CN106613358B - Tremella fuciformis strain extraction method - Google Patents
Tremella fuciformis strain extraction method Download PDFInfo
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- CN106613358B CN106613358B CN201611252893.4A CN201611252893A CN106613358B CN 106613358 B CN106613358 B CN 106613358B CN 201611252893 A CN201611252893 A CN 201611252893A CN 106613358 B CN106613358 B CN 106613358B
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- A01G18/00—Cultivation of mushrooms
Abstract
The invention discloses a tremella strain extraction method, which comprises the following steps: selecting the ear, disinfecting, rinsing, wiping, placing under the humidity condition of 55-65% for 4-5 hours, air-drying, and inactivating the incense ash hyphae on the ear. And cutting thin ear pieces at the air-dried ear piece growing points, inoculating the thin ear pieces into a culture medium, and culturing until thick white hyphae with the thickness of 2-4 mm appear at the inoculating points. And (3) performing tube transfer culture on the concentrated white fungus threads until white hair balls are formed, so as to obtain the white fungus strains. According to the method for extracting the tremella fuciformis strains, the incense ash hyphae of the tremella fuciformis are inactivated by controlling the humidity, and meanwhile, the tremella fuciformis strains are extracted by a tissue separation method, only one tube rotation is needed, the purification time is short, and the operation is simple and convenient; meanwhile, the strain characteristics inherit parents, and the quality is stable.
Description
Technical Field
The invention relates to the field of tremella cultivation, and particularly relates to a tremella strain extraction method.
Background
Tremella contains abundant pectin, multiple vitamins, multiple amino acids and glycogen, and has high edible value. The key point for cultivating tremella is to obtain high-yield and high-quality strains. The traditional seed production separation can not solve the problem of strain stability well all the time, and the abnormal conditions of the female parent strain can generally cause poor production or even no harvest in the whole batch every time, thus causing great loss to farmers or enterprises. And the tremella strain matrix is too complex to separate, is not easy to obtain, and has unstable quality. For example, a matrix separation method is commonly used for preparing pure tremella strains, and the method is used for air drying 30-45 days for each separation. Because the strain is separated out and has the fragrant gray hypha due to insufficient air drying time, the activity of the tremella strain is influenced due to overlong air drying time, and the number of times of transferring the tremella sawdust is reduced, so that the number of transferring test tubes is large each time.
Disclosure of Invention
The invention provides a tremella strain extraction method, which aims to solve the technical problems of complex operation and unstable quality of the conventional strain.
The technical scheme adopted by the invention is as follows:
a tremella strain extraction method comprises the following steps:
selecting the ear, disinfecting, rinsing, wiping, placing under the humidity condition of 55-65% for 4-5 hours, air-drying, and inactivating the incense ash hyphae on the ear.
And cutting thin ear pieces at the air-dried ear piece growing points, inoculating the thin ear pieces into a culture medium, and culturing until thick white hyphae with the thickness of 2-4 mm appear at the inoculating points.
And (3) performing tube transfer culture on the concentrated white fungus threads until white hair balls are formed, so as to obtain the white fungus strains.
Further, the air drying conditions of the ear are as follows: and placing the lug in a superclean workbench for 4-5 hours under the condition of gentle wind.
Further, the culture medium comprises potatoes, glucose, agar and water, and is prepared by adding 200g of potatoes, 20g of glucose and 24-26 g of agar into wood chip culture medium incense ash hypha decoction water extract to reach a constant volume of 1000ml, sterilizing and cooling.
Further, the culture medium is prepared by adding 200g of potato, 20g of glucose and 25g of agar into the wood chip culture medium incense ash hypha boiling water extract to reach the volume of 1000ml, sterilizing and cooling.
Furthermore, the size of the thin ear is 0.2mm multiplied by 0.05mm, and the thickness of the thin ear is 0.1-1 mm.
Further, the thin ear is inoculated into a culture medium in a test tube, and 2-4 pieces are inoculated in each test tube.
Further, the thin ear is cultivated at 22-25 ℃ for 15-17 days to obtain thick white hyphae.
Further, dividing the thick white hyphae into 2mm multiplied by 3mm, rotating the tubes, and culturing for 7-12 days to obtain white hair clusters.
Further, the ear is disinfected by 73-78% alcohol, rinsed for 4-6S by 73-78% alcohol, and rinsed twice by sterile water for 4-6S each time.
