CN102965328A - Preparation method of Fuzhuan tea golden flower fungus spore powder - Google Patents
Preparation method of Fuzhuan tea golden flower fungus spore powder Download PDFInfo
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- CN102965328A CN102965328A CN2012104704777A CN201210470477A CN102965328A CN 102965328 A CN102965328 A CN 102965328A CN 2012104704777 A CN2012104704777 A CN 2012104704777A CN 201210470477 A CN201210470477 A CN 201210470477A CN 102965328 A CN102965328 A CN 102965328A
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Abstract
The invention discloses a preparation method of Fuzhuan tea golden flower fungus spore powder, which comprising the steps of: inoculating golden flower fungus to a CZG slant culture medium, culturing for 5days at a temperature of 26-30 DEG C to obtain a seed culture; adding sterile water in the seed culture, washing down golden flower fungus spores, fully oscillating to obtain a uniformly-dispersed spore suspension; inoculating the spore suspension on the culture medium, carrying out constant-temperature culture at 26-30 DEG C, covering with a sterile breathable sealing film before white hypha germinates, removing the sterile breathable sealing film when yellow hypha germinates, continuously culturing for 3-5 days to obtain a yeast seed; then carrying out enlargement to obtain mouldy bran; and crushing the mouldy bran and then sieving to obtain the Fuzhuan tea golden flower fungus spore powder. The artificially cultured Fuzhuan tea golden flower fungus spore powder can be used for extracting effective components of the golden flower fungus and inoculating fermented Fuzhuan tea; and the fermentation period can be shortened and the pollution of infectious microbe is reduced.
Description
Technical field
The invention belongs to the spawn culture technical field, relate to a kind of preparation method of Fu brick tea golden flower bacterium spore powder.
Background technology
The golden flower bacterium be the Fu brick tea grow dim self-assembling formation in the process a kind of have beneficial natural disposition can dominant bacteria, formal name used at school is the loose capsule bacterium (Eurotium cristatum) of coronoid process.The Fu brick tea is the ancient tea product of a kind of stub border areas, has promoting the production of body fluid to quench thirst, the benefit such as fat-reducing that stagnates of disappearing gives birth to effect, studies show that this existence with the golden flower bacterium is inseparable.The multiple miscellaneous bacterias such as easy growth aspergillus, mould have affected the normal growth of golden flower bacterium, thereby reduced the quality of Fu brick tea, and fermentation period are longer in the Fu brick tea of spontaneous fermentation.If can manually cultivate the spore powder that obtains the golden flower bacterium, in order to inoculation fermentation Fu brick tea, not only can shorten fermentation period, and reduce the pollution of miscellaneous bacteria, thereby improve the quality of Fu brick tea.
Summary of the invention
The problem that the present invention solves is to provide a kind of preparation method of Fu brick tea golden flower bacterium spore powder, to obtain the spore powder of artificial culture golden flower bacterium, in order to inoculation fermentation Fu brick tea, not only can shorten fermentation period, and reduces the pollution of miscellaneous bacteria.
The present invention is achieved through the following technical solutions:
A kind of preparation method of Fu brick tea golden flower bacterium spore powder may further comprise the steps:
1) the golden flower bacterium is seeded to the seed culture medium inclined-plane, cultivates 5d, obtain inoculum for 26～30 ℃;
2) add sterilized water to inoculum, wash golden flower bacterium spore, fully after the vibration, obtain homodisperse spore suspension;
3) wheat bran and water are fully mixed with the ratio of 100g:80～100mL after as substratum, in culture vessel, pave, thickness is 0.8～1.5cm, moist heat sterilization;
After the sterilization, volume ratio inoculating spores suspension with 5～15%, fully shake up, 26～30 ℃ of constant temperature culture, the outlet gauze capping of culture vessel after the inoculation, add a cover aseptic breathable sealing film at gauze when treating that white hypha begins to sprout, sprout at white hypha and finish to remove aseptic breathable sealing film when yellow mycelia begins to sprout, obtain the sort of quyi after cultivating 6～8d;
4) wheat bran and water are fully mixed with the ratio of 100g:80～100mL after as substratum, in culture vessel, pave, thickness is 2.0～3.0cm, moist heat sterilization;
After the sterilization, the mass ratio inoculation sort of quyi with 5～10%, 26～30 ℃ of constant temperature culture, the outlet gauze capping of culture vessel in 0～36h after the inoculation, and sprout to finish to remove when yellow mycelia has just sprouted aseptic breathable sealing film at white hypha, stop to cultivate after continuing to cultivate 3～4d, obtain wheat bran;
5) with the wheat bran grinding and sieving, obtain golden flower bacterium spore powder.
