CN107267398B - Ganoderma lucidum liquid strain culture medium and ganoderma lucidum liquid strain culture method - Google Patents
Ganoderma lucidum liquid strain culture medium and ganoderma lucidum liquid strain culture method Download PDFInfo
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Abstract
The invention discloses a culture medium and a culture method for ganoderma lucidum liquid strains. The ganoderma lucidum liquid strain culture medium comprises: egg6g of white peptone, 15g of glucose, 0.5g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of potato starch, 3g of corn and cassava starch and vitamin B110mg, agar 1g and purified water 1000 ml. And the culture method of the ganoderma lucidum liquid strain comprises the following steps: based on the formula of the ganoderma lucidum liquid strain culture medium, performing high-temperature sterilization, inoculating the ganoderma lucidum liquid strain into a mother strain, and performing shake culture to obtain the ganoderma lucidum liquid strain. The formula of the ganoderma lucidum liquid strain culture medium and the ganoderma lucidum liquid strain reduce culture steps, shorten spawn running period, shorten growth period, have low contamination rate, low cost and high yield, and are suitable for large-scale and industrial production.
Description
Technical Field
The invention relates to the technical field of edible fungus cultivation, in particular to a ganoderma lucidum liquid strain culture medium and a ganoderma lucidum liquid strain culture method.
Background
At present, the culture mode of the ganoderma lucidum strain is mainly solid culture, namely the traditional culture mode, and the ganoderma lucidum produced by adopting the traditional culture mode is difficult to meet the requirements of domestic and foreign markets in the aspects of quality and yield. The ganoderma lucidum cultured by solid strain has the defects of slow fungus growth, inconsistent fungus age, weak disease resistance, weak vitality, easy infectious microbe infection and the like.
Disclosure of Invention
The invention provides a lucid ganoderma liquid strain culture medium and a lucid ganoderma liquid strain culture method, and solves the technical problems that lucid ganoderma produced and cultured by solid strains is slow in spawn running, inconsistent in fungus age, weak in disease resistance, weak in vitality, easy to be infected by mixed bacteria and the like.
In order to solve the technical problems, the invention provides a ganoderma lucidum liquid strain culture medium which is prepared from 6g of peptone, 15g of glucose, 0.5g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of potato starch, 3g of corn and cassava starch and vitamin B110mg, agar 1g and purified water 1000 ml.
The invention provides another ganoderma lucidum liquid strain culture medium which is prepared from 250g of peptone, 1000g of glucose, 40g of magnesium sulfate, 20g of monopotassium phosphate, 100g of potato starch, 200g of corn cassava starch and vitamin B1600mg, 100g of agar, 5g of antifoaming agent and 60L of purified water.
The invention also provides a method for culturing the ganoderma lucidum liquid strains, which comprises the following steps:
(1) preparing a plurality of triangular flasks with the volume of 500ml, respectively filling 200 ml/flask of culture medium prepared from Ganoderma lucidum liquid strain culture medium into the triangular flasks, sterilizing the triangular flasks in a high-pressure steam cooker, taking out, and cooling;
(2) taking one ganoderma lucidum slant test tube mother seed, selecting 1 or 2 broad bean strains with the size, inoculating the broad bean strains into each triangular flask, and standing for 24 hours;
(3) after 24 hours, a plurality of triangular flasks were placed on a rotary shaker, and cultured on the shaker for 6 days to prepare small-vial liquid strains.
As a preferred scheme of the method for culturing the liquid strains of the lucid ganoderma, the high-pressure steam kettle in the step (1) is sterilized at the high temperature of 120-126 ℃ for 30 minutes.
As a preferable embodiment of the method for culturing liquid strains of Ganoderma lucidum of the present invention, the rotary speed of the shaker in step (3) is 120-130 r/min, and the temperature is 25-30 ℃.