The invention has the following beneficial effects: according to the method for extracting the tremella fuciformis strains, the incense ash hyphae of the tremella fuciformis are inactivated by controlling the humidity, and meanwhile, the tremella fuciformis strains are extracted by a tissue separation method, only one tube rotation is needed, the purification time is short, and the operation is simple and convenient; meanwhile, the strain characteristics inherit parents, and the quality is stable.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph of thin ear plates of a preferred embodiment of the present invention inoculated into the medium at day 5;
FIG. 2 is a graph of thin ear plates inoculated into the medium at day 10 in accordance with a preferred embodiment of the present invention
FIG. 3 is a graph of a thin tab of a preferred embodiment of the present invention inoculated into the medium at day 15;
FIG. 4 is a diagram showing the culture on day 6 after the transfer of the hyphae of the mycelia according to the preferred embodiment of the present invention.
Detailed Description
The embodiments of the invention will be described in detail below with reference to the drawings, but the invention can be implemented in many different ways as defined and covered by the claims.
The preferred embodiment of the invention provides a tremella strain extraction method, which comprises the following steps:
selecting the ear, disinfecting, rinsing, wiping, placing under the humidity condition of 55-65% for 4-5 hours, air-drying, and inactivating the incense ash hyphae on the ear.
And cutting thin ear pieces at the air-dried ear piece growing points, inoculating the thin ear pieces into a culture medium, and culturing until thick white hyphae with the thickness of 2-4 mm appear at the inoculating points.
And (3) performing tube transfer culture on the concentrated white fungus threads until white hair balls are formed, so as to obtain the white fungus strains.
Selecting white tremella with round shape, uniform and white leaves and fast growth and 3-5 cm diameter as a separation seed source, and selecting 3-5 tremella pieces with strong vitality from the tremella pieces to extract tremella strains. The ears may be conventionally sterilized and rinsed and wiped dry. Sterile gauze can be used for wiping. The incense ash hypha is associated hypha of Tremella, and is inactivated to obtain pure Tremella strain. The incense ash hyphae need to survive under a high humidity condition, and when the incense ash hyphae are placed under a humidity condition of 55-65% for 4-5 hours for air drying, the incense ash hyphae can be dried and dead, and the tremella fuciformis strains cannot be affected.
The appearance of the ear is air-dried, the ear is taken down and cut into small pieces at the growing point, and the growing point is cut into thin points as much as possible. The thin ear pieces are fully adsorbed on the culture medium to absorb the nutrition of the culture medium and stimulate hyphae to germinate.
The thin ear is quickly placed into a test tube by using a sterile inoculation hook, and the cut surface is tightly attached to the surface of the culture medium. The culture medium can be the medium commonly used for culturing Tremella fuciformis berk strains, such as PAD medium. Referring to fig. 1, fig. 1 is a pictorial view of a thin tab inoculated into the medium at day 5.
The test tube strains are usually cultured for 15-17 days, colloid bodies are arranged in the middle of the inoculation points, and the tubes are rotated when thick white hyphae grow for 2-4 mm around the inoculation points. The method comprises two conditions of growing tremella hypha and not growing tremella hypha.
Refer to fig. 2 and 3. FIGS. 2 and 3 clearly show the process of germinating white fungus hyphae from thin ear. And (3) when the tremella fuciformis is cultured for 15 days, most of the tremella fuciformis germinates tremella hyphae, and a small part of the tremella fuciformis grows up and grows thick and does not germinate tremella hyphae. The test tube for tremella hyphae is counted to be more than 90.25%, so that the hyphae can be extracted from the test tube for growing the thick tremella hyphae and transferred to the test tube. The traditional substrate block purification method has complicated strain growth conditions, and some test tubes only grow incense ash hyphae, some test tubes grow incense ash hyphae and tremella hyphae, some test tubes only grow tremella hyphae, some test tubes grow mixed bacteria, and some test tubes do not grow any hyphae. Therefore, the strong white pure tremella hyphae need to be selected from a large number of test tubes, and the purity is not easy to guarantee.
The method comprises selecting thick, strong and fast tremella hyphae from test tubes, transferring into tubes, cutting mycelia around the colloid block about 2mm × 3mm, transferring into culture container such as test tube or triangular flask, allowing small hair ball to grow on the hyphae the next day, and transferring into tubes for 6 days to continue growth of tremella hyphae, referring to FIG. 4. The tube can be used after 7-12 days, and the white hair ball (pure white fungus strain) is round, full, dense and white.
