CN102154407B - Corayceps militaris polysaccharide two-stage fermentation synthesis process - Google Patents
Corayceps militaris polysaccharide two-stage fermentation synthesis process Download PDFInfo
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Abstract
The invention belongs to the microorganism field, particularly relates to a corayceps militaris polysaccharide two-stage fermentation synthesis process; the process comprises the following steps: corayceps militaris strains are vaccinated on a potato slant culture medium, are cultured at the constant temperature and then are put in a conical flash containing a seed culture medium for shaking culture; then the corayceps militaris strains are put in a fermentation tank containing a fermentation medium, the fermentation process is as follows: the corayceps militaris is cultured at constant temperature for four to five days in ventilating and stirring condition, which is mainly used for providing a culture condition suitable for growing of corayceps militaris thallus so that the corayceps militaris grows rapidly; then the corayceps militaris is cultured for two to three days at the constant temperature in the condition of ventilation and slightly stirring, a culture solution is centrifuged, and ethanol in two times of volume of is added into an obtained liquid supernatant which is mixed and put up for the night at the temperature of 4 DEG C; and precipitates obtained through secondary centrifugation is dried so as to obtain the corayceps militaris. The process is reasonable in design and simple in operation, is suitable for large-scale industrial production and can solve the situation of drug source shortage and has obvious benefits.
Description
Technical field
The present invention relates to the synthetic Cordyceps militaris (L.) Link. polysaccharide process of artificial culture, is exactly specifically a kind of method that adopts the synthetic Cordyceps militaris (L.) Link. polysaccharide of biological two stage fermentation methods.
Background technology
Cordyceps militaris (L.) Link. bacterium [Cordyceps militaris] (common name Cordyceps militaris (L.) Link.) is the entomogenous fungi complex body that lepidopteran Hepialidae Genus Hepialus larva is formed afterwards by China pilose spore parasitism; the rare medicinal herbs that belongs to national second class protection, is listed as one of China " Chinese medicine three is large precious " with ginseng, pilose antler.It contains plurality of active ingredients (as Cordyceps mycelium, polysaccharide, cordycepin etc.), has strengthening immunity, antitumor, anti-oxidant and hypoglycemic effect, and the market requirement is very big.
But wild cordyceps growth cycle is long, and due to wild Chinese caterpillar fungus dug excessively to coyoting, havoc ecotope not only, and cause natural cs endangered.Therefore, must find out the effective ways that can meet the need of market and can not destroy wild Chinese caterpillar fungus resource.At present, the artificial culture method of Chinese caterpillar fungus is mainly contained to artificial culture and two kinds of methods of deep liquid cultivation.The artificial culture cycle is long, and the Chinese caterpillar fungus of acquisition yields poorly, and is difficult to meet Production requirement.It is short that deep liquid cultivation has the cycle, and plant factor advantages of higher, is more conducive to large-scale industrial production.But be mainly to produce thalline, and biologically active substance (polysaccharide) output is relatively low.Therefore, if can develop a kind of cultural method that can obtain a large amount of moving winter worm summer herb thallus, can produce again large number of biological active substance (polysaccharide), will be for improving Chinese caterpillar fungus production efficiency, the worm grass product of exploitation high added value, realizes large-scale industrialization and produces significant.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques, and its operating procedure is simple, is applicable to large-scale industrial production.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques of the present invention, it comprises the following steps:
A. preserve bacterial classification: the Coragyceps militaris bacterium that is grown in potato slant medium is kept in 4 ℃ of refrigerators standby.
B. slant strains activation:
The Coragyceps militaris bacterium of preserving in above-mentioned steps a is transferred to potato slant medium with the inoculation shovel after sterilizing, in 25 ℃ of thermostat containers, cultivates the Coragyceps militaris bacterium after being activated for 7 days, standby.
C. seed culture: the Coragyceps militaris bacterium after above-mentioned steps b activation is cut to about 1cm2 with the inoculation hook of sterilizing and proceed in the 250mL triangular flask that 50mL seed culture medium is housed with the inclined-plane mycelia of substratum, in shaking table, 25 ℃, 150r/min concussion cultivation 4-5 days, obtain seed liquor standby.
Nutritive ingredient and the add-on of described seed culture medium are as follows:
By said components and add-on, be made into the seed culture medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by.
