CN108504621B - Culture medium for paecilomyces hepiali Cs-4 and preparation method thereof - Google Patents
Culture medium for paecilomyces hepiali Cs-4 and preparation method thereof Download PDFInfo
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Abstract
The invention relates to aThe culture medium for paecilomyces hepiali Cs-4 comprises a seed culture medium and a liquid fermentation culture medium, wherein the seed culture medium comprises glucose, peptone, bran, inorganic salt and amino acid, and the liquid fermentation culture medium comprises hot pressed bean cake powder, glucose, sucrose, inorganic salt, amino acid and vitamin B1Adenine and notoginseng powder. The preparation method of the culture medium is simple and is suitable for industrial mass production. At different stages, different media are used to facilitate growth of the strains. Adding amino acid and vitamin B into culture medium1And mycelia of adenine and Cs-4 grow vigorously, and the prepared cordyceps sinensis powder is high in adenosine, total amino acid, ergosterol and mannitol. The notoginseng powder can be used by the strain after being added into the culture medium, the notoginsenoside and the ginsenoside can be detected in the fungus powder, simultaneously, the content of the original active ingredients in the fungus powder is also improved, and the medicinal value of the cordyceps fungus powder is obviously improved.
Description
Technical Field
The invention belongs to the field of microbiology, and particularly relates to a culture medium for paecilomyces hepiali Cs-4 and a preparation method thereof.
Background
Cordyceps sinensis is a medicinal name, is a complex of animals and fungi, and is a complex of stroma of Cordyceps sinensis of Clavipitaceae and larva of larva such as insect, rhizoma Dioscoreae Septemlobae and swift moth of host Hepialidae. Cordyceps sinensis is a complex composed of stroma (i.e., grass part) and sclerotium (i.e., cadaver part of worm). In winter, the larva hibernates in soil, fungi are parasitized in the soil, nutrition is absorbed, and the larva is filled with hyphae to die; in summer, seedlings grow from the bodies of the young insect corpses, are similar to grass, and are collected before and after summer. The main active components of Cordyceps sinensis are cordycepin and Cordyceps adenosine, and have effects of regulating immunity, resisting tumor, and relieving fatigue. Modern researches find that the cordyceps sinensis contains protein, fat, crude fiber, carbohydrate, various vitamins, cordycepin, cordycepic acid, various amino acids, iron, phosphorus, calcium, zinc, manganese, selenium and other elements necessary for human bodies. Pharmacological tests prove that the cordyceps sinensis has the effects of regulating the immunity of organisms, enhancing the physical quality, delaying aging, enhancing the hypoxia tolerance, resisting kidney injury, resisting pathogenic microorganisms, relieving asthma, eliminating phlegm, increasing white blood cells and the like. Meanwhile, the cordyceps sinensis has better effects of calming and easing pain, and can relieve pain during tumor attack and adverse reactions of chemotherapy.
Along with the improvement of the living standard of people, the attention on the big health industry is higher and higher, the pharmacological efficacy of the cordyceps sinensis is widely known, and the demand of the cordyceps sinensis also reaches the state of short supply. Cordyceps powder as a substitute of Cordyceps sinensis is produced by culturing Paccilomyces hepiali Cs-4 with culture medium under enlarged condition, and further fermenting. The quality of the cordyceps sinensis powder mainly depends on the quality of cordyceps sinensis mycelia, the mycelia are products obtained after the strain is subjected to amplification culture and fermentation culture, and a culture medium is very important for the growth of the Cs-4 strain.
Disclosure of Invention
The invention aims to provide a culture medium for paecilomyces hepiali Cs-4, which provides rich nutrition for the growth of a Cs-4 strain, promotes the formation of Cs-4 cordyceps mycelium, and improves the content of active ingredients such as adenosine and ergosterol in the mycelium. Meanwhile, the culture medium is added with the pseudo-ginseng powder, and the obtained Cs-4 mycelium not only has the original medicinal effect of the cordyceps sinensis, but also has the pharmacological application of the pseudo-ginseng saponin, particularly the ginsenoside.
