CN106967617A - A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and numerous use culture medium and preparation method thereof soon - Google Patents
A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and numerous use culture medium and preparation method thereof soon Download PDFInfo
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- CN106967617A CN106967617A CN201710176481.5A CN201710176481A CN106967617A CN 106967617 A CN106967617 A CN 106967617A CN 201710176481 A CN201710176481 A CN 201710176481A CN 106967617 A CN106967617 A CN 106967617A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention belongs to field of microbial culture technology there is provided a kind of separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and fast numerous use culture medium, by being prepared including following raw material components：Potato, quinoa, glucose, agar, distilled water.Invention also provides the preparation method of the culture medium.Learnt from result of the test, the agalloch eaglewood endogenetic fungus quantity showed increased that culture medium of the present invention is separated to, agalloch eaglewood endogenetic fungus can reach maximum growth amount in a short time in liquid shakes bacterium culture, and the bacterium time is shaken so as to substantially reduce.And this medium nutrient content all is from natural food materials food materials（Potato, quinoa）Plus glucose, without any other additive, to as entrance cure the disease medicinal material agalloch eaglewood Edgeworthia chrysantha bacterium culture it is particularly important, environmental protection, and preparing easy to be economical and practical.
Description
Technical field
The invention belongs to field of microbial culture technology, and in particular to one kind is used to separate agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and real
The culture medium now quickly bred, is a kind of environmental protection culture medium suitable for agalloch eaglewood Edgeworthia chrysantha bacterium industrialization production, is related to simultaneously
The preparation method of the culture medium.
Background technology
Agalloch eaglewood is that suspension culture of Aquilaria sinensis tree Aquilaria sinensis (Lour.) Gilg that Isolated From Thymelaeaceae Species agalloch eaglewood belongs to contains resin
Timber.Since ancient times, agalloch eaglewood is considered a kind of rare spice and Chinese medicine, has had in the Song Dynasty " one or two agalloch eaglewood one or two gold medals "
Saying, with very high medical value and commercial value.Agalloch eaglewood has the effect of promoting qi circulation and relieving pain, warming middle energizer to arrest vomiting, gas of receiving are relievingd asthma.Its
It is distributed in the ground such as Guangdong, Hainan, Guangxi, Fujian, happiness is born in the mountain region of low altitude area, at hills and roadside sun in sparse woods.In recent years
Come, due to the reasons such as agallochum natural propagation rate is low, artificial predation formula felling, Home-made occluder yield wretched insufficiency, and market is needed
Continuous expansion is asked, imbalance between supply and demand is protruded.Agalloch eaglewood has been put into national second class protection endangered species.
Prevailing paradigm is thought typically produce agallochum fat naturally in the suspension culture of Aquilaria sinensis tree of nature, only by physico
Damage or some biological stimulations, suspension culture of Aquilaria sinensis tree can secret out of special tree for the immune repair mechanism of itself, cell
Fat protects oneself, with endophyte secondary metabolite together form agalloch eaglewood, and its natural cumulative process needs very long tens of
Year, and quality is uncontrollable.With going deep into for research, many researchs existing at present confirm the formation and the infection of its endophyte of agalloch eaglewood
Relevant, these agalloch eaglewood endophytes can promote suspension culture of Aquilaria sinensis tree Edgeworthia chrysantha.We are from wild agallochum Edgeworthia chrysantha wood tissue, healthy whitewood's group
Knit and agalloch eaglewood endogenetic fungus is extracted and isolated in leaf tissue, providing fungi flora for next step research agalloch eaglewood Edgeworthia chrysantha mechanism supplies
Try material.The composition of most of fungi culture medium announced at present addition is a lot, wherein some compositions added come from
Chemical synthesis, and the one of important use of agalloch eaglewood is that, as rare Chinese medicine, these process obtained composition by chemical extraction
Whether harmful effect can be produced to health on the knees of the gods.As Chinese patent (CN105331560A) discloses a kind of for luring
The endophyte fluid nutrient medium of agalloch eaglewood Edgeworthia chrysantha is led, is formulated and is：Potato 70%, wood chip 17%, wheat bran 5%, corn flour 4%, Huang
Bean powder 1%, peptone 0.5%, glucose 1%, phosphoric acid the second light industry bureau potassium 0.2%, magnesium sulfate 0.2%, vitamin 0.1%.