D. fermentation culture: by the cultured seed liquor of above-mentioned steps c with 10% inoculum size access the fermentation cylinder for fermentation containing fermention medium, fermentation culture process comprises two stages:
First stage fermentation condition comprises: 25 ℃ of fermentation jar temperatures, and ventilation 1vvm, mixing speed 100-150rpm, cultivates 4-5 days under the condition of fermentation tank pressure 0.05MPa.
Subordinate phase fermentation condition: 25 ℃ of fermentor tank tank temperature, ventilation 0vvm (stuffiness), cultivates 2-3 days under mixing speed 30-50rpm condition, obtains winter worm summer herb thallus nutrient solution.
Nutritive ingredient and the add-on of described fermention medium are as follows:
By said components and add-on, be made into the fermention medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by.
E. filtering separation thalline: the winter worm summer herb thallus nutrient solution after above-mentioned steps d fermentation ends is filtered to winter worm summer herb thallus with stainless steel sheet frame filter, obtain winter worm summer herb thallus and supernatant liquor, filter winter worm summer herb thallus distilled water flushing three times of collecting, in 60-70 ℃ dry, Cordyceps fungus body water content is controlled at 7-8%.
F. alcohol chromatography: the supernatant liquor that above-mentioned steps e is obtained adds 95% alcohol chromatography of two volumes, stirs after 30 minutes on magnetic stirrer with 200r/m, spends the night at 4 ℃.
G. centrifugal drying: the liquid after above-mentioned steps f is spent the night carries out that 12000rpm is centrifugal, and collecting precipitation thing, dries at 70-80 ℃ of constant temperature oven, and water content is controlled at 7-8%, obtains dry Cordyceps militaris (L.) Link. polysaccharide.
H. crush storage: the dried Cordyceps militaris (L.) Link. polysaccharide of above-mentioned steps g is pulverized and sieved, be placed in air seasoning place and store, obtain product Cordyceps militaris (L.) Link. polysaccharide of the present invention.
Described a kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques, the preparation of described potato slant medium comprises:
First 200g potato decortication is cut into small pieces, adds water 1.0L to boil 30min, with 6 layers of cloth, filter, get filtrate, obtain potato juice, then the potato juice preparing is settled to 1.0L, standby 121 ℃ of sterilizings 20 minutes; Add again afterwards glucose 20g and agar 20g, obtain potato slant medium.
Preferably, described a kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques,
Nutritive ingredient and the add-on of described fermention medium are as follows:
Preferably, described a kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques, in described fermentation culture process, seed liquor contains in the fermentor tank of fermention medium with 5% inoculum size access, fermentation culture process comprises two stages: first stage fermentation condition comprises: 25 ℃ of fermentation jar temperatures, ventilation 1vvm, mixing speed 100-150rpm, cultivates 6-7 days under the condition of fermentation tank pressure 0.05MPa; Subordinate phase fermentation condition: 25 ℃ of fermentor tank tank temperature, ventilation 0vvm, cultivates 1-2 days under mixing speed 20-30rpm condition.
Cordyceps militaris (L.) Link. bacterium used be preserved in " Chinese industrial microbial strains preservation management " center ", preservation registration number is: CICC 14015; Classification And Nomenclature: Cordyceps militaris.
The present invention is seeded in constant temperature culture on potato slant medium by Coragyceps militaris bacterium and within 7 days, accesses afterwards containing concussion cultivation 4-5 days in the triangular flask of seed culture medium.Then access is containing in the fermentor tank of fermention medium, and fermenting process is as follows: under constant temperature ventilation agitation condition, cultivate Cordyceps militaris (L.) Link. 4-5 days, this stage is mainly to provide to the culture condition that is applicable to Cordyceps militaris (L.) Link. thalli growth, makes a large amount of breeding of Cordyceps militaris (L.) Link.; Then stuffy at constant temperature, under gentle agitation condition, cultivate 2-3 days, this process is in order to make Cordyceps militaris (L.) Link. synthetic polysaccharide in a large number; Medium centrifugal, adds the ethanol of two volumes in the supernatant liquor of acquisition, stir 4 ℃ and spend the night, and the throw out that recentrifuge obtains obtains Cordyceps militaris (L.) Link. polysaccharide through super-dry.