Specifically, aiming at the defects of the prior art, the invention provides the following technical scheme:
a culture medium for paecilomyces hepiali Cs-4, comprising a seed culture medium and a liquid fermentation culture medium, the seed culture medium comprising: glucose, peptone, bran, inorganic salts and amino acids, the liquid fermentation medium comprising: hot pressed bean cake powder, glucose, sucrose, inorganic salt, amino acid, and vitamin B1And adenine.
Amino acid is added into the seed culture medium to promote the growth of the strain and ensure the stability of passage. The compositions of all levels of fermentation culture media are slightly different, and the nutritional requirements of the strains at different growth stages are met. Adenine is added into the culture medium, and the Cs-4 strain is fermented and cultured, so that the adenosine content in the prepared cordyceps sinensis powder is obviously improved, and the contents of ergosterol, total amino acid and mannitol are also improved.
Preferably, the inorganic salt is selected from one or more of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, zinc sulfate, zinc chloride, sodium chloride and calcium chloride, and the amino acid is selected from one or more of histidine, arginine, glycine and L-glutamic acid.
Preferably, the seed culture medium comprises a slant culture medium and a shake flask culture medium, wherein the slant culture medium comprises, in g/mL by weight volume: 2 to 4 percent of glucose, 0.2 to 0.4 percent of peptone, 0.4 to 0.8 percent of bran, 0.1 to 0.3 percent of monopotassium phosphate, 0.03 to 0.05 percent of magnesium sulfate, 0.1 to 0.3 percent of histidine, 2 to 4 percent of agar and the balance of water; the shake flask culture medium comprises: 1 to 5 percent of glucose, 0.2 to 0.4 percent of peptone, 0.4 to 0.6 percent of bran, 0.2 to 0.3 percent of fishbone powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.03 to 0.05 percent of magnesium sulfate, 0.03 to 0.05 percent of zinc sulfate, 0.1 to 0.3 percent of histidine and the balance of water.
Preferably, the liquid fermentation medium further contains pseudo-ginseng powder. After the pseudo-ginseng powder is added, the cordyceps sinensis powder obtained by fermentation culture has the original cordyceps sinensis efficacy, and also has the efficacy of pseudo-ginseng saponin and ginsenoside. In addition, after adenine and pseudo-ginseng powder are added into the fermentation medium at the same time, the content of ergosterol is obviously improved, and the content of notoginsenoside is also improved compared with that when pseudo-ginseng powder is added alone and adenine is not added.
Preferably, the liquid fermentation medium comprises a seeding tank medium, a propagation tank medium and a fermentation tank medium, wherein the seeding tank medium comprises the following components in g/mL by weight volume: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.2-0.4% of potassium dihydrogen phosphate, 0.05-0.1% of magnesium sulfate, 0.05-0.1% of zinc sulfate, 0.1-0.2% of histidine, vitamin B10.02-0.3%, adenine 0.05-0.2% and0.1-0.2% of soybean oil and the balance of water.
Preferably, the liquid fermentation medium comprises a seeding tank medium, a propagation tank medium and a fermenter medium, wherein the propagation tank medium comprises, in g/mL by weight volume: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.05-0.2%, soybean oil 0.2-0.3%, and water in balance.
Preferably, the liquid fermentation medium comprises a seeding tank medium, a propagation tank medium and a fermenter medium, wherein the fermenter medium comprises, in g/mL by weight volume: 3-4% of hot pressed bean cake powder, 1-2% of glucose, 1-2% of sucrose, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.1-0.2%, soybean oil 0.2-0.3%, and water in balance.
Preferably, the liquid fermentation medium contains pseudo-ginseng powder, wherein the composition of the fermentation tank medium is as follows: 4% of hot pressed bean cake powder, 2% of glucose, 1% of sucrose, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.03% of zinc sulfate, 0.1% of histidine, 0.1% of glycine, and vitamin B10.05%, adenine 0.15%, soybean oil 0.2%, notoginseng powder 1% and water in balance.
