WO2019223288A1 - 一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺 - Google Patents

一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺 Download PDF

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WO2019223288A1
WO2019223288A1 PCT/CN2018/119032 CN2018119032W WO2019223288A1 WO 2019223288 A1 WO2019223288 A1 WO 2019223288A1 CN 2018119032 W CN2018119032 W CN 2018119032W WO 2019223288 A1 WO2019223288 A1 WO 2019223288A1
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culture
fermentation
culture medium
production process
tank
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French (fr)
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吴彪
万义斌
葛友群
左飞鸿
杨明
何琳
李进进
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江西国药有限责任公司
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Priority to US17/105,438 priority Critical patent/US20210079339A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/16—Solid state fermenters, e.g. for koji production

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  • the invention belongs to the technical field of microorganisms and relates to a fermentation production process of Paecilomyces hepiali Cs-4.
  • Cordyceps sinensis is only one of more than 190 kinds of Cordyceps sinensis in China. It is mainly produced on the Qinghai-Tibet Plateau, also referred to as "Cordyceps sinensis".
  • Cordyceps is a complex of ergot fungus Cordyceps sinensis larvae and larvae carcass parasitic on bat moth insect larvae.
  • the main active ingredients are cordycepin, cordycepin, cordyceps polysaccharides and other substances that are good for human body absorption.
  • Cordyceps sinensis is traditional in China.
  • Precious medicated dietary tonic which is flat and sweet, has the functions of nourishing lungs and kidneys, relieving cough, benefiting from deficiency, and nourishing essence.
  • Cordyceps sinensis has the effects of nourishing the lungs and yin, nourishing the kidney and yang, relieving cough and reducing phlegm, anti-cancer and anti-aging effects.
  • Cordyceps sinensis can be used for lung cough and blood cough, impotence and nocturnal emission. Its irreplaceable pharmacological effects and harsh ecological environment make it precious and scarce. With the continuous improvement of human living standards, the demand for cordyceps sinensis is increasing.
  • Paecilomyces hepiali Cs-4 is a strain isolated from fresh Cordyceps collected from Qinghai. The strain was identified at the Institute of Microbiology of the Chinese Academy of Sciences. The fermentation product has similar components to natural Cordyceps, adenine, nucleoside, urine The active ingredients such as pyrimidine, mannitol, and ergosterol are similar to natural Cordyceps, and their pharmacological effects are better than natural Cordyceps, with less toxicity. Therefore, the large-scale, high-quality industrial fermentation of Paecilomyces hepiali Cs-4 is of great significance to promote people's health and improve their quality of life.
  • the fermentation of Cordyceps militaris strains is a prerequisite for the preparation of Cordyceps militaris products.
  • the traditional fermentation process of Cordyceps militaris strains has the problems of low fermentation yield, long fermentation time and high cost. Therefore, processes such as static fermentation, batch fermentation, and fed-batch fermentation have appeared, but these fermentation processes still have various problems, such as high bacteria infection rate, long fermentation cycle, high cost, and low content of active ingredients in the fermentation product. Wait. Therefore, it is necessary to study the fermentation process with high yield, low cost, and high content of active ingredients in the Cordyceps powder product obtained by fermentation.
  • the purpose of the present invention is to provide a fermentation production process of Paecilomyces hepiali Cs-4, which can improve the yield of fermentation products and the content of active ingredients therein, shorten the fermentation time and reduce the cost.
  • the present invention provides the following technical solutions:
  • a fermentation production process of Paecilomyces hepiali Cs-4 includes the following steps:
  • Slant culture inoculate the Cs-4 strain on slant culture medium, and culture at a constant temperature of 15-20 ° C for 7-9 days.
  • the slant culture medium contains histidine;
  • Shake flask culture Under aseptic conditions, insert the strain cultured in step S1 into the shake flask medium, and place it in a constant temperature shaker at 15-20 ° C under light conditions for 7-9 days. Contains histidine and vitamin B 1 ;
  • Seed pot culture under aseptic conditions, according to the inoculation amount of 3-5%, the bacteria cultured in step S2 is inserted, and the seed pot medium contains purine;
  • breeding tank culture under aseptic conditions, insert 9-9% of the inoculation amount into the bacteria cultured in step S3, and the breeding tank culture medium contains purines and pyrimidines;
  • Fermenter culture under aseptic conditions, 5-20% of the inoculation amount is used to access the bacteria cultured in step S4, and the fermentor medium contains purines and pyrimidines; after the fermenter culture is completed, a fermentation broth is obtained;
  • step S5 the fermentation broth of step S5 is connected to the flask culture medium according to the inoculation amount of 1-2%, and the flask culture is performed according to the conditions of step S2, and then steps S3-S5 are repeated to ferment and culture the Cs-4 strain.
  • the purine is selected from one or two of adenine and guanine, and the pyrimidine is uracil.
  • the composition of the slant culture medium includes 2-4% glucose, 0.2-0.4% peptone, 0.4-0.8% bran, 0.1-0.3% potassium dihydrogen phosphate, and 0.03-magnesium sulfate in terms of weight-volume ratio g / mL. 0.05%, histidine 0.1-0.3%, agar 2-4%, water balance, natural pH; shake flask culture medium composition includes glucose 1-5%, peptone 0.2-0.4%, bran 0.4-0.6%, Potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.03-0.05%, zinc sulfate 0.03-0.05%, histidine 0.1-0.3%, vitamin B 1 0.05-0.1%, water balance, natural pH.
  • the seed pot culture medium, the propagation pot culture medium and the fermentation pot culture medium contain panax notoginseng powder.
  • the cordyceps fungus powder obtained by fermentation and cultivation has the effects of notoginsenosides and ginsenosides.
  • the content of ergosterol was significantly increased after purine and panax notoginseng were added to the fermentation medium at the same time.
  • the composition of the seed tank culture medium includes hot pressed beancake powder 2-4%, glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.2-0.4%, sulfuric acid Magnesium 0.05-0.1%, zinc sulfate 0.05-0.1%, histidine 0.1-0.2%, vitamin B 1 0.02-0.3%, soybean oil 0.1-0.2%, adenine 0.05-0.2%, and panax notoginseng 0.4-0.6%, Water balance, natural pH.
  • the composition of the culture medium of the breeding tank includes 2-4% of hot-pressed soybean cake powder, 2-4% of glucose, 2-4% of sucrose, 0.1-0.2% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, and 0.03 of zinc sulfate. -0.05%, histidine 0.1-0.2%, vitamin B 1 0.1-0.2%, soybean oil 0.2-0.3%, adenine 0.03-0.1%, guanine 0.03-0.08%, uracil 0.05-0.2% and panax notoginseng 0.5-0.7%, water balance, natural pH.