Cordyceps militaris (L.) Link. bacterium used is inclined-plane strain characteristics: on potato slant medium, cultivate and within three days, can see white suede trichothallic, cultivate and within seven days, substantially cover with inclined-plane, the Cordyceps militaris (L.) Link. polysaccharide pharmaceutical use that the method that the present invention provides is produced is all identical with natural Cordyceps militaris (L.) Link. with nutritive value, and this technique can significantly be alleviated the situation to Cordyceps sinensis medicine resource anxiety.
The beneficial effect that the present invention compared with prior art has is:
The present invention according to Cordyceps militaris (L.) Link. metabolism and growth characteristic Design the synthetic polysaccharide of two stage fermentation culture process, utilize technique of the present invention can obtain a large amount of Cordyceps militaris (L.) Link. thalline, can obtain again the Cordyceps militaris (L.) Link. polysaccharide of high yield.Therefore, the efficiency of the synthetic Cordyceps polysaccharide of two stage deep liquid fermentation process of the present invention is far above traditional deep liquid fermentation process.Than traditional liquid deep layer culture process Cordyceps militaris (L.) Link. polysaccharide yield, improve 75%.
In addition, technological design of the present invention is reasonable, simple to operate, is applicable to large-scale industrial production, can solve medicine source situation in short supply, meet the need of market, and remarkable benefit.
Embodiment
Below the present invention is described in further detail.
Embodiment mono-
A kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques of the present invention, it comprises the following steps:
A. preserve bacterial classification: the Coragyceps militaris bacterium that is grown in potato slant medium is kept in 4 ℃ of refrigerators standby.
B. slant strains activation:
Actication of culture: the Coragyceps militaris bacterium of preserving in above-mentioned steps a is transferred to potato slant medium with the inoculation shovel after sterilizing, in 25 ℃ of thermostat containers, cultivates the Coragyceps militaris bacterium after being activated for 7 days, standby.
C. seed culture: the Coragyceps militaris bacterium after above-mentioned steps b activation is cut to about 1cm2 with the inoculation hook of sterilizing and proceed in the 250mL triangular flask that 50mL seed culture medium is housed with the inclined-plane mycelia of substratum, in shaking table, 25 ℃, 150r/min concussion cultivation 4-5 days, obtain seed liquor standby.
Nutritive ingredient and the add-on of described seed culture medium are as follows:
By said components and add-on, be made into the seed culture medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by.
D. fermentation culture: by the cultured seed liquor of above-mentioned steps c with 10% inoculum size access the fermentation cylinder for fermentation containing fermention medium, fermentation culture process comprises two stages:
First stage fermentation condition comprises: 25 ℃ of fermentation jar temperatures, and ventilation 1vvm, mixing speed 100-150rpm, cultivates 4-5 days under the condition of fermentation tank pressure 0.05MPa.
Subordinate phase fermentation condition: 25 ℃ of fermentor tank tank temperature, ventilation 0vvm (stuffiness), cultivates 2-3 days under mixing speed 30-50rpm condition.
Nutritive ingredient and the add-on of described fermention medium are as follows:
By said components and add-on, be made into the fermention medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by.
E filtering separation thalline: by stainless steel sheet frame filter (SHXBCRA150-10C for the winter worm summer herb thallus nutrient solution after above-mentioned steps d fermentation ends, strolling in Shanghai, Science and Technology Ltd. produces) filtration winter worm summer herb thallus, obtain winter worm summer herb thallus and supernatant liquor, filter winter worm summer herb thallus distilled water flushing three times of collecting, in 60-70 ℃ dry, Cordyceps fungus body water content is controlled at 7-8%.
F alcohol chromatography: the supernatant liquor that above-mentioned steps e is obtained adds 95% alcohol chromatography of two volumes stirs after 30 minutes with 200r/m on magnetic stirrer, at 4 ℃, spends the night.
G centrifugal drying: the liquid after above-mentioned steps f is spent the night carries out the centrifugal (SS600 of 12000rpm, 12000rpm, the whizzer that Machinery Plant of Shenyang Chemical Engineering College produces), collecting precipitation thing, at 70-80 ℃ of constant temperature oven, dry, water content is controlled at 7-8%, obtains dry Cordyceps militaris (L.) Link. polysaccharide.