The invention also provides a preparation method of the culture medium, wherein the preparation method of the slant culture medium comprises the following steps:
(1) weighing bran, adding water, boiling, and filtering to obtain a filtrate;
(2) adding peptone into the filtrate, heating, and stirring until dissolved;
(3) adding other culture medium components except agar, heating and/or dissolving in water, and adding water to desired volume;
(4) under the heating condition, adding agar into the solution prepared in the step (3);
(5) subpackaging the prepared culture medium, and then sterilizing;
the preparation method of the shake flask culture medium is different from the preparation method of the slant culture medium in that the agar is not added in the step (4);
the liquid fermentation culture medium is prepared by weighing 50% water, adding each culture medium component, heating for dissolving, adding water to desired volume, and sterilizing.
Preferably, before the bran is weighed, the bran is sieved by a 40-mesh sieve, and the sterilization is high-pressure steam sterilization, wherein the pressure is 0.09-0.12MPa, the temperature is 120 +/-1 ℃, and the time is 30-40 minutes.
Compared with the prior art, the invention has the advantages that:
(1) the compositions of the seed culture medium and the fermentation culture medium are different, and experiments prove that the slant culture medium is suitable for the growth of the Cs-4 strain, ensures the stability of passage and provides excellent seeds for the subsequent fermentation culture. The compositions of all levels of fermentation culture media are slightly different, so that the nutrition supply for the growth of strains at different stages is ensured, and the obtained mycelium has high yield and high nutritive value.
(2) Compared with the common bean cake powder, the hot-pressed bean cake powder has low lipid content, the Cs-4 strain can better utilize the protein in the hot-pressed bean cake powder, and the utilization rate of raw materials is high. The liquid fermentation culture medium is added with amino acids and vitamin B in addition to basic nutrition1And adenine, wherein the mycelium of the Cs-4 strain cultured by fermentation is vigorous in growth and higher in yield than the mycelium cultured by a common culture medium. Because adenine is a precursor substance for adenosine biosynthesis, the adenosine content in cordyceps mycelia obtained by fermentation by using a culture medium added with adenine is obviously increased. The detection result shows that the total content of ergosterol, mannitol and amino acid in the cordyceps mycelia obtained by fermentation culture is also improved.
(3) After the panax notoginseng powder is added into the culture medium, the total arasaponin in the panax notoginseng powder is subjected to biotransformation in the growth process of the Cs-4 strain, and after the fermentation is finished, the monomer saponin can be detected in the fermentation product. Therefore, the Cs-4 mycelium not only has the original medicinal effect of the cordyceps, but also has the pharmacological application of the monomer saponin, particularly the ginsenoside. After adenine and pseudo-ginseng powder are simultaneously added into a fermentation medium, the saponin content of the cordyceps sinensis powder is higher than that of the cordyceps sinensis powder which is singly added, and the ergosterol content is higher than that of the cordyceps sinensis powder which is singly added with adenine.
Detailed Description
The invention provides a culture medium for paecilomyces hepiali Cs-4, which comprises a seed culture medium and a liquid fermentation culture medium, wherein the seed culture medium is divided into a slant culture medium and a shake flask culture medium, and the liquid fermentation culture medium is divided into a seeding tank culture medium, a propagation tank culture medium and a fermentation tank culture medium.
The slant medium comprises, in g/mL by weight volume: 2-4% of glucose, 0.2-0.4% of peptone, 0.4-0.8% of bran, 0.1-0.3% of monopotassium phosphate, 0.03-0.05% of magnesium sulfate, 0.1-0.3% of histidine and 2-4% of agar, and adding water to a constant volume, wherein the pH value is natural.
