CN107582604A - A kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its preparation method and application - Google Patents
A kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its preparation method and application, the preparation method of Cordyceps militaris conversion bark of eucommia solid fermentation product comprises the following steps：（1）It is prepared by actication of culture and fermented liquid strain；（2）Solid fermentation culture medium prepares and optimization；（3）Solid fermentation and product processing.The present invention is cultivated the bark of eucommia as main property of medicine matrix inoculation Cordyceps militaris, and optimize and with the addition of the effective factor, and then obtain culture, it is easy to operate, work well, substantially increase the content of cordycepin in tunning, there is provided with enhancing is immune, strengthening the bones and muscles, strengthening the essence gas, three high drop and other effects and with the tunning for developing health food and medicine potentiality, there is preferable economy and social value.
Description
Technical field
The present invention relates to a kind of preparation method using the bark of eucommia as the Cordyceps militaris microbe conversion product of one of main substrate component
And application, especially Cordyceps militaris conversion bark of eucommia solid fermentation product preparation method and application, belong to microbial fermentation technology and
Technical field of biotransformation.
Background technology
On the basis of inheriting tradition herb fermenting method, Chinese medicine is developed with reference to Modern microbiological fermentation engineering
Utilize, turn into an important channel in health products, functional food and new drug development.Biotransformation method have stereoselectivity and
Regioselectivity, reaction condition are gentle, high catalytic efficiency, reaction diversity, accessory substance is few, free from environmental pollution, post processing is simple
The advantages that and be used widely.Fungal transformation Chinese medicine does not need other catalyst, as long as fungi bacterium needed for first culture is a large amount of
Kind, unit chemosynthesis reaction then is carried out to substrate, so biotransformation method is more single-minded than chemical reagent reaction method, more has
Effect.Fungal transformation Chinese medicine typically all carries out catalytic reaction under the conditions of normal temperature and natural pH, runs safety simple to operate, caused
Public hazards are few, and post processing is also very simple, and conversion results include improving herbal medicine efficacy, increase effective component of chinese medicine and fungi activity product
Content, weaken herbal toxic effect etc..
The bark of eucommiaEucommia ulmoides Oliv., it can be traditional rare in China as the Chinese medicine of health food to be
Tonic herb, there is the title of " living fossil plant ".《Sheng Nong's herbal classic》With《Compendium of Materia Medica》The bark of eucommia is classified as top grade, and calls it
" cure mainly waist and knee, bowl spares, strengthening the essence gas, hard muscles and bones, except wet, dribbling urination of itching under the moon.Long term usage, make light of one's life by commiting suicide resistance to old "；" all lower Jiao's
Consider, the non-bark of eucommia is not mended；Lower Jiao's is wet, and the non-bark of eucommia is unfavorable；The acid of sufficient shin, the non-bark of eucommia are not gone；Waist and knee pain, the non-bark of eucommia are not removed ".It is existing
Show for pharmaceutical research, the bark of eucommia has decompression, reducing blood lipid blood glucose, antitumor, anti-bacteria and anti-virus, anti-inflammatory, anti-oxidant, liver protection is sharp
The effects such as courage, kidney tonifying diuresis, anti-osteoporosis, antiabortive, anti-aging, raising collagen synthesis, available for soreness of waist and knee joint, muscles and bones
The diseases such as impotence is weak, impotence, frequent micturition, threatened abortion are intended to fall, uncontrolled urination.
Cordyceps militarisCordyceps militaris(L.) Link is the new resource food of Ministry of Public Health's approval, and China's name
One of your Chinese medicine.According to《Xinhua's Compendium of Materia Medica》Record：" Cordyceps militaris is sweet, mild-natured, there is liver and kidney benefiting, mends marrow, hemostasia and dissipation blood stasis work(
Energy." pharmacological evaluation for many years and clinical practice prove, Cordyceps militaris have adjust it is immune, antitumor, antiviral, anti-infective, anti-
A variety of pharmacological activity such as oxidation, antifatigue, anti-aging, hypoglycemic, protection hepatic and renal function, its main component cordycepin, which has, exempts from
Epidemic disease regulation, hypoglycemic etc. act on.