The invention has the following beneficial effects: according to the method for extracting the tremella fuciformis strains, the incense ash hyphae of the tremella fuciformis are inactivated by controlling the humidity, and meanwhile, the tremella fuciformis strains are extracted by a tissue separation method, only one tube rotation is needed, the purification time is short, and the operation is simple and convenient; meanwhile, the strain characteristics inherit parents, and the quality is stable.
Optionally, the ear is air-dried under the conditions: and placing the lug in a superclean workbench for 4-5 hours under the condition of gentle wind. The clean bench is not easy to be infected with mixed bacteria, the air drying speed of the ear pieces is proper under the condition of gentle wind, and the incense ash hypha can be effectively inactivated.
Optionally, the culture medium comprises potato, glucose, agar and water, and is prepared by adding 200g of potato, 20g of glucose and 24-26 g of agar into wood chip culture medium incense ash mycelium, decocting water extractive solution to volume of 1000ml, sterilizing, and cooling.
The culture medium is PAD culture medium, and the components and the preparation method are basically the same as those of the common PAD culture medium. The difference is that the water adopted in the invention is the boiled water extracting solution of the wood chip culture medium incense ash hypha. Culturing incense ash hypha on a wood chip culture medium for culturing tremella till the whole culture medium is covered, and boiling water together with the incense ash hypha and the wood chip culture medium to prepare PAD culture medium constant volume water. In the PAD culture medium with the amount, the using amount of the wood chip culture medium incense ash hyphae is 2 bottles, the bottle size is 750ML, namely the mass of each bottle of wood chip culture medium (calculated by mass percent, 68% of wood chips, 30% of wheat bran, 1% of sugar and 1% of gypsum) incense ash hyphae is about 320g, and the wood chip culture medium incense ash hyphae is added into 2000ML of water to be boiled for 30 minutes to obtain an extracting solution. Sterilizing at 121 deg.C for 30 min after constant volume, placing the slant, and cooling for 3 days.
The tremella hyphae can germinate only by being mixed with the tremella hyphae, and the applicant finds that the extract obtained by boiling the wood chip culture medium incense ash hyphae with water can provide nutrient components for the growth of the tremella hyphae. Therefore, the operation is more convenient.
Optionally, the culture medium is prepared by adding potato 200g, glucose 20g, agar 25g into wood flour culture medium incense ash mycelium decoction extractive solution to volume of 1000ml, sterilizing, and cooling.
Optionally, the size of the thin ear is 0.2mm × 0.05mm, and the thickness of the thin ear is 0.1-1 mm.
The thin ear piece with the size and the thickness can absorb the nutrition of the culture medium to the maximum extent and stimulate hypha to germinate.
Optionally, the thin tabs are inoculated into the medium in tubes, 2-4 pieces per tube. Under the condition, the distribution density of the thin lugs is proper, and the growth speed is high.
Optionally, the thin ear is cultured at 22-25 deg.C for 15-17 days to obtain thick white hyphae. Under the condition, hyphae germinate and grow fast, and the seed production time is saved.
Optionally, the thick white hyphae are divided into 2mm × 3mm and transferred into tubes, and cultured for 7-12 days to obtain white hair clusters. Under the condition, the growth speed of the thick hypha is high, and the seed production time is saved.
Optionally, the tab is disinfected by 73-78% alcohol, rinsed with 73-78% alcohol for 4-6S, and rinsed twice with sterile water for 4-6S each time.
And 3, adopting 73-78% alcohol for disinfection to kill other mixed bacteria and ensure the purity of the tremella strain. Preferably 75% alcohol. Rinsing with 73-78% alcohol for 4-6S, and rinsing with sterile water twice for 4-6S each time to remove redundant alcohol.
Example 1
1. Preparation of biological PDA culture Medium
PDA is potato 200g, glucose 20g, agar 25g, wood chip culture medium incense ash hypha 2 bottles of boiling water extract with constant volume of 1000ml, sterilizing at 121 deg.C for 30 min, placing on inclined plane, cooling and using for 3 days.
2. Tremella seed source treatment
And (A) selecting tremella fuciformis with round and regular shape, uniformly unfolded leaves, white color, fast growth and diameter of 3-5 cm as a separation seed source.
And B, selecting 3 strong-vitality lugs, disinfecting by using 73% alcohol, and putting into an ultra-clean workbench.
C rinse 4S with 75% alcohol and twice with 4S sterile water.
D, taking out and sucking the water on the surface of the lug with sterile gauze.