The shake flask medium comprises, in g/mL by weight volume: 1-5% of glucose, 0.2-0.4% of peptone, 0.4-0.6% of bran, 0.2-0.3% of fishbone powder, 0.1-0.3% of monopotassium phosphate, 0.03-0.05% of magnesium sulfate, 0.03-0.05% of zinc sulfate and 0.1-0.3% of histidine, adding water to a constant volume, and keeping the pH value natural.
The seeding tank medium comprises, in weight volume g/mL: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.2-0.4% of potassium dihydrogen phosphate, 0.05-0.1% of magnesium sulfate, 0.05-0.1% of zinc sulfate, 0.1-0.2% of histidine, vitamin B10.02-0.3%, adenine 0.05-0.2% and soybean oil 0.1-0.2%, adding water to constant volume, and pH is natural.
The propagation tank culture medium comprises the following components in g/mL by weight volume: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.05-0.2% and soybean oil 0.2-0.3%, adding water to constant volume, and pH is natural.
The fermenter medium comprises, in g/mL weight volume: 3-4% of hot pressed bean cake powder, 1-2% of glucose, 1-2% of sucrose, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.1-0.2% andsoybean oil 0.2-0.3%, water and pH value.
In a preferred embodiment, the liquid fermentation medium further comprises notoginseng powder in an amount of 0.5-1%.
The general method for preparing the seed culture medium comprises the following steps: boiling bran in water, and filtering to obtain filtrate; under heating condition, adding peptone, sequentially adding other components, heating or dissolving in water, adding water to desired volume, and sterilizing. The liquid fermentation culture medium is prepared by weighing 50% water, adding each culture medium component, heating for dissolving, adding water to desired volume, and sterilizing.
After the bran is sieved by a 40-mesh sieve, the bran is weighed, then 6g of bran is added into every 100mL of water for feeding, the mixture is heated to be boiled and kept for 30-40 minutes, and then the filtrate is obtained by filtration. The filtrate contains various trace elements and vitamins, and is combined with amino acid in a seed culture medium, so that the strain has high growth speed and stable passage.
The invention is further illustrated by the following specific examples.
Materials and equipment related to the invention are commercially available, and the paecilomyces hepiali Cs-4 strain is preserved in the China general microbiological culture Collection center at 10-21 th of 1997 with the preservation number of CGMCC NO. 0327. The strain Cs-4 is isolated from fresh Cordyceps sinensis, or obtained commercially.
Example 1
The composition of the slant culture medium is as follows: 2% of glucose, 0.2% of peptone, 0.4% of bran, 0.1% of monopotassium phosphate, 0.03% of magnesium sulfate, 0.1% of histidine and 2% of agar, adding water to a constant volume of 1000mL, and keeping the pH value natural.
The preparation method comprises the following steps: weighing bran which is sieved by a 40-mesh sieve according to the calculated amount, adding water with the corresponding volume (calculated by adding 6g of bran into every 100mL of water for feeding), heating to boil, maintaining boiling for 30 minutes, and filtering to obtain filtrate. Adding peptone, heating, stirring to dissolve peptone, adding other components except agar, heating, adding water to dissolve all components, and adding water to desired volume. Heating, adding agar, melting agar, subpackaging the prepared culture medium into eggplant-shaped bottles, plugging with cotton plug, bundling, sterilizing with high pressure steam at 120 deg.C under 0.09Mpa in a sterilizing cabinet for 30 min, cooling, and making into slant. The Cs-4 strain was inoculated on a slant and cultured at 15 ℃ for 7 days. The strain grows vigorously on the inclined plane and has more spores.
The composition of the shake flask culture medium was: 1% of glucose, 0.2% of peptone, 0.4% of bran, 0.2% of fishbone powder, 0.1% of monopotassium phosphate, 0.03% of magnesium sulfate, 0.03% of zinc sulfate, 0.1% of histidine and the balance of water.
