CN109136098A - A kind of culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium and method - Google Patents
A kind of culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium and method Download PDFInfo
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Abstract
The invention discloses a kind of culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium and methods.Contain tea water extract, potato leachate, distilled water, trehalose, CaCl in culture medium2And NaCl；The volume ratio of the tea water extract, potato leachate and distilled water is (1.5-2.5): (2.5-3.5): (2.5-3.5) composition；The content of trehalose is 0.4-0.6g in every 1000mL culture medium；CaCl in every 1000mL culture medium2Content be 0.5-1.5g；The content of every 1000mL NaCl in medium is 25-35g；The tea water extract the preparation method comprises the following steps: 150-250g green teas are boiled 10-20min with 1000mL distilled water, filter and remove residue is up to tea water extract；The potato leachate the preparation method comprises the following steps: 150-250g potatoes are boiled 10-20min with 1000mL distilled water, filter and remove residue is up to potato leachate.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of culture for being suitable for being separately cultured coronoid process dissipate capsule bacterium
Base and method.
Background technique
Dark green tea is one of six big basic teas, belongs to post-fermented tea, can passage at any time be aged at leisure, be aromatic,
There is aid digestion weight-reducing and supplements each biostearin, microelement.Dark green tea sold to northwest, southwestern city as border-sale tea majority in the past
, the crowd of drinking is mainly the food rich in fat such as utility beef, mutton, cheese and the borderland minority people for lacking veterinary antibiotics
Race.Such as Fu tea hides tea, Pu'er stub border areas always.Side pin feature most will significantly count Fu-brick tea.Recently as right
The lipid-loweringing of dark green tea, the understanding and research of blood pressure lowering and effect of weight reducing, dark green tea TaiWan, China, Japan and China's wide geographic area by
Consumer payes attention to and likes.
Dark green tea is in numerous tea types, the coronoid process dissipate capsule bacterium (Eurotium cristatum) that is shown unique characteristics with it and prominent
Lower blood-fat and reduce weight effect and well-known, the size of coronoid process dissipate capsule bacterium out, how much, it widely distributed or not is most directly to judge to obtain brick tea
The standard of quality.Due to the presence of coronoid process dissipate capsule bacterium, so that the color for obtaining brick tea is able to be different from other teas, and have
There are anti-oxidant rush digestion lower blood-fat and reduce weight, antibacterial, anticancer health-care effect, makes the functional study of people's pay attention to day by day coronoid process dissipate capsule bacterium.
Coronoid process dissipate capsule bacterium in dark green tea ----it is commonly called as " golden flower ", the one kind for belonging to Eurotiale Trichocomaceae Eurotium is true
Bacterium, can be grown in the substratess such as soil, tealeaves, cordyceps sinensis, Chinese medicine tablet and Fu-brick tea is under specific temperature and humidity conditions, lead to
Cross the naturally prebiotic thallus that " floating " technique grows up to.In national standard, Fu-brick tea be sole requirement " coronoid process dissipate capsule bacterium " this
The dark green tea veriety of index.Coronoid process dissipate capsule bacterium is the epigamous of coronule aspergillus (Aspergilus cristatellus), in tealeaves
" golden flower " of middle formation has a kind of light faint scent taste of chrysanthemum；And when steeping drink, that fragrance of a flower just incorporates among millet paste, is turned into
The flavour of tea and make the more mellow micro-puckery of millet paste, it is pure it is not thick, mouthfeel is powerful.
A large amount of research and document prove, in tea " golden flower ", energy dissolved fat, and defatting beauty.It is new old with promoting to adjust
Metabolism, health care and pathology prevention effect.Because of its drug effect such as smilax, in addition the special mouthfeel of Fu tea, is named, therefore with " Fu " word
Someone takes for the ingredient for having Poria cocos in Fu tea, is arohid flavour and its unique pharmacological action of " golden flower " in fact." golden flower " is right
Post-fermented tea quality does not influence not only, and " golden flower " can secrete amylase and oxidizing ferment in tealeaves instead, can be catalyzed in tealeaves
Starch Conversion be monosaccharide, catalysis polyphenol compound matter oxidation makes tealeaves soup look become reddish brown, eliminates thick green taste.On taste
More pure and mild tasty and refreshing, sweet tea slides back to sweet.Digestion is promoted for coronoid process dissipate capsule bacterium in recent years, reducing blood lipid, dissolved fat, adjusts carbohydate metabolism
Become hot spot etc. various efficacy studies.The physiological property of separation, culture, bacterium in relation to coronoid process dissipate capsule bacterium, function and related
The research of component metabolic product is more, and for dark green tea industry, the breeding and industrialization of the coronoid process dissipate capsule bacterium of high-quality and more effects are trained
It supports and is also important research contents.
