CN116171798A - Method for preparing cordyceps militaris by taking sea cucumber processing byproducts as culture medium - Google Patents

Method for preparing cordyceps militaris by taking sea cucumber processing byproducts as culture medium Download PDF

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Publication number
CN116171798A
CN116171798A CN202310156736.7A CN202310156736A CN116171798A CN 116171798 A CN116171798 A CN 116171798A CN 202310156736 A CN202310156736 A CN 202310156736A CN 116171798 A CN116171798 A CN 116171798A
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sea cucumber
cordyceps militaris
culture medium
processing byproducts
matrix
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Inventor
米锐
周遵春
李亚洁
李学军
都兴范
陈星合
郑杰
李龙
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Liaoning Academy Of Marine And Fisheries Sciences Dalian Institute Of Biotechnology Liaoning Academy Of Agricultural Sciences Liaoning Marine Environment Monitoring Station
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Liaoning Academy Of Marine And Fisheries Sciences Dalian Institute Of Biotechnology Liaoning Academy Of Agricultural Sciences Liaoning Marine Environment Monitoring Station
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Priority to CN202310156736.7A priority Critical patent/CN116171798A/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/40—Cultivation of spawn

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  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Environmental Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium, which is characterized in that sea cucumber processing byproducts (viscera or boiled liquid) are prepared into sea cucumber matrixes, the single sea cucumber matrixes are used for replacing a liquid potato culture medium, and cordyceps militaris cultivation seed culture solution is prepared from cordyceps militaris original seed culture solution in a strain activation transfer mode, so that subsequent cordyceps militaris fruiting body cultivation is carried out. The cultivated Cordyceps militaris has the advantages of regular shape, high yield, increased content of effective components such as Cordyceps polysaccharide and cordycepin, and effectively improved product quality. Meanwhile, the comprehensive utilization range of sea cucumber processing byproducts is further enlarged, the process is simple, the operability is high, and large-scale industrial production can be realized.