  • the composition of the fermenter culture medium comprises 3-4% of hot-pressed soybean cake powder, 1-2% of glucose, 1-2% of sucrose, 0.2-0.3% of potassium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, and 0.04 of zinc sulfate. -0.06%, histidine 0.2-0.3%, vitamin B 1 0.2-0.3%, soybean oil 0.2-0.3%, adenine 0.05-0.12%, guanine 0.03-0.08%, uracil 0.05-0.2% and panax notoginseng 0.5-1.0%, water balance, natural pH.
  • the shaker described in step S2 is a reciprocating shaker, the shaking frequency of the shaker is 140 ⁇ 5 times / minute, and the light used for illumination is red light; the condition of the step S3 seed pot culture is a constant temperature of 16 ⁇ 3 ° C, The tank pressure is 0.02-0.05 MPa, the ventilation volume (v / v ⁇ min) is 1: 0.5-1.8, and the culture is allowed to stand for 4-5 days.
  • the breeding tank is cultured at a constant temperature of 16 ⁇ 3 ° C., the tank pressure is 0.02-0.05 MPa, the ventilation (v / v ⁇ min) is 1: 0.15-0.4, and the culture is allowed to stand for 5-6 days.
  • the fermentation tank is cultured at a constant temperature of 16 ⁇ 3 ° C., the tank pressure is 0.02-0.05 MPa, the air flow rate (v / v ⁇ min) is 1: 0.65-0.9, preferably 1: 0.75, and the mixture is stirred at a speed of 40-60 rpm. Cultivate for 4-5 days.
  • the Cs-4 strain after shake flask culture grows vigorously and produces a lot of spores, providing excellent strains for seed tank culture.
  • the composition of the fermentation medium at each level is also slightly different, which ensures the nutritional supply of the strains at different stages of growth, and the resulting mycelium has a high yield and high nutritional value. Seed tanks and breeding tanks do not require stirring, which saves costs.
  • the fermentation tank culture After the fermentation tank culture is completed, a part of the fermentation broth is inserted into the shake flask culture medium as seeds, and the shake flask culture is performed again, and then the step-up scale culture of the seed tank, the breeding tank, and the fermentation tank is sequentially performed, and the oblique surface is omitted.
  • the cultivation process shortens the fermentation process cycle and reduces costs.
  • the fermentation broth was used as a seed to perform shake flask culture and step-up culture, and the content of each active ingredient in the Cordyceps fungus powder obtained by the treatment was high.
  • the entire fermentation process has a high degree of automation, simple operation, and is suitable for industrial production.
  • the fermentation production process of Paecilomyces hepiali Cs-4 includes slant culture, shake flask culture, seed tank culture, breeding tank culture and fermentation tank culture. After the fermentation tank culture is completed, a fermentation broth is obtained. The inoculation amount was connected to the shake flask culture medium, and the remaining fermentation broth was filtered and dried to prepare Cs-4 Cordyceps powder. The fermentation bottle is used as a seed to repeat the process of shake flask culture and step-up scale-up cultivation, to obtain the fermentation broth again, and then partly insert the shake flask medium, and repeat the fermentation process again.
  • the first time fermentation is carried out by using a slant culture strain as a shake flask culture to obtain a fermentation broth
  • the fermentation broth can be used as a seed for shake flask culture at least 10 times in a row to ferment and produce Cs-4 mycelium, and further Preparation of Cordyceps powder.
  • the seed tank culture medium contains adenine 0.05-0.2% and panax notoginseng powder 0.4-0.6%
  • the breeding tank culture medium contains adenine 0.03-0.1%, guanine 0.03-0.08%, uracil 0.05-0.2% and Panax notoginseng powder 0.5-0.7%
  • fermentor culture medium contains adenine 0.05-0.12%, guanine 0.03-0.08%, uracil 0.05-0.2% and panax notoginseng 0.5-1.0%.
  • the pH of each medium is natural, and generally ranges from 5.0 to 6.5. Shake flask culture is carried out under the irradiation of red light, and the wavelength range of the red light is 622 to 760 nm.
  • the preparation method of the slant and shake flask culture medium is: Weigh the bran that passed through a 40 mesh sieve according to the calculated amount, calculate the amount of water according to 6 g of bran per 100 mL of water, heat to boiling, maintain boiling for 30 minutes, and filter to obtain the filtrate. Then add other components, heat and / or add water to dissolve, add water to make up the volume, dispense and sterilize.
  • the preparation method for the culture medium of the seed tank, the breeding tank and the fermentation tank is: measuring 50% of water, adding each medium component thereto, heating and dissolving, adding water to make up the volume, and then sterilizing.
  • the sterilization methods of various culture media are high-pressure steam sterilization, steam pressure 0.09-0.12Mpa, temperature 121 ⁇ 1 °C, and sterilization time 30-40 minutes.
  • the composition of the slanted medium is: glucose 2%, peptone 0.2%, bran 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.03%, histidine 0.1%, agar 2%, water balance, and natural pH.
  • the Cs-4 strain which has been preserved at about 1 cm 2 with an inoculating shovel, is uniformly coated on the inclined surface, and cultured at a temperature of 15 ° C. and a humidity of 85% for 7 days. After the growth is mature, the appearance inspection is performed, and the culture characteristics are full: the spores and mycelia are plump and white or pale yellow plush. Remove the slanted surface with poor appearance, and store the slanted surface with good growth in the refrigerator at 1-8 °C for future use.
  • the composition of the shake flask medium is: glucose 1%, peptone 0.2%, bran 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.03%, zinc sulfate 0.03%, histidine 0.1%, and vitamin B 1 0.05%, Water balance, natural pH.
  • 1 cm 2 of the vigorously growing slant strain was taken with an inoculating shovel and inserted into the shake flask medium.
  • the frequency of the reciprocating shaker is 135 beats / min.
  • red light with a wavelength of 622 to 760 nm is used for irradiation.
  • the cultured seeds of each shake flask are combined into an inoculation steel bottle.
  • the composition of the seed tank culture medium is: hot pressed bean cake powder 2%, glucose 2%, sucrose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, zinc sulfate 0.05%, histidine 0.1%, vitamin B 1 0.02 %, Soybean oil 0.1%, adenine 0.05%, water balance, natural pH.
  • the pressure-difference method is used to insert the cultured bacteria in the shake flask into the seed tank according to the inoculation amount of 3%.
  • a temperature of 16 ° C, a tank pressure of 0.02 MPa, and a ventilation volume of 1: 0.5 (v / v ⁇ min) the cells were allowed to stand for 4 days.
  • the composition of the breeding tank culture medium is: hot pressed bean cake powder 2%, glucose 2%, sucrose 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, zinc sulfate 0.03%, histidine 0.1%, and vitamin B 1 0.1 %, Soybean oil 0.2%, adenine 0.05%, uracil 0.05%, water balance, natural pH.
  • a pressure difference method is used to insert the cultured bacteria in the seed tank into the propagation tank according to the 9% inoculation amount.