As a preferred scheme of the method for culturing the liquid strains of the lucid ganoderma, the method further comprises the following steps after the step (3):
(4) preparing a large-capacity triangular flask, inoculating the small-bottle liquid strain prepared in the step (3) into the large triangular flask, filling 4 liters of liquid strain culture medium per bottle, putting the large-capacity triangular flask into a rotary shaking table, and culturing for 36 hours on the shaking table;
(5) inoculating liquid strain with a content of 4L/bottle into a 60L stainless steel aeration fermentation tank, and culturing for 36 hr in the aeration fermentation tank to obtain Ganoderma liquid strain;
(6) injecting Ganoderma liquid strain in the fermentation tank with injector, inoculating into bag mouth part of Ganoderma solid bag material culture strain, and performing large-scale production.
As a preferable scheme of the method for culturing the liquid strains of the lucid ganoderma, in the step (5), the air supplied to the ventilating fermentation tank is sterile air which is subjected to a series of purification treatments by an air sterilization and filtration system.
The lucid ganoderma liquid strain culture medium and the lucid ganoderma liquid strain culture method have the beneficial effects that: the ganoderma lucidum liquid strain is efficient, convenient, safe and pollution-free, the quality of the ganoderma lucidum strain is stable, the production period is short, the spawn running is fast, the production is not influenced by seasons, the ganoderma lucidum is fast to grow, the disease resistance is strong, the yield is high, and the large-scale and industrial production can be carried out. According to the suitable growth conditions of the lucid ganoderma, an optimal environment control mode is adopted, large-scale and large-batch cultivation and production are carried out in an industrial mode, the lucid ganoderma is not influenced by seasons and environments, and the goal of controllable, stable and efficient production all the year around is achieved.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the above objects, features and advantages more apparent and understandable.
Example 1
1000ml of liquid strain medium was prepared: 6g of peptone, 15g of glucose, 0.5g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of potato starch, 3g of corn and cassava starch and vitamin B11 tablet (10 mg), agar 1g and purified water 1000 ml.
The vial liquid strain culture method comprises the following steps:
preparing a triangular flask with the volume of 500ml, respectively filling the triangular flask into 200 ml/bottle of liquid strain culture medium, placing the triangular flask in a high-pressure steam pot for sterilization, sterilizing at the high temperature of 120-126 ℃ for 30 minutes, taking out and cooling, taking one test tube stock of a ganoderma lucidum inclined plane, selecting 1 or 2 broad beans in size, inoculating the broad beans into the triangular flask, and standing for 24 hours. After 24 hours, the triangular flask is put into a rotary shaking table and cultured on the shaking table, the quality transmission effect of oxygen can be influenced by the characteristics of the shaking table, and the growth of hypha is further influenced, so the rotating speed is controlled to be 120-130 r/min, the temperature is optimally controlled to be 25-30 ℃, and the shaking table is cultured for 6 days.
Example 2
Preparation of 60 liters of liquid strain culture medium: 250g of peptone, 1000g of glucose, 40g of magnesium sulfate, 20g of monopotassium phosphate, 100g of potato starch, 200g of corn and cassava starch and vitamin B160 tablets, 100g of agar, 1 spoon (5 g) of antifoaming agent and 60L of purified water.
The culture method of inoculating into the fermentation tank comprises the following steps:
1. preparing a triangular flask with the volume of 500ml, respectively filling the triangular flask into 200 ml/bottle of liquid strain culture medium, placing the triangular flask in a high-pressure steam pot for sterilization, sterilizing at the high temperature of 120-126 ℃ for 30 minutes, taking out and cooling, taking one test tube stock of a ganoderma lucidum inclined plane, selecting 1 or 2 broad beans in size, inoculating the broad beans into the triangular flask, and standing for 24 hours. After 24 hours, the triangular flask is placed into a rotary shaking table and cultured on the shaking table, the quality transmission effect of oxygen can be influenced by the characteristics of the shaking table, and the growth of hyphae is further influenced, so that the rotation speed is controlled to be 120-130 r/min, the temperature is optimally controlled to be 25-30 ℃, and the shaking table is cultured for 6 days.
2. Preparing a large-capacity triangular flask, inoculating small-bottle liquid strain into the large triangular flask, filling 4 liters of liquid strain culture medium per bottle, putting the triangular flask into a rotary shaking table, and culturing for 36 hours on the shaking table.