Therefore, the bark of eucommia is converted using Cordyceps militaris solid fermentation, improves Cordyceps militaris activity substance content, there is provided exempt from enhancing
The tunning of epidemic disease, strengthening the bones and muscles, strengthening the essence gas, three high drop and other effects has preferable application prospect.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its system
Preparation Method, cordycepin content is improved using the Cordyceps militaris solid fermentation conversion bark of eucommia, obtains that there is immune enhancing, strengthening the bones and muscles, benefit
The tunning of vital essence, three high drop effect, produce preferable economy and social value.It is another object of the present invention to provide
The application of above-mentioned tunning.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of preparation method of Cordyceps militaris conversion bark of eucommia solid fermentation product, this method comprise the following steps：
（1）It is prepared by actication of culture and fermented liquid strain：The Cordyceps militaris spawn of preservation is inoculated on PDA culture medium flat board, so
After be put into 25 DEG C of incubators, dark culturing 4-5d, liquid seeds Shaking culture is inoculated in when strain covers with 2/3 scope of flat board,
Shaking table is put into, 4-5d is cultivated under dark condition, treats that strain is covered with bottle, takes out, obtains Cordyceps militaris fermented liquid strain；
（2）Solid fermentation culture medium prepares and optimization：, will be in advance with 20% according to the mass percent of bark of eucommia powder 20%, rice 80%
The rice of murphy juice immersion and the mixing of bark of eucommia powder, 0.4% vitamin B2 is added loaded in can, stirs, is put into height
Autoclave sterilizing 30min is pressed, cooling, obtains solid fermentation culture medium；
（3）Solid fermentation and product processing：Treat step（2）The step of 20% being inoculated with after the cooling of solid fermentation culture medium（1）Obtain
Cordyceps militaris fermented liquid strain, stirs, tightens bottle cap, is put into 25 DEG C of incubators, dark culturing 18d, after the completion of culture
Tunning is taken out, is packaged after weighing, 60 DEG C of air dry ovens is put into and dries, powder is beaten and crosses 80 mesh sieves powder is made.
A kind of Cordyceps militaris converts bark of eucommia solid fermentation product, is obtained according to above-mentioned preparation method, the tunning, contains worm
The mg/g of lavendustin 285.4, contains Pinoresinol diglucoside（PDG）25.2 mg/g.
Above-mentioned Cordyceps militaris conversion bark of eucommia solid fermentation product, can apply to prepare immune enhancing, strengthening the bones and muscles, strengthening the essence
The health food or medicine of gas, three high drop.
Beneficial effects of the present invention：Compared with prior art, the bark of eucommia is inoculated with pupa worm by the present invention
Grass is cultivated, and obtains culture, easy to operate, is worked well, and substantially increases the content of cordycepin in tunning,
There is provided with enhancing is immune, strengthening the bones and muscles, strengthening the essence gas, three high drop and other effects and with developing health food and medicine potentiality
Tunning, there is preferable economy and social value.
With reference to embodiment, the present invention is further illustrated.
Embodiment
Embodiment 1：Cordyceps militaris converts the preparation method of bark of eucommia solid fermentation product, comprises the following steps：
（1）It is prepared by actication of culture and fermented liquid strain：The Cordyceps militaris spawn of preservation is inoculated on PDA culture medium flat board, so
After be put into 25 DEG C of incubators, dark culturing 4-5d, liquid seeds Shaking culture is inoculated in when strain covers with 2/3 scope of flat board,
Shaking table is put into, 4-5d is cultivated under dark condition, treats that strain is covered with bottle, takes out, obtains Cordyceps militaris fermented liquid strain.