  • a temperature of 16 ° C Under the conditions of a temperature of 16 ° C, a tank pressure of 0.02 MPa, and a ventilation rate of 1: 0.15 (v / v ⁇ min), the cells were allowed to stand for 5 days.
  • the composition of the fermenter culture medium is: hot pressed bean cake powder 3%, glucose 1%, sucrose 1%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.04%, zinc sulfate 0.04%, histidine 0.2%, and vitamin B 1 0.2 %, Soybean oil 0.2%, adenine 0.05%, uracil 0.05%, water balance, natural pH.
  • the pressure culture method is used to transfer the cultured bacteria in the breeding tank into the fermentation tank according to the 5% inoculation amount.
  • the culture was carried out at a temperature of 16 ° C., a tank pressure of 0.02 MPa, an air flow rate of 1: 0.75 (v / v ⁇ min), and a stirring speed of 40 rpm for 4 days.
  • samples of the fermentation broth were taken every 24 hours, and the pH value, reducing sugar, and amino nitrogen were measured.
  • pH ⁇ 7, reducing sugars ⁇ 1.5%, amino nitrogen ⁇ 0.4mg / mL, and bacterial concentration ⁇ 14% the fermentation was completed. Put it in a tank, separate the solid and liquid from the fermentation liquid, and dry and pulverize the solid to obtain Cs-4 Cordyceps powder.
  • the nucleoside content in the fungal powder obtained in this embodiment is significantly increased, and the ergosterol content is also increased.
  • Example 2 The difference between Example 2 and Example 1 is that the seed pot medium contains 0.4% of Panax notoginseng powder, the breeding pot medium contains 0.5% of Panax notoginseng powder, and the fermentor medium contains 0.5% of Panax notoginseng powder.
  • the Cs-4 strain was fermented and cultured according to the same process conditions as in Example 1, and finally a Cs-4 Cordyceps powder was obtained.
  • the ergosterol content in the fungal powder prepared in this example is significantly increased, and the fungal powder contains saponin.
  • composition of the slanting medium is: glucose 3%, peptone 0.3%, bran 0.6%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.04%, histidine 0.2%, agar 3%, water balance, and natural pH.
  • the composition of the flask culture medium is: glucose 3%, peptone 0.3%, bran 0.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.04%, zinc sulfate 0.04%, histidine 0.2%, and vitamin B 1 0.07%. Water balance.
  • composition of the seed tank culture medium is: hot pressed bean cake powder 3%, glucose 3%, sucrose 3%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.08%, zinc sulfate 0.06%, histidine 0.15%, vitamin B 1 0.2 %, Soybean oil 0.1%, adenine 0.1%, panax notoginseng powder 0.5%, water balance.
  • the composition of the breeding tank culture medium is: hot pressed bean cake powder 3%, glucose 3%, sucrose 3%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, zinc sulfate 0.04%, histidine 0.1%, vitamin B 1 0.15 %, Soybean oil 0.25%, adenine 0.03%, guanine 0.08%, uracil 0.1% and panax notoginseng powder 0.6%, water balance.
  • the composition of the fermenter culture medium is: hot pressed bean cake powder 3%, glucose 2%, sucrose 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, zinc sulfate 0.05%, histidine 0.3%, and vitamin B 1 0.2 %, Soybean oil 0.25%, adenine 0.1%, guanine 0.03%, uracil 0.1% and panax notoginseng powder 0.6%, water balance.
  • the Cs-4 strain was fermented and cultured according to the same process conditions as in Example 1, and finally a Cs-4 Cordyceps powder was obtained.
  • the content of the active ingredient in the Cordyceps powder prepared in this example is slightly higher than that in Example 2, which is the result of the interaction between guanine and adenine.
  • the composition of the slanted medium is: glucose 4%, peptone 0.4%, bran 0.8%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, histidine 0.3%, agar 4%, water balance, and natural pH.
  • the Cs-4 strain which was stored at about 1 cm 2 with an inoculating shovel, was uniformly coated on the inclined surface, and cultured at a temperature of 20 ° C. and a humidity of 90% for 9 days.
  • the composition of the shake flask medium is: glucose 5%, peptone 0.4%, bran 0.6%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, zinc sulfate 0.05%, histidine 0.3%, vitamin B 1 0.1%, Water balance.
  • 1 cm 2 of the vigorously growing slant strain was taken with an inoculating shovel and inserted into the shake flask medium.
  • the shaking culture was performed on a reciprocating shaker at 20 ° C for 9 days, and the shaking frequency of the reciprocating shaker was 140 beats / minute. During the whole shake flask cultivation process, red light was used for irradiation. After the shake flask culture is completed, the cultured seeds of each shake flask are combined into an inoculation steel bottle.
  • the composition of the seed tank culture medium is: 4% hot-pressed soybean cake powder, 4% glucose, 4% sucrose, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.1% zinc sulfate, 0.2% histidine, and vitamin B 1 0.3. %, Soybean oil 0.2%, adenine 0.2%, panax notoginseng powder 0.6%, water balance.
  • the pressure-difference method is used to insert the cultured bacteria in the shake flask into the seed tank according to the 5% inoculation amount.
  • a temperature of 19 ° C a tank pressure of 0.05 MPa, and a ventilation volume of 1: 1.8 (v / v ⁇ min)
  • the cells were allowed to stand for 5 days.
  • the composition of the breeding tank culture medium is: hot pressed soybean cake powder 4%, glucose 4%, sucrose 4%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, zinc sulfate 0.05%, histidine 0.2%, and vitamin B 1 0.2 %, Soybean oil 0.3%, adenine 0.1%, guanine 0.03%, uracil 0.2% and panax notoginseng powder 0.7%, water balance.
  • a pressure difference method is used to insert the cultured bacteria in the seed tank into the propagation tank according to the 12% inoculation amount.
  • a tank pressure of 0.05 MPa, and a ventilation volume of 1: 0.4 (v / v ⁇ min) the cells were allowed to stand for 6 days.
  • the composition of the fermenter culture medium is: hot pressed soybean cake powder 4%, glucose 1%, sucrose 1%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, zinc sulfate 0.06%, histidine 0.2%, vitamin B 1 0.3 %, Soybean oil 0.3%, adenine 0.12%, guanine 0.08%, uracil 0.2% and panax notoginseng powder 1.0%, water balance.
  • the pressure culture method is used to transfer the cultured bacteria in the breeding tank into the fermentation tank according to the inoculation amount of 15%.
  • the culture was carried out at a temperature of 19 ° C., a tank pressure of 0.05 MPa, an aeration rate of 1: 0.9 (v / v ⁇ min), and a stirring speed of 60 rpm for 5 days.
  • samples of the fermentation broth were taken every 24 hours, and the pH value, reducing sugar, and amino nitrogen were measured.
  • pH ⁇ 7, reducing sugars ⁇ 1.5%, amino nitrogen ⁇ 0.4mg / mL, and bacterial concentration ⁇ 14% the fermentation was completed. Put it in a tank, separate the solid and liquid from the fermentation liquid, and dry and pulverize the solid to obtain Cs-4 Cordyceps powder.
