CN106983134A - The preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder - Google Patents
The preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder Download PDFInfo
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- CN106983134A CN106983134A CN201710230253.1A CN201710230253A CN106983134A CN 106983134 A CN106983134 A CN 106983134A CN 201710230253 A CN201710230253 A CN 201710230253A CN 106983134 A CN106983134 A CN 106983134A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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Abstract
The invention discloses the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder, comprise the following steps：Coragyceps militaris bacterium is subjected to Multiplying culture, Multiplying culture liquid is obtained, the Multiplying culture liquid is subjected to concentration, mycelium concentrate is obtained, freeze drying protectant is added into the mycelium concentrate, is mixed, pre-freeze, is freezed, packaging is produced.Glucose of the invention by using Coragyceps militaris bacterium and containing certain content; trehalose, the freeze drying protectant of the compounding such as skimmed milk powder and β CD, with reference to Vacuum Freezing ＆ Drying Technology; strain survival rate is effectively improved, the squamous subculture degenerate problem of strain is fundamentally solved.
Description
Technical field
The present invention relates to a kind of preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder, be conducive to the effective of Coragyceps militaris bacterium
Preservation.
Background technology
Northern Chinese caterpillar Fungus bacteriumCordyceps militaris , ( L . ex Fr. )Link.Also referred to as pupa grass, pupa worm
Grass, Cordceps militaris.Northern Chinese caterpillar Fungus, is a kind of precious Chinese herbal medicine with higher nourishing and medical value.Cordyceps militaris is in China
The existing more than ten years history of artificial cultivation, its medical value and have won fame both at home and abroad cordyceps sinensis ( Cordyceps sinensis) phase
Seemingly.
On the one hand the maturation of Artificial Planting Method of C. militarles and industrialized development illustrate the wilderness demand in market, on the other hand
Also the rare present situation of cordyceps sinensis wild resource is reflected.Rising suddenly and sharply year after year for wild cordyceps price, makes increasing people
Pay close attention to the artificial cultivation of Cordyceps militaris.The Cordyceps militaris original strain of artificial cultivation is all from worm winter, entomogenous fungi summer with field acquisition, leads to
It is often annual field acquisition, then is used to produce by the complicated stable strain of acquired character that isolates and purifies.But cordyceps compared with
Other fungies be more easy to make a variation, degenerate, it is actual prove in Cordyceps Militaris by generative propagation after, tube is passed on more than more than three times,
Strain goes out careless performance and can significantly decreased.Asexual passage is more than 5 times, and cordyceps species can lose careless performance.It is general and
Speech generally requires 6-9 month preservation terms from original parent species are prepared to cultivating and growing, and production unit has to every 1-2 months i.e.
Passage preservation is carried out, so as to cause strain performance seriously to degenerate.To ensure strain quality, generally require to adopt again every year
Collection, repeated isolation purification work not only adds workload, and the difference of annual production strain can also cause product quality
It is unstable.Spawn degeneration can cause the mistake that drops in production over a large area to be received, and great risk is brought to batch production.How effectively
The excellent cordyceps of preservation, solve the problems, such as the spawn degeneration of Cordyceps militaris turns into the focus of domestic and foreign scholars research.It is wide at present
The method of the preservation cordyceps of general application is low temperature test tube preserving process, but Fang Huazhou et al. result of study shows to use this side
Method preservation, the time characters of progenies of the cordyceps species in 30 d is relatively stable, does not appear as degenerating, the strain after the d of preservation 60 is lost
Transmissibility shape shows obvious difference, as the delay of preservation time its mycelial growth rate is slack-off and colonial morphology changes
Become, the angle variability of bacterium colony is significantly improved.Existing method for preserving obviously can not solve the problems, such as actual production, still need to explore more effective
Method for preserving.
Excellent Cordyceps militaris spawn ensure that the quality and quality of worm grass product.Tradition is needed to utilizing for Cordyceps militaris spawn
Culture is enlarged to strain, cumbersome, the cycle is long, many drawbacks such as strain easily pollutes, degenerated, making a variation.To solve this
One production practical problem, this research is on the basis of analysis spawn degeneration reason, by the Coragyceps militaris bacterium of separated acquisition
By optimization culture, centrifugation, add protective agent, again through vacuum freeze-drying technique, be made into freeze-dried powder, in guarantor at -20 DEG C
Deposit, to reach the purpose of long term storage strain.This preparation method is preserving its vigor by techniques such as vacuum, freezing, dryings
In the case of create anoxic, low temperature, drying condition to cordyceps, effectively control the reproductive order of generation of Cordyceps Militaris, Er Qieneng
Production is enough directly used in, the squamous subculture degenerate problem of strain is fundamentally solved.Not only efficiently solve strain long-term
The problem of preservation, and annual heavy strain improvement work is eliminated, reduce cost, it is ensured that product quality, be pupa worm
Grassland industryization development further provides technical support, with far-reaching realistic meaning.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, effectively strain can be kept to live it is an object of the invention to provide one kind
Power, facilitates the preparation method of the northern Chinese caterpillar Fungus bacterium freeze-dried powder of production application, and the northern Chinese caterpillar Fungus bacterium freeze-dried powder prepared is protected
In the presence of at -20 DEG C, without passage, strain still keeps relatively being higher by careless and goes out that grass is healthy and strong after the storage several months, and annesl is normal, without abnormal
Shape.