Description

Method for preparing cordyceps militaris by taking sea cucumber processing byproducts as culture medium
Technical Field
The invention belongs to the technical field of comprehensive utilization of aquatic products and processing byproducts, and particularly relates to a method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium.
Background
Cordyceps militarisCordyceps militaris (L.) Link) The Cordyceps militaris is a model seed of Cordyceps genus of Clavipitaceae ascomycetes, is different from the Cordyceps genus, is a dual-purpose fungus for medicine and food with higher nutritive value and medicinal efficacy, contains not only protein, fat and other nutritive components, but also cordycepin, adenosine, cordyceps polysaccharide and other active components. Cordycepin is a special nucleoside active substance in Cordyceps, and has anticancer, antibacterial, and antiviral effects; adenosine is an endogenous nucleoside distributed throughout human cells, participates in myocardial energy metabolism, and has physiological effects on the cardiovascular system and many systems and tissues of the body; cordyceps sinensis polysaccharide is considered as one of very good immune promoters, and can enhance the immunity of organisms. The artificial cultivation of Cordyceps militaris strain is obtained by separating and domesticating wild Cordyceps militaris strain, and wild Cordyceps militaris is usually parasitic on larva or pupa of Lepidoptera insect, so the culture medium used for artificial cultivation of Cordyceps militaris is a key factor for determining cultivation success or failure. The existing artificial cultivation method of the cordyceps militaris is that the stock seeds and the cultivated seeds are cultivated by adopting a liquid culture medium (liquid potato culture medium) to obtain a cordyceps militaris cultivated seed culture solution, and then the cordyceps militaris cultivated seed culture solution is inoculated into solid culture media such as living pupa and rice or wheat for diapause relief and is used for cultivating cordyceps sinensis fruiting bodies. The potato liquid culture medium is prepared by peeling potato 200 g, cutting into small pieces, adding 500 mL distilled water, boiling for 30 min, filtering with 4 layers of gauze to obtain filtrate, and adding MgSO into the filtrate 4 1.5 g、KH 2 PO 4 3 g, VB1 10 mg, glucose 20. 0g, constant volume to 1000 mL, and natural pH. Although a small amount of exogenous nutrient components are added to the strain medium and the cultivation medium in order to improve the quality of the cordyceps militaris, the cordyceps militaris cannot be separated from the liquid potato medium, the solid medium such as rice or wheat and the like.
The deep processing of sea cucumbers mainly aims at the application of sea cucumber body walls, and a plurality of byproducts including sea cucumber viscera and sea cucumber decoction are generated in the processing process. Sea cucumber viscera are the parts of digestion, respiration, water pipe, reproductive system and the like remained after body wall processing; the sea cucumber water boiling liquid is liquid containing a large amount of water-soluble sea cucumber dissolution substances generated by inactivating enzyme of fresh sea cucumber by boiling water to prevent autolysis. Research shows that the sea cucumber processing byproducts contain functional active substances similar to body walls, so that a great deal of research is devoted to the development and utilization of the sea cucumber processing byproducts. If the viscera of sea cucumber are processed into products such as seasonings, enzymolysis powder, bioactive peptides and the like, the boiled liquid of sea cucumber is also processed into defatted powder and nutritional beverage, or the related researches of extracting active ingredients such as polysaccharide, saponin and the like are also carried out.
As the sea cucumber byproducts contain a large amount of salt, so far, no related report exists on the use of sea cucumber viscera enzymolysis liquid or sea cucumber decoction liquid to replace a liquid potato culture medium after filtration, sterilization and concentration, and the expansion culture of cordyceps militaris bacterial stock into cultivated species and further the subsequent culture of cordyceps militaris bacterial fruiting bodies.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium.
The technical scheme of the invention is as follows: a method for preparing Cordyceps militaris by using sea cucumber processing byproducts as culture medium comprises the following steps:
step 1, preparation of sea cucumber matrixes: adding distilled water into sea cucumber viscera, crushing, adding protease for enzymolysis to obtain sea cucumber viscera enzymolysis liquid, and sterilizing to obtain sea cucumber matrix A; or collecting sea cucumber decoction, filtering to remove impurities, concentrating, sterilizing, and collecting as sea cucumber matrix B;
step 2, preparing cordyceps militaris original strain culture solution: transferring Cordyceps militaris strain into liquid culture to obtain Cordyceps militaris strain culture medium;
step 3, adding the cordyceps militaris original strain culture solution into the sea cucumber matrix A or/and the sea cucumber matrix B, and fermenting and culturing to obtain cordyceps militaris cultivated strain culture solution;
and step 4, inoculating the obtained cordyceps militaris strain culture medium to a rice or wheat culture medium, and culturing cordyceps militaris fruiting bodies.
The specific preparation method of the sea cucumber matrix A in the step 1 comprises the following steps of: 8-10, adding distilled water for homogenating, adding protease with the mass ratio of 1-3%, controlling the temperature to be 45-55 ℃, magnetically stirring for enzymolysis for 2-3 hours, filtering for impurity removal, and sterilizing for 30 minutes at 115 ℃.
The concentration of the sea cucumber matrix B prepared in the step 1 is 1/5-1/2 of the original volume.
And 2, taking fungus blocks of the cordyceps militaris inclined surface preservation mother strain fully covered with mycelia, inoculating the fungus blocks into a liquid potato culture medium, and shake-flask culturing for 5-7 d at the temperature of 18-22 ℃ at the speed of 130-150 r/min to obtain cordyceps militaris original strain culture solution.
The step 3 is to transfer the cordyceps militaris original strain culture solution into the sea cucumber matrix A or/and the sea cucumber matrix B according to the proportion of 2-5%, and perform aeration stirring fermentation for 4-7 d at the temperature of 18-22 ℃ to obtain the cordyceps militaris cultivated strain culture solution.
Inoculating 4-8% of cordyceps militaris strain culture medium to a rice or wheat culture medium according to the mass ratio, performing dark culture for 6-8 d under the ventilation condition of 18-22 ℃, and then irradiating with 300 lx scattered light; after fruiting body protrusion appears, the illumination intensity is 400 lx, intermittent illumination culture is realized in a mode of 1 h irradiation and 80 minutes in an intermittent manner, the room temperature is 18-22 ℃ and the humidity is 70-90% until the fruiting body top ascus shell of the Cordyceps sinensis is cultivated to expand, and then harvesting is carried out, so that the high-quality Cordyceps sinensis fruiting body is obtained.