Described golden flower bacterium is that separation and purification obtains in the Fu brick tea, and its separation purification method is: get Fu brick tea tealeaves 10g, add the 90mL sterilized water, 110rmin
-1Concussion 10min obtains bacteria suspension, and carries out gradient dilution, and the bacteria suspension of gradient concentration is applied to respectively on CZG (5%NaCl) substratum 28 ℃ of constant temperature culture; Get the bacterium colony of suitable weaker concn, the separation of ruling, picking list bacterium colony behind 28 ℃ of cultivation 5d, line separates again, until microscopy obtains the pure strain of golden flower bacterium without miscellaneous bacteria behind the picking list bacterium colony, is forwarded to the inclined-plane in 4 ℃ of preservations.
Used seed culture medium is CZG substratum (5%NaCl) in the described step 1).
Described step 2) in, add sterilized water to inoculum, wash golden flower bacterium spore, aseptic technique is poured in the Erlenmeyer flask that the equal-volume granulated glass sphere is housed, with 100～120rmin
-1Vibration 5～15min obtains homodisperse spore suspension.
In the described step 3), during described moist heat sterilization, in packing Erlenmeyer flask behind wheat bran and the abundant mixing of water, 110～121 ℃ of sterilization 20～30min; After sterilization finished, loose substratum was shaken in immediately vibration.Place aseptic operating platform to be cooled to room temperature.
The cleistothecium quantity of golden flower bacterium is abundant behind described also continuation cultivation 3～5d, is even golden yellow.
Before white hypha is sprouted, add a cover aseptic breathable sealing film in the described step 4), remove before the mycelia flavescence, be cultured to golden yellow and stop to cultivate.
Wheat bran is crossed 100 mesh sieves after crushed in the described step 5); Perhaps wheat bran is pulverized and is placed in the airtight sterile chamber cryopreservation.
Compared with prior art, the present invention has following useful technique effect:
The preparation method of Fu brick tea golden flower bacterium spore powder provided by the invention, with the selected bran mass of having used of thick generation, not only cost is low, method stable, easy handling, and the spore that obtains is loose and dense; And at the white hypha duration of germination, by adding a cover aseptic breathable sealing film, control on the one hand air flow, control on the other hand the moisture of substratum, the humidity that has guaranteed media surface is suitable, avoid the golden flower bacterium to sprout with asexual spore at the white hypha duration of germination, reduced living contaminants, thereby improved required anisosporous quality.In the golden flower bacterium spore powder that obtains, its spore content is 8 * 10
8Individual/g.
The preparation method of Fu brick tea golden flower bacterium spore powder provided by the invention, short period of time can obtain a large amount of golden flower bacterium spore powder, the golden flower bacterium spore powder that obtains can be in order to the extraction of microorganism effective constituent, also can directly be used as the bacterial classification of artificial fermentation Fu brick tea, greatly shorten Fu brick tea fabrication cycle, reduce the pollution of miscellaneous bacteria, thereby improve Fu brick tea quality.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
Golden flower bacterium of the present invention can be adopted the preservation of bacteria strain of existing each DSMZ, also voluntarily separation and purification.Carry out separation and purification such as adopting method of scoring:
Get Fu brick tea tealeaves 10g, add the 90ml sterilized water, 110rmin
-1Concussion 10min obtains bacteria suspension, and carries out gradient dilution (to 10
6Doubly), the bacteria suspension of gradient concentration is applied to respectively on CZG (5%NaCl) substratum 28 ℃ of constant temperature culture; Get the bacterium colony of suitable weaker concn, the separation of ruling, picking list bacterium colony behind 28 ℃ of cultivation 5d, line separates again, until microscopy obtains the pure strain of golden flower bacterium without miscellaneous bacteria behind the picking list bacterium colony, is forwarded to the inclined-plane in 4 ℃ of preservations.