The preparation method of the shake flask culture medium is different from the preparation method of the slant culture medium in that agar is not added, water is added for constant volume, and the shake flask culture medium is subpackaged into conical flasks of 750mL and sterilized according to the same conditions. Taking 1cm of the culture medium under aseptic conditions by using an inoculating shovel2The slant culture colonies were inoculated into a shake flask medium, placed on a shaker, and cultured at 15 ℃ for 7 days. And after the test is qualified, combining the subpackaged bacterial liquid obtained after each shake-flask culture into an inoculation steel cylinder, and then inoculating into a seed tank for culture.
The composition of the seeding tank culture medium is as follows: 2% of hot pressed bean cake powder, 2% of glucose, 2% of sucrose, 0.2% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.05% of zinc sulfate, 0.1% of histidine, and vitamin B10.02%, adenine 0.05%, soybean oil 0.1%, and water in balance. The preparation method comprises measuring 50% water, adding each culture medium component, heating for dissolving, adding water to desired volume, and adjusting pH naturally. The prepared culture medium is put into a sterilization cabinet for high-pressure steam sterilization, the steam pressure is 0.10Mpa, the temperature is 121 ℃, and the sterilization time is 35 minutes. After 4 days of seeding tank culture, inoculating into a propagation tank for culture.
The composition of the culture medium of the propagation tank is as follows: 2% of hot pressed bean cake powder, 2% of glucose, 2% of sucrose, 0.1% of potassium dihydrogen phosphate, 0.04% of magnesium sulfate, 0.03% of zinc sulfate, 0.1% of histidine, 0.1% of glycine, and vitamin B10.02%, adenine 0.05%, soybean oil 0.2%, and water in balance. The preparation method and sterilization conditions of the culture medium are the same as those of a seeding tank culture medium. Culturing in a propagation tank for 5 days, and inoculating into a fermentation tank for culturing.
The composition of the fermenter medium was: 3% of hot pressed bean cake powder and grapes1% of sugar, 1% of cane sugar, 0.1% of monopotassium phosphate, 0.04% of magnesium sulfate, 0.03% of zinc sulfate, 0.1% of histidine, 0.1% of glycine and vitamin B10.02%, adenine 0.1%, soybean oil 0.2%, and water in balance. The preparation method and sterilization conditions of the culture medium are the same as those of a seeding tank culture medium. And (4) taking out the fermentation tank after culturing for 4 days, carrying out solid-liquid separation on the fermentation liquor, and drying and crushing the solid to obtain the Cs-4 cordyceps sinensis powder.
Example 2
The composition of the slant culture medium is as follows: 3% of glucose, 0.3% of peptone, 0.6% of bran, 0.2% of monopotassium phosphate, 0.04% of magnesium sulfate, 0.2% of histidine and 3% of agar, adding water to a constant volume of 1000mL, and keeping the pH value natural.
The composition of the shake flask culture medium was: 3% of glucose, 0.3% of peptone, 0.5% of bran, 0.2% of fishbone powder, 0.2% of monopotassium phosphate, 0.04% of magnesium sulfate, 0.04% of zinc sulfate, 0.2% of histidine and the balance of water.
The preparation method, total preparation amount and other conditions of inoculation and culture of the slant culture medium and the shake flask culture medium are the same as those of example 1. However, the bran boiled time was 40 minutes.
The composition of the seeding tank culture medium is as follows: 3% of hot pressed bean cake powder, 3% of glucose, 3% of sucrose, 0.3% of potassium dihydrogen phosphate, 0.08% of magnesium sulfate, 0.06% of zinc sulfate, 0.15% of histidine, and vitamin B10.15%, adenine 0.1%, soybean oil 0.1%, and water in balance.
The composition of the culture medium of the propagation tank is as follows: 3% of hot pressed bean cake powder, 3% of glucose, 3% of sucrose, 0.15% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.04% of zinc sulfate, 0.1% of histidine, 0.1% of glycine, and vitamin B10.15%, adenine 0.1%, soybean oil 0.25%, and water in balance.
The composition of the fermenter medium was: 3% of hot pressed bean cake powder, 1% of glucose, 1% of sucrose, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.04% of zinc sulfate, 0.15% of histidine, 0.1% of glycine, and vitamin B10.2%, adenine 0.15%, soybean oil 0.25%, and water in balance.