  • the content of the Cordyceps fungus powder prepared in this embodiment is similar to that of the fungus powder prepared in Example 3.
  • Example 4 The difference between this embodiment and Example 4 is that the seed source of the shake flask culture medium is the fermentation broth after the fermentation of Example 4 and the inoculation amount is 1%.
  • the Cs-4 strain was fermented and cultured according to the same process conditions as in Example 4, and Cs-4 Cordyceps powder was finally obtained.
  • the content of nucleoside, ergosterol and saponin in the bacterial powder is higher than that of the bacterial powder in Example 4, because the bacterial species have stronger biotransformation ability to purine, pyrimidine and panax notoginseng powder.
  • This embodiment has the same composition of each medium as in Example 4, except that the seed source of the shake flask medium is the fermentation broth after the fermentation in Example 4, and the inoculation amount is 2%.
  • the shake flask culture conditions are shaking culture on a reciprocating shaker at 18 ° C. for 8 days, and the shaking frequency of the reciprocating shaker is 145 times / minute. During the whole shake flask cultivation process, red light is used for irradiation.
  • the seed pot was cultured at a constant temperature of 13 ° C, a pot pressure of 0.03 MPa, and a ventilation volume of 1: 1.0 (v / v ⁇ min). The culture was allowed to stand for 4 days.
  • the breeding tank was cultured at a constant temperature of 13 ° C, a tank pressure of 0.03 MPa, and a ventilation volume of 1: 0.2 (v / v ⁇ min). The culture was allowed to stand for 5 days.
  • the fermenter was cultured at a constant temperature of 13 ° C, a tank pressure of 0.03 MPa, an air flow rate of 1: 0.65 (v / v ⁇ min), and a culture rate of 50 rpm for 5 days.
  • This comparative example differs from Example 1 in that adenine is not contained in the seed tank culture medium, adenine and uracil are not contained in the propagation tank culture medium, and adenine and uracil are not contained in the fermentation tank culture medium.
  • the Cs-4 strain was fermented and cultured according to the same process conditions as in Example 1, and finally a Cs-4 Cordyceps powder was obtained.
  • the content of nucleosides and ergosterol in the Cordyceps fungus powder prepared in this comparative example is lower than that in Example 1.
  • Example 2 The difference between this comparative example and Example 2 is that adenine is not contained in the seed tank culture medium, adenine and uracil are not contained in the propagation tank culture medium, and adenine and uracil are not contained in the fermentation tank culture medium.
  • the Cs-4 strain was fermented and cultured in accordance with the same process conditions as in Example 1 to obtain Cs-4 Cordyceps powder.
  • the nucleoside, ergosterol and saponin contents in the bacterial powder prepared in this comparative example are lower.
  • the low saponin content indicates that purines and pyrimidines can promote the utilization of Panax notoginseng powder by the strain.
  • the ergosterol content in the powder obtained by this comparative example was increased, indicating that the addition of Panax notoginseng powder could increase the content of ergosterol.
  • This comparative example differs from Example 3 only in that the shake flask culture is performed under dark conditions, and the content of active ingredients in the Cs-4 Cordyceps powder obtained is low. This shows that shake flask culture needs to be performed under light conditions.
  • Example 3 The difference between this comparative example and Example 3 is only that the shake flask culture is performed under the condition of natural light.
  • the content of active ingredients in the Cs-4 Cordyceps powder obtained is higher than that of Comparative Example 3, but lower than that of Example 3. This shows that the flask culture under red light conditions is more conducive to strain growth.
  • the difference between this comparative example and Example 3 is that the shake flask culture conditions are: shaking culture on a reciprocating shaker at 25 ° C. for 8 days, and a reciprocating shaker shaking frequency of 120 times / minute.
  • the seed tank was cultured at a constant temperature of 20 ° C, a tank pressure of 0.03 MPa, and a ventilation volume of 1: 0.4 (v / v ⁇ min), and the cells were allowed to stand for 4 days.
  • the breeding tank was cultured at a constant temperature of 20 ° C, a tank pressure of 0.03 MPa, and a ventilation volume of 1: 0.2 (v / v ⁇ min). The culture was allowed to stand for 5 days.
  • the culture conditions of the fermenter were constant temperature at 20 ° C., the tank pressure was 0.03 MPa, and the air flow rate was 1: 0.65 (v / v ⁇ min).
  • the culture was allowed to stand for 5 days. After the fermentation was completed, the pot was placed to prepare Cs-4 Cordyceps powder.
  • the content of active ingredients in Cordyceps fungus powder obtained in this comparative example is low, the main reason is that the culture conditions are not suitable, especially the fermenter culture cannot use static culture.
  • Cs-4 Cordyceps powder prepared in Examples 1-6 and Comparative Examples 1-5 were sampled, and the contents of nucleosides (adenosine, guanosine and uridine), ergosterol and saponin were measured.
  • the test results are shown in Table 1. According to the test results, it can be clearly seen that the Cs-4 Cordyceps powder produced by the fermentation production process of the Cs-4 strain provided by the present invention has high medicinal value.