Embodiment 1
A kind of preparation method of Fu brick tea golden flower bacterium spore powder may further comprise the steps:
Step 1) actication of culture: take from the golden flower bacterium that separation and purification obtains in the Fu brick tea and be seeded to CZG (5%NaCl) slant medium (test tube 18 * 180mm), 28 ℃ of cultivations (aerobic), bacterium colony presents golden yellow when being cultured to 5d, namely obtains inoculum, and is for subsequent use.
Step 2) preparation spore suspension: above-mentioned cultured inoculum is added the 30mL sterilized water, wash golden flower bacterium spore, aseptic technique is poured in the Erlenmeyer flask that the equal-volume granulated glass sphere is housed, with 100rmin
-1Vibration 10min obtains homodisperse spore suspension, and is for subsequent use.
The step 3) spore is cultivated: get wheat bran, add water 80mL preparation solid medium with every 100g, in the packing 250mL Erlenmeyer flask, paving thickness is 1.0～1.3cm behind the abundant mixing, binds up with gauze 121 ℃ of sterilization 30min with eight layers.After sterilization finished, loose substratum was shaken in immediately vibration.Place aseptic operating platform to be cooled to room temperature, inoculation step 2) spore suspension 1mL, and cultivate in 28 ℃ of constant incubators after fully shaking up.Wait to be cultured to the 2d white hypha and sprout, add a cover aseptic breathable sealing film in the outlet of Erlenmeyer flask, white hypha covers with gradually substratum and removes when cultivating 3d.Continue in 28 ℃ of constant incubators and be cultured to 7d, substratum presents the pie that drying is compacted, and the cleistothecium quantity of golden flower bacterium is abundant, and is evenly golden yellow, namely obtains the sort of quyi, for subsequent use.
The step 4) enlarged culturing: after above-mentioned amount of water material moistening evenly, get koji tray, shakeouing thickness is 2.0～3.0cm, and eight layers of gauze cover, and are cooled to room temperature after the sterilization, and the inoculum size with 10% is seeded to koji tray, cultivates 4～5d.Add a cover aseptic breathable sealing film after wherein cultivating 12h, remove before the mycelia flavescence to cultivating behind the 36h, continue in 28 ℃ of constant incubators and be cultured to 4～5d, stop to cultivate.
Step 5): spore is collected: above-mentioned wheat bran is crossed 100 mesh sieves after crushed, namely obtain golden flower bacterium spore powder; Perhaps directly pulverize and be placed in the airtight sterile chamber low temperature standing storage.
Embodiment 2
Step 1): actication of culture: take from separation and purification obtains in the Fu brick tea golden flower bacterium and be seeded to CZG (5%NaCl) slant medium (bacterium colony presents golden yellow when being cultured to 5d, namely obtains inoculum, and is for subsequent use for test tube 15 * 150mm), 30 ℃ of cultivations.
Step 2): the preparation spore suspension: above-mentioned cultured inoculum is added the 20mL sterilized water, wash golden flower bacterium spore, aseptic technique is poured in the Erlenmeyer flask that the equal-volume granulated glass sphere is housed, with 110rmin
-1Vibration 10min obtains homodisperse spore suspension, and is for subsequent use.
Step 3): spore is cultivated: get wheat bran, add water 100mL preparation solid medium with every 100g, in the packing 500mL Erlenmeyer flask, paving thickness is 0.8～1.0cm behind the abundant mixing, binds up with gauze 121 ℃ of sterilization 30min with eight layers.After sterilization finished, loose substratum was shaken in immediately vibration.Place aseptic operating platform to be cooled to room temperature, inoculation step 2) spore suspension 2mL, and fully vibration shakes up 29 ℃ of constant incubators of rear placement, cultivates 2d and adds a cover aseptic breathable sealing film, and removes in cultivating 3d.Continuation is being cultured to 7d, and substratum presents the pie that drying is compacted, and the cleistothecium quantity of golden flower bacterium is abundant, and is evenly golden yellow, namely obtains the sort of quyi, for subsequent use.
