holes of the cover plate gradually. When the culture grows to a certain height, cutting the culture under the cover plate along the cover plate, cutting off the culture medium at the bottom of the cover plate, putting the culture medium into a container for containing wine, injecting the wine and storing the wine. Thus not only ensuring the uniform growth of the cordyceps sinensis fruiting body, but also removing the turbidity phenomenon of the wine caused by soaking the culture medium in the wine for a long time. Ensures the quality of the cordyceps wine.
The purpose of the invention is realized by the following technical scheme:
step 1, pressing cordyceps sinensis strains on the surface of a culture medium by using a porous cover plate in a solid culture process;
step 2, when the sporophores grow to a proper height from the small holes, separating the sporophores at the contact part of the cover plate and the culture medium from the culture medium;
step 3, placing the cover plate and the sporophore bundles growing in the cover plate into a container together;
step 4, injecting wine into the container; and
and 5, storing.
Further, in the solid culture process in the step 1, after the dark culture is finished, pressing the porous cover plate on the surface of the culture medium; further, when the mycelium is fully distributed with the culture medium, the dark culture is ended, and the dark culture is changed into illumination culture; further, the porous cover plate is pressed under a medium selected to have good mycelium growth on the surface.
Further, the cover plate in the step 1 is made of materials which have density higher than that of the base wine and do not react with the base wine, such as gypsum boards, glass plates, stainless steel plates and the like; the shape of the cover plate depends on the shape of the container opening, and the area of the cover plate is slightly smaller than that of the container opening; the aperture of the small hole of the cover plate is 4-6 mm, and the hole distance is 1-2 mm; furthermore, the aperture of the small hole of the cover plate is 5-6 mm.
Further, the proper height in the step 2 is 4-8 cm; further, selecting the sporophore bundles which are exuberant in growth and uniform for separation; furthermore, the height of the selected coremium bundles is determined by the height of the container, the distance from the container opening is 2-3 cm, and the height difference of the coremium bundles is less than 2 cm; still further, the difference in height of the selected bundles of sporophores is less than 1 cm.
Further, the wine injection amount in the step 4 is a coreopsis: base liquor (w/w) 10-20: 80-90.
Further, the wine injection method in the step 4 comprises the following steps: injecting base liquor to a position 1-2 cm higher than the sporophores, standing for 30-60 min, and injecting liquor again to a position 1-2 cm away from the bottle mouth.
Further, the storage conditions in step 5 are as follows: the relative humidity is 50-70%, and the temperature is 10-30 ℃.
Further, the storage days in step 5 were 45 days or more.
The culture process of the cordyceps sinensis comprises the following steps: amplifying Cordyceps strains by liquid culture; and performing solid culture on the amplified strains. The liquid culture and solid culture methods can refer to the culture method of Cordyceps disclosed in the prior art. The liquid culture aims at strain amplification, and can adopt a conventional strain amplification culture method, including any one or combination of several methods of slant culture, shake culture, seeding tank culture and fermentation tank culture; the culture medium is conventional liquid culture medium in the art, such as PSA culture medium or yeast extract powder-compound amino acid-sucrose culture medium, yeast extract powder-white sugar-soybean protein hydrolysate culture medium, bran decoction-white sugar culture medium, etc.; further preferably a PSA culture medium or a yeast extract powder-compound amino acid-sucrose culture medium; furthermore, the PSA culture medium comprises the following components in proportion: 15-25% of potato, 1-5% of cane sugar and 1-5% of agar; the yeast extract powder-compound amino acid-sucrose culture medium comprises the following components in proportion: 0.5-2% of yeast extract powder, 0.2-1% of compound amino acid and 2-5% of cane sugar. Furthermore, the PSA culture medium comprises the following components in proportion: 20% of potato, 2% of cane sugar and 2% of agar; the yeast extract powder-compound amino acid-sucrose culture medium comprises the following components in proportion: 1% of yeast extract powder, 0.5% of compound amino acid and 3.5% of cane sugar.