3. Inoculating liquid strain with a content of 4L/bottle into a stainless steel fermentation tank with a volume of 60L, culturing for 36 hours in an aerated fermentation tank, wherein the supplied air is required to be sterile in the whole culture process, and purifying the air into sterile air to enter the fermentation tank through a series of purification treatments of an air sterilization and filtration system. The function of the sterile air in the fermentation culture is to provide oxygen to promote the culture solution to turn over, and the mycelium is continuously mixed in the culture medium, thereby improving the absorption and the effect on nutrient substances, maintaining the uniform diffusion of metabolites and preventing the contamination of bacteria in the fermentation process. During the culture process, observing the temperature, pressure, stirring speed, gas and liquid flow, liquid viscosity, turbidity, ventilation and exhaust, and Ganoderma lucidum pellet state, and paying attention to the appearance of Ganoderma lucidum liquid culture solution, such as clarification, turbidity, flocculence, etc., and paying attention to color, smell, etc., such as Ganoderma lucidum liquid strain has special fragrance, and mold has wine taste, bacterial sourness, peculiar smell or no taste.
4. Injecting Ganoderma liquid strain in the fermentation tank with injector, inoculating into bag mouth part of Ganoderma solid bag material culture strain, and performing large-scale production.
In the above examples, the mycelia of Ganoderma lucidum grow in the liquid medium, and absorb water and nutrients in the liquid medium to culture mycelia, which are secondary mycelia, stronger than primary mycelia formed by spore germination and have a higher growth rate. The hyphae cultured in the liquid are observed in appearance to have two forms: one is a normal filament and the other is a spheroid. As the hyphae grow radially and extend around, the hyphae gradually grow in the original culture medium under the action of aeration or stirring in the liquid culture medium, or branch grows out, and the hyphae are criss-cross and intertwined to form a sphere, which is called as a mycelial ball. The mycelium pellet has various forms, is generally radial, has burrs, and has a diameter of about 4-8 MM. Some of the bacteria balls have smooth surfaces, some of the bacteria balls have rough surfaces, and some of the bacteria balls are similar to the acanthosphere. The culture medium (liquid) is a place where hyphae can live, exchange substances and produce metabolites, and the composition of the culture medium has important influence on the growth and the propagation of the hyphae and the synthesis of the metabolites, and is related to the yield and the quality of the fermentation product. In order to obtain good harvest of ganoderma lucidum, the propagation of mycelium pellets is important, and the culture solution is clear when a large amount of mycelium pellets are formed, and is turbid when no mycelium pellets or a small amount of mycelium pellets are formed. From this result, it was found that the turbid culture solution is less favorable for the growth of hyphae.
The mycelium pellet morphology of ganoderma is also influenced by the components of the culture medium, pH value and culture conditions:
firstly, the culture medium contains starch components, the higher the viscosity of the culture medium is, the smaller the mycelium pellet is, the shorter the bristles are, the fastest the ganoderma lucidum mycelium grows in soluble starch such as potato starch, corn tapioca starch and the like, and the other is worse.
Secondly, the mycelium can be filamentous or spherical under different pH values; the pH value of the culture medium in the scheme is 5.0-6.0, the pH range which is the optimum growth range of the ganoderma lucidum mycelia is 0.1% of phosphate concentration in the culture medium, the ganoderma lucidum mycelia grow slowly, the mycelia are large, and if the concentration is reduced, the mycelia are small.