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Abstract

提供了一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺，包括斜面培养、摇瓶培养、种子罐培养、繁殖罐培养和发酵罐培养，发酵结束后的发酵液可作为摇瓶培养的种子。斜面培养基中含组氨酸，摇瓶培养基中含组氨酸和维生素B1，种子罐培养基中含嘌呤，繁殖罐培养基中含有嘌呤和嘧啶，发酵罐培养基中含有嘌呤和嘧啶，培养得到的菌粉中核苷含量高。

Description

一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺 技术领域
本发明属于微生物技术领域，涉及一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺。
背景技术
冬虫夏草仅仅是我国190多种虫草中的一种，主产于青藏高原上，又简称"虫草"。虫草是麦角菌科真菌冬虫夏草寄生在蝙蝠蛾科昆虫幼虫上的子座及幼虫尸体的复合体，主要活性成分为虫草素、虫草酸、虫草多糖等有益于人体吸收的物质，冬虫夏草是我国传统的名贵药膳滋补品，它性平味甘，具有补肺肾、止咳嗽、益虚损、养精气之功能。据临床研究报道，冬虫夏草具有养肺阴，补肾阳、止咳化痰、抗癌防老的功效，为平补阴阳之品，诸无所忌。冬虫夏草可用于肺痨咳血，阳痿遗精等症；其药理药效的不可替代作用、生态环境恶劣等使其珍贵而稀少，随着人类生活水平的不断提高，人们对冬虫夏草的需求量越来越大，而资源量却越来越少，供求矛盾越来越突出。为了解决这一问题，近几年，我国生物医学界的研究人员先后分离出多株虫草真菌菌株，试图利用人工发酵方式生产虫草菌丝体，用以代替天然冬虫夏草。经过不懈的努力，有几家已生产出在药理、毒理及功效成份等方面与天然虫草相似的产品，并能实现现代化条件下的大规模生产。
蝙蝠蛾拟青霉Cs-4是从青海采集的新鲜虫草上分离获得的菌株，在中科院微生物研究所进行了菌种鉴定，其发酵物与天然虫草有相似的成份，腺嘌呤、核苷、尿嘧啶、甘露醇、麦角甾醇等有效成份均和天然虫草相似，且药理作用优于天然虫草，毒性更小。因此，蝙蝠蛾拟青霉Cs-4的大规模、高质量的工业发酵生产对于促进人们身体健康，提高生活质量有重要意义。
发酵培养虫草菌株是制备虫草产品的前提，传统的虫草菌株发酵工艺存在发酵得率低、发酵时间长和成本高等问题。因此，出现了静置发酵、间歇发酵和补料分批发酵等工艺，但这些发酵工艺仍存在各种问题，比如染菌率高、发酵周期长、成本高和发酵产物中的活性成分含量低等。因此，研究高收率、低成本，且发酵得到的虫草菌粉产品中的活性成分含量高的发酵工艺十分必要。
发明内容
本发明的目的在于提供一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺，提高发酵产物的收率及其中的活性成分含量，缩短发酵时间、降低成本。
具体来说，针对现有技术的不足，本发明提供了如下技术方案：
一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺，该发酵生产工艺包括以下步骤：
S1.斜面培养：将Cs-4菌株接种到斜面培养基上，在15-20℃恒温下培养7-9天，斜面培养基中含有组氨酸；
S2.摇瓶培养：无菌条件下，往摇瓶培养基内接入步骤S1培养好的菌种，置于15-20℃恒温摇床、光照条件下培养7-9天，摇瓶培养基中含有组氨酸和维生素B 1；
S3.种子罐培养：无菌条件下，按照3-5％的接种量接入步骤S2培养好的菌种，种子罐培养基中含有嘌呤；
S4.繁殖罐培养：无菌条件下，按照9-12％的接种量接入步骤S3培养好的菌种，繁殖罐培养基中含有嘌呤和嘧啶；
S5.发酵罐培养：无菌条件下，按照5-20％的接种量接入步骤S4培养好的菌种，发酵罐培养基中含有嘌呤和嘧啶；发酵罐培养结束后，得到发酵液；
或者，按照1-2％的接种量将步骤S5的发酵液接至摇瓶培养基内，按照步骤S2的条件进行摇瓶培养，然后重复步骤S3-S5，发酵培养Cs-4菌株。
液体发酵培养基中添加嘌呤和嘧啶，发酵制备的虫草菌粉中核苷含量明显提高。
优选地，所述嘌呤选自腺嘌呤和鸟嘌呤中的一种或两种，所述嘧啶为尿嘧啶。
优选地，以重量体积比g/mL计，斜面培养基的组成包括葡萄糖2-4％、蛋白胨0.2-0.4％、麸皮0.4-0.8％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、组氨酸0.1-0.3％、琼脂2-4％，水余量，pH自然；摇瓶培养基的组成包括葡萄糖1-5％、蛋白胨0.2-0.4％、麸皮0.4-0.6％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、硫酸锌0.03-0.05％、组氨酸0.1-0.3％，维生素B 1 0.05-0.1％，水余量，pH自然。
优选地，种子罐培养基、繁殖罐培养基和发酵罐培养基中含有三七粉。添加三七粉后，发酵培养得到的虫草菌粉在具备原有虫草功效的同时，还具备三 七皂苷、人参皂苷的功效。此外，发酵培养基中同时添加嘌呤和三七粉后，麦角甾醇的含量明显提高。
优选地，以重量体积比g/mL计，种子罐培养基的组成包括热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.2-0.4％、硫酸镁0.05-0.1％、硫酸锌0.05-0.1％、组氨酸0.1-0.2％、维生素B 1 0.02-0.3％、豆油0.1-0.2％、腺嘌呤0.05-0.2％和三七粉0.4-0.6％，水余量，pH自然。
优选地，繁殖罐培养基的组成包括热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、维生素B 1 0.1-0.2％、豆油0.2-0.3％、腺嘌呤0.03-0.1％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-0.7％，水余量，pH自然。
优选地，发酵罐培养基的组成包括热榨豆饼粉3-4％、葡萄糖1-2％、蔗糖1-2％、磷酸二氢钾0.2-0.3％、硫酸镁0.04-0.06％、硫酸锌0.04-0.06％、组氨酸0.2-0.3％、维生素B 1 0.2-0.3％、豆油0.2-0.3％、腺嘌呤0.05-0.12％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-1.0％，水余量，pH自然。
优选地，步骤S2所述的摇床为往复式摇床，摇床振荡频率为140±5次/分，光照使用的光为红光；步骤S3种子罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.5-1.8，静置培养4-5天。
优选地，繁殖罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.15-0.4，静置培养5-6天。
优选地，发酵罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.65-0.9，优选1：0.75，在40-60rpm转速下搅拌培养4-5天。