The solid culture medium is subjected to dark culture at the growth stage of the mycelia, the temperature is 18-23 ℃, the relative humidity is 65-85%, and the culture time is generally 6-8 days until the mycelia are fully distributed in the culture medium; the growth stage of the coremium is changed into illumination culture, the culture temperature is 18-20 ℃, the relative humidity is 80-85%, the circulation and exchange of indoor air are gradually increased in the late stage of the coremium culture, the culture temperature is increased to 23-25 ℃ until the coremium grows to 4-8cm, and the culture time is generally 35-50 days. The solid culture medium is a culture medium which takes grains, crop straws, crop husks, economic forest branches or plant stems as main components; wherein a grain culture medium is preferred; the grain can be selected from one or more of semen Tritici Aestivi, semen Maydis, rice, semen Setariae, semen glycines powder, semen Fagopyri Esculenti, fructus Hordei vulgaris, herba Avenae Fatuae, brown rice, and semen oryzae Sativae. Furthermore, the solid culture medium also comprises adjuvants, such as white sugar, yeast extract powder, vitamin B1, soybean protein, compound amino acids, etc.
The Cordyceps militaris (cordyces militaris (Vuill.) Fr.) used in the present invention is a broad-spread species. The Cordyceps militaris is distributed in provinces such as Hubei, Hebei, Jilin, Shaanxi, Yunnan, Guangxi, Guizhou, Sichuan and Taiwan. The strain can be obtained according to the collection method of common Chinese medicinal materials, is a strain known in the prior art, and can be purchased from commercial approaches (such as edible fungus factories and research institutes).
The invention has the beneficial effects that:
① the appearance is beautiful, the invention fixes Cordyceps militaris by using a cover plate with holes, so that the Cordyceps militaris is in a vertical state, the character of the Cordyceps militaris wine is stable and consistent, the appearance is beautiful, ② quality is high, substances in a culture can not be separated out in the storing process of the Cordyceps militaris wine due to the removal of a solid culture medium, the wine color is clear and clear, and the commercial value is higher, ③ effective component content is high, the content of adenosine in the Cordyceps militaris wine prepared by the method reaches 20 mu g/ml, the content of HEA (N6- (2-hydroxyethyl) adenosine, also called comycin, which is a special component of Cordyceps militaris and is used as one of quality control indexes of Cordyceps militaris products) reaches 42 mu g/ml, and the content is obviously higher than that of the common wine soaking method.
Drawings
FIG. 1A cordyceps wine prepared by the invention
FIG. 2 porous cover plate
Experimental examples of Effect
Experimental example 1 comparison of the effects of the cover method of the present invention and the conventional wine soaking method
First, experiment method
The cordyceps wine is prepared by the following two methods, wherein each method is provided with 6 groups of repetition:
the first method, the conventional brewing method: taking the cordyceps sinensis strain obtained by the enlarged culture in the embodiment 1, directly inoculating the cordyceps sinensis strain into a wine container for solid culture according to the embodiment 4, and after the culture is finished, injecting wine and storing according to the method in the embodiment 10;
the second method, the cover plate method of the invention: the Cordyceps sinensis strain obtained by the enlarged culture in example 1 was subjected to solid culture in example 4, after the dark culture was completed, pressing and cutting in example 9 were carried out, and the cover plate and the bundle of sporophores were placed together in a container, and wine was poured and stored in the same manner as in example 10.
And (3) inspecting the appearance and the effective component content of the cordyceps sinensis wine prepared by different methods.
Second, experimental results
The results are shown in Table 1.
TABLE 1 comparison of the effect of Cordyceps wine prepared by different methods (n ═ 6)
Experimental example 2 comparison of effects of different cover plate methods
First, experiment method
The cordyceps wine is prepared by the following two methods, wherein each method is provided with 6 groups of repetition:
the method comprises the following steps: taking the cordyceps sinensis strain obtained by the enlarged culture in the embodiment 1, carrying out solid culture according to the embodiment 4, collecting the luxuriant and orderly and consistent sporophore bundles after the culture is finished, inserting the sporophore bundles into the small holes of the cover plate, placing the cover plate and the sporophore bundles into a container, injecting wine according to the method in the embodiment 10 and storing the wine;
the second method comprises the following steps: the Cordyceps sinensis strain obtained by the enlarged culture in example 1 was subjected to solid culture in example 4, after the dark culture was completed, pressing and cutting in example 9 were carried out, and the cover plate and the bundle of sporophores were placed together in a container, and wine was poured and stored in the same manner as in example 10.
And (5) inspecting the difference of the preparation effects of different methods.
Second, experimental results
The results are shown in Table 2.