The preparation method and total preparation amount of the culture medium of the seeding tank, the propagation tank and the fermentation tank are the same as those of the embodiment 1, the sterilization pressure of the culture medium is 0.12MPa, the temperature is 119 ℃, and the sterilization is carried out for 40 minutes. The inoculation method, the fermentation culture conditions and the treatment method after the strain is taken out of the tank are the same as those in the embodiment 1, and the Cs-4 cordyceps sinensis powder is finally obtained.
Example 3
The composition of the slant culture medium is as follows: glucose 4%, peptone 0.4%, bran 0.8%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, histidine 0.3%, and agar 4%, adding water to a constant volume of 1000mL, and adjusting pH to natural.
The composition of the shake flask culture medium was: 5% of glucose, 0.4% of peptone, 0.6% of bran, 0.3% of fishbone powder, 0.3% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.05% of zinc sulfate, 0.3% of histidine and the balance of water.
The preparation method, total preparation amount and other conditions of inoculation and culture of the slant culture medium and the shake flask culture medium are the same as those of example 1.
The composition of the seeding tank culture medium is as follows: 4% of hot pressed bean cake powder, 4% of glucose, 4% of sucrose, 0.4% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.1% of zinc sulfate, 0.2% of histidine and vitamin B10.3 percent, adenine 0.2 percent, soybean oil 0.2 percent and the balance of water.
The composition of the culture medium of the propagation tank is as follows: 4% of hot pressed bean cake powder, 4% of glucose, 4% of sucrose, 0.2% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate, 0.05% of zinc sulfate, 0.2% of histidine, 0.2% of glycine, and vitamin B10.3 percent, adenine 0.2 percent, soybean oil 0.3 percent and the balance of water.
The composition of the fermenter medium was: 4% of hot pressed bean cake powder, 2% of glucose, 2% of sucrose, 0.2% of potassium dihydrogen phosphate, 0.06% of magnesium sulfate, 0.05% of zinc sulfate, 0.2% of histidine, 0.2% of glycine, and vitamin B10.3 percent, adenine 0.2 percent, soybean oil 0.3 percent and the balance of water.
The preparation method and the total preparation amount of the culture medium of the seeding tank, the propagation tank and the fermentation tank are the same as those in the embodiment 1, the inoculation method, the fermentation culture conditions and the treatment method after the culture medium is taken out of the tank are the same as those in the embodiment 1, and finally the Cs-4 cordyceps sinensis powder is obtained.
Example 4
This implementationThe example differs from example 1 in that: the seeding tank culture medium also contains 0.6% of radix Notoginseng powder, the propagation tank culture medium also contains 0.8% of radix Notoginseng powder, and the fermentation tank culture medium comprises hot pressed bean cake powder 4%, glucose 2%, sucrose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, zinc sulfate 0.03%, histidine 0.1%, glycine 0.1%, vitamin B010.05%, adenine 0.15%, soybean oil 0.2%, notoginseng powder 1% and water in balance. And (3) fermenting and culturing the Cs-4 strain according to the same method to finally obtain the Cs-4 cordyceps sinensis powder.
Example 5
The difference between this example and example 2 is that the seeding tank culture medium also contains 0.5% of notoginseng powder, the propagation tank culture medium also contains 0.8% of notoginseng powder, and the fermentation tank culture medium also contains 1% of notoginseng powder. And (3) fermenting and culturing the Cs-4 strain according to the same method to finally obtain the Cs-4 cordyceps sinensis powder.
Comparative example 1
(slant medium without amino acids, liquid fermentation medium without amino acids, VB1 and adenine)
This comparative example was different from the medium composition of example 1, and the preparation method of the medium and other steps such as strain culture were the same as those of example 1.
The composition of the slant culture medium is as follows: 2% of glucose, 0.2% of peptone, 0.4% of bran, 0.1% of monopotassium phosphate, 0.03% of magnesium sulfate and 2% of agar, and adding water to a constant volume of 1000mL, wherein the pH value is natural.