Microbial growth speed is relevant with strain, cell age, inoculum concentration and condition of culture, if the nutrition of culture medium into
Divide the need for meeting growth, then strain is easily adapted to very much, can play a part of shortening lag phase, its growth rate can add
It hurry up, the time into exponential phase (growth rate reaches highest period, and bacterium number is increased with geometric progression) can greatly shorten.
Although microorganism for when it is different because of kind, but same kind is also influenceed by medium component.In natural environment, many bacterium
The growth rate planted can not be increased with geometric progression, and be increased with arithmetic series, because these strains need certain must
The growth factor needed, such as vitamin, these growth factors can influence growth rate.And in natural environment, growth factor
Concentration is extremely low, is spent once by bacterial reproduction after a part, will influence the bacterial growth rate of next step, thus causes arithmetic stage
Number growth phenomenon.
Quinoa, originates in South America andes region, is print plus the main conventional food of aborigines, there is 5000-7000
Edible and plantation history for many years, because it has unique abundant, comprehensive nutritive value, has brought up print plus nationality, ancient times
Inca is referred to as " mother of grain ".Quinoa is used for the space food of astronaut by NASA in the 1980's.The United Nations
Food and agricultural organization thinks that quinoa is that a kind of unique solitary plant can substantially meet the food of human body basic nutrition demand, formal recommendation
Quinoa is the perfect wholefood of the optimum mankind.Many countries were all greatly developing quinoa plantation in recent years, Chinese
Also some quinoa specialized companies cooperate with South America for specialized farming company, and similar South America quinoa grows the region of weather at home
Large-scale plantation lamb's-quarters is started.Quinoa plumule accounts for the 30% of seed, and with nutritional activities, the protein content of quinoa is up to
16%-22% (beef 20%), quality is suitable with milk powder and meat, rich in several amino acids, wherein having whole 9 necessary to human body
Kind of essential amino acid, ratio is appropriate and is easy to absorb, and is especially enriched in the lysine lacked in plant, calcium, magnesium, phosphorus, potassium, iron, zinc,
The mineral matter nutritional such as selenium, manganese, copper content is high, rich in unrighted acid, flavonoids, B family vitamin and E family vitamins, choline,
The multiple beneficial compound such as glycine betaine, folic acid, alpha-linolenic acid, beta glucan.The characteristic of quinoa can be applied to microorganism for it
Laid a good foundation in culture medium, because quinoa is nutritious comprehensively, a variety of conventional constituents in conventional medium can be substituted and met
Microorganism is quickly the need for breeding.As Chinese patent (CN104718987A) discloses a kind of with quinoa puffing material and quinoa crushing
The method for expecting to carry out fermenting and producing for culture medium, inoculation cordyceps sinensis and Cordyceps militaris seed liquor, expanded quinoa and mechanical crushing lamb's-quarters
Fermented after wheat mixing, on the one hand allow thalline quickly to utilize the nutriment produced by puffing process, on the other hand
Mechanical crushing quinoa is the equal of that solid state fermentation carrier makes fermentation substrate keep loose favourable thalli growth, when thalli growth is vigorous
When thalline produce a large amount of ectoenzymes and can further decompose utilization mechanical crushing quinoa again.The process need not add other materials,
Ensure that the quality of fermented product is highly controllable.
The content of the invention
It is an object of the invention to provide a kind of environmental protection, health it is harmless to humans, suitable for agalloch eaglewood Edgeworthia chrysantha Nei Shengzhen
Bacterium separates and fast numerous culture medium, and the culture medium is mainly equipped with other auxiliary materials by natural material potato, quinoa and is prepared from, makes
It is standby easy, economical and practical.
Specifically, agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation of the present invention and fast numerous use culture medium, by including following parts by weight
The raw material components of number ratio are prepared：100-200 parts of potato, 15-40 parts of quinoa, 15-25 parts of glucose, 0-20 parts of agar,
700-900 parts of distilled water.