The dried Cordyceps militaris (L.) Link. polysaccharide of h crush storage: above-mentioned steps g pulverizes and sieves, and is placed in air seasoning place and stores, and obtains product Cordyceps militaris (L.) Link. polysaccharide of the present invention.
Embodiment 2
The two stage fermentation culture process methods of the present embodiment, with the difference of embodiment 1 be the nutritive ingredient of described fermention medium and add-on as follows:
Embodiment 3
The two stage fermentation culture process methods of the present embodiment, be in fermentation culture process with the difference of embodiment 1, seed liquor contains in the fermentor tank of fermention medium with 5% inoculum size access, fermentation culture process comprises two stages: first stage fermentation condition comprises: 25 ℃ of fermentation jar temperatures, ventilation 1vvm, mixing speed 100-150rpm, cultivates 6-7 days under the condition of fermentation tank pressure 0.05MPa; Subordinate phase fermentation condition: 25 ℃ of fermentor tank tank temperature, ventilation 0vvm (stuffiness), cultivates 1-2 days under mixing speed 20-30rpm condition.
The advantage that the present invention compared with prior art has is: the present invention is according to Cordyceps militaris (L.) Link. metabolism and growth feature, the synthetic Cordyceps militaris (L.) Link. polysaccharide of two stage fermentation culture process has been set up in design, utilize technique of the present invention can obtain a large amount of Cordyceps militaris (L.) Link. thalline, can obtain again the Cordyceps militaris (L.) Link. polysaccharide of high yield.In the first stage of fermentation culture process, owing to ventilating and stirring action, in fermented liquid, dissolved oxygen is in liberal supply, Cordyceps militaris (L.) Link. thalli growth is fast, connect bacterium after 4 days glucose substantially clean by consumption, in this stage, Cordyceps militaris (L.) Link. thalline Fast Growth, but this Cordyceps militaris (L.) Link. polysaccharide is synthetic slower.In subordinate phase, glucose in this stage substratum is consumed in a large number, Cordyceps militaris (L.) Link. thalline enters stationary phase substantially, now stop ventilating, and mixing speed is reduced, dissolved oxygen in fermented liquid is declined, Cordyceps militaris (L.) Link. thalline is in this not regrowth of stage, start the outer Cordyceps militaris (L.) Link. polysaccharide of a large amount of synthetic born of the same parents, and low dissolved oxygen environment is conducive to the synthetic of Cordyceps militaris (L.) Link. polysaccharide in fermented liquid, therefore, the efficiency of the synthetic Cordyceps militaris (L.) Link. polysaccharide of two stage deep liquid fermentation process of the present invention is far above traditional deep liquid fermentation process.Than traditional liquid deep layer culture process Cordyceps militaris (L.) Link. polysaccharide yield, improve 75%.The present invention and traditional liquid deep layer are cultivated Cordyceps militaris (L.) Link. polysaccharide process and are relatively seen the following form.
The table 1 liang comparison that stage deep liquid is cultivated Cordyceps militaris (L.) Link. polysaccharide and traditional liquid deep layer culture process
Claims (2)
1. Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques, is characterized in that, it comprises the following steps:
A. preserve bacterial classification: the Coragyceps militaris bacterium that is grown in potato slant medium is kept in 4 ℃ of refrigerators standby;
B. slant strains activation:
The Coragyceps militaris bacterium of preserving in above-mentioned steps a is transferred to potato slant medium with the inoculation shovel after sterilizing, in 25 ℃ of thermostat containers, cultivates 7 days, the Coragyceps militaris bacterium after being activated, standby;
C. seed culture: the Coragyceps militaris bacterium after above-mentioned steps b activation is cut to 1cm with the inoculation hook of sterilizing
2inclined-plane mycelia with substratum proceeds in the 250mL triangular flask that 50mL seed culture medium is housed, and in shaking table, 25 ℃, 150r/min concussion cultivation 4-5 days, obtain seed liquor standby;
Nutritive ingredient and the add-on of described seed culture medium are as follows:
By said components and add-on, be made into the seed culture medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by;