Liu Shiquan etc. using improvement PDA culture medium plate gradient dilution method to " Jin Xiangyi ', board obtain the fungi in brick tea into
It has gone and has isolated and purified and count.Experiment isolates to have obtained 5 kinds of fungies, and wherein dominant bacteria is wax leaf bulk bacteria (Eurotium
herbariorum ).Huang Hao etc. is trained " golden flower " bacterium isolated and purified in brick tea is obtained from golden Hunan benefit board in 2008 in PDA
It supports and is cultivated on base, and choose preferable " golden flower " bacterium of growing way and carry out squamous subculture.It is transferred and is cultivated in 20% sucrose Cha Shi later
On base and Czapek's medium, 28 DEG C of constant temperature are cultivated and observe, record, and are finally accredited as scattered Saccharomyces (Eurotium link
Ex Fires) coronoid process dissipate capsule bacterium.Liu, which makees easily to wait, uses PDA, and CZqM3Y, M40Y and 40% sucrose albumen arteries and veins culture medium dissipate capsule to coronoid process
Bacterium is cultivated, and observation colony characteristics are passed through, it was demonstrated that and coronoid process dissipate capsule bacterium has secreted brown water colo(u)r in reproductive process,
These pigments can improve millet paste color, while strong fragrance is generated in well-grown, reduce tea while increasing tea perfume
Grass smell in leaf improves tea leaf quality.Poplar comforts woods etc. and the growth conditions of coronoid process dissipate capsule bacterium is studied and illustrated, right
Research is optimized in " golden flower " bacteria liquid zymotechnique, provides theories integration for coronoid process dissipate capsule bacterium correlative study from now on.
Cao Cong etc. has studied the condition of culture of coronoid process dissipate capsule bacterium solid culture, and discovery PDA and CZG culture medium is suitable for coronoid process dissipate capsule bacterium
Culture.
It is more to the coronoid process dissipate capsule bacterium research in dark green tea in recent years, including the separation to the coronoid process dissipate capsule bacterium in dark green tea, sieve
Choosing, culture even industrialization have more research.But coronoid process dissipate capsule bacterium is separately cultured, use conventional methods mostly and
Means, although what is had has carried out some improvement to culture medium, effect is less desirable.It is summed up the separation of coronoid process dissipate capsule bacterium
Culture there are problems that the following aspects it is difficult with and.
1. from separation screening coronoid process dissipate capsule bacterium is an important job in dark green tea or in relevant environment.Due to the tealeaves having
Storage for many years, is influenced by environmental conditions, the coronoid process dissipate capsule bacterium in tealeaves is in a kind of relative dormancy or is damaged state, to bacterium
Resurrection culture and reparation seem quite important, some need cultivate for a long time or repeatedly culture.Most of separation trainings at present
It supports, traditional agar cultural method of use, effect is bad, and efficiency is lower, and will be too many for mass propgation and screening
It is many and diverse.Need a kind of suitable large-scale training method.
2. coronoid process dissipate capsule bacterium is a kind of bacterium of resource existing for nature, the more ground in China and tea growing areas even lake
The dark green tea of the ground such as south, Guangxi, Yunnan, Hubei, Sichuan, Shaanxi type is all distributed.Type is more, and geographical distribution characteristic is obvious,
Even the areal, coronoid process dissipate capsule bacterium variety classes characteristic the case where.Type or physiology strain for bacterium different in tealeaves
Type, the research of geographical strains model or identification are all badly in need of studying a kind of easy to be suitable, the training method of fast high-flux.
3. the quality of dark green tea is related with the quantity of golden flower floating, on the other hand, the good coronoid process dissipate capsule bacterium of separation screening, and directly
It is inoculated into dark green tea or relevant production environment, plays an important role for improving tea leaf quality.Studying one kind can carry out largely
Bacteria selection, culture, the method for growth test are necessary.
4, currently used concave slide moisturizing culture, the volume of occupancy is big, long time cultivation and control pollution, it is difficult to control
Liquid volume, and cultivated using culture bottle, the space occupied and facility are larger, it is difficult to while progress tens is even up to a hundred
A sample is separately cultured.
5. storing the dark green tea of many years, containing precious good coronoid process dissipate capsule bacterium, physiological activity is poor, is separately cultured more
It is difficult.