Invention also provides agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and the preparation method of fast numerous use culture medium, including
Following steps：
(1) potato is peeled, cut into slices after mixed with water, be beaten, screenings separation, obtain potato juice；Or cut potato
Add water and boil after skin, stripping and slicing, screenings separation obtains potato juice；
(2) quinoa is soaked in water overnight, mashing, screenings separation obtains quinoa juice；
(3) potato juice and quinoa juice are mixed, adds glucose sugar, agar (being added when preparing solid medium) and distillation
Water, is mixed, and regulation pH is 5.8-6.0,121 DEG C of sterilizing 20min, is produced.
Separate and further illustrated with numerous preparation method with culture medium soon as agalloch eaglewood Edgeworthia chrysantha endogenetic fungus of the present invention, institute
The weight ratio for stating potato and water is 1:3-8；The potato is peeled, the boiling time that adds water after stripping and slicing is 25-35min；It is described
Quinoa is soaked in water the time for 8-24h；The quinoa and the weight of water ratio are 1:3-8.
The quinoa added in culture medium of the present invention, it is nutritious comprehensive, rich in more than 40 kinds of nutriments, including a variety of ammonia
Base acid, mineral matter, the compound of vitamin and multiple beneficial, can provide the growth of agalloch eaglewood endogenetic fungus comprehensively and quickly breed
Need.Learnt from result of the test, the agalloch eaglewood endogenetic fungus quantity showed increased that culture medium of the present invention is separated to, bacterium training is shaken in liquid
Agalloch eaglewood endogenetic fungus can reach maximum growth amount in a short time in supporting, and the bacterium time is shaken so as to substantially reduce.And this training
Support base nutritional ingredient and all be from natural food materials food materials (potato, quinoa) plus glucose, without any other additive, to doing
For entrance cure the disease the agalloch eaglewood Edgeworthia chrysantha bacterium of medicinal material culture it is particularly important, environmental protection, and preparing easy to be economical and practical.
Embodiment
The present invention is described in further detail with reference to embodiments, the present embodiment is only to make clearer to the present invention
Illustrate, rather than limitation of the present invention.It should be pointed out that for the person of ordinary skill of the art, not departing from this hair
On the premise of bright design, various modifications and improvements can be made, these are within the scope of the present invention.
The separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and fast numerous use culture medium in embodiment, by the original for including following ratio of weight and number
Material component is prepared：100-200 parts of potato, 15-40 parts of quinoa, 15-25 parts of glucose, 0-20 parts of agar, distilled water
700-900 parts.Preparation method comprises the following steps：
(1) potato is peeled, cut into slices after with water by weight be 1:3-8 is mixed, mashing, screenings separation, obtains potato
Juice；Or potato is peeled, after stripping and slicing plus the water of 3-8 times of weight boils 25-35min, screenings separation obtains potato juice；
(2) quinoa is soaked into 8-24h with the water of 3-8 times of weight, mashing, screenings separation obtains quinoa juice；
(3) potato juice and quinoa juice are mixed, adds glucose sugar, agar (being added when preparing solid medium) and distillation
Water, is mixed, and regulation pH is 5.8-6.0,121 DEG C of sterilizing 20min, is produced.
Below by more specific embodiment, the present invention will be described.
Embodiment 1
A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and fast numerous use culture medium, preparation method comprise the following steps：
(1) potato is peeled, cut into slices, it is 1 by weight with water to weigh 200g:6 mixing, are beaten with breaking-wall cell machine,
Screenings is separated by filtration, potato juice is obtained；
(2) 10g quinoas are weighed and soak 12h with the water of 6 times of weight, then are beaten with breaking-wall cell machine, screenings is separated by filtration, obtains
Quinoa juice；
(3) potato juice and quinoa juice are mixed, adds glucose sugar 20g, agar 15g, it is fixed with distilled water fully after dissolving
Hold to 1000ml, mix, regulation pH is 5.8,121 DEG C of sterilizing 20min, is produced.