Above-mentioned purpose of the present invention was achieved the field acquisition production traits excellent worm summer in winter by following technical solution
Careless bacterium obtains test tube slant pure culture by isolating and purifying, and is inoculated in progress concussion Multiplying culture in proliferated culture medium, obtains liquid
Body strain.Lower floor's mycelium is collected by centrifugation in Multiplying culture liquid under proper condition, compound freeze drying protectant is added, mixes, dress
Bottle, freezes, produces northern Chinese caterpillar Fungus bacterium freeze-dried powder.
Step 1：The preparation of proliferated culture medium
Proliferated culture medium：Peeled potatoes 200g, glucose 20g, potassium dihydrogen phosphate 3.0g, magnesium sulfate 1.5g, Vb10mg, albumen
Peptone（N sources are provided）5g, is settled to 1000ml, and pH is naturally standby.
Step 2：Aweto Multiplying culture
24 DEG C of temperature, shakes frequency 130-150rpm, incubation time 72h, to can see uniform mycelium pellet.
Step 3：Medium centrifugal collects mycelium
Centrifugation time 10min, centrifugal speed, 6000rpm.
Step 4：It is prepared by freeze drying protectant
Freeze drying protectant, by mass percentage, including following component：
Skimmed milk powder 10%, trehalose 5%, glycerine 10%, glucose 5%, β-CD 5%, mannitol 15%.
The preparation method of the freeze drying protectant, comprises the following steps：
Said components are dissolved and balanced standby after 30min according to proportioning.
Step 5：Freeze drying protectant is added by certain volume ratio, is mixed with mycelium concentrate.
Step 6：Pre-freeze temperature to needed for freezing.Pre-freezing temperature is -80 DEG C, and the time is 60min.
Step 7：The condition of vacuum freeze drying is：Degree vacuum is 20Pa absolutely, and temperature is -40 DEG C, and freeze-drying time is
24h。
Step 8：Seal with wax, it is closed.
The present invention compared with prior art, has the advantages that
The low temperature test tube preserving process used at present, need to be enlarged culture, cumbersome, the cycle is long, strain to Cordyceps militaris spawn
Many drawbacks such as easy pollution, degeneration, variation.This preparation method is preserving the feelings of its vigor by techniques such as vacuum, freezing, dryings
Anoxic, low temperature, drying condition are created to cordyceps under condition, the problem of strain is preserved for a long time is efficiently solved, from basic
On solve the squamous subculture degenerate problem of strain.
Embodiment 1
(1) preparation and its culture of proliferated culture medium
A:Its component includes：Potato 200g, glucose 20g, potassium dihydrogen phosphate 3.0g, magnesium sulfate 1.5g, Vb10mg, peptone
（N sources are provided）5g.
Said components are dissolved and balance 30min by B according to proportioning, in 121.3 DEG C of conditions of temperature, 30min autoclavings.I.e.
Obtain culture medium.
C:Fungus block is inoculated in proliferated culture medium, in 20 DEG C, concussion frequency 130rpm cultivates 72h, uniform to that can see
Mycelium pellet.
(2) above-mentioned nutrient solution is poured into centrifuge tube, in 6000rpm, 15min centrifuge, is centrifuged, take concentrate
It is standby.
(3) by mass percentage, freeze drying protectant includes following component：Skimmed milk powder 5%, trehalose 7%, glycerine 10%, Portugal
Grape sugar 5%, β-CD 7%, mannitol 10%.Said components are dissolved and balanced after 30min according to proportioning, by concentrate and protective agent
Than for 1：5 mix with mycelium concentrate.
(4) (3) mixed liquor is subjected to pre-freeze to lyophilized required temperature, its pre-freezing temperature is -80 DEG C, and the time is 60min.Most
Afterwards, vacuum freeze drying is carried out, its condition is that exhausted degree vacuum is 20Pa, and temperature is -40 DEG C, and freeze-drying time is 24h.
Embodiment 2
(1) preparation and its culture of proliferated culture medium
A:Its component includes：Potato 200g, glucose 20g, potassium dihydrogen phosphate 3.0g, magnesium sulfate 1.5g, Vb10mg, peptone
（N sources are provided）5g.
Said components are dissolved and balance 30min by B according to proportioning, in 121.3 DEG C of conditions of temperature, 30min autoclavings.I.e.
Obtain culture medium.
C:Fungus block is inoculated in proliferated culture medium, in 24 DEG C, concussion frequency 140rpm cultivates 72h, uniform to that can see
Mycelium pellet.