The sea cucumber matrix A and/or the sea cucumber matrix B are/is added into the rice or wheat cultivation medium in the step 4, and the addition amount is 0.1-0.5% of the mass of the cultivation medium.
The invention prepares sea cucumber substrate from sea cucumber processing byproducts (viscera or boiled liquid), replaces liquid potato culture medium with single sea cucumber substrate, prepares cordyceps militaris cultivation seed culture solution from cordyceps militaris original seed culture solution in a strain activation transfer mode, and further cultures subsequent cordyceps militaris fruiting bodies. The cultivated Cordyceps militaris has the advantages of regular shape, high yield, increased content of effective components such as Cordyceps polysaccharide and cordycepin, and effectively improved product quality. Meanwhile, the comprehensive utilization range of sea cucumber processing byproducts is further enlarged, the process is simple, the operability is high, and large-scale industrial production can be realized.
Drawings
FIG. 1 is a diagram showing the growth of fruiting bodies of Cordyceps militaris for 30 days in examples and comparative examples of the invention.
FIG. 2 is a diagram showing the state of growth of fruiting bodies of Cordyceps militaris in examples and comparative examples for 50 days.
Description of the embodiments
Examples
The invention relates to a method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium, which sequentially comprises the following steps:
step 1, preparation of sea cucumber matrixes: the viscera of sea cucumber are mixed according to the mass ratio of 1:8, adding distilled water for homogenating, adding protease with the mass ratio of 3%, controlling the temperature to be 45-55 ℃, magnetically stirring for enzymolysis to be 2 h, filtering for impurity removal, and sterilizing for 30 min at 115 ℃ to obtain a sea cucumber matrix A;
step 2, preparing cordyceps militaris original strain culture solution: taking fungus blocks of Cordyceps militaris (strain sources are commercially available) inclined surface preservation mother strains which are fully distributed with mycelia, inoculating the fungus blocks into a liquid potato culture medium (the preparation method is the same as that described in the background of the invention), and shake-culturing for 5 d at the temperature of 18-22 ℃ in a 150 r/min shaking bottle to obtain a Cordyceps militaris stock culture solution;
step 3, transferring the cordyceps militaris original strain culture solution into a sea cucumber matrix A according to a proportion of 3%, and carrying out aeration stirring fermentation for 5 d at 18-22 ℃ to obtain cordyceps militaris cultivated strain culture solution;
inoculating 6% of cordyceps militaris strain culture medium to a rice culture medium according to the mass ratio, culturing in a dark way at 18-22 ℃ under the ventilation condition until the strain is 6-8 days, namely, culturing until mycelia grow fully in the culture medium, and irradiating with 300 lx scattered light; after fruiting body protrusion appears, the illumination intensity is 400 lx, intermittent illumination culture is realized in a mode of 1 h irradiation and 80 minutes in an intermittent manner, the room temperature is 18-22 ℃ and the humidity is 70-90% until the fruiting body top ascus shell of the cordyceps sinensis begins to expand, and then harvesting is carried out, so that the cordyceps militaris fruiting body S1 is obtained.
Examples
The cultivation method is basically the same as that of example 1, except that the sea cucumber substrate A is replaced by the sea cucumber substrate B, and the cordyceps militaris fruiting body S2 is obtained by taking wheat as a cultivation medium. The sea cucumber matrix B is prepared by removing impurities in sea cucumber decoction by gauze or a filter screen, concentrating to 1/5-1/2 of the original volume, and sterilizing.
Examples
The cultivation method is basically the same as that of example 1, except that the sea cucumber substrate A is added into the rice cultivation medium in the step 4, and the addition amount is 0.3% of the mass of the cultivation medium, so as to obtain the cordyceps militaris fruiting body S3.
Examples
The cultivation method is basically the same as that of example 2, except that the sea cucumber substrate B is added into the wheat cultivation medium in the step 4, and the addition amount is 0.3% of the mass of the cultivation medium, so as to obtain the cordyceps militaris fruiting body S4.
Comparative example:
the cultivation method is basically the same as that of example 1, except that step 3 is to obtain a cordyceps militaris cultivation seed culture solution by using a liquid potato culture medium (the preparation method is the same as that of the background technology of the invention) according to the method of the prior art, and obtain a cordyceps militaris fruiting body C1.
Experiment:
1. comparison of growth status of Cordyceps militaris fruiting body
Photographing is carried out on 30 days and 50 days of culturing the cordyceps militaris fruiting body in examples 1-4 and comparative examples, and the photographing is shown in fig. 1 and 2.
In fig. 1 and 2, the fruiting bodies C1, S2, S3, and S4 are respectively from left to right. As can be seen from fig. 1 and 2, the fruiting bodies S1, S2, S3, and S4 of Cordyceps militaris are uniform in shape, high in yield, and free from white fungus film formation, compared with the fruiting body of comparative example C1.
2. Comparison of functional ingredients of Cordyceps militaris fruiting body
Cordyceps militaris is prepared according to the methods of examples 1-4 and comparative examples, after culturing for 50 days, fruiting bodies C1, S2, S3 and S4 of Cordyceps militaris are obtained by synchronously harvesting according to the growth state of the fruiting bodies, and the fruiting bodies are dried. Calculating to obtain yield according to average fruit body dry weight; detecting cordycepin, adenosine and pravastatin by high performance liquid chromatography, and determining Cordyceps polysaccharide by UV method.
The measurement results are shown in Table 1.
TABLE 1
Yield g/bottle Cordycepin mg/kg Cordyceps polysaccharide g/100g Adenosine mg/kg Pranostat Ding g/kg
C1 3.06 2.53×10 3 8.47 1.85×10 3 5876.83
S1 4.89 2.72×10 3 9.84 2.38×10 3 6678.85
S2 3.73 2.65×10 3 9.80 2.32×10 3 6432.56
S3 4.23 2.63×10 3 9.45 2.25×10 3 6412.13
S4 3.42 2.55×10 3 9.21 2.13×10 3 6245.24
As shown in Table 1, compared with the comparative example C1, the average yield of single bottle of the test group S1, S2, S3 and S4 is improved by 11.76-59.8%, and the contents of cordycepin, cordyceps polysaccharide, adenosine and pravastatin are also greatly improved, which indicates that the cultivation method of the invention effectively improves the quality of Cordyceps militaris.