Embodiment 2
A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and fast numerous use culture medium, preparation method comprise the following steps：
(1) potato is peeled, stripping and slicing, weigh 200g plus 8 times of water and boil 30min, be then beaten with breaking-wall cell machine, mistake
Filter separation screenings, obtains potato juice；
(2) 20g quinoas are weighed and soak 16h with the water of 5 times of weight, then are beaten with breaking-wall cell machine, screenings is separated by filtration, obtains
Quinoa juice；
(3) potato juice and quinoa juice are mixed, adds glucose sugar 20g, agar 10g, it is fixed with distilled water fully after dissolving
Hold to 1000ml, mix, regulation pH is 6.0,121 DEG C of sterilizing 20min, is produced.
Example 3 below -8 and the preparation process of above-described embodiment 1,2 are basically identical, the difference is that only raw material components
The difference of consumption, therefore, preparation process is not repeated.
Embodiment 3：Potato 200g, quinoa 30g, agar 15g, glucose 20g, distilled water complement to 1000ml.
Embodiment 4：Potato 150g, quinoa 15g, agar 15g, glucose 20g, distilled water complement to 1000ml.
Embodiment 5：Potato 150g, quinoa 25g, agar 20g, glucose 20g, distilled water complement to 1000ml.
Embodiment 6：Potato 150g, quinoa 35g, agar 15g, glucose 20g, distilled water complement to 1000ml.
Embodiment 7：Potato 100g, quinoa 30g, agar 15g, glucose 20g, distilled water complement to 1000ml.
Embodiment 8：Potato 100g, quinoa 40g, agar 20g, glucose 20g, distilled water complement to 1000ml.
Solid medium obtained by embodiment 1-8 as isolation medium, with add agar solid PDA medium and
SDA agar mediums are as control medium, using tissue isolation to agalloch eaglewood Edgeworthia chrysantha wood tissue, healthy whitewood's tissue and leaf
The endogenetic fungus of piece is separated, and the wood tissue of surface sterilization is removed into outer weekly form with sterile razor blade on superclean bench
Layer wood tissue, is cut into long a width of 3cm × 2cm flake, long a width of 2cm × 2cm sizes is cut into after blade sterilization, are put into
State on culture medium flat plate, 28 DEG C of lucifuge cultures, 3 repetitions are set.Separated endogenetic fungus is purified repeatedly, to obtaining
Single bacterium colony is counted, and is counted isolated endogenetic fungus quantity, be the results are shown in Table 1.
Separating effect of the embodiment 1-8 culture mediums of table 1 to agalloch eaglewood endogenetic fungus
The fluid nutrient medium conduct as made from embodiment 1-8 preparation process, raw material components consumption (except without agar)
Fast breeding culture medium, correspondence embodiment 9-16.
The endogenetic fungus 3 being separated in wild agallochum Edgeworthia chrysantha wood tissue is randomly selected as examination strain is supplied, each
Picking single bacterium colony is inoculated into (liquid in embodiment 9-16 culture mediums and two control mediums respectively after the independent plate streaking of strain
PDA culture medium and SDA culture mediums), 50ml centrifuge tube with cover dress 20ml culture mediums (every part do two parallel), 200 turns 28 DEG C are shaken
Bacterium is cultivated 7 days.Its respective biomass, specific practice are determined with dry weight method：After nutrient solution is filtered with filter paper, the bacterium stayed on paper
Filament is first washed with proper amount of clear water, and 80 DEG C of drying are weighed.This experiment carries out three repetitions, averages.It the results are shown in Table 2.
The embodiment 9-16 culture mediums of table 2 compare the increment of different strain
Embodiment 1-8 culture mediums are measured by 3 different parts endogenetic fungus isolation and purification cultures as seen from Table 1
Isolated strains sum apparently higher than control medium, improve more than 60%.Embodiment 9-16 culture mediums pass through 3 as seen from Table 2
Plant different fungies to carry out shaking within 7 days bacterium culture, measured thalli growth amount improves about 20- apparently higher than control medium
40% or so.
Claims (6)
1. a kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and fast numerous use culture medium, it is characterised in that by including following ratio of weight and number
Raw material components be prepared：100-200 parts of potato, 15-40 parts of quinoa, 15-25 parts of glucose, 0-20 parts of agar, distillation
700-900 parts of water.
2. agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation as claimed in claim 1 and the preparation method of fast numerous use culture medium, it is characterised in that
Comprise the following steps：
（1）Potato is peeled, cut into slices after mixed with water, be beaten, screenings separation, obtain potato juice；Or potato is peeled, cut
Add water and boil after block, screenings separation obtains potato juice；
（2）Quinoa is soaked in water overnight, mashing, screenings separation obtains quinoa juice；
（3）Potato juice and quinoa juice are mixed, glucose sugar, agar and distilled water is added, mixes, regulation pH is 5.8-6.0,
121 DEG C of sterilizing 20min, are produced.
3. the separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and the soon preparation method of numerous use culture medium according to claim 2, its feature exist
In the weight ratio of the potato and water is 1:3-8.
4. the separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and the soon preparation method of numerous use culture medium according to claim 2, its feature exist
In the potato is peeled, the boiling time that adds water after stripping and slicing is 25-35min.
5. the separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and the soon preparation method of numerous use culture medium according to claim 2, its feature exist
In the quinoa is soaked in water the time for 8-24h.
6. the separation of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus and the soon preparation method of numerous use culture medium according to claim 2, its feature exist
In the weight ratio of the quinoa and water is 1:3-8.
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| CN201710176481.5A CN106967617A (en) | 2017-03-23 | 2017-03-23 | A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and numerous use culture medium and preparation method thereof soon |
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| CN201710176481.5A CN106967617A (en) | 2017-03-23 | 2017-03-23 | A kind of agalloch eaglewood Edgeworthia chrysantha endogenetic fungus separation and numerous use culture medium and preparation method thereof soon |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894475A (en) * | 2019-12-31 | 2020-03-20 | 广东海洋大学 | Method for separating agilawood bark rot pathogenic bacteria by using host matrix |
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| CN101731282A (en) * | 2009-12-02 | 2010-06-16 | 中国医学科学院药用植物研究所 | Tambac inducer and preparation method thereof |
| CN102696690A (en) * | 2012-06-15 | 2012-10-03 | 唐显 | Flora and method for producing Chinese eaglewood wood on Aquilaria senensis (Lour.) Gilg by bottle interpolation method |
| CN103027081A (en) * | 2012-12-25 | 2013-04-10 | 常州亚当生物技术有限公司 | Artificial Chinese eaglewood forming agent |
| CN104718987A (en) * | 2015-03-20 | 2015-06-24 | 江南大学 | Cordyceps sinensis chenopodium quinoa |
| US20150305249A1 (en) * | 2014-04-23 | 2015-10-29 | Functional Fungi, Llc. | Nutritionally and botanically enhanced mycelial mass |
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2017
- 2017-03-23 CN CN201710176481.5A patent/CN106967617A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101731282A (en) * | 2009-12-02 | 2010-06-16 | 中国医学科学院药用植物研究所 | Tambac inducer and preparation method thereof |
| CN102696690A (en) * | 2012-06-15 | 2012-10-03 | 唐显 | Flora and method for producing Chinese eaglewood wood on Aquilaria senensis (Lour.) Gilg by bottle interpolation method |
| CN103027081A (en) * | 2012-12-25 | 2013-04-10 | 常州亚当生物技术有限公司 | Artificial Chinese eaglewood forming agent |
| US20150305249A1 (en) * | 2014-04-23 | 2015-10-29 | Functional Fungi, Llc. | Nutritionally and botanically enhanced mycelial mass |
| CN104718987A (en) * | 2015-03-20 | 2015-06-24 | 江南大学 | Cordyceps sinensis chenopodium quinoa |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894475A (en) * | 2019-12-31 | 2020-03-20 | 广东海洋大学 | Method for separating agilawood bark rot pathogenic bacteria by using host matrix |
| CN110894475B (en) * | 2019-12-31 | 2022-09-27 | 广东海洋大学 | Method for separating agilawood bark rot pathogenic bacteria by using host matrix |
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Application publication date: 20170721 |