TABLE 2 comparison of the effect of Cordyceps wine prepared by different methods (n ═ 6)
Detailed Description
Example 1 expanded culture of Cordyceps species
Slant culture: inoculating the separated and purified Cordyceps strain in a slant test tube, and placing the slant test tube inoculated with the strain in an incubator at 22 deg.C for 7 days until the test tube is full of mycelia;
and (3) shake flask culture: placing 200m1 liquid culture medium into a 500m1 triangular flask, sterilizing for 30 minutes under 0.11Mpa, cooling to room temperature, inoculating strains of 1 inclined test tube to the culture medium of the 500m1 triangular flask, and culturing in a constant temperature shaking incubator at 22 +/-1 ℃ and 150 rpm for 3 days;
culturing in seed tank by charging 20L liquid culture medium into 50L airlift fermentation tank, adding edible defoaming agent at a culture medium temperature of more than 95 deg.C, adding 0.03 wt% of the culture medium, heating to 121 deg.C under 0.11Mpa with steam, maintaining the pressure for 30-45 min, sterilizing, cooling to 20 deg.C, inoculating 4 bottles of the cultured strain in 500m1 shake flask into the culture medium at a culture temperature of 22 + -1 deg.C, and maintaining the pressure for 4.903-7.0845 × 10 min at an aeration rate of 1: 0.5V/V min4Pa, after 3 days aeration culture, the culture volume is 20L after reaching the logarithmic growth phase.
Culturing in a fermentation tank: charging 200L liquid culture medium into 500L airlift fermentation tank, wherein the temperature of the culture medium is above 95 deg.C, adding edible defoaming agent in an amount of 0.03 wt% of the culture medium, introducing steam under 0.11Mpa, heating to 121 deg.C, and maintaining the pressure for 3Sterilizing for 0-45 min, cooling to 20 deg.C, inoculating all the seeds in 200L seed tank, culturing at 22 + -1 deg.C with ventilation of 1: 0.5V/V min, and maintaining pressure for 4.903-7.0845 × 104Pa, after 3 days aeration culture, the logarithmic growth phase is reached. The final culture volume was 200L.
Slant tube medium: 20% of potato cooking juice, 3.5% of white granulated sugar and 0.5% of yeast extract powder;
liquid culture medium in shake flasks, seed tanks and fermentors: contains yeast extract powder 0.5%, compound amino acid 1%, white granulated sugar 3.5%, and water to 100% and has pH of 6.5.
Example 2 expanded culture of Cordyceps species
Slant culture: inoculating the separated and purified Cordyceps militaris strain in a slant test tube, and placing the slant test tube inoculated with the strain in an incubator at 22 deg.C for 7 days until the test tube is full of mycelia;
and (3) shake flask culture: placing 200m1 liquid culture medium into a 500m1 triangular flask, sterilizing for 30 minutes under 0.11Mpa, cooling to room temperature, inoculating strains of 1 inclined test tube to the culture medium of the 500m1 triangular flask, and culturing in a constant temperature shaking incubator at 22 +/-1 ℃ and 150 rpm for 3 days;
seeding tank culture, namely filling 20L of liquid culture medium into a 50L airlift fermentation tank, wherein the temperature of the culture medium is higher than 95 ℃, adding edible antifoaming agent, the adding amount is 0.03 percent of the weight of the culture medium, introducing steam under the pressure of 0.11Mpa, heating to 121 ℃, maintaining the pressure for 30-45 minutes, sterilizing, cooling to 20 ℃, inoculating 4 bottles of cultured 500m1 shake flask strains into the culture medium, wherein the culture temperature is 22 +/-1 ℃, the ventilation amount is 1: 0.5V/V min, and the pressure is maintained for 4.903-7.0845 × 104Pa, after 3 days aeration culture, the culture volume is 20L after reaching the logarithmic growth phase.
Slant tube medium: every 1 liter of liquid contains 200 g of potato decoction, 20 g of cane sugar, 20 g of agar and the balance of water to 1000ml, and the pH value is 6.5;
shake flask and seed tank culture medium: each 1 liter of liquid contains 30 g of yeast extract powder, 30 g of white granulated sugar and 5 g of soybean protein hydrolysate, and water is added to supplement the volume to 1000ml, and the pH value is 6.5.
Example 3 expanded culture of Cordyceps species
Slant culture: inoculating the separated and purified Cordyceps strain in a slant test tube, and placing the slant test tube inoculated with the strain in an incubator at 22 deg.C for 7 days until the test tube is full of mycelia;
culturing in seeding tank by filling 20L liquid culture medium into 50L airlift seeding tank, adding edible defoaming agent at a culture medium temperature of more than 95 deg.C, adding steam at 0.03 wt% of the culture medium under 0.11Mpa, heating to 121 deg.C, maintaining the pressure for 30-45 min, sterilizing, cooling to 20 deg.C, inoculating strains in 4 bottles of the above slant test tubes into the culture medium at a culture temperature of 22 + -1 deg.C, and maintaining the pressure for 4.903-7.0845 × 10 min at an air flow of 1: 0.5V/V min4Pa, after 3 days aeration culture, the culture volume is 20L after reaching the logarithmic growth phase.