The shake flask medium was of the same composition and amount as the slant medium except that the medium was free of agar.
The seed tank, the propagation tank and the culture medium in the fermentation tank in the liquid fermentation culture medium have the same composition and all comprise: 2% of bean cake powder, 2% of glucose, 2% of sucrose, 0.2% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.1% of soybean oil and the balance of water. And (3) fermenting and culturing the Cs-4 strain according to the same method to finally obtain the Cs-4 cordyceps sinensis powder.
Comparative example 2
(adenine is not contained in the liquid fermentation medium)
The slant and shake flask culture media of this example were the same as in example 1, and the seedpot, propagation tank and fermentor media differed from example 1 in the absence of adenine. And (3) fermenting and culturing the Cs-4 strain according to the same method to finally obtain the Cs-4 cordyceps sinensis powder.
Comparative example 3
(the content of amino acids and adenine in the medium was too low)
This comparative example differs from example 1 in that: the content of histidine in the slant culture medium and the shake flask culture medium is 0.05 percent, the content of histidine in the seeding tank culture medium is 0.06 percent, and the content of adenine in the seeding tank culture medium is 0.02 percent; histidine 0.03%, glycine 0.06%, adenine 0.03% in the culture medium of the propagation tank; histidine 0.04%, glycine 0.05%, adenine 0.04% in the fermenter medium. And (3) fermenting and culturing the Cs-4 strain according to the same method to finally obtain the Cs-4 cordyceps sinensis powder.
Comparative example 4
(liquid fermentation medium without adenine and containing notoginseng powder)
This comparative example differs from example 4 in that no adenine was added to the liquid fermentation medium.
Example 6
Samples were taken from the Cs-4 Cordyceps sinensis powders prepared in examples 1 to 5 and comparative examples 1 to 4, and the total amino acid, mannitol, adenosine, ergosterol and saponin contents were determined. The results are shown in Table 1.
As can be seen from Table 1, the Cordyceps powder obtained by fermentation in examples 1-3 has high contents of total amino acids, mannitol, adenosine and ergosterol, especially adenosine, which is much higher than that of the Cordyceps powder prepared in comparative example 1 using conventional medium. After the liquid fermentation culture medium of the embodiments 4 and 5 is added with the pseudo-ginseng powder, the contents of total amino acids, mannitol, adenosine and ergosterol in the cordyceps sinensis powder are still high, particularly the contents of the ergosterol are greatly improved compared with the embodiments 1 to 3, and in addition, pseudo-ginseng saponin and ginsenoside can be detected in the cordyceps sinensis powder. The liquid fermentation culture medium used in the comparative example 2 is not added with adenine, but various culture media are added with amino acid, and the detection result shows that compared with the examples 1-3, the prepared cordyceps sinensis powder has obviously reduced adenosine content, and the content of other components is slightly lower than that of the example 1. The detection data of the comparative example 3 show that the medicinal value of the cordyceps sinensis powder can be obviously improved only when the amino acid and the adenine in the culture medium reach certain amounts. The content of saponin in the cordyceps sinensis powder prepared in the comparative example 4 is lower than that in the cordyceps sinensis powder prepared in the example 4, which shows that the addition of adenine is also beneficial to the utilization of the panax notoginseng powder by the strain and can improve the content of saponin in the cordyceps sinensis powder.
