CN111937678B - Antrodia camphorata culture medium and preparation method thereof - Google Patents
Antrodia camphorata culture medium and preparation method thereof Download PDFInfo
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- CN111937678B CN111937678B CN202010884812.2A CN202010884812A CN111937678B CN 111937678 B CN111937678 B CN 111937678B CN 202010884812 A CN202010884812 A CN 202010884812A CN 111937678 B CN111937678 B CN 111937678B
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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
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Abstract
The invention belongs to the technical field of fungal bioengineering, and particularly relates to an antrodia culture medium and a preparation method thereof. The culture medium provided by the invention comprises the following raw materials: 30-60g of grain substrate; 0.3-0.6g of gypsum powder; 20-60ml of nutrient solution; shaking flask for 10-30ml of strain; 0.2-0.6ml of exogenous additive; the exogenous additive is soybean oil; the shake flask strain is obtained by culturing carrot. The culture medium of the invention adopts a grain matrix with lower price, especially wheat grains, is suitable for the growth of antrodia camphorata, has simple preparation and is suitable for the large-scale culture and production of enterprises; according to the invention, soybean oil is selected as an exogenous additive, so that the cost performance is high, the absorption and utilization rate is high, and the triterpenoid content of antrodia camphorata can be increased; the carrot is selected as the nutrient solution in the shake flask to replace the traditional potato, and a three-stage culture mode of constant temperature, temperature reduction and temperature rise is adopted, so that the growth and germination of antrodia cinnamomea mycelia are facilitated.
Description
Technical Field
The invention belongs to the technical field of fungal bioengineering, and particularly relates to an antrodia culture medium and a preparation method thereof.
Background
Antrodia camphorata is a unique rare medicinal fungus which is discovered by scientists in Taiwan in 1990, in Aphyllophorales, Polyporaceae and Botrytis. The camphor tree grows in mountainous areas with the altitude of 450-2000 meters, and only grows on rotten heartwood inner walls of the stems of the camphor trees which are more than one hundred years and are special in Taiwan, and the stems are difficult to collect due to the fact that the surfaces of the stems are moist. Taiwan is currently called the most expensive wild fungus, Taiwan folk is called "ruby in forest" and in Hongkong and Macau is also called "Shenzhi".
Antrodia camphorata contains abundant physiological active ingredients, including polysaccharide, triterpenes, superoxide dismutase, vitamins, trace elements, etc. The effective components of the triterpenoid compound are most specific, more than 200, and are incomparable with other mushroom fungi, and the triterpenoid compound has outstanding effects of resisting cancer, protecting liver and the like. In recent years, the market attention of antrodia camphorata is higher and higher, but wild antrodia camphorata grows slowly and has low natural yield, and the antrodia camphorata is the only host of antrodia camphorata and is the national conservation type tree species, so that the antrodia camphorata is cultured by adopting an artificial culture method at present.
At present, the artificial culture method mainly comprises a basswood culture method, a solid culture method and a liquid culture method. The basswood culture method can culture the antrodia cinnamomea fruiting bodies with similar active ingredients to wild antrodia cinnamomea, but has high cost and slow period and cannot meet the market demand. The liquid culture method is an effective way for realizing industrial production of a plurality of medicinal fungi, has the main advantages of short period, controllable quality and the like, but a large number of experiments of scientific research units prove that the method has lower content of effective components in culture; the solid culture method has moderate growth period, but the preparation process of the space bag is complicated, and the culture cost is high.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide an antrodia culture medium.
The invention also provides a preparation method of the antrodia culture medium.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides an antrodia culture medium, which consists of the following raw materials: 30-60g of grain substrate; 0.3-0.6g of gypsum powder; 20-60ml of nutrient solution; shaking flask for 10-30ml of strain; 0.2-0.6ml of exogenous additive; the exogenous additive is soybean oil; the shake flask strain is obtained by culturing carrot.
The nutrient solution used by the invention is composed of the following raw materials by weight percent: glucose 0.5-1.5%, NH4Cl 0.2-0.5%，MgSO4·7H2O 0.02-0.05%，KH2PO4 0.1-0.3%, and the balance of water; the grain matrix is one of rice, wheat, corn grit, millet, highland barley and oat.