Culturing in a fermentation tank, namely, filling 200L of liquid culture medium into a 500L airlift fermentation tank, wherein the temperature of the culture medium is higher than 95 ℃, adding edible antifoaming agent, the adding amount of which is 0.03 percent of the weight of the culture medium, introducing steam under the pressure of 0.11Mpa, heating to 121 ℃, maintaining the pressure for 30-45 minutes, sterilizing, cooling to 20 ℃, inoculating all the cultured 200L seed tanks to culture, wherein the culture temperature is 22 +/-1 ℃, the ventilation amount is 1: 0.5V/V min, and the pressure is maintained for 4.903-7.0845 × 104Pa, after 3 days aeration culture, the logarithmic growth phase is reached. The final culture volume was 200L.
Slant tube medium: every 1 liter of liquid contains 200 g of potato decoction, 20 g of cane sugar, 20 g of agar and the balance of water to 1000ml, and the pH value is 6.5;
seeding tank and fermentation tank culture medium: every 1L of the liquid contains 40 g of bran cooking juice, 30 g of white granulated sugar and the balance of supplementary water to 1000ml, and the pH value is 6.5.
Example 4 solid culture
Solid medium: preparing a nutrient solution according to the proportion of 3.5 percent of white granulated sugar, 0.5 percent of yeast extract powder and 110mg/1000ml of vitamin B, and preparing the rice and the nutrient solution according to the proportion of 1: 1.6. Placing the culture container filled with culture medium in a sterilizing pot, and sterilizing at 121 deg.C for 30-50 min; after sterilization, the culture vessel was transferred into a buffer chamber, naturally cooled to 24 ℃ or lower, and transferred into an inoculation chamber.
Inoculation: irradiating the culture container with ultraviolet light in a superclean bench or a hundred-level laminar flow hood (below) for 0.5h before inoculation; the strain obtained by the scale-up culture in examples 1 to 3 was inoculated onto a solid medium in an inoculum size of 7% per culture vessel, and the inoculated vessel was placed in a culture chamber for culture.
The culture conditions are as follows: and (3) hypha culture stage: the temperature of the spawn running room is preferably 18-23 ℃, the relative humidity of air is 65-85%, and the spawn running room is shielded from light; culturing for 6-8 days indoors, and growing hyphae; and (3) a sporophyte bundle growth stage: when hyphae are fully distributed on the whole box surface and deeply pricked to the bottom of the box, light exposure is started, but direct sunlight is avoided, the culture temperature is adjusted to 18-20 ℃, and the humidity is 80% -85%; in the late stage of culturing the coremium, gradually increasing the circulation and exchange of indoor air, and raising the culture temperature to 23-25 ℃. The contamination should be removed immediately during the cultivation stage. And (5) finishing the culture when the sporophore bundle grows to 4-8 cm.
EXAMPLE 5 solid culture
Solid medium: preparing nutrient solution according to the proportion of 3.5 percent of white granulated sugar, 0.5 percent of yeast extract powder and 110mg percent of vitamin B/1000 ml, and preparing the millet and the nutrient solution according to the proportion of 1: 1.6. Placing the culture container filled with culture medium in a sterilizing pot, and sterilizing at 121 deg.C for 30-50 min; after sterilization, the culture vessel was transferred into a buffer chamber, naturally cooled to 24 ℃ or lower, and transferred into an inoculation chamber.
The culture conditions were the same as in example 4.
Example 6 solid culture
Solid medium: after brown rice is cleaned and water is controlled, a proper amount of water is added, and the weight ratio of brown rice is as follows: 1 part of water: 1.5, mixing uniformly; placing the culture container filled with culture medium in a sterilizing pot, and sterilizing at 121 deg.C for 30-50 min; after sterilization, the culture vessel was transferred into a buffer chamber, naturally cooled to 24 ℃ or lower, and transferred into an inoculation chamber.
The culture conditions were the same as in example 4.