与现有技术相比，本发明的效果和益处在于：
(1)摇瓶培养后的Cs-4菌株生长旺盛，产孢子多，为种子罐培养提供优良的菌种。各级发酵培养基的组成也略有不同，保证了不同阶段菌株生长的营养供应，得到的菌丝体产量高，营养价值高。种子罐和繁殖罐培养不需要搅拌，节省成本。
(2)培养基中添加了氨基酸、嘌呤和嘧啶，发酵培养的Cs-4菌株的菌丝体生长旺盛，收率高，制备得到的虫草菌粉中的核苷含量明显提高。再往培养基中添加三七粉后，从菌粉中能够检测到皂苷，且麦角甾醇的含量明显提高。 本发明还发现，嘌呤和嘧啶能促进菌株对三七粉的利用，提高菌粉中皂苷的含量。
(3)发酵罐培养结束后，将部分发酵液作为种子接入摇瓶培养基内，再次进行摇瓶培养，然后依次进行种子罐、繁殖罐和发酵罐的逐级放大培养，省去了斜面培养的过程，缩短了发酵工艺周期，降低成本。此外，以发酵液为种子进行摇瓶培养、逐级放大培养，处理得到的虫草菌粉中的各活性成分含量高。整个发酵工艺过程的自动化程度高，操作简单，适合工业化生产。
具体实施方式
本发明提供的蝙蝠蛾拟青霉Cs-4的发酵生产工艺包括斜面培养、摇瓶培养、种子罐培养、繁殖罐培养和发酵罐培养，发酵罐培养结束得到发酵液，按照1-2％的接种量将发酵液接至摇瓶培养基内，剩余发酵液过滤、干燥制备Cs-4虫草菌粉。以发酵液为种子重复摇瓶培养和逐级放大培养的过程，再次得到发酵液，然后再部分接入摇瓶培养基，再次重复发酵工艺过程。一般，在第一次以斜面培养菌株为摇瓶培养的菌种进行发酵，得到发酵液后，可以至少连续10次使用发酵液作为摇瓶培养的种子，发酵生产Cs-4菌丝体，进而制备虫草菌粉。
在一优选实施方式中，种子罐培养基中含有腺嘌呤0.05-0.2％和三七粉0.4-0.6％，繁殖罐培养基中含有腺嘌呤0.03-0.1％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-0.7％，发酵罐培养基中含有腺嘌呤0.05-0.12％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-1.0％。各培养基的pH为自然，一般在5.0-6.5。摇瓶培养在红光照射下进行，红光的波长范围在622～760nm。
斜面和摇瓶培养基的制备方法为：按计算量称量过40目筛的麸皮，按照每100mL水中加6g麸皮计算加水量，加热至沸腾，维持沸腾30分钟，过滤得滤液。然后加入其它组分，加热和/或加水溶解，加水定容，分装、灭菌。种子罐、繁殖罐和发酵罐培养基的制备方法为：量取50％的水，往其中加入各培养基组分，加热溶解后，加水定容，然后灭菌。
各种培养基的灭菌方法均为高压蒸汽灭菌，蒸汽压力0.09-0.12Mpa，温度121±1℃，灭菌时间30-40分钟。
下面以具体的实施例来说明本发明。
需说明的是，本发明方法中涉及的材料和设备均为市售，其中，蝙蝠蛾拟 青霉Cs-4菌株于1997年10月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC NO.0327。菌株Cs-4分离自青海新鲜冬虫夏草，也可以通过商业途径获取。
实施例1
斜面培养基的组成为：葡萄糖2％、蛋白胨0.2％、麸皮0.4％、磷酸二氢钾0.1％、硫酸镁0.03％、组氨酸0.1％、琼脂2％，水余量，pH自然。用接种铲取1cm 2左右保存好的Cs-4菌株，均匀涂布在斜面上，在温度15℃、湿度85％下培养7天。待生长成熟，进行外观检查，培养特征：孢子及菌丝丰满，呈白色或淡黄色毛绒状。剔除外观生长不良的斜面，将生长良好的斜面置于冰箱1-8℃保存备用。
摇瓶培养基的组成为：葡萄糖1％、蛋白胨0.2％、麸皮0.4％、磷酸二氢钾0.1％、硫酸镁0.03％、硫酸锌0.03％、组氨酸0.1％和维生素B 1 0.05％，水余量，pH自然。在无菌条件下，用接种铲子取1cm 2生长旺盛的斜面菌株接入摇瓶培养基内。在15℃往复式摇床上振荡培养7天，往复式摇床振荡频率为135次/分，整个摇瓶培养过程中，使用波长在622～760nm的红光照射。摇瓶培养结束后，将各摇瓶培养好的种子合并到接种钢瓶内。
种子罐培养基的组成为：热榨豆饼粉2％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.2％、硫酸镁0.05％、硫酸锌0.05％、组氨酸0.1％、维生素B 1 0.02％、豆油0.1％、腺嘌呤0.05％，水余量，pH自然。无菌条件下，采用压差法按照3％的接种量将摇瓶培养好的菌种接入种子罐内。在温度16℃，罐压0.02MPa，通气量为1:0.5(v/v·min)的条件下，静置培养4天。
繁殖罐培养基的组成为：热榨豆饼粉2％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.1％、硫酸镁0.04％、硫酸锌0.03％、组氨酸0.1％、维生素B 1 0.1％、豆油0.2％、腺嘌呤0.05％、尿嘧啶0.05％，水余量，pH自然。无菌条件下，采用压差法按照9％的接种量将种子罐内培养好的菌种接入繁殖罐内。在温度16℃，罐压0.02MPa，通气量为1:0.15(v/v·min)的条件下，静置培养5天。
发酵罐培养基的组成为：热榨豆饼粉3％、葡萄糖1％、蔗糖1％、磷酸二氢钾0.2％、硫酸镁0.04％、硫酸锌0.04％、组氨酸0.2％、维生素B 1 0.2％、豆油0.2％、腺嘌呤0.05％、尿嘧啶0.05％，水余量，pH自然。无菌条件下，采用压差法按 照5％的接种量将繁殖罐内培养好的菌种接入发酵罐内。在温度16℃，罐压0.02MPa，通气量为1:0.75(v/v·min)，搅拌速度为40rpm的条件下，培养4天。开始发酵后，每隔24小时取发酵液样，测定pH值、还原糖、氨基氮。当pH＜7，还原糖＜1.5％，氨基氮＜0.4mg/mL，菌浓≧14％时，发酵结束。放罐，发酵液固液分离，将固体烘干、粉碎得到Cs-4虫草菌粉。
相比液体发酵培养基中不添加嘌呤和嘧啶制备的虫草菌粉，本实施例得到的菌粉中核苷含量明显提高，麦角甾醇含量也提高。
实施例2
实施例2与实施例1的区别仅在于，种子罐培养基中含有三七粉0.4％，繁殖罐培养基中含有三七粉0.5％，发酵罐培养基中含有三七粉0.5％。按照与实施例1相同的工艺条件发酵培养Cs-4菌株，最后得到Cs-4虫草菌粉。相比实施例1的虫草菌粉，本实施例制备的菌粉中的麦角甾醇含量明显提高，菌粉中含有皂苷。
实施例3
斜面培养基的组成为：葡萄糖3％、蛋白胨0.3％、麸皮0.6％、磷酸二氢钾0.2％、硫酸镁0.04％、组氨酸0.2％、琼脂3％，水余量，pH自然。
摇瓶培养基的组成为：葡萄糖3％、蛋白胨0.3％、麸皮0.5％、磷酸二氢钾0.2％、硫酸镁0.04％、硫酸锌0.04％、组氨酸0.2％，维生素B 1 0.07％，水余量。
种子罐培养基的组成为：热榨豆饼粉3％、葡萄糖3％、蔗糖3％、磷酸二氢钾0.3％、硫酸镁0.08％、硫酸锌0.06％、组氨酸0.15％、维生素B 1 0.2％、豆油0.1％、腺嘌呤0.1％，三七粉0.5％，水余量。
繁殖罐培养基的组成为：热榨豆饼粉3％、葡萄糖3％、蔗糖3％、磷酸二氢钾0.15％、硫酸镁0.05％、硫酸锌0.04％、组氨酸0.1％、维生素B 1 0.15％、豆油0.25％、腺嘌呤0.03％、鸟嘌呤0.08％、尿嘧啶0.1％和三七粉0.6％，水余量。