（2）Solid fermentation culture medium prepares and optimization：, will be advance according to the mass percent of bark of eucommia powder 20%, rice 80%
The rice and bark of eucommia powder soaked with 20% murphy juice mixes, and adds 0.4% vitamin B2 loaded in can, stirs,
High-pressure sterilizing pot sterilizing 30min is put into, cooling, obtains solid fermentation culture medium.
（3）Solid fermentation and product processing：Treat step（2）The step of 20% being inoculated with after the cooling of solid fermentation culture medium（1）
The Cordyceps militaris fermented liquid strain arrived, stirs, tightens bottle cap, is put into 25 DEG C of incubators, dark culturing 18d, has cultivated
Into rear taking-up tunning, packaged after weighing, be put into 60 DEG C of air dry ovens and dry, beaten powder and cross 80 mesh sieves powder is made.
In the embodiment of the present invention, in order to obtain the optimised process of the Cordyceps militaris solid fermentation conversion bark of eucommia, related process is joined
Number is screened and investigated, and to improve the content of specific active ingredient in tunning, is studied it has also been found that this fermentation process
The content of cordycepin in product can be significantly improved, is described in detail below：
1. materials and methods
1.1 material
1.1.1 strain：Cordyceps militarisCordyceps militaris B1528；
1.1.2 culture medium mainly forms：Bark of eucommia powder, rice, potato.
1.2 method
1.2.1 prepared by need testing solution
After culture terminates, tunning is taken out into 60 DEG C of drying, powder is beaten, crosses 40 mesh sieves.Precision weighs the g of sample 4.0, adds 40
ML chloroform solutions, 30 min of ultrasound, discard chloroform solution, and the dregs of a decoction volatilize, and add 40 mL methanol, 45 min of ultrasound, extract solution evaporation first
Alcohol is moved in 10 mL measuring bottles, adds methanol constant volume to graduation mark to stop, shake up to appropriate, 0.22 μm of filter membrane filtration, takes subsequent filtrate,
Produce.
1.2.2 prepared by mixed reference substance solution
Take Pinoresinol diglucoside some from reference substance bottle, accurately weighed 0.0027 g, add methanol constant volume to 10 mL capacity
In bottle, shake up, reference substance storing solution mg/mL containing Pinoresinol diglucoside 0.2700 is made；Cordycepin is taken from reference substance bottle
0.0023 g is accurately weighed, puts in 25 mL volumetric flasks plus reference substance storing solution is made per mL mg containing cordycepin 0.0920 in methanol.
The mL of Pinoresinol diglucoside reference substance storing solution 1 and the mL of cordycepin reference substance storing solution 0.5 are placed in together after precision measures
In one 10 mL volumetric flask, addition methanol obtains mixed reference substance solution to graduation mark（Pinoresinol diglucoside（PDG）
0.0270 mg/mL, the mg/mL of cordycepin 0.0046）, 4 DEG C save backup.
1.2.3 chromatographic condition
Chromatographic column：Agilent TC-C18（250 mm×4.6 mm,5 μm)；Column temperature:20℃；Detection wavelength:277 nm；Stream
Speed:1.0 mL/min；Sampling volume:10 μL；Mobile phase：Water（A）- acetonitrile（B）, condition of gradient elution：0-10 min, 95%-
85.5%A；10-23 min, 85.5%-85.4%A；23-33 min, 85.4%-95%A.On this condition, each measure mixing pair
According to product and need testing solution, collection of illustrative plates such as Fig. 1.Two kinds of compositions are above 1.5 with adjacent element separating degree, and theoretical cam curve is not then low
In 1000.
1.2.4 optimizing factors experimental design
1.2.4.1 incubation time
In total amount 25g culture medium（Similarly hereinafter）The middle addition 7.5g barks of eucommia, sampling survey in 3,6,9,12,15,18 days after 25 DEG C of cultures are warm
The cordycepin and PDG contents of tunning are measured, investigates the Cordyceps militaris conversion bark of eucommia with the change of incubation time its tunning
The changing rule of principal product content.