Claims (7)

1. A method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium is characterized by sequentially comprising the following steps:
step 1, preparation of sea cucumber matrixes: adding distilled water into sea cucumber viscera, crushing, adding protease for enzymolysis to obtain sea cucumber viscera enzymolysis liquid, and sterilizing to obtain sea cucumber matrix A; or collecting sea cucumber decoction, filtering to remove impurities, concentrating, sterilizing, and collecting as sea cucumber matrix B;
step 2, preparing cordyceps militaris original strain culture solution: transferring Cordyceps militaris strain into liquid culture to obtain Cordyceps militaris strain culture medium;
step 3, adding the cordyceps militaris original strain culture solution into the sea cucumber matrix A or/and the sea cucumber matrix B, and fermenting and culturing to obtain cordyceps militaris cultivated strain culture solution;
and step 4, inoculating the obtained cordyceps militaris strain culture medium to a rice or wheat culture medium, and culturing cordyceps militaris fruiting bodies.
2. The method for preparing cordyceps militaris by using sea cucumber processing byproducts as a culture medium according to claim 1, wherein the specific preparation method of the sea cucumber matrix A in the step 1 is that sea cucumber viscera are prepared according to a mass ratio of 1: 8-10, adding distilled water for homogenating, adding protease with the mass ratio of 1-3%, controlling the temperature to be 45-55 ℃, magnetically stirring for enzymolysis for 2-3 hours, filtering for impurity removal, and sterilizing for 30 minutes at 115 ℃.
3. The method for preparing cordyceps militaris by using sea cucumber processing byproducts as a culture medium according to claim 2, wherein the concentration of the sea cucumber substrate B prepared in the step 1 is 1/5-1/2 of the original volume.
4. The method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium, which is characterized in that the step 2 is to take fungus blocks of cordyceps militaris inclined surfaces which are fully covered with mycelia and store mother strains, connect the fungus blocks into a liquid potato culture medium, shake flask culture for 5-7 d at the temperature of 18-22 ℃ at 130-150 r/min, and obtain cordyceps militaris stock culture solution.
5. The method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium according to claim 4, wherein the step 3 is characterized in that cordyceps militaris original strain culture solution is transferred into a sea cucumber substrate A or/and a sea cucumber substrate B according to the proportion of 2-5%, and is aerated and stirred for fermentation for 4-7 d at the temperature of 18-22 ℃ to obtain cordyceps militaris cultivar culture solution.
6. The method for preparing cordyceps militaris by taking sea cucumber processing byproducts as a culture medium, which is characterized in that in the step 4, a cordyceps militaris strain culture medium is inoculated onto a rice or wheat culture medium according to the mass ratio of 4-8%, and under the ventilation condition of 18-22 ℃, the cordyceps militaris strain culture medium is subjected to dark culture for 6-8 d, and then is irradiated by scattered light of 300 lx; after fruiting body protrusion appears, the illumination intensity is 400 lx, intermittent illumination culture is realized in a mode of 1 h irradiation and 80 minutes in an intermittent manner, the room temperature is 18-22 ℃ and the humidity is 70-90% until the fruiting body top ascus shell of the Cordyceps sinensis is cultivated to expand, and then harvesting is carried out, so that the high-quality Cordyceps sinensis fruiting body is obtained.
7. The method for preparing Cordyceps militaris by using sea cucumber processing byproducts as culture medium according to claim 6, wherein the rice or wheat culture medium in step 4 is added with sea cucumber matrix A and/or sea cucumber matrix B in an amount of 0.1-0.5% of the mass of the culture medium.
CN202310156736.7A 2023-02-23 2023-02-23 Method for preparing cordyceps militaris by taking sea cucumber processing byproducts as culture medium Pending CN116171798A (en)

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