6. the screening operation amount of a large amount of populations is big, in production process, as the breeding, holding and evaluation of germplasm, need
Carry out large batch of multiple batches of culture screening.Need to study a kind of suitable Simple culture method.
7. coronoid process dissipate capsule bacterium evaluation and confirmation in pair dark green tea, will be the important indicators of evaluation volt brick tea quality, from tealeaves
It is middle to separate a certain number of coronoid process dissipate capsule bacteriums and carry out characteristic confirmation, it is a job.
8. observation and cultural method can be carried out to incubation in real time by also lacking a kind of, the life situation of bacterium can truly reflect
(avoiding sampling bring error).
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium
And method.
In order to achieve the above object, technical solution provided by the invention are as follows:
Contain tea water extract, potato leachate, distilled water, sea in the culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium
Algae sugar, CaCl2And NaCl；The volume ratio of the tea water extract, potato leachate and distilled water is (1.5-2.5): (2.5-
3.5): (2.5-3.5) composition；The content of trehalose is 0.4-0.6g in every 1000mL culture medium；In every 1000mL culture medium
CaCl2Content be 0.5-1.5g；The content of every 1000mL NaCl in medium is 25-35g；The system of the tea water extract
Preparation Method are as follows: 150-250g green teas are boiled into 10-20min with 1000mL distilled water, filter and remove residue is up to tea water extract；
The potato leachate the preparation method comprises the following steps: 150-250g potatoes are boiled 10-20min with 1000mL distilled water, filter off
Slag is up to potato leachate.
Preferably, the volume ratio of the tea water extract, potato leachate and distilled water is 2:3:3 composition.
Preferably, the content of trehalose is 0.5g in culture medium described in every 1000mL；In culture medium described in every 1000mL
CaCl2Content be 1g；The content of NaCl in medium described in every 1000mL is 30g.
Preferably, the tea water extract the preparation method comprises the following steps: 200g green tea is boiled with 1000mL distilled water
15min, filter and remove residue is up to tea water extract；The potato leachate the preparation method comprises the following steps: 200g potato is steamed with 1000mL
Distilled water boils 15min, and filter and remove residue is up to potato leachate.
The cultural method for being suitable for being separately cultured coronoid process dissipate capsule bacterium includes the following steps:
(1) prepare the culture cup assembly, transparent sealing cup film and magnetic bead for cultivating coronoid process dissipate capsule bacterium；The culture assembly includes bottom
Seat, the pedestal are equipped with several culture cups；A height of 8-the 10mm of the cup of the culture cup, the diameter of culture cup are 6-7mm,
The material of the culture cup is polystyrene；
(2) magnetic powder suspension is added into each culture cup, adds culture medium described in any one of claim 1 to 5；The magnetic
The volume ratio of powder suspension and culture medium is 5:(60-80)；
(3) the golden flower bacterium colony of picking, tea dust powder to inoculation of medium coronoid process dissipate capsule bacterium liquid or from tealeaves；Coronoid process is inoculated with to dissipate
When capsule bacterium solution, the coronoid process dissipate capsule bacterium liquid is 10:(65-85 with the volume ratio of magnetic powder suspension and the mixture of culture medium)；
(4) culture cup for being vaccinated with coronoid process dissipate capsule bacterium liquid is sealed with transparent sealing cup film, culture cup assembly is placed in magnetic force and is stirred
It mixes on device, 2-4d or more is cultivated under the conditions of magnetic agitation rotating speed is 50-70r/min, 26-28 DEG C.
In the process of culture, the sprouting of coronoid process dissipate capsule bacterium, mycelia growth conditions etc. can be observed at any time, and can be done corresponding
Test.Culture cup is taken out from incubator directly, is not required to tear transparent sealing cup film, keeps the sterile isolation in cup,
Culture mesh cup is placed directly on stereomicroscope or inverted microscope objective table, is directly observed in cup with the camera lens of different multiplying
The growing state of coronoid process dissipate capsule bacterium.
When carrying out microexamination, magnetic bead can first be adsorbed to the side of cup in place with bar magnet, can also in observation bar magnet
It is adsorbed, to exclude influence of the magnetic bead to field of view in culture solution.
After having observed, culture cup can be optionally placed again into incubator and be cultivated, it can be according to circumstances and demand real
When truly observe the growth of bacterium in culture cup.