(2) above-mentioned nutrient solution is poured into centrifuge tube, in 7000rpm, 10min centrifuge, is centrifuged, pour out supernatant
Liquid is standby.
(3) by mass percentage, freeze drying protectant includes following component：Skimmed milk powder 10%, trehalose 3%, glycerine 10%,
Glucose 10%, β-CD 3%, mannitol 12%.Said components are dissolved and balanced after 30min according to proportioning, by concentrate and guarantor
It is 1 to protect agent ratio：8 mix with mycelium concentrate.
(4) it is -80 DEG C (3) mixed liquor to be carried out into pre-freeze to lyophilized its pre-freezing temperature of required temperature, and the time is 60min.Most
Afterwards, vacuum freeze drying is carried out, its condition is that exhausted degree vacuum is 20Pa, and temperature is -40 DEG C, and freeze-drying time is 24h.
Embodiment 3
(1) preparation and its culture of proliferated culture medium
A:Its component includes：Potato 200g, glucose 20g, potassium dihydrogen phosphate 3.0g, magnesium sulfate 1.5g, Vb10mg, peptone
（N sources are provided）5g.
Said components are dissolved and balance 30min by B according to proportioning, in 121.3 DEG C of conditions of temperature, 30min autoclavings.I.e.
Obtain culture medium.
C:Fungus block is inoculated in proliferated culture medium, in 22 DEG C, concussion frequency 150rpm cultivates 72h, uniform to that can see
Mycelium pellet.
(2) above-mentioned nutrient solution is poured into centrifuge tube, in 8000rpm, 5min centrifuge, is centrifuged, pour out supernatant
Liquid is standby.
(3) by mass percentage, freeze drying protectant includes following component：Skimmed milk powder 15%, trehalose 5%, glycerine 10%,
Glucose 8%, β-CD 5%, mannitol 15%.Said components are dissolved and balanced after 30min according to proportioning, by concentrate and protection
Agent ratio is 1：10 mix with mycelium concentrate.
It is -80 DEG C that (3) mixed liquor is carried out into pre-freeze to lyophilized its pre-freezing temperature of required temperature, and the time is 60min.Finally,
Vacuum freeze drying is carried out, its condition is that exhausted degree vacuum is 20Pa, and temperature is -40 DEG C, and freeze-drying time is 24h.
Claims (8)
1. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder, it is characterised in that comprise the following steps：By Coragyceps militaris bacterium
Bacterium solution carries out Multiplying culture, obtains nutrient solution, and the nutrient solution is carried out into centrifugal treating, mycelium concentrate is obtained, to described
Freeze drying protectant is added in mycelium concentrate, is mixed, pre-freeze and lyophilized, packaging produce the northern winter worm summer deposited suitable for normal temperature
Careless freeze-dried powder.
2. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that culture medium propagation temperature
20 ~ 24 DEG C of degree, concussion frequency is 130 ~ 150, and the time is 72h；The wherein composition of culture medium：Potato 200g, glucose 20g,
Potassium dihydrogen phosphate 3.0g, magnesium sulfate 1.5g, Vb10mg, peptone（N sources are provided）5g.
3. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that the centrifugal treating
Condition be：6000rmp,15min;7000rmp,10min;800rmp, 5min.
4. the preparation method of the northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1 suitable for long-term storage, its feature exists
In the freeze drying protectant, by percent by volume, including following component：
Skimmed milk powder 5 ~ 15%
Trehalose 3 ~ 7%
Glycerine 10%
Glucose 5 ~ 10%
β-CD 3~7%
Mannitol 10 ~ 15%
The preparation method of the freeze drying protectant, including, said components are dissolved according to proportioning, 115 DEG C is warming up to and sterilizes 15 points
Clock, produces freeze drying protectant.
5. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that the mycelium is dense
Contracting liquid is 1 with adding protectant volume ratio:5~10.
6. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that the pre-freeze refers to
It is placed in cryogenic refrigerator and carries out pre-freeze, its pre-freeze time is 60min, and pre-freezing temperature is -80 DEG C.
7. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that described freeze refers to
It is placed in freeze drier and carries out frozen dried, lyophilized exhausted degree vacuum is 20Pa, temperature is -40 DEG C, and freeze-drying time is
24h。
8. the preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder according to claim 1, it is characterised in that the packaging be by
Obtained freeze-drying powder is less than in the environment of 35% in relative humidity, seals with wax.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394263A (en) * | 2020-05-28 | 2020-07-10 | 中华全国供销合作总社南京野生植物综合利用研究所 | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method |
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
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| CN101129414A (en) * | 2007-09-10 | 2008-02-27 | 王辉 | Chinese caterpillar fungus freeze-dried powder produced by artificial zymosis method and method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394263A (en) * | 2020-05-28 | 2020-07-10 | 中华全国供销合作总社南京野生植物综合利用研究所 | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method |
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
| CN115838635B (en) * | 2022-10-08 | 2023-08-22 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermented cordyceps sinensis powder |
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