Further, the shake flask strain is prepared by the following preparation method: inoculating 5-6 pieces of thin mycelia with diameter of 6mm × 6mm and diameter of 2mm under mycelia into shake flask nutrient solution, culturing at 25-30 deg.C for 3-5 days at rotation speed of 100-.
Further, the shake flask nutrient solution is prepared by the following preparation method: cutting 230g of carrot 160-4Cl 2-4g，KH2PO4 0.2-0.5g，MgSO4·7H20.2-0.5g of O and water to 1000ml, subpackaging 100ml in a 250ml triangular flask, and sterilizing at 115 ℃ for 30 min.
The addition amount of propyl gallate is 0.1% of total amount of rhizoma Solani Tuber osi and radix Dauci Sativae.
The invention provides a preparation method of an antrodia culture medium, which comprises the following steps:
(1) adding tartaric acid into soybean oil serving as an exogenous additive, heating to 80-85 ℃, stirring for 3-5h, and then adjusting the pH value to be neutral to obtain the treated exogenous additive;
(1) putting the grain matrix in parts by weight into a 450ml transparent can, adding gypsum powder, nutrient solution and exogenous additives, repeating the treatment for 5 times, shaking up, soaking for 24 hours, and sterilizing for 1 hour at 121 ℃;
(2) adding shake flask strain into the culture medium.
Further, the mass ratio of the soybean oil to the tartaric acid is 1: 0.15.
further, the culture is carried out for 15-30d at the temperature of 25-30 ℃; the culture is carried out under the condition of keeping out light.
Compared with the prior art, the invention has the beneficial effects that:
(1) the culture medium of the invention adopts a low-price grain substrate, especially wheat grains, is suitable for the growth of antrodia camphorata, is simple to prepare, and is suitable for large-scale culture and production of enterprises.
(2) The invention selects soybean oil as an exogenous additive, has high cost performance and high absorption and utilization rate, and can improve the triterpenes content of the antrodia camphorata.
(3) The carrot is selected as the nutrient solution in the shake flask to replace the traditional potato, and a three-stage culture mode of constant temperature, temperature reduction and temperature rise is adopted, so that the growth and germination of antrodia cinnamomea mycelia are facilitated.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
Example 1 preparation of Antrodia camphorata Shake flask strains
The Antrodia camphorata strain (BCRC 35396) used in this example was provided by Taiwan institute of food industry development and research, and was cultured in a slant medium (containing 15% carrot, 2% glucose, 1.6% agar, 0% agar) at 25 deg.C.05% MgSO4·7H2O、0.05% KH2PO4、0.001% VB1) Culturing until the mycelia of Antrodia camphorata grow full. Inoculating the slant culture with Antrodia Camphorata mycelium into the flat culture medium (the components are the same as the slant culture medium) for continuous culture until the Antrodia Camphorata mycelium grows into a circle with radius of about 4 cm. Equally dividing the mycelium of the plate culture medium into 6mm multiplied by 6mm and 2mm below the mycelium, adding 5-6 mycelium blocks into each bottle of liquid culture, culturing for 4 days at 28 ℃ at the rotating speed of 150r/min, then cooling to 16 ℃ for culturing for 8 hours, then heating to 25 ℃ and standing for 15 hours.
The shake flask nutrient solution adopts the following preparation method: cutting 230g of carrot into slices, adding 0.23g of propyl gallate and 500ml of water, boiling for 15-25min, filtering with gauze to remove residues, adding 10-20g of glucose and NH into the filtrate4Cl 2-4g，KH2PO4 0.2-0.5g，MgSO4·7H20.2-0.5g of O, supplementing water to 1000ml, subpackaging 100ml in a 250ml triangular flask, and sterilizing at 115 ℃ for 30 min;
the preparation method of the culture medium comprises the following steps:
(1) adding exogenous additive soybean oil according to a mass ratio of 1: adding tartaric acid at a ratio of 0.15, heating to 80-85 deg.C, stirring for 5 hr, and adjusting pH to neutral to obtain processed soybean oil;
(1) putting 50 parts of grain matrix into a 450ml transparent can, adding 0.5 part of gypsum powder, 30ml of nutrient solution and 0.4 ml of processed soybean oil, repeating each process for 5 times, shaking up, soaking for 24 hours, and sterilizing for 1 hour at 121 ℃;
(2) adding shake flask strain into culture medium, and culturing at 25-30 deg.C in dark condition for 25 d.