Example 7 solid culture
Solid medium: after wheat is cleaned and water is controlled, a proper amount of water is added, and the weight ratio of the wheat to the water is 1: 1.3, uniformly mixing; placing the culture container filled with culture medium in a sterilizing pot, and sterilizing at 121 deg.C for 30-50 min; after sterilization, the culture vessel was transferred into a buffer chamber, naturally cooled to 24 ℃ or lower, and transferred into an inoculation chamber.
The culture conditions were the same as in example 4.
Example 8 solid culture
Solid medium: 69% of corncobs, 16% of cottonseed hulls, 11% of sawdust, 3% of gypsum, 1% of lime and solid materials: water 1: 1.5 (depending on the water content of the raw materials), and uniformly mixing; placing the culture container filled with culture medium in a sterilizing pot, and sterilizing at 121 deg.C for 30-50 min; after sterilization, the culture vessel was transferred into a buffer chamber, naturally cooled to 24 ℃ or lower, and transferred into an inoculation chamber.
The culture conditions were the same as in example 4.
Example 9 pressing plate and cutting
Carrying out solid culture on the cordyceps sinensis strains subjected to the expanded culture in the embodiments 1-3 according to any one of the methods in the embodiments 4-8, taking a porous gypsum board (the diameter is slightly smaller than the mouth of a wine container, the aperture is 5mm, the hole spacing is 1mm, see figure 2) after dark culture is finished, and selecting a solid culture medium with luxuriant surface hyphae and good growth to press downwards so as to enable the sporophor bundles to grow from the small holes. After the solid culture is finished, selecting a cover plate with regular and consistent surface peduncle bundles and about 4-5 cm of height, cutting the culture medium along the edge, removing the culture medium at the lower part of the cover plate along the contact surface of the cover plate and the culture medium, and placing the cover plate and the peduncle bundles growing in the cover plate into a container.
EXAMPLE 10 wine filling and storage
And selecting a clean and sterilized table top, adopting an aseptic operation method, enabling the base wine to slowly flow down along the inner wall of the wine bottle, injecting the base wine to a position 1-2 cm higher than the sporophores, standing for 30-60 min, and injecting the base wine again to a position 1-2 cm away from the bottle mouth.
Selecting a place which is dry, clean, bright and well ventilated, wherein the relative environment humidity is about 70 percent and the temperature is about 25 ℃; the sealing of the container is tight, so that wine leakage and 'running degree' are prevented; avoiding direct irradiation of strong light. Storing in cellar for about 45 days until the wine color becomes orange to orange yellow, and taking out of cellar to obtain final product (see figure 1).
Claims (6)
1. The preparation method of the beautiful cordyceps militaris wine is characterized by comprising the following steps:
step 1, selecting a culture medium with good mycelium growth on the surface of a porous cover plate and pressing the culture medium downwards after dark culture is finished in the solid culture process of cordyceps militaris strains; the porous cover plate is made of a material which has a density larger than that of base liquor and does not react with the base liquor, the aperture of each small hole is 4-6 mm, and the hole distance is 1-2 mm;
step 2, when the sporophores grow to a proper height from the small holes, separating the sporophores at the contact part of the cover plate and the culture medium from the culture medium;
step 3, placing the cover plate and the sporophore bundles growing in the cover plate into a container together;
step 4, injecting wine into the container; and
step 5, storing;
wherein, the wine injection method in the step 4 comprises the following steps: injecting base liquor to a position 1-2 cm higher than the sporophores, standing for 30-60 min, and injecting liquor again to a position 1-2 cm away from the bottle mouth.
2. The method of claim 1, wherein the cover sheet is any one of a gypsum board, a glass plate, or a stainless steel plate.
3. The method of claim 1, wherein the suitable height of step 2 is 4 to 8 cm.
4. The method of claim 1, wherein the step 2 comprises selecting the bundles of the strong and uniform stalks for separation, wherein the height difference of the bundles is less than 2 cm.
5. The method of claim 1, wherein the step 4 comprises: the weight ratio of the coremium to the base wine is (10-20): (80-90) injecting wine.
6. The method of claim 1, wherein the storage conditions in step 5 are: the relative humidity is 50-70%, and the temperature is 10-30 ℃; the storage days are more than 45 days.
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Effective date of registration: 20191224 Address after: 314200 Building 2, No. 1938, xinqun Road, Pinghu Economic Development Zone, Jiaxing City, Zhejiang Province Applicant after: Zhejiang Panya Health Food Co., Ltd. Address before: 314200 No. 1938, group road, Pinghu Economic Development Zone, Zhejiang, Jiaxing Applicant before: Zhejiang Faya Biological Pharmaceutical Co., Ltd. |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |