CN114836289A - Preparation method of cordyceps militaris wine with high cordycepin and pentostatin content - Google Patents
Preparation method of cordyceps militaris wine with high cordycepin and pentostatin content Download PDFInfo
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Abstract
The invention discloses a preparation method of cordyceps militaris wine with high cordycepin and pentostatin content, which comprises the following steps: obtaining strains; preparing a culture medium; preparing and inoculating liquid strains; preparing cordyceps militaris wine. The invention has the beneficial effects that: the strain used in the invention is low-dose 60 Co-gamma ray irradiation breeding to obtain mutant strain with high yield of cordycepin and pentostatin; the culture medium is not added with any auxiliary materials or bacteriostatic agents except the rice/wheat and water, so that the production cost is greatly reduced, the culture material does not need to be cured, and the energy conservation and emission reduction are realized; non-aged culture materialThe growth of hyphae is facilitated, and the production period is greatly shortened; the cordyceps militaris sporocarp and the fermentation mycoplasm are used as raw materials, cordyceps militaris compound wine with high-efficiency active substance release is obtained through base wine soaking and irradiation ageing, the primary soaked wine obtained after primary cleaning and soaking can be used as seasoning wine for preparing other compound wine, and the active cordyceps militaris compound wine obtained after secondary soaking can be directly drunk.
Description
Technical Field
The invention relates to the technical field of edible fungus cultivation and processing, in particular to a preparation method of cordyceps militaris wine with high cordycepin and pentostatin content.
Background
Cordyceps militaris (Cordyceps militaris) also called Cordyceps militaris, Cordyceps militaris and Cordyceps militaris, belongs to Ascomycotina, Pyrenomycetes, Clavipitaceae and Cordyceps, and is a famous and precious edible and medicinal fungus because its stroma grows on pupa of insects of Lepidoptera. Since the artificial cultivation of the cordyceps militaris, researches show that the cordyceps militaris not only can nourish the human body, but also has the pharmacological functions of tonifying the lung, tonifying the kidney and strengthening yang, and has the pharmacological functions of strengthening deficiency, benefiting vital essence and qi, stopping bleeding, reducing phlegm, calming, immunizing and the like. In 3 months in 2009, the Ministry of health issued No. 3 announcement in 2009, and approved cordyceps militaris as a new resource food. No. 10 announcements in 2014 changed the edible amount, quality index requirements and application range, and no longer made requirements on the edible amount and application range, which opens a door for the application of the cordyceps militaris industry in the field of food processing.
Cordycepin (cordycepin) is a marked metabolite generated by cordyceps militaris, and a large number of researches show that cordycepin has the effects of regulating the immunity of organisms, resisting tumors, fungi, viruses and leukemia, reducing blood sugar and blood fat and the like. Has been approved by the FDA for clinical studies in the treatment of leukemia. However, the purified cordycepin is easily and rapidly degraded by the deamination of Adenosine Deaminase (ADA) after entering the human body, and the 3' -deoxyinosine without biological activity is formed to be ineffective. Xia et al [ Cell Chemical Biology,2017,24(12):1479-1489] research finds that cordycepin as a protective molecule of cordycepin in cordyceps militaris can be tightly bound with ADA, inhibit the activity of ADA and protect the stability of cordycepin structure, thereby effectively preventing the degradation of cordycepin. Furthermore, pentostatin is marketed as an antineoplastic agent in the united states as early as 1992 and is used for the treatment of hair stem cell leukemia.
The variety of the edible fungi is one of the key factors for determining the content of the active substances, so that in order to realize the effective utilization of the active substances in the edible fungi, firstly, strains with high content of the active substances are selected for production. At present, various breeding techniques are applied to the breeding of strains with high cordycepin, high polysaccharide and high protein yield in cordyceps militaris, such as ultraviolet mutagenesis [ fir, etc., Chinese brewing, 2012,31(7): 57-61)]、 60 Co radiation mutagenesis [ Shixiaofeng et al, edible and medicinal fungi, 2015,23(6):371-]Atmospheric room temperature plasma mutagenesis (ARTP) [ Liu Guang Jian et al, domestic fungus 2020,42(5):12-14,18]Cesium 137 radiation mutagenesis (CN102599005B, a method for producing cordyceps militaris for improving cordycepin yield) and heavy carbon ion beam injection mutagenesis (CN108220172B, a cordyceps militaris mutant strain with high cordycepin yield and application). In addition, there are reports of increasing the content of cordycepin by adding an inducer to a medium, such as "a method for increasing the content of cordycepin in mycelia of Cordyceps militaris" (CN109136112B) and "a method for obtaining high-yield cordycepin using a solid medium" (CN 111826410A). However, the breeding of the strain with high pentostatin content is not reported, and the related research of the combined breeding of the cordycepin and the pentostatin is not available.
At present, the cordyceps militaris can be cultured artificially in a solid culture mode on a large scale, liquid strains are widely applied to industrial production of the cordyceps militaris by virtue of the advantages of short culture time, quick strain growth, regular fungus age, convenience in inoculation and the like, and the strain activity is also concerned as an important factor for ensuring the yield and quality of fruiting bodies. Many research reports judge the strain activity by indexes such as biomass, pellet density, pellet diameter, odor, color and the like, but at present, no scientific basis for correlation between the indexes and the strain activity exists. In order to find a biomarker capable of rapidly determining the activity of a strain, Neilong and the like detect the enzyme activities and hypha growth activities of laccase, amylase and protease generated by a needle mushroom liquid strain in different culture days, find that the laccase can be used as the biomarker for detecting the activity of the needle mushroom liquid strain [ Neilong and the like, edible fungi 2014(2):11-12], and develop 'a rapid detection kit for the activity of the needle mushroom liquid strain' according to the research result (CN 104101700B). However, extracellular enzymes are secreted to the outside during the hypha growth process, the secretion and action processes are not instantaneous, although research shows that the activity of some extracellular enzymes is obviously and positively correlated with the strain activity, the result is influenced by the limitation of sampling time, and only the activity of the enzymes during sampling can be shown, and the death and the activity of cells cannot be judged. Ergosterol is an important component of eukaryotic cell membranes and can be used as an indicator of living cells [ Bermingham, et al, Mycol. Res.,1995,99(4): 479-484] for evaluating the viability of strains.
After the cultivation of cordyceps militaris is finished, the fruiting bodies and the culture medium are mostly utilized separately through harvesting. The fruiting body is mainly eaten fresh or sold in dry market. The residual culture medium (about 40000 tons/year) is mostly discarded except a small part of the culture medium used as animal feed, which not only pollutes the environment but also causes great resource waste. Therefore, in recent years, there has been a case where a cordyceps militaris wine is disclosed by using both a fruit body and a culture medium. As patents in the prior art disclose some solutions:
1. a production process of fresh Cordyceps militaris wine (publication No. CN104312879B) comprises filling fresh yellow wine into a bottle, allowing culture medium and fruiting body of fresh Cordyceps militaris to inhabit with yellow wine, and aging for 85 days to obtain a new compound beverage, fresh Cordyceps militaris wine.
2. A method for preparing Cordyceps militaris alcoholic beverage (publication No. CN110468005A) comprises leaching fruiting body under high pressure, adding Saccharomyces cerevisiae activating solution 8-20 parts, fermenting and culturing at 23-30 deg.C for 7-9 days; if the required cordyceps militaris alcoholic drink is less than or equal to 4 degrees, adding sterile distilled water into the culture container to be prepared to the target concentration; if the required cordyceps militaris alcoholic drink is more than 4 degrees, adding a mixed solution of 38-72 degrees of white spirit and sterile distilled water into a culture container to be prepared to a target degree; then carrying out secondary leaching; aging for 1-2 months to obtain the final product.
3. A Cordyceps militaris wine and its preparation method (publication No. CN109439499A), highly wine soaking living Cordyceps plant with culture medium is prepared by (1) placing culture medium into glass wine bottle to culture Cordyceps militaris, adding wine into wine bottle when Cordyceps militaris fruiting body grows out and spore powder reaches 80% -90% maturity, soaking until the alcoholic strength is stable, pouring out to obtain primary soaking wine, blending primary soaking wine of different batches to required alcoholic strength to obtain primary aging wine; (2) refilling the primary cured wine into the glass wine bottle, covering the bottle cap but not screwing, and standing to obtain secondary cured wine; (3) and (4) inspecting the secondary cured wine, inspecting the qualified product, screwing a bottle cap, sealing, packaging and warehousing to obtain the cordyceps militaris wine.
4. A culture method of fresh Cordyceps militaris wine (publication number: CN109486622A) sequentially comprises the following steps: (1) putting a cordyceps militaris culture medium into a wine bottle, performing steam high-temperature sterilization on the wine bottle and the culture medium, (2) sowing cordyceps militaris seeds on the culture medium when the culture medium is cooled to 20-25 ℃, and (3) filling wine into the wine bottle full of cordyceps militaris when cordyceps militaris sporocarp grows to 5-10 cm after cultivation for 50-60 days, sealing the glass wine bottle, soaking for 30 days, and drinking, wherein the wine can also be used as a cordyceps militaris display artwork.
Although the prior art discloses a preparation method of cordyceps militaris wine, active substances such as cordycepin and pentostatin in the cordyceps militaris wine are not taken as production process optimization and screening indexes, so that the preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content is needed to solve the problems. A small amount of active substances are obtained by primary soaking and cleaning, and are used for producing other compound wine, secondary soaking and irradiation aging is accelerated, and the release of active substances such as cordycepin, pentostatin and the like is accelerated.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of cordyceps militaris wine with high cordycepin and pentostatin content, so as to solve the problems.
A preparation method of cordyceps militaris wine with high cordycepin and pentostatin content comprises the following steps:
step S1, obtaining strains;
step S2, preparing a culture medium;
step S3, liquid strain preparation and inoculation;
and step S4, preparing cordyceps militaris wine.
The specific method of step S1 is as follows: at 50-800Gy 60 Cordyceps militaris YF-026 with high growth rate and high cordycepin and pentostatin content is bred by Co-gamma ray irradiation for production.
The specific method of step S2 is as follows: selecting intact and mildew-free pure rice or wheat as culture material, adding 0.5-1.3 times of water, stirring, and packaging into special wine bottle (figure 1), adding 4000-8000Gy 60 Co-gamma ray irradiation sterilization.
The specific method of step S3 is as follows: the activity of liquid culture extracellular enzyme and the ergosterol content in mycelia are used as strain activity evaluation indexes, seed liquid with strong activity is screened and inoculated into a wine bottle culture medium after irradiation sterilization in a sterile environment, and low-temperature culture is carried out.
The specific method of step S4 is as follows: culturing the fruiting body of Cordyceps militaris in wine bottle to 5-10 cm, filling the base wine into special wine bottle, soaking and cleaning the fruiting body and culture medium for 3-5 days at 15-25 deg.C, and keeping out of the sun. Pouring out the cleaning wine liquid, filtering and clarifying to obtain the cordyceps militaris primary soaking wine for preparing other prepared wines; and (4) filling the base wine again, and soaking for the second time for 15-30d at 15-25 ℃ in the dark to prepare the cordyceps militaris in-situ growth compound wine.
Wherein the step S4 further comprises processing the Cordyceps militaris in-situ growth mixed liquor again through 1000-3000Gy 60 Co-gamma ray irradiation accelerates aging and improves the dissolution rate of cordycepin and pentostatin, and then the high-activity cordyceps militaris compound wine is obtained after storage for 30-50 days.
Wherein the inoculation amount of the seed liquid in the step S3 is 6-15%, the culture temperature is 4-15 ℃, and the culture time is 14-25 d.
Compared with the prior art, the invention has the beneficial effects that:
(1) the strain used in the invention is low-dose 60 Co-gamma ray irradiation breeding to obtain high yield mutant strain of cordycepin and pentostatin.
(2) The culture medium used in the invention does not contain any auxiliary materials or bacteriostatic agents except rice/wheat and water, so that the production cost is greatly reduced, the culture material does not need to be cured, and the energy conservation and emission reduction are realized; in addition, the culture material without curing is more beneficial to the growth of hypha, and the production period is greatly shortened.
(3) The cordyceps militaris sporocarp and the fermentation mycoplasm are used as raw materials, the cordyceps militaris compound wine with high-efficiency active substance release is obtained through base wine soaking and irradiation ageing acceleration, the primary soaking wine obtained after primary cleaning and soaking can be used as seasoning wine for preparing other compound wines, and the active cordyceps militaris compound wine obtained after secondary soaking can be directly drunk.
Detailed Description
The following detailed description of embodiments of the invention, but the invention can be practiced in many different ways, as defined and covered by the claims.
Example 1
A cultivation method of non-aged and non-added cordyceps militaris raw materials comprises the following specific steps:
(1) at 500Gy 60 Cordyceps militaris strains obtained by Co-gamma ray mutagenesis are used as starting strains, and are inoculated to a liquid culture medium after being activated to prepare seed liquid;
(2) After the liquid culture is finished, measuring the activities of laccase and amylase in fermentation liquor and the content of ergosterol in hyphae, and screening high-activity strains;
(3) inoculating the strain to a pure rice culture medium according to a proportion of 10%, and culturing at 10 ℃, wherein the addition amount of water in the culture medium is 0.5 times of the dry weight of rice.
Example 2
A cultivation method of non-aged and non-added cordyceps militaris raw materials comprises the following specific steps:
(1) at 100Gy 60 Performing Co-gamma ray mutagenesis to obtain pupa Cordyceps strain as starting strain, activating, and inoculating to liquid culture medium to prepare seed solution;
(2) after the liquid culture is finished, measuring the activities of laccase and amylase in fermentation liquor and the content of ergosterol in hyphae, and screening high-activity strains;
(3) inoculating the strain to a pure rice culture medium according to the proportion of 8 percent, and culturing at the temperature of 13 ℃, wherein the addition amount of water in the culture medium is 1.0 time of the dry weight of rice.
Example 3
A cultivation method of non-aged and non-added cordyceps militaris raw materials comprises the following specific steps:
(1) at 800Gy 60 Cordyceps militaris strains obtained by Co-gamma ray mutagenesis are used as starting strains, and are inoculated to a liquid culture medium after being activated to prepare seed liquid;
(2) after the liquid culture is finished, measuring the activities of laccase and amylase in fermentation liquor and the content of ergosterol in hyphae, and screening high-activity strains;
(3) Inoculating the strain to a pure wheat culture medium according to a proportion of 15%, and culturing at 5 ℃, wherein the addition amount of water in the culture medium is 1.5 times of the dry weight of wheat.
Example 4
The preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 3d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) adding base liquor again, keeping out of the sun, soaking at 20 deg.C for 20d, and soaking once every 5d 60 Co-gamma ray irradiation is carried out, and the doses are 1000Gy,2000Gy,2000Gy and 2000Gy respectively, so as to obtain the active cordyceps militaris wine.
Example 5
The preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 4d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) adding base wine again, soaking at 15 deg.C for 20 days in dark place, and soaking every 5 days 60 And (3) performing Co-gamma ray irradiation with the doses of 3000Gy,2000Gy,1000Gy and 1000Gy respectively to obtain the active cordyceps militaris wine.
Example 6
A preparation method of cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 4d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) Adding base wine again, soaking at 25 deg.C for 25d in dark place, and soaking every 5d 60 And (3) performing Co-gamma ray irradiation with the doses of 3000Gy,2500Gy,2000Gy,1500Gy and 1000Gy respectively to obtain the active cordyceps militaris wine.
Example 7
The preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 5d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) adding base wine again, keeping out of the sun, soaking at 20 deg.C for 25d, and soaking every 5d 60 And (3) performing Co-gamma ray irradiation with the doses of 3000Gy,1000Gy,1000Gy,1000Gy and 1000Gy respectively to obtain the active cordyceps militaris wine.
Comparative example 1: a cultivation method of non-aged and non-added cordyceps militaris raw materials comprises the following specific steps:
(1) at 1000Gy 60 Cordyceps militaris strains obtained by Co-gamma ray mutagenesis are used as starting strains, and are inoculated to a liquid culture medium after being activated to prepare seed liquid;
(2) after the liquid culture is finished, measuring the activities of laccase and amylase in fermentation liquor and the content of ergosterol in hyphae, and screening high-activity strains;
(3) inoculating the strain to a pure rice culture medium according to a proportion of 10%, and culturing at 10 ℃, wherein the addition amount of water in the culture medium is 0.5 times of the dry weight of rice.
Comparative example 2: a cultivation method of non-aged and non-added cordyceps militaris raw materials comprises the following specific steps:
(1) at 200Gy 60 Cordyceps militaris strains obtained by Co-gamma ray mutagenesis are used as starting strains, and are inoculated to a liquid culture medium after being activated to prepare seed liquid;
(2) after the liquid culture is finished, measuring the activities of laccase and amylase in fermentation liquor and the content of ergosterol in hyphae, and screening high-activity strains;
(3) inoculating the strain to a pure rice culture medium according to a proportion of 10%, and culturing at 10 ℃, wherein the addition amount of water in the culture medium is 0.5 times of the dry weight of rice.
Comparative example 3: the preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 4d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) adding base wine again, soaking at 15 deg.C for 20 days in dark place, and soaking every 5 days 60 Co-gamma ray irradiation with the doses of 500Gy,500Gy,600Gy and 1000Gy respectively to obtain the active cordyceps militaris wine.
Comparative example 4: the preparation method of the cordyceps militaris wine with high cordycepin and pentostatin content comprises the following specific steps:
(1) cleaning base liquor, soaking Cordyceps militaris fruiting body and fermentation mycoplasm in dark for 4d, removing impurities from the base liquor, and filtering to obtain primary soaking liquor;
(2) Adding base wine again, soaking at 15 deg.C for 20 days in dark place, and soaking every 5 days 60 And (3) performing Co-gamma ray irradiation with doses of 4000Gy,300Gy,3000Gy and 2500Gy respectively to obtain the active cordyceps militaris wine.
The contents of cordycepin and pentostatin in the cordyceps militaris fruiting bodies cultivated by the raw material of the example 1-3 are shown in the following table 1:
TABLE 1 content of cordycepin and pentostatin in the fruiting bodies of different processed Cordyceps militaris
| Treatment of | Cordycepin (mg/mL) | Pentostatin (mg/mL) |
| Example 1 | 811.46 | 161.26 |
| Example 2 | 800.38 | 150.01 |
| Example 3 | 852.11 | 180.15 |
| Comparative example 1 | 436.12 | 80.11 |
| Comparative example 2 | 435.09 | 79.12 |
1. In combination with comparative examples 1 to 3 it can be concluded that, 60 the higher the Co-gamma ray dosage, the higher the contents of the obtained cordycepin and pentostatin, and the higher the ratio of the added strains, the contents of the obtained cordycepin and pentostatin.
2. By combining example 1, comparative example 1 and comparative example 2, 60 too high or too low Co-gamma radiation dose can affect and reduce the content of cordycepin and pentostatin.
The contents of cordycepin and pentostatin in the cordyceps militaris wine prepared in examples 4-7 were compared, and the results are shown in the following table:
TABLE 2 content of cordycepin and pentostatin in Cordyceps militaris wine prepared by different processing techniques
| Treatment of | Cordycepin (mg/mL) | Pentostatin (mg/mL) |
| Example 4 | 932.46 | 181.26 |
| Example 5 | 1211.38 | 221.01 |
| Example 6 | 852.11 | 102.15 |
| Example 7 | 684.15 | 89.88 |
| Comparative example 3 | 623.31 | 65.02 |
| Comparative example 4 | 625.23 | 66.21 |
3. In combination with examples 4-7 it can be concluded that, 60 the content of the cordycepin and the pentostatin can be improved by gradually increasing or decreasing the irradiation dose of the Co-gamma rays, 60 the dosage of Co-gamma ray radiation is rapidly increased or decreasedAt a lesser level, the content of cordycepin and pentostatin will decrease.
4. Combining example 5 with comparative example 3 and comparative example 4 results, 60 both high and low Co-gamma ray doses affect the content of cordycepin and pentostatin in the cordyceps militaris wine.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. A preparation method of cordyceps militaris wine with high cordycepin and pentostatin content is characterized in that: the method comprises the following steps:
step S1, obtaining strains;
step S2, preparing a culture medium;
step S3, liquid strain preparation and inoculation;
and step S4, preparing cordyceps militaris wine.
2. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: wherein the strains in the step S1 are: at 50-800Gy 60 Cordyceps militaris YF-026 with high growth rate and high cordycepin and pentostatin content is bred by Co-gamma ray irradiation for production.
3. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: the specific method of step S2 is as follows: screening intact and mildew-free pure rice or wheat as culture material, adding 0.5-1.3 times of water, stirring, filling into special wine bottle, adding 4000-8000Gy 60 Co-gamma ray irradiation sterilization.
4. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: the specific method of step S3 is as follows: the activity of liquid culture extracellular enzyme and the ergosterol content in mycelia are used as strain activity evaluation indexes, seed liquid with strong activity is screened and inoculated into a wine bottle culture medium after irradiation sterilization in a sterile environment, and low-temperature culture is carried out.
5. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: the specific method of step S4 is as follows: culturing the fruiting body of Cordyceps militaris in wine bottle to 5-10 cm, filling the base wine into special wine bottle, soaking and cleaning the fruiting body and culture medium for 3-5 days at 15-25 deg.C, and keeping out of the sun. Pouring out the cleaning wine liquid, filtering and clarifying to obtain the cordyceps militaris primary soaking wine for preparing other prepared wines; and (4) filling the base wine again, and soaking for the second time for 15-30d at 15-25 ℃ in the dark to prepare the cordyceps militaris in-situ growth compound wine.
6. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: wherein the step S4 further comprises processing the Cordyceps militaris in-situ growth mixed liquor again through 1000-3000Gy 60 Co-gamma ray irradiation accelerates aging and improves the dissolution rate of cordycepin and pentostatin, and then the high-activity cordyceps militaris compound wine is obtained after storage for 30-50 days.
7. The method for preparing cordyceps militaris wine with high cordycepin and pentostatin content according to claim 1, wherein the method comprises the following steps: wherein the inoculation amount of the seed liquid in the step S3 is 6-15%, the culture temperature is 4-15 ℃, and the culture time is 14-25 d.
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