D. fermentation culture: by the cultured seed liquor of above-mentioned steps c with 10% inoculum size access the fermentation cylinder for fermentation containing fermention medium, fermentation culture process comprises two stages:
First stage fermentation condition comprises: 25 ℃ of fermentation jar temperatures, and ventilation 1vvm, mixing speed 100-150rpm, cultivates 4-5 days under the condition of fermentation tank pressure 0.05MPa;
Subordinate phase fermentation condition: 25 ℃ of fermentor tank tank temperature, ventilation 0vvm (stuffiness), cultivates 2-3 days under mixing speed 30-50rpm condition, obtains winter worm summer herb thallus nutrient solution;
Nutritive ingredient and the add-on of described fermention medium are as follows:
By said components and add-on, be made into the fermention medium of liquid, at 121 ℃, under 0.1MPa, heat sterilization is cooling after 20 minutes, stand-by;
E. filtering separation thalline: the winter worm summer herb thallus nutrient solution after above-mentioned steps d fermentation ends is filtered to winter worm summer herb thallus with stainless steel sheet frame filter, obtain winter worm summer herb thallus and supernatant liquor, filter winter worm summer herb thallus distilled water flushing three times of collecting, in 60-70 ℃ dry, Cordyceps fungus body water content is controlled at 7-8%;
F. alcohol chromatography: the supernatant liquor that above-mentioned steps e is obtained adds 95% alcohol chromatography of two volumes, stirs after 30 minutes on magnetic stirrer with 200r/m, spends the night at 4 ℃;
G. centrifugal drying: the liquid after above-mentioned steps f is spent the night carries out that 12000rpm is centrifugal, and collecting precipitation thing, dries at 70-80 ℃ of constant temperature oven, and water content is controlled at 7-8%, obtains dry Cordyceps militaris (L.) Link. polysaccharide;
H crush storage: the dried Cordyceps militaris (L.) Link. polysaccharide of above-mentioned steps g is pulverized and sieved, be placed in air seasoning place and store, obtain product Cordyceps militaris (L.) Link. polysaccharide.
2. a kind of Cordyceps militaris (L.) Link. polysaccharide two stage fermentation synthesis techniques as claimed in claim 1, is characterized in that,
The preparation of described potato slant medium comprises: first 200g potato decortication is cut into small pieces, adds water 1.0L to boil 30min, with 6 layers of cloth, filter, get filtrate, obtain potato juice, then the potato juice preparing is settled to 1.0L, standby 121 ℃ of sterilizings 20 minutes; Add again afterwards glucose 20g and agar 20g, obtain potato slant medium.
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| CN102559768A (en) * | 2012-02-09 | 2012-07-11 | 湖南大学 | Two-step fermentation production method of microbial flocculant |
| CN102605005B (en) * | 2012-04-16 | 2013-09-18 | 山东省农业科学院农业资源与环境研究所 | Method for producing cordycepic acid by fermentation of cordyceps militaris liquid |
| CN102618598B (en) * | 2012-04-16 | 2013-08-21 | 山东省农业科学院农业资源与环境研究所 | Liquid fermentation method for improving yield of cordyceps sinensis polysaccharide by utilizing expansin |
| CN102936609B (en) * | 2012-11-05 | 2014-12-17 | 南京农业大学 | Preparation method of swift moth paecilomyces varioti extracellular acid glycoprotein |
| CN103262798B (en) * | 2013-06-04 | 2014-08-06 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps fungus and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps fungus |
| CN103564194B (en) * | 2013-10-17 | 2015-04-22 | 广东海洋大学 | Cordyceps polysaccharide composition, as well as preparation method and application of cordyceps polysaccharide composition |
| CN104497160B (en) * | 2014-12-31 | 2017-03-29 | 华南师范大学 | A kind of Polysaccharides in Cultured Cordyceps militaris extract and preparation method and application |
| CN105420124A (en) * | 2015-12-30 | 2016-03-23 | 福建省顺昌新新农业科技有限公司 | Preparation method for cordyceps militaris strains |
| CN109517083A (en) * | 2018-11-01 | 2019-03-26 | 佛山科学技术学院 | The preparation method and applications of Cordyceps hawkesii Gary intracellular polyse |
| CN113564097A (en) * | 2021-07-22 | 2021-10-29 | 重庆市中药研究院 | Method for producing conidia by liquid fermentation of cordyceps sinensis |
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| CN1148623A (en) | 1996-10-04 | 1997-04-30 | 海南兆隆实业有限公司 | North cordyceps mycelium fermentation technology |
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