TABLE 1 Total amino acid, mannitol, adenosine, ergosterol and saponin content in Cs-4 Cordyceps powder
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (4)
1. A culture medium for paecilomyces hepiali Cs-4, which comprises a seed culture medium and a liquid fermentation culture medium, and is characterized in that the seed culture medium comprises the following components: glucose, peptone, bran, inorganic salt and amino acid, wherein the liquid fermentation medium comprises the following components: hot pressed bean cake powder, glucose, sucrose, inorganic salt, amino acid, and vitamin B1And adenine; the inorganic salt is selected from one or more of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, zinc sulfate, zinc chloride, sodium chloride and calcium chloride, and the amino acid is selected from one or more of histidine, arginine, glycine and L-glutamic acid;
the seed culture medium comprises a slant culture medium and a shake flask culture medium;
the liquid fermentation culture medium comprises a seeding tank culture medium, a propagation tank culture medium and a fermentation tank culture medium;
the composition of the slant culture medium comprises the following components in weight volume g/mL: 2 to 4 percent of glucose, 0.2 to 0.4 percent of peptone, 0.4 to 0.8 percent of bran, 0.1 to 0.3 percent of monopotassium phosphate, 0.03 to 0.05 percent of magnesium sulfate, 0.1 to 0.3 percent of histidine, 2 to 4 percent of agar and the balance of water;
the composition of the shake flask culture medium in g/mL by weight volume comprises: 1 to 5 percent of glucose, 0.2 to 0.4 percent of peptone, 0.4 to 0.6 percent of bran, 0.2 to 0.3 percent of fishbone powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.03 to 0.05 percent of magnesium sulfate, 0.03 to 0.05 percent of zinc sulfate, 0.1 to 0.3 percent of histidine and the balance of water;
the composition of the seeding tank medium in g/mL by weight volume comprises: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.2-0.4% of potassium dihydrogen phosphate, 0.05-0.1% of magnesium sulfate, 0.05-0.1% of zinc sulfate, 0.1-0.2% of histidine, vitamin B10.02-0.3%, adenine 0.05-0.2%, soybean oil 0.1-0.2%, and water in balance;
the composition of the propagation tank culture medium comprises the following components in g/mL by weight volume: 2-4% of hot pressed bean cake powder, 2-4% of glucose, 2-4% of cane sugar, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.05-0.2%, soybean oil 0.2-0.3%, and water in balance;
the composition of the fermenter medium in g/mL weight volume comprises: 3-4% of hot pressed bean cake powder, 1-2% of glucose, 1-2% of sucrose, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.03-0.05% of zinc sulfate, 0.1-0.2% of histidine, 0.1-0.2% of glycine, vitamin B10.02-0.3%, adenine 0.1-0.2%, soybean oil 0.2-0.3%, and water in balance;
the liquid fermentation culture medium also contains 0.5-1% of Notoginseng radix powder.
2. The culture medium of claim 1, wherein the composition of the fermentor medium is: 4% of hot pressed bean cake powder, 2% of glucose, 1% of sucrose, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.03% of zinc sulfate, 0.1% of histidine, 0.1% of glycine, and vitamin B10.05%, adenine 0.15%, soybean oil 0.2%, notoginseng powder 1% and water in balance.
3. The method for preparing the culture medium of claim 2, wherein the method for preparing the slant culture medium comprises the steps of:
(1) weighing bran, adding water, boiling, and filtering to obtain a filtrate;
(2) adding peptone into the filtrate, heating, and stirring until dissolved;
(3) adding other culture medium components except agar, heating and/or dissolving in water, and adding water to desired volume;
(4) under the heating condition, adding agar into the solution prepared in the step (3);
(5) subpackaging the prepared culture medium, and then sterilizing;
the preparation method of the shake flask culture medium is different from the preparation method of the slant culture medium in that the agar is not added in the step (4);
the liquid fermentation culture medium is prepared by measuring 50% water, adding each culture medium component,
heating to dissolve, adding water to desired volume, and sterilizing.
4. The method for preparing culture medium according to claim 3, wherein before the bran is weighed, the bran is sieved with a 40-mesh sieve, and the sterilization is high-pressure steam sterilization, wherein the pressure is 0.09-0.12MPa, the temperature is 120 +/-1 ℃, and the time is 30-40 minutes.
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| CN114276937B (en) * | 2021-12-28 | 2023-11-24 | 江苏神华药业有限公司 | Method for fermenting paecilomyces hepialid by using Chinese yam as carbon source |
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