Comparative example 1
The other points are the same as example 1: carrot is replaced by equal amount of potato, and the same potato in the shake flask nutrient solution is treated by the same treatment method.
Comparative example 2
The medium formulation was essentially the same as in example 1.
The preparation method of the culture medium comprises the following steps:
(1) putting 50 parts of cereal matrix into a 450ml transparent can, adding 0.5 part of gypsum powder, 30ml of nutrient solution and 0.4 ml of soybean oil (without treatment), repeating each treatment for 5 times, shaking up, soaking for 24 hours, and sterilizing for 1 hour at 121 ℃;
(2) adding shake flask strain into culture medium, and culturing at 25-30 deg.C in dark condition for 25 d.
Comparative example 3
The formulation of the medium and the preparation method are essentially the same as in example 1.
The shake flask nutrient solution adopts the following preparation method: cutting 230g carrot into slices, adding 500ml water, boiling for 15-25min, filtering with gauze to remove residue, adding 10-20g glucose and NH into the filtrate4Cl 2-4g，KH2PO4 0.2-0.5g，MgSO4·7H20.2-0.5g of O and water to 1000ml, subpackaging 100ml in a 250ml triangular flask, and sterilizing at 115 ℃ for 30 min.
In the culture media prepared in comparative examples 2 and 3, in the culture process, the layering phenomenon occurs in part of parallel tests, rancidity and peculiar smell are easy to occur, and the culture process fails.
Effects of the embodiment
First, the growth of the mycelia of Antrodia camphorata in example 1, comparative example 1 and comparative example 3 was compared, and the specific results are shown in Table 1.
TABLE 1 growth of slant, plate, Shake flask mycelia of Antrodia camphorata
Note: 1.+ means not busy, + means relatively busy, and, + + + means busy; 2. the time to full tube is the number of days from germination to full tube.
Determination of (di) triterpenes content
The determination method comprises the following steps: oleanolic acid is used as a reference substance, and the measurement is carried out by adopting a vanillin-perchloric acid colorimetric method. Oven drying Antrodia Camphorata grain culture medium at 40 deg.C, pulverizing, weighing 2g, placing into triangular flask with plug, adding ethanol 40ml, performing ultrasonic treatment for 30min, filtering, adding ethanol 30ml into residue, performing ultrasonic treatment for 30min, filtering, washing with ethanol, and filteringThe residue was taken several times and the filtrate was taken up to 100 ml. 0.2ml of the above solution was pipetted into a 15ml stoppered tube and the solvent was evaporated. Precisely weighing 0.5g of vanillin, and adding glacial acetic acid to the solution to be dissolved in 10ml to obtain a vanillin-glacial acetic acid solution. Adding vanillin-glacial acetic acid solution 0.2ml and perchloric acid 0.8ml into solution test tube, shaking, heating in 70 deg.C water bath for 15min, taking out, immediately cooling in ice bath for 5min, taking out, adding ethyl acetate 4ml, shaking, measuring absorbance at 546nm wavelength with ultraviolet spectrophotometer, drawing standard curve with absorbance (A) as ordinate and concentration (C) as abscissa, and regression equation is A =0.0409 XC-0.0105, R2=0.9992。
(1) Analysis of grain culture Medium composition
Removing impurities, respectively weighing 50g of each grain (rice, wheat, corn grit, millet, highland barley, oat) in 450ml transparent can, adding 0.5g of Gypsum Fibrosum powder, and nutrient solution (glucose 1%, NH)4Cl 0.2%，MgSO4·7H2O 0.05%，KH2PO4 0.1%) of 30ml, 5 times of each treatment, shaking up and soaking for 24 hours, sterilizing at 121 ℃ for 1 hour, shaking the flask to obtain 20ml of strain, and culturing at 28 ℃ in the dark for 25 days, wherein the specific results are shown in Table 2.
TABLE 2 comparison of triterpene content in different grain media
The three grains have larger grains, are not suitable for absorbing liquid and caking, particularly the wheat is better, is easy to purchase and has higher performance-price ratio, and is suitable for industrial amplification production.