E, hanging the lug plates in the superclean workbench by using a hook, and drying the incense ash hyphae on the surfaces of the lug plates to death after opening the soft air for 4H.
3. Inoculation of
The appearance of the ear piece A is air-dried, the ear piece is taken down and cut into 0.2mmX0.05mm at the growing point, and the thickness is 1 mm.
B, the thin lug is quickly placed into the test tube by using a sterile inoculation hook, and the cut surface is tightly attached to the surface of the culture medium. Each test tube is connected with 3 blocks.
4. Cultivation of
And (3) culturing the test tube strains at 22 ℃ for 17 days, rotating the tube when the middle of an inoculation point is gelatinous and thick white hyphae grow around the inoculation point by 2-4 mm.
5. Rotating pipe
Selecting concentrated white fungus mycelia to grow densely and robustly, rotating the tube quickly, cutting 2mmX3mm size of the mycelia around the colloidal block, transferring into a test tube or a triangular flask, growing small hair clusters on the mycelia in the next day, and obtaining white hair clusters, namely pure white fungus strains, in 7 days.
Example 2
Example 2 differs from example 1 in that the thin ear has a thickness of 0.8 mm. The temperature in the incubation step was 24 ℃ and the incubation time was 16 days. The incubation time in the tube transfer step was 9 days.
Example 3
Example 2 differs from example 1 in that the thin ear has a thickness of 0.5 mm. The temperature in the incubation step was 25 ℃ and the incubation time was 15 days. The incubation time in the tube transfer step was 7 days.
Comparative example 1
PDA is potato 200g, glucose 20g, agar 18g, and the used water is common sterile water. Sterilizing at 121 deg.C for 30 min, placing on inclined plane, cooling, and using for 3 days.
Selecting white fungus with round shape, uniformly spread leaves, white color and fast growth, removing fruiting bodies of the white fungus, shoveling out a white fungus substrate block by using an inoculating shovel, cutting off soft incense ash hypha around the substrate block by using a scalpel, exposing a white hardened white fungus hypha block, cutting the substrate block into a square shape of 2-3 cm, and drying the square shape at a small fan air inlet at room temperature for 35-45 days.
Soaking the dried matrix block in 75% alcohol for 30 s, removing excessive alcohol on the surface layer with sterile gauze, cutting the matrix block with sterile scalpel in ultraclean bench, and inoculating 0.1-0.2 cubic mm of matrix block in the middle part with inoculating needle to PDA culture medium.
Then culturing for 5-6 days at room temperature of 22-25 deg.C to obtain white Tremella mycelium. When pure tremella hyphae germinate and grow on PDA medium in a test tube. The growth of the cells in light was observed. Some test tubes only grow incense ash hypha, some test tubes grow incense ash hypha and tremella hypha, some test tubes only grow tremella hypha, some test tubes grow sundry fungi, and some test tubes do not grow any hypha. Taking the tip of the tremella hyphae from the test tube with the tremella hyphae only, and inoculating the tremella hyphae tip on the culture medium in a new test tube again, and repeating the operation for 2 times or more to complete the purification of the tremella hyphae which is newly separated.
Comparative example 2
PDA is potato 200g, glucose 20g, agar 18g, and the water is common sterile water. Bottling with 15ml each, and sterilizing at 121 deg.C for 30 min.
Placing 4 beakers, stainless steel hooks, inoculating needles, scissors, tweezers, sterile water, sterile gauze, alcohol lamps, 0. l% mercuric chloride solution, a triangular flask filled with potato agar culture medium, and a seed ear in a super-clean bench for disinfection for 30 minutes.
Firstly, 3 beakers are disinfected by alcohol, then a plurality of sterile water is respectively poured into the beakers, 0.1% mercuric chloride solution is poured into the other beakers, a plurality of ear lobes with thick meat and large pieces are cut by scissors, the beakers are soaked in the mercuric chloride solution for 5 to 10 seconds, then the beakers are quickly and sequentially placed into 3 sterile water beakers, each is soaked for 1 minute, then sterile gauze is used for absorbing water, the beakers are quickly hung in a triangular flask by a steel hook, and a cotton plug is plugged. The distance between the ear and the culture medium is about 3 cm for preventing the mixed bacteria infection.
Placing in a constant temperature box, maintaining the temperature at 23-25 deg.C, and culturing for 24 hr to obtain atomized spore eggs on the surface layer of the culture medium.