发酵罐培养基的组成为：热榨豆饼粉3％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.3％、硫酸镁0.05％、硫酸锌0.05％、组氨酸0.3％、维生素B 1 0.2％、豆油0.25％、腺嘌呤0.1％、鸟嘌呤0.03％、尿嘧啶0.1％和三七粉0.6％，水余量。
按照与实施例1相同的工艺条件发酵培养Cs-4菌株，最后得到Cs-4虫草菌粉。本实施例制备的虫草菌粉中的活性成分含量比实施例2略高，这是鸟嘌呤 和腺嘌呤相互作用的结果。
实施例4
斜面培养基的组成为：葡萄糖4％、蛋白胨0.4％、麸皮0.8％、磷酸二氢钾0.3％、硫酸镁0.05％、组氨酸0.3％、琼脂4％，水余量，pH自然。用接种铲取1cm 2左右保存好的Cs-4菌株，均匀涂布在斜面上，在温度20℃、湿度90％下培养9天。
摇瓶培养基的组成为：葡萄糖5％、蛋白胨0.4％、麸皮0.6％、磷酸二氢钾0.3％、硫酸镁0.05％、硫酸锌0.05％、组氨酸0.3％，维生素B 1 0.1％，水余量。在无菌条件下，用接种铲子取1cm 2生长旺盛的斜面菌株接入摇瓶培养基内。在20℃往复式摇床上振荡培养9天，往复式摇床振荡频率为140次/分，整个摇瓶培养过程中，使用红光照射。摇瓶培养结束后，将各摇瓶培养好的种子合并到接种钢瓶内。
种子罐培养基的组成为：热榨豆饼粉4％、葡萄糖4％、蔗糖4％、磷酸二氢钾0.4％、硫酸镁0.1％、硫酸锌0.1％、组氨酸0.2％、维生素B 1 0.3％、豆油0.2％、腺嘌呤0.2％，三七粉0.6％，水余量。无菌条件下，采用压差法按照5％的接种量将摇瓶培养好的菌种接入种子罐内。在温度19℃，罐压0.05MPa，通气量为1:1.8(v/v·min)的条件下，静置培养5天。
繁殖罐培养基的组成为：热榨豆饼粉4％、葡萄糖4％、蔗糖4％、磷酸二氢钾0.2％、硫酸镁0.06％、硫酸锌0.05％、组氨酸0.2％、维生素B 1 0.2％、豆油0.3％、腺嘌呤0.1％、鸟嘌呤0.03％，尿嘧啶0.2％和三七粉0.7％，水余量。无菌条件下，采用压差法按照12％的接种量将种子罐内培养好的菌种接入繁殖罐内。在温度19℃，罐压0.05MPa，通气量为1:0.4(v/v·min)的条件下，静置培养6天。
发酵罐培养基的组成为：热榨豆饼粉4％、葡萄糖1％、蔗糖1％、磷酸二氢钾0.2％、硫酸镁0.06％、硫酸锌0.06％、组氨酸0.2％、维生素B 1 0.3％、豆油0.3％、腺嘌呤0.12％、鸟嘌呤0.08％、尿嘧啶0.2％和三七粉1.0％，水余量。无菌条件下，采用压差法按照15％的接种量将繁殖罐内培养好的菌种接入发酵罐内。在温度19℃，罐压0.05MPa，通气量为1:0.9(v/v·min)，搅拌速度为60rpm的条件下，培养5天。开始发酵后，每隔24小时取发酵液样，测定pH值、还原糖、氨基氮。当pH＜7，还原糖＜1.5％，氨基氮＜0.4mg/mL，菌浓≧14％时，发酵 结束。放罐，发酵液固液分离，将固体烘干、粉碎得到Cs-4虫草菌粉。
本实施例制备的虫草菌粉与实施例3制得的菌粉中的活性成分含量相近。
实施例5
本实施例与实施例4的区别仅在于，摇瓶培养基的种子来源是实施例4发酵结束后的发酵液，接种量为1％。按照与实施例4相同的工艺条件发酵培养Cs-4菌株，最后得到Cs-4虫草菌粉。该菌粉中的核苷、麦角甾醇及皂苷含量比与实施例4中的菌粉更高，原因在于菌种对嘌呤、嘧啶及三七粉的生物转化能力更强。
实施例6
本实施例与实施例4的各培养基组成相同，区别在于，摇瓶培养基的种子来源是实施例4发酵结束后的发酵液，接种量为2％。摇瓶培养条件为在18℃往复式摇床上振荡培养8天，往复式摇床振荡频率为145次/分，整个摇瓶培养过程中，使用红光照射。
种子罐培养条件为13℃恒温，罐压0.03MPa，通气量为1:1.0(v/v·min)的条件下，静置培养4天。繁殖罐培养条件为13℃恒温，罐压0.03MPa，通气量为1:0.2(v/v·min)的条件下，静置培养5天。发酵罐培养条件为13℃恒温，罐压0.03MPa，通气量为1:0.65(v/v·min)的条件下，搅拌速度为50rpm的条件下，培养5天。发酵结束后，放罐，制备Cs-4虫草菌粉。本实施例得到的虫草菌粉中活性成分含量与实施例5相近，说明本实施例的工艺参数条件与实施例5一样适用。
对比例1
本对比例与实施例1的区别在于，种子罐培养基中不含腺嘌呤，繁殖罐培养基中不含腺嘌呤和尿嘧啶，发酵罐培养基中不含腺嘌呤和尿嘧啶。按照与实施例1相同的工艺条件发酵培养Cs-4菌株，最后得到Cs-4虫草菌粉。本对比例制得的虫草菌粉中的核苷和麦角甾醇含量低于实施例1。
对比例2
(液体发酵培养基中添加三七粉)
本对比例与实施例2的区别在于，种子罐培养基中不含腺嘌呤，繁殖罐培养基中不含腺嘌呤和尿嘧啶，发酵罐培养基中不含腺嘌呤和尿嘧啶。按照与实 施例1相同的工艺条件发酵培养Cs-4菌株，最后得到Cs-4虫草菌粉。相比实施例2的虫草菌粉，本对比例制备的菌粉中的核苷、麦角甾醇和皂苷含量更低，皂苷含量低说明嘌呤和嘧啶能促进菌株对三七粉的利用。相比对比例1的虫草菌粉，本对比例得到的菌粉中麦角甾醇含量有提高，说明添加三七粉能提高麦角甾醇含量。
对比例3
本对比例与实施例3的区别仅在于摇瓶培养是在黑暗的条件下进行，制得的Cs-4虫草菌粉中的活性成分含量低。由此说明，摇瓶培养需要在光照条件下进行。
对比例4
本对比例与实施例3的区别仅在于摇瓶培养是在自然光的条件下进行，制得的Cs-4虫草菌粉中的活性成分含量高于对比例3，但低于实施例3。由此说明，红光条件下进行摇瓶培养更有利于菌株生长。
对比例5
本对比例与实施例3的区别在于：摇瓶培养条件为在25℃往复式摇床上振荡培养8天，往复式摇床振荡频率为120次/分。种子罐培养条件为20℃恒温，罐压0.03MPa，通气量为1:0.4(v/v·min)的条件下，静置培养4天。繁殖罐培养条件为20℃恒温，罐压0.03MPa，通气量为1:0.2(v/v·min)的条件下，静置培养5天。发酵罐培养条件为20℃恒温，罐压0.03MPa，通气量为1:0.65(v/v·min)的条件下，静置培养5天。发酵结束后，放罐，制备Cs-4虫草菌粉。本对比例得到的虫草菌粉中活性成分含量低，主要原因是培养条件不适合，尤其发酵罐培养不能采用静置培养。
实施例7
从实施例1-6和对比例1-5制备得到的Cs-4虫草菌粉取样，测定其中的核苷(腺苷、鸟苷和尿苷)、麦角甾醇和皂苷的含量。检测结果见表1，根据检测结果可明显看到，采用本发明提供的Cs-4菌株发酵生产工艺生产得到的Cs-4虫草菌粉的药用价值高。
Figure PCTCN2018119032-appb-000001
表1.制备的虫草菌粉中的核苷、麦角甾醇和皂苷含量
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺，其特征在于，所述发酵生产工艺包括以下步骤：
    S1.斜面培养：将Cs-4菌株接种到斜面培养基上，在15-20℃恒温下培养7-9天，斜面培养基中含有组氨酸；