(3) The content of triterpenes in the antrodia camphorata cultured in the example 1 and the comparative examples 1-2 is detected, a control group is arranged at the same time, the control group is respectively replaced by equal amount of olive oil, peanut oil, soybean oil and corn oil, other preparation methods are the same as the example 1, and the specific results are shown in the table 3.
TABLE 3 comparison of triterpene content of different exogenous additives
The influence of different exogenous additives on the triterpenoid content of the antrodia camphorata cultured by grains is different, the soybean oil is more than olive oil and more than peanut oil and corn oil, and the soybean oil has higher price ratio and is safe and suitable for industrial large-scale production.
All of the above mentioned intellectual property rights are not intended to be restrictive to other forms of implementing the new and/or new products. Those skilled in the art will take advantage of this important information, and the foregoing will be modified to achieve similar performance. However, all modifications or alterations are based on the new products of the invention and belong to the reserved rights.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (3)
1. The antrodia culture medium is characterized by comprising the following raw materials: 30-60g of grain substrate; 0.3-0.6g of gypsum powder; 20-60ml of nutrient solution; shaking flask for 10-30ml of strain; 0.2-0.6ml of exogenous additive; the exogenous additive is soybean oil; the shake flask strain is obtained by culturing carrots;
the mass ratio of the soybean oil to the tartaric acid is 1: 0.15;
the shake flask strain adopts the following preparation method: culturing Antrodia camphorata strains on a slant culture medium at 25 ℃ until the Antrodia camphorata mycelia overgrow, and then inoculating the slant strains with the Antrodia camphorata mycelia to an Antrodia camphorata plate culture medium with the same components as the slant culture medium for continuous culture until the Antrodia camphorata mycelia grow into a circle with the radius of 4 cm; equally dividing the mycelium of the plate culture medium into 6mm multiplied by 6mm and 2mm below the mycelium, adding 5-6 mycelium blocks into each bottle of liquid culture, culturing for 3-5 days at the rotation speed of 100-200r/min and the temperature of 25-30 ℃, then cooling to 10-20 ℃, culturing for 5-10 hours, then heating to 25-27 ℃, and standing for 10-20 hours;
the slant culture medium is 15% of carrot, 2% of glucose, 1.6% of agar and 0.05% of MgSO4·7H2O、0.05% KH2PO4、0.001% VB1；
The shake flask nutrient solution adopts the following preparation method: cutting 230g of carrot into slices, adding 0.23g of propyl gallate and 500ml of water, boiling for 15-25min, filtering with gauze to remove residues, adding 10-20g of glucose and NH into the filtrate4Cl 2-4g，KH2PO4 0.2-0.5g，MgSO4·7H20.2-0.5g of O, supplementing water to 1000ml, subpackaging 100ml in a 250ml triangular flask, and sterilizing at 115 ℃ for 30 min;
the nutrient solution is composed of the following raw materials in percentage by weight: glucose 0.5-1.5%, NH4Cl 0.2-0.5%，MgSO4·7H2O 0.02-0.05%，KH2PO4 0.1-0.3%, and the balance of water;
the grain matrix is one of rice, wheat, corn grit, millet, highland barley and oat.
2. The method for preparing the culture medium of antrodia according to claim 1, comprising the following steps:
(1) adding tartaric acid into soybean oil serving as an exogenous additive, heating to 80-85 ℃, stirring for 3-5h, and then adjusting the pH value to be neutral to obtain the treated exogenous additive;
(2) putting the grain matrix into a 450ml transparent can, adding gypsum powder, nutrient solution and processed exogenous additives, repeating each process for 5 times, shaking up, soaking for 24 hours, and sterilizing for 1 hour at 121 ℃;
(3) adding shake flask strain into the culture medium.
3. The method according to claim 2, wherein the culturing is carried out at 25-30 ℃ for 15-30 d; the culture is carried out under the condition of keeping out light.
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| CN103125270A (en) * | 2013-02-28 | 2013-06-05 | 深圳市仁泰生物科技有限公司 | High-yield antrodia cinnamomea mycelium fermentation method for triterpenoids |
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