In addition, the inoculation amount and the ventilation amount during the culture process, and the change of the shape of the thalli is also influenced by the speed of stirring. When the inoculation amount is large, the growth can be generally in a dispersion type, and when the inoculation amount is small, the formation of the bacterial balls is facilitated. Generally speaking, the larger the inoculation amount, the smaller the diameter of the bacterial pellet, and the smaller the inoculation amount, the larger the diameter of the bacterial pellet. In the using process of a common shaking table, an oscillation method is used, and the disadvantage is that the shaking table is turned upside down to achieve the up-and-down uniformity but not achieve the uniformity of the same horizontal plane, so in the technical scheme, the shaking table is improved, a stirring shaft is added to horizontally stir the shaking table, the diameter of the bacteria balls is small when the stirring speed is high or the ventilation volume is large, and the diameter of the bacteria balls is large when the stirring speed is low or the ventilation volume is small. Therefore, the stirring speed of the shaking table is controlled at the rotating speed of 120-130 r/min, so that the bacterial liquid is dynamically cultured on the liquid of the shaking table at the upper layer, and the mycelium pellets are cultured at the lower layer, which is more favorable for the growth of the strains.
In addition, the concentration of sugar in the culture medium is generally 2%, and when the sugar in the culture medium is excessive, the dissolution of oxygen in the air is often hindered, and the growth of submerged mycelia is not facilitated, so in the technical scheme, the concentration of sugar is emphatically controlled, the content of sugar in the conventional laboratories is higher than 2%, and the conventional laboratories can only be practically applied to experimental culture under laboratory conditions, are not suitable for mass production of agriculture, and have no practical operation prospect.
In addition, the growth of the ganoderma lucidum mycelia needs the action of a certain trace amount of growth factors, the growth factors are substances which cannot be synthesized in the growth process of the mycelia and are beneficial to the growth and the propagation of the mycelia and are required to be obtained from the outside, and the addition of the vitamin B1 in the ganoderma lucidum liquid culture medium is proved by practice to be capable of obviously promoting the growth of the mycelia.
Finally, the ganoderma lucidum liquid culture medium contains protein substances, and foams are easily formed under the condition of ventilation and stirring, so that the culture solution rises, and a large amount of solution escaping can be caused if the culture solution is not controlled, and the opportunity of bacterial contamination is increased. Meanwhile, the normal operation of ventilation and stirring can be influenced due to excessive foam, so that the successful cultivation of ganoderma lucidum liquid strains is influenced, and the cultivation can be ensured only by adding the defoaming agent.
The plantation adopting the technical scheme has the advantages of large-scale and industrial production, and the production cost is reduced by 37-60% when the volume of the fermentation tank is increased by 10 times.
It should be noted that, the fermentation tank in the present technical solution is further equipped with an air sterilization filtration system (air purification system), for the aerobic liquid ganoderma lucidum strain cultivation pure fermentation, air is required to be purified to be sterile, and natural air contains countless dust and microorganisms, and must be subjected to a series of purification treatments to become sterile air before entering the fermentation tank. The role of sterile air in fermentation production: firstly, oxygen is provided, secondly, culture liquid is promoted to turn over, mycelia are continuously mixed in a culture medium, absorption and operation of nutrient substances are improved, thirdly, uniform diffusion of metabolites is maintained, and fourthly, positive pressure operation in the fermentation process is maintained, so that bacteria contamination is prevented.
The air treated by the air sterilizing and filtering system achieves the following conditions: several indexes of sterility, no dust, no impurity, no water, no oil, pressure, etc.
Experimental studies show that the use effects of the solid culture medium and the liquid strain culture medium of Ganoderma lucidum of the present invention (example 1-2) are shown in Table 1.
In conclusion, the invention reduces the culture steps, shortens the spawn running period, shortens the growth period, and has low contamination rate, low cost and high yield.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (6)
1. A culture medium for liquid strains of lucid ganoderma is characterized by comprising 6g of peptone, 15g of glucose, 0.5g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of potato starch, 3g of corn and cassava starch and vitamin B110mg, agar 1g and purified water 1000 mL.
2. A culture medium for liquid strains of lucid ganoderma is characterized by comprising 250g of peptone, 1000g of glucose, 40g of magnesium sulfate, 20g of monopotassium phosphate, 100g of potato starch, 200g of corn and cassava starch, and vitaminBiotin B1600mg, 100g of agar, 5g of antifoaming agent and 60L of purified water.