The present invention will be further described below:
The present invention designs and using the culture cup for coronoid process dissipate capsule bacterium culture, can carry out townhouse or yoke plate culture.And it uses
The mode of transparent sealer closing rim of a cup avoids moisture drain evaporation, can be satisfied under coronoid process dissipate capsule bacterium culture long-time controlled condition
Culture.Culture cup selects high-quality polystyrene manufacturing hole cup, and circular is flat, and townhouse or yoke plate is made, incidentally identifies block.Cup
High 8-10mm, diameter 6-7mm.Wall thickness 1mm or so, it is colorless and transparent.Transparent sealing cup film: transparent, presser sensor adherency, it can be ensured that close
Envelope and minimum evaporation, 75-100 DEG C of operating temperature.Rim of a cup is cultivated for closing.
The present invention controllably may be used to culture solution implementation using the method for adding particle magnetic bead in culture cup, with magnetic agitation mode
The microcirculation of tune can meet coronoid process dissipate capsule bacterium culture growth and need micro- oxygen supply condition.Magnetic-particle: stability is good, monodispersed
Magnetic corpuscular, ferromagnetic to nanoscale by micron order or Ferrimagnetic is constituted, magnetic particle is suspended in carrier solution, is a kind of exist
Become that there is very ferromagnetic liquid under externally-applied magnetic field effect, it is not only with new function material magnetic but also with mobility
Material.Magnetic bead is uniformly dispersed, and has superparamagnetism, saturation magnetization 70-80emu/g, magnetic response time < 10s；Partial size is more equal
One, it is in monodisperse；It is good with preferable suspension: magnetic bead partial size 400-600 nm, sedimentation time > 8min；It need to only jiggle
It can quickly mix.It can be used with Magnetic Isolation coordinative composition of equipments；With 20% ethyl alcohol, preservation can be stablized at 2-8 DEG C °.
The present invention provides the method and conditions of suitable coronoid process dissipate capsule bacterium fast high-flux culture being separately cultured.The present invention
Method can once carry out the culture and fast and convenient observation of multisample.Than conventional sampling, smear methods improve 3-5 times of work efficiency
More than.And do not have to sampling, the pollution to culture is avoided, the growth shape of coronoid process dissipate capsule bacterium also can be more really intuitively understood
Condition.Relative to the heat insulating culture of concave slide, the present invention can be difficult to control and keep moisture to avoid slide culture, avoid marginal zone
The desiccation in domain makes condition of culture be easier to control and standardize, and preferably control condition especially needs to cultivate for a long time,
The state that can accomplish closed sterile soft simultaneously, prevents other living contaminants.Especially for the biological and functional conditions of bacterium
Property survey, multisample, many condition and how duplicate culture can be carried out.Control condition is easy to accomplish consistent.Collection magnetic force microballon stir,
Culture and microscope inspection integration can repeatedly carry out culture body, it can be achieved that be not required to sampling and the changes culture body state such as sample preparation
True observation in real time.It is easy to operate quick.
In addition, the present invention can carry out tens to the thousands of simple and effective culture of isolated with normality condition of culture simultaneously,
For separation screening coronoid process dissipate capsule bacteriums a large amount of from dark green tea or the screening test of the related biological nature to coronoid process dissipate capsule bacterium.
Detailed description of the invention
Fig. 1 is not inhale the coronoid process dissipate capsule bacterium growing state observation figure in magnetic powder culture solution；
Fig. 2 is that the observation of coronoid process dissipate capsule bacterium growing state is schemed in culture solution after adsorbing magnetic powder.
Specific embodiment
Coronoid process dissipate capsule bacterium is separately cultured in 1 Fu tea of embodiment.
1. preparing coronoid process dissipate capsule bacterium culture medium: taking 200g tealeaves, boil 15min, filter and remove residue, system with 1000mL distilled water
At tea water extract.200g potato chips is taken, boils 15min, filter and remove residue with 1000mL distilled water.Difference tea water extract: soil
Beans immersion liquid: distilled water is configured to mixed liquor by the volume ratio of 2:3:3, is made into 1000mL, then weighs 0.5g trehalose, 1.0g
The NaCl of CaCl2 and 30.0g.115 DEG C of 20min high pressure sterilizations after dissolution.
The culture hole cup that 2 rows are sterilized is taken, the culture solution that 705 μ L are added and have been sterilized with sterile every glass of pipettor.It adds
Sterile 5 μ L of magnetic bead,
With pincet, the picking golden flower from the benefit Fu-brick tea of Hunan takes out a golden flower bacterium sample, is put into 2.0 centrifuge tubes, adds 1mL physiology
Sample bacteria suspension is made in salt water, vortex oscillation 3min.Again with physiological saline by sample bacteria suspension dilute 5 times, 10 times, 50 times,
100 times.Every kind of dilution adds 2 glasss respectively, every glass plus 10 μ l bacteria suspensions, while being carried out pair with culture tube and slide moisturizing culture
According to.