1.2.4.2 most suitable filling amount
In total amount 25g culture medium（Similarly hereinafter）Middle bark of eucommia addition is set as 2.5,5.0,7.5,10,12.5 g, 25 DEG C, dark
Lower culture 18d, carry out the influence experiment of cordycepin and PDG yield in different filling amount Cordyceps militaris conversion bark of eucommia tunnings.With
Filter out most suitable filling amount and carry out follow-up test.
1.2.4.3 cultivation temperature
Suitable factor that is filtering out and using is tested with 1.2.4.2 and prepares solid medium and inoculation, be respectively placed in 15 DEG C, 20
DEG C, 25 DEG C, 18d is cultivated in 30 DEG C of constant incubators, take out cordycepin and PDG content in detection tunning.
1.2.4.4 growth factor
The most suitable factor that is being filtered out with 1.2.4.3 and using manufactures solid medium, and addition VA, VB1, VB2, VC, VE make a living
The long factor, the same 1.2.4.3 of condition of culture, carry out different growth factors to Cordyceps militaris convert bark of eucommia tunning in cordycepin and
The influence of PDG yield.Follow-up test is carried out to filter out the optimum growh factor.
1.2.4.5 most suitable inoculum concentration
Factor of influence that is being filtered out using 1.2.4.4 and using as culture medium prescription, set inoculum concentration be 20%, 40%, 60%,
80%, the influence that different vaccination amount converts cordycepin and PDG contents in bark of eucommia tunning to Cordyceps militaris is carried out, condition of culture is same
1.2.4.4。
1.2.5 data statistics and analysis
Measurement result carries out significance difference analysis using SPSS16.0 softwares.
2 experimental results
2.1 incubation times optimize
At first 9 days of culture, cordycepin was in increasing trend, then first drops and rise afterwards.Reduced at the 12nd day.Cordycepin content was at the 18th day
Highest, up to 29.3mg/g.From to looking after, the addition bark of eucommia also significantly increases the content of cordycepin（It is shown in Table 1）.It follows that
It is to improve the effective ways of cordycepin that the bark of eucommia is added in culture matrix.
The investigation that 1 different incubation times of table influence on cordycepin content
2. 2 fillers（The bark of eucommia）Amount optimization
The result of the test of amount of filler optimization shows（Table 2）, when inoculum concentration is 2.5（10%）When, cordycepin content highest, it is
141.4mg/g, it is significantly higher than other groups（P ＜ 0.05）, the growing way of strain is preferably also, and hyphae length is most, but in view of in product
Another active component PDG, therefore subsequent experimental selection amount of filler is 5g（20%）, cordycepin is also significantly greater than when amount of filler is 7.5
Control（P ＜ 0.05）.
The investigation that 2 different amount of filler of table influence on cordycepin and PDG contents
The optimization of 2.3 cultivation temperatures
Influence of the cultivation temperature to cordycepin in product is very big.Cultivation temperature from when being increased to 25 DEG C for 15 DEG C, cordycepin content with
The rise of temperature and increase, cordycepin content is up to 159.2mg/g when cultivation temperature is 25 DEG C, more obvious than control yield
Increase（P ＜ 0.05）, be significantly higher than control and other.Continue to raise with cultivation temperature, cordycepin content is reduced therewith（See
Table 3）.
The investigation that the different temperatures of table 3 influences on cordycepin and PDG contents
2.4 growth factors optimize
Influence of the growth factor to cordycepin in tunning is shown in Table 4.Growth factor has significant promote to cordycepin synthesis
Effect, wherein vitamin B2（VB2）It is the optimum growh factor of cordycepin synthesis, under its effect, cordycepin contains in product
Amount also has good rush cordycepin synthesis up to 199.1mg/g, VE.From to looking after, worm is also greatly facilitated in VA addition
The generation of careless element, generally speaking generation of the different growth factors to cordycepin have active influence, and highly significant.