3. A method for culturing liquid strains of lucid ganoderma is characterized by comprising the following steps:
(1) preparing a plurality of 500 ml-capacity triangular flasks, respectively filling 200 ml/flask of the ganoderma lucidum liquid strain culture medium according to claim 1 or 2 into the triangular flasks, sterilizing the triangular flasks in a high-pressure steam cooker, taking out, and cooling;
(2) taking one ganoderma lucidum slant test tube mother seed, selecting 1 or 2 broad bean strains with the size, inoculating the broad bean strains into each triangular flask, and standing for 24 hours;
(3) after 24 hours, a plurality of flasks were placed on a rotary shaker for horizontal agitation, and cultured on the shaker for 6 days to prepare small-vial liquid strains.
4. The method for culturing liquid strains of ganoderma lucidum as claimed in claim 3, wherein: the high-pressure steam boiler sterilization in the step (1) is specifically high-temperature sterilization for 30 minutes at the temperature of 120-126 ℃.
5. The method for culturing liquid strains of ganoderma lucidum as claimed in claim 3, wherein: the rotary speed of the shaking table in the step (3) is 120-130 r/min, and the temperature is 25-30 ℃.
6. The method for culturing liquid strains of ganoderma lucidum as claimed in claim 3, wherein: the method also comprises the following steps after the step (3):
(4) preparing a large-capacity triangular flask, inoculating the small-bottle liquid strain prepared in the step (3) into the large triangular flask, filling 4 liters of liquid strain culture medium per bottle, putting the large-capacity triangular flask into a rotary shaking table for horizontal stirring, and culturing for 36 hours on the shaking table;
(5) inoculating a liquid strain with the loading capacity of 4 liters per bottle into a 60-liter stainless steel ventilation fermentation tank, culturing for 36 hours in the ventilation fermentation tank, wherein the air supplied to the ventilation fermentation tank is sterile air which is subjected to a series of purification treatment by an air sterilization filtration system, and preparing a ganoderma lucidum liquid strain;
(6) injecting Ganoderma liquid strain in the fermentation tank with injector, inoculating into bag mouth part of Ganoderma solid bag material culture strain, and performing large-scale production.
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| CN107637390A (en) * | 2017-11-03 | 2018-01-30 | 江苏农林职业技术学院 | A kind of live body silkworm and the method for the wild cicada fungus of silkworm chrysalis culture |
| CN110669678A (en) * | 2019-10-22 | 2020-01-10 | 赤峰制药股份有限公司 | Griseofulvin strain solid culture medium |
| CN114836492A (en) * | 2021-02-02 | 2022-08-02 | 中粮生物科技（北京）有限公司 | Method for producing ganoderan and ganoderic acid by fermentation |
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| CN1097025A (en) * | 1993-06-30 | 1995-01-04 | 云南省农业科学院粮食作物研究所 | Prescription of culture liquid for germanium-rich glossy ganoderma |
| CN1055723C (en) * | 1998-01-14 | 2000-08-23 | 李宝健 | Ganoderma lucidum 10 ton tank fermentation technology |
| CN1266897A (en) * | 1999-03-10 | 2000-09-20 | 上海中祥生物制品有限公司 | Process for fermenting lucid ganoderma in 18-ton fermentor |
| CN104082038A (en) * | 2014-07-10 | 2014-10-08 | 乳山市华隆生物科技有限公司 | Method for preparing high-purity lucid ganoderma hyphostroma powder through liquid submerged fermentation |
| CN104584859A (en) * | 2015-01-01 | 2015-05-06 | 曾建斌 | Production method for soft edible lucid ganoderma |
| CN105981591A (en) * | 2015-04-14 | 2016-10-05 | 城口县天星灵芝种植有限公司 | Cultivation method of lucid ganoderma |
| CN106350456A (en) * | 2016-08-24 | 2017-01-25 | 孝感市应用技术研究所 | Manufacture and detection method of lucid ganoderma liquid strains |
| CN106434368A (en) * | 2016-09-24 | 2017-02-22 | 云南福保农业科技开发有限公司 | Culture method of ganoderma leucocontextum liquid strain |
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