At this time, the steel hook and the lug can be taken out from the superclean bench, the cotton plug is plugged, and after the culture is continued for 2-3 days, white pasty colony with smooth edge and convex middle can be seen on the surface of the culture medium, namely the tremella spores. If no mixed bacteria exists, a repeated marking method or a dilution method can be adopted, and pure spores are obtained and then the tube is rotated and expanded.
The differences between examples 1 to 3, comparative example 1 and comparative example 2 are shown in the following table.
| Item | Purification according to the invention | Purification of the substrate block | Spore purification |
| Number of purification tube transfer times | 1 | ≥1 | ≥3 |
| Difficulty of extraction operation | Simple and easy | In general | Height of |
| Stable situation | Genetic parent | Genetic instability | Susceptibility to variation |
| Purity of extraction | Height of | In general | Sea separation |
| Purification time (Tian) | 22-29 | 45-60 | ≥35 |
| Applications of | Is ready for use | Is ready for use | Through trial production stabilization |
| Probability of mixed bacteria | Very small | In general | Height of |
The extraction method of the tremella fuciformis strain is convenient to operate, stable in hereditary shape, high in tremella fuciformis strain purity, short in purification time and low in probability of mixed bacteria.
The fragrant ash hyphae of the ear are inactivated by controlling the humidity, and the tremella strain is extracted by a tissue separation method, so that tube rotation is only needed once, the purification time is short, and the operation is simple and convenient; meanwhile, the strain characteristics inherit parents, and the quality is stable.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. A tremella strain extraction method is characterized by comprising the following steps:
selecting the ear pieces, disinfecting, rinsing and wiping the ear pieces, placing the ear pieces under a humidity condition of 55-65% for 4-5 hours, and air-drying the ear pieces to inactivate incense ash hyphae on the ear pieces, wherein the air-drying condition of the ear pieces is as follows: placing the lug in a superclean workbench, and placing for 4-5 hours under the condition of gentle wind; the ear is disinfected by 73-78% alcohol, rinsed for 4-6 seconds by 73-78% alcohol, and rinsed twice by sterile water for 4-6 seconds each time;
cutting thin auricles at the growing points of the air-dried auricles, inoculating the thin auricles into culture media in test tubes, inoculating 2-4 blocks of the thin auricles in each test tube, culturing until thick white hyphae with the thickness of 2-4 mm appears at the inoculating points, wherein the size of the thin auricles is 0.2mm multiplied by 0.05mm, the thickness of the thin auricles is 0.1-1 mm, the culture media comprise potatoes, glucose, agar and water, the culture media are obtained by adding 200g of potatoes, 20g of glucose and 24-26 g of agar into wood chip culture medium incense ash hyphae, boiling the water extracting solution to a constant volume of 1000ml, sterilizing and cooling; cultivating the thin ear slices at 22-25 ℃ for 15-17 days to obtain the thick white hyphae;
and dividing the thick white fungus threads into 2mm multiplied by 3mm, rotating the tubes, and culturing for 7-12 days until white hair balls are formed, thereby obtaining the white fungus strains.
2. The method for extracting tremella fuciformis strains as claimed in claim 1, wherein the culture medium is prepared by adding 200g of potatoes, 20g of glucose and 25g of agar into a wood chip culture medium, boiling water extract of incense ash hypha to a volume of 1000ml, sterilizing and cooling.
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| KR100252384B1 (en) * | 1997-11-19 | 2000-04-15 | 대한민국 | ASI 6046 strain of Hypoxylon Lujibino Island, which is a symbiotic fungus of white-necked mushroom, and artificial cultivation method of white-headed mushroom |
| CN102037849B (en) * | 2009-10-12 | 2012-08-08 | 江寿根 | Tremella fuciformis strain culture method |
| CN102657024A (en) * | 2012-02-20 | 2012-09-12 | 古田县晟农食用菌农民专业合作社 | Method for culturing tremella fuciformis berk strains |
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| 银耳菌种规范化生产公益讲座(三)-银耳两个纯菌种的单独分离与混合育成母种的技术;丁湖光;《浙江食用菌》;20100719;第9-12页 * |
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Address after: 417000 Loudi City, Hunan province Louxing District Di Xing Road No. 38 Applicant after: Hunan mushroom square biological Polytron Technologies Inc Address before: 417000 Loudi City, Hunan province Louxing District Di Xing Road No. 38 Applicant before: HUNAN VIGOR FOODS BIO-TECHNOLOGY CO., LTD. |
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