Culture cup is sealed with transparent sealing cup film, places on magnetic stirring apparatus, is placed in 27 ± 1 DEG C of biochemical cultivation cases together
In, magnetic agitation rotating speed 70r/min, constant temperature incubation.
Culture 36,48,72,96 hours, culture cup is put on inverted microscope observes respectively, at the same with observe
Compare the upgrowth situation of culture.
As a result: respectively with 5,10,20,40 eyepieces, can clearly observe the ascus cultivated into cup and spore germination, bacterium
Silk growth conditions.The sprouting of coronoid process dissipate capsule bacterium, mycelia growth conditions etc. can be observed at any time.Magnetic bead is adsorbed into cup in place with bar magnet
Side, magnetic bead do not influence field of view.See Fig. 1 and Fig. 2.
After having observed, culture cup can be optionally placed again into incubator and be cultivated, it can be according to circumstances and demand real
When truly observe the growth of bacterium in culture cup.
Claims (5)
1. a kind of culture medium for being suitable for being separately cultured coronoid process dissipate capsule bacterium, which is characterized in that soaked in the culture medium containing tealeaves
Liquid, potato leachate, distilled water, trehalose, CaCl out2And NaCl；The tea water extract, potato leachate and distilled water
Volume ratio is (1.5-2.5): (2.5-3.5): (2.5-3.5) composition；The content of trehalose is in every 1000mL culture medium
0.4—0.6g；CaCl in every 1000mL culture medium2Content be 0.5-1.5g；The content of every 1000mL NaCl in medium is
25—35g；The tea water extract the preparation method comprises the following steps: 150-250g green teas are boiled 10-with 1000mL distilled water
20min, filter and remove residue is up to tea water extract；The potato leachate the preparation method comprises the following steps: by 150-250g potatoes use
1000mL distilled water boils 10-20min, and filter and remove residue is up to potato leachate.
2. being suitable for being separately cultured the culture medium of coronoid process dissipate capsule bacterium as described in claim 1, which is characterized in that the tealeaves leaching
The volume ratio of liquid, potato leachate and distilled water is 2:3:3 composition out.
3. being suitable for being separately cultured the culture medium of coronoid process dissipate capsule bacterium as described in claim 1, which is characterized in that every 1000mL institute
The content for stating trehalose in culture medium is 0.5g；CaCl in culture medium described in every 1000mL2Content be 1g；Described in every 1000mL
The content of NaCl in medium is 30g.
4. being suitable for being separately cultured the culture medium of coronoid process dissipate capsule bacterium as described in claim 1, which is characterized in that the tealeaves leaching
Out liquid the preparation method comprises the following steps: 200g green tea is boiled 15min with 1000mL distilled water, filter and remove residue is up to tea water extract；
The potato leachate the preparation method comprises the following steps: 200g potato is boiled 15min with 1000mL distilled water, filter and remove residue is up to potato
Leachate.
5. a kind of cultural method for being suitable for being separately cultured coronoid process dissipate capsule bacterium, which is characterized in that described method includes following steps:
(1) prepare the culture cup assembly, transparent sealing cup film and magnetic bead for cultivating coronoid process dissipate capsule bacterium；The culture assembly includes bottom
Seat, the pedestal are equipped with several culture cups；A height of 8-the 10mm of the cup of the culture cup, the diameter of culture cup are 6-7mm,
The material of the culture cup is polystyrene；
(2) magnetic powder suspension is added into each culture cup, adds culture medium described in any one of claim 1 to 5；The magnetic
The volume ratio of powder suspension and culture medium is 5:(60-80)；
(3) the golden flower bacterium colony of picking, tea dust powder to inoculation of medium coronoid process dissipate capsule bacterium liquid or from tealeaves；Coronoid process is inoculated with to dissipate
When capsule bacterium solution, the coronoid process dissipate capsule bacterium liquid is 10:(65-85 with the volume ratio of magnetic powder suspension and the mixture of culture medium)；
(4) culture cup for being vaccinated with coronoid process dissipate capsule bacterium liquid is sealed with transparent sealing cup film, culture cup assembly is placed in magnetic force and is stirred
It mixes on device, 2-4d or more is cultivated under the conditions of magnetic agitation rotating speed is 50-70r/min, 26-28 DEG C.
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