The investigation that 4 different growth factors of table influence on cordycepin and PDG contents
2.5 inoculum concentrations optimize
How much the inoculum concentration of solid fermentation culture influences humidity in culture medium, dissolved oxygen amount etc., and then influences the conjunction of metabolite
Into.The influence that inoculum concentration synthesizes to cordycepin in tunning is shown in Table 5, and inoculum concentration is excessively unfavorable for the synthesis of cordycepin, inoculation
Measure for 20% when most beneficial for cordycepin generation, can reach 285.4mg/g.
The investigation that the different vaccination amount of table 5 influences on cordycepin and PDG contents
3. experiment conclusion
The property of medicine matrix bark of eucommia is added in Cordyceps militaris solid culture, it is possible to achieve conversion to the bark of eucommia and to yield of Cordycepin
Effect is obviously promoted, the active ingredient of the bark of eucommia had both been remained in tunning, also substantially increases the content of cordycepin, wherein
Different condition and acting for experiment factor pair cordycepin are significantly different, are trained by adding the bark of eucommia, growth factor and optimization
Foster condition can be at double increase cordycepin content, such as addition 10% the bark of eucommia when, the output increased of cordycepin 13 times
More, the addition bark of eucommia+vitamin B2 is even more the content that cordycepin greatly improved, and reaches 199.1 mg/g, finally by optimization
Yield of Cordycepin be up to 285.4mg/g, wherein addition vitamin B2 effect it is larger, significantly carried than having before optimization
Rise, this result provides technology and theories integration to produce the solid fermentation product of high quality.
Claims (3)
1. a kind of preparation method of Cordyceps militaris conversion bark of eucommia solid fermentation product, it is characterised in that comprise the following steps：
It is prepared by actication of culture and fermented liquid strain：The Cordyceps militaris spawn of preservation is inoculated on PDA culture medium flat board, Ran Houfang
Enter 25 DEG C of incubators, dark culturing 4-5d, liquid seeds Shaking culture is inoculated in when strain covers with 2/3 scope of flat board, is put into
Shaking table, 4-5d is cultivated under dark condition, treats that strain is covered with bottle, take out, obtain Cordyceps militaris fermented liquid strain；
Solid fermentation culture medium prepares and optimization：, will be in advance with 20% soil according to the mass percent of bark of eucommia powder 20%, rice 80%
The rice of fermented bean drink immersion and the mixing of bark of eucommia powder, 0.4% vitamin B2 is added loaded in can, stirs, is put into high pressure
Autoclave sterilizing 30min, cooling, obtains solid fermentation culture medium；
Solid fermentation and product processing：Treat step（2）The step of 20% being inoculated with after the cooling of solid fermentation culture medium（1）Obtained pupa
Chinese caterpillar fungus fermentation liquid strain, stirs, tightens bottle cap, is put into 25 DEG C of incubators, dark culturing 18d, is taken after the completion of culture
Go out tunning, packaged after weighing, be put into 60 DEG C of air dry ovens and dry, beat powder and cross 80 mesh sieves powder is made.
2. a kind of Cordyceps militaris converts bark of eucommia solid fermentation product, it is characterised in that：According to claim 1, method obtains, should
In tunning, containing cordycepin 285.4mg/g, contain Pinoresinol diglucoside 25.2mg/g.
3. Cordyceps militaris conversion bark of eucommia solid fermentation product described in claim 2 prepare enhancing immune, strengthening the bones and muscles, strengthening the essence gas,
Application in three high drop health food or medicine.
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| CN111494255A (en) * | 2020-06-03 | 2020-08-07 | 广东芭薇生物科技股份有限公司 | Composition containing ginseng cordyceps sinensis fermentation extract and application of composition in cosmetics |
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