    S2.摇瓶培养：无菌条件下，往摇瓶培养基内接入步骤S1培养好的菌种，置于15-20℃恒温摇床、光照条件下培养7-9天，摇瓶培养基中含有组氨酸和维生素B 1；
    S3.种子罐培养：无菌条件下，按照3-5％的接种量接入步骤S2培养好的菌种，种子罐培养基中含有嘌呤；
    S4.繁殖罐培养：无菌条件下，按照9-12％的接种量接入步骤S3培养好的菌种，繁殖罐培养基中含有嘌呤和嘧啶；
    S5.发酵罐培养：无菌条件下，按照5-20％的接种量接入步骤S4培养好的菌种，发酵罐培养基中含有嘌呤和嘧啶；发酵罐培养结束后，得到发酵液；
    或者，按照1-2％的接种量将步骤S5发酵液接至摇瓶培养基内，按照步骤S2的条件进行摇瓶培养，然后重复步骤S3-S5，发酵培养Cs-4菌株。
  2. 根据权利要求1所述的发酵生产工艺，其特征在于，所述嘌呤选自腺嘌呤和鸟嘌呤中的一种或两种，所述嘧啶为尿嘧啶。
  3. 根据权利要求1所述的发酵生产工艺，其特征在于，以重量体积比g/mL计，斜面培养基的组成包括葡萄糖2-4％、蛋白胨0.2-0.4％、麸皮0.4-0.8％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、组氨酸0.1-0.3％、琼脂2-4％，水余量；摇瓶培养基的组成包括葡萄糖1-5％、蛋白胨0.2-0.4％、麸皮0.4-0.6％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、硫酸锌0.03-0.05％、组氨酸0.1-0.3％，维生素B 1 0.05-0.1％，水余量。
  4. 根据权利要求1所述的发酵生产工艺，其特征在于，种子罐培养基、繁殖罐培养基和发酵罐培养基中含有三七粉。
  5. 根据权利要求1-4任一项所述的发酵生产工艺，其特征在于，以重量体积比g/mL计，种子罐培养基的组成包括热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.2-0.4％、硫酸镁0.05-0.1％、硫酸锌0.05-0.1％、组氨酸0.1-0.2％、维生素B 1 0.02-0.3％、豆油0.1-0.2％、腺嘌呤0.05-0.2％和三七粉 0.4-0.6％，水余量。
  6. 根据权利要求1-4任一项所述的发酵生产工艺，其特征在于，以重量体积比g/mL计，繁殖罐培养基的组成包括热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、维生素B 1 0.1-0.2％、豆油0.2-0.3％、腺嘌呤0.03-0.1％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-0.7％，水余量。
  7. 根据权利要求1-4任一项所述的发酵生产工艺，其特征在于，以重量体积比g/mL计，发酵罐培养基的组成包括热榨豆饼粉3-4％、葡萄糖1-2％、蔗糖1-2％、磷酸二氢钾0.2-0.3％、硫酸镁0.04-0.06％、硫酸锌0.04-0.06％、组氨酸0.2-0.3％、维生素B 1 0.2-0.3％、豆油0.2-0.3％、腺嘌呤0.05-0.12％、鸟嘌呤0.03-0.08％、尿嘧啶0.05-0.2％和三七粉0.5-1.0％，水余量。
  8. 根据权利要求7所述的发酵生产工艺，其特征在于，步骤S2所述的摇床为往复式摇床，摇床振荡频率为140±5次/分，光照使用的光为红光；步骤S3种子罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.5-1.8，静置培养4-5天。
  9. 根据权利要求8所述的发酵生产工艺，其特征在于，繁殖罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.15-0.4，静置培养5-6天。
  10. 根据权利要求9所述的发酵生产工艺，其特征在于，发酵罐培养的条件为16±3℃恒温，罐压0.02-0.05MPa，通气量(v/v·min)为1：0.65-0.9，优选1：0.75，在40-60rpm转速下搅拌培养4-5天。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277301A (zh) * 2010-06-10 2011-12-14 中国农业大学 一株绿青色拟青霉及其应用
CN106148200A (zh) * 2016-07-22 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 一种富硒低铅、砷的冬虫夏草菌丝体的培养基及其培植方法
CN107164237A (zh) * 2017-06-09 2017-09-15 中国农业大学 富硒蛹虫草及其栽培方法与应用
CN108504621A (zh) * 2018-05-25 2018-09-07 江西国药有限责任公司 用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法
CN108676730A (zh) * 2018-05-25 2018-10-19 江西国药有限责任公司 一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1095103A (zh) * 1993-05-07 1994-11-16 中国中医研究院 虫草菌丝发酵生产工艺
CN1215754A (zh) * 1997-10-24 1999-05-05 中国医学科学院药物研究所 冬虫夏草发酵产物的制备方法及其应用
CN103627770A (zh) * 2012-08-23 2014-03-12 云南云百草实验室有限公司 两种拟青霉属真菌对三七的生物转化
CN104911109A (zh) * 2015-05-28 2015-09-16 吉林大学 一种高产蝙蝠蛾拟青霉诱变菌株及培育方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277301A (zh) * 2010-06-10 2011-12-14 中国农业大学 一株绿青色拟青霉及其应用
CN106148200A (zh) * 2016-07-22 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 一种富硒低铅、砷的冬虫夏草菌丝体的培养基及其培植方法
CN107164237A (zh) * 2017-06-09 2017-09-15 中国农业大学 富硒蛹虫草及其栽培方法与应用
CN108504621A (zh) * 2018-05-25 2018-09-07 江西国药有限责任公司 用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法
CN108676730A (zh) * 2018-05-25 2018-10-19 江西国药有限责任公司 一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺

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