Step 4): enlarged culturing: after above-mentioned amount of water material moistening evenly, get koji tray, shakeouing thickness is 2.0～2.5cm, and eight layers of gauze cover, and are cooled to room temperature after the sterilization, and the inoculum size with 5% is seeded to koji tray, cultivates 4d.Wherein 14h adds a cover aseptic breathable sealing film, and removes before the mycelia flavescence behind continuation cultivation 15～20h, is cultured to golden yellow and stops to cultivate.
Step 5): spore is collected: above-mentioned wheat bran is crossed 100 mesh sieves after crushed, namely obtain golden flower bacterium spore powder; Perhaps directly pulverize and be placed in the airtight sterile chamber low temperature standing storage.
Embodiment 3
Step 1): actication of culture: take from separation and purification obtains in the Fu brick tea golden flower bacterium and be seeded to CZG (5%NaCl) slant medium (bacterium colony presents golden yellow when being cultured to 5d, namely obtains inoculum, and is for subsequent use for test tube 20 * 200mm), 28 ℃ of cultivations.
Step 2): the preparation spore suspension: above-mentioned cultured inoculum is added the 30mL sterilized water, wash golden flower bacterium spore, aseptic technique is poured in the Erlenmeyer flask that the equal-volume granulated glass sphere is housed, with 110rmin
-1Vibration 5min obtains homodisperse spore suspension, and is for subsequent use.
Step 3): spore is cultivated: get wheat bran, add water 80mL preparation solid medium with every 100g, in the packing 500mL Erlenmeyer flask, paving thickness is 0.8～1.0cm behind the abundant mixing, binds up with gauze 121 ℃ of sterilization 30min with eight layers.After sterilization finished, loose substratum was shaken in immediately vibration.Place aseptic operating platform to be cooled to room temperature, inoculation step 2) spore suspension 2mL, and fully shake up in 28 ℃ of constant incubators and cultivate, and cultivate 2d and add a cover aseptic breathable sealing film, mycelia gradually flavescence is removed when 3d.Continuation is cultured to 6d in 28 ℃ of constant incubators, substratum presents the pie that drying is compacted, and the cleistothecium quantity of golden flower bacterium is abundant, and is evenly golden yellow, namely obtains the sort of quyi, for subsequent use.
Step 4): enlarged culturing: after above-mentioned amount of water material moistening evenly, get koji tray, shakeouing thickness is 2.0～3.0cm, and eight layers of gauze cover, and are cooled to room temperature after the sterilization, and the inoculum size with 10% is seeded to koji tray, cultivates 4d.Wherein 12h adds a cover aseptic breathable sealing film, and removes before the mycelia flavescence behind continuation cultivation 20～24h, is cultured to golden yellow and stops to cultivate.
Step 5): spore is collected: above-mentioned wheat bran is crossed 100 mesh sieves after crushed, namely obtain golden flower bacterium spore powder; Perhaps directly pulverize and be placed in the airtight sterile chamber low temperature standing storage.
Claims (8)
1. the preparation method of a Fu brick tea golden flower bacterium spore powder is characterized in that, may further comprise the steps:
1) the golden flower bacterium is seeded to the seed culture medium inclined-plane, cultivates 5d, obtain inoculum for 26～30 ℃;
2) add sterilized water to inoculum, wash golden flower bacterium spore, fully after the vibration, obtain homodisperse spore suspension;
3) wheat bran and water are fully mixed with the ratio of 100g:80～100mL after as substratum, in culture vessel, pave, thickness is 0.8～1.5cm, moist heat sterilization;
After the sterilization, volume ratio inoculating spores suspension with 5～15%, fully shake up, 26～30 ℃ of constant temperature culture, the outlet gauze capping of culture vessel after the inoculation, add a cover aseptic breathable sealing film at gauze when treating that white hypha begins to sprout, sprout at white hypha and finish to remove aseptic breathable sealing film when yellow mycelia begins to sprout, obtain the sort of quyi after cultivating 6～8d;
4) wheat bran and water are fully mixed with the ratio of 100g:80～100mL after as substratum, in culture vessel, pave, thickness is 2.0～3.0cm, moist heat sterilization;
After the sterilization, the mass ratio inoculation sort of quyi with 5～10%, 26～30 ℃ of constant temperature culture, the outlet gauze capping of culture vessel in 0～36h after the inoculation, and add a cover aseptic breathable sealing film at gauze, and when the yellow mycelia of white hypha sprouting end has just sprouted, remove aseptic breathable sealing film, and continue to stop to cultivate behind cultivation 3～4d, obtain wheat bran;
5) with the wheat bran grinding and sieving, obtain golden flower bacterium spore powder.
2. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that described golden flower bacterium is that separation and purification obtains in the Fu brick tea, and its separation purification method is: get Fu brick tea tealeaves 10g, add the 90mL sterilized water, 110rmin
-1Concussion 10min obtains bacteria suspension, and carries out gradient dilution, and the bacteria suspension of gradient concentration is applied to respectively on CZG (5%NaCl) substratum 28 ℃ of constant temperature culture; Get the bacterium colony of suitable weaker concn, the separation of ruling, picking list bacterium colony behind 28 ℃ of cultivation 5d, line separates again, until microscopy obtains the pure strain of golden flower bacterium without miscellaneous bacteria behind the picking list bacterium colony, is forwarded to the inclined-plane in 4 ℃ of preservations.
3. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that, used seed culture medium is CZG substratum (5%NaCl) in the described step 1).
4. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that described step 2) in, add sterilized water to inoculum, wash golden flower bacterium spore, aseptic technique is poured in the Erlenmeyer flask that the equal-volume granulated glass sphere is housed, with 100～120rmin
-1Vibration 5～15min obtains homodisperse spore suspension.
5. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that, in the described step 3), and during described moist heat sterilization, in packing Erlenmeyer flask behind wheat bran and the abundant mixing of water, 110～121 ℃ of sterilization 20～30min; After sterilization finished, loose substratum was shaken in immediately vibration, places aseptic operating platform to be cooled to room temperature.
6. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that, in the described step 3), remove aseptic breathable sealing film after, the cleistothecium quantity of continue cultivating 3～5d golden flower bacterium is abundant, is even golden yellow.
7. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that, adds a cover aseptic breathable sealing film in the described step 4) before white hypha is sprouted, and removes before the mycelia flavescence, is cultured to golden yellow and stops to cultivate.
8. the preparation method of Fu brick tea golden flower bacterium spore powder as claimed in claim 1 is characterized in that, wheat bran is crossed 100 mesh sieves after crushed in the described step 5); Perhaps wheat bran is pulverized and is placed in the airtight sterile chamber cryopreservation.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865810A (en) * | 2014-03-26 | 2014-06-18 | 湖南城市学院 | Method for culturing golden flower fungus powder by utilizing tea leftovers |
| CN103865811A (en) * | 2014-03-26 | 2014-06-18 | 湖南城市学院 | Process for cyclically preparing eurotium cristarum by adding nutrient solution into tea residues |
| CN104738256A (en) * | 2013-12-31 | 2015-07-01 | 深圳华大基因科技有限公司 | Fu tea and preparation method and use thereof |
| CN104739888A (en) * | 2013-12-31 | 2015-07-01 | 深圳华大基因科技有限公司 | Eurotium cristatum powder as well as preparation method and application thereof |
| CN105831311A (en) * | 2016-04-05 | 2016-08-10 | 咸阳泾渭茯茶有限公司 | Processing method of Fuzhuan tea using summer and autumn raw tea |
| CN106318877A (en) * | 2016-08-29 | 2017-01-11 | 刘翔 | Method for separating and purifying eurotium cristatum from fu-brick tea and liquid culture method of eurotium cristatum |
| CN106358765A (en) * | 2016-11-16 | 2017-02-01 | 陕西北极宫茶叶有限公司 | Method of cultivating eurotium cristatum on solid-state medium |
| CN107862697A (en) * | 2017-10-27 | 2018-03-30 | 陕西科技大学 | A kind of golden flower bacterium method of counting for Fu tea quality testings |
| CN108142984A (en) * | 2017-12-12 | 2018-06-12 | 湖北中烟工业有限责任公司 | A kind of method for preparing tobacco leaf clout fragrance using the golden flower bacterium fermentation of tea source |
| CN109294929A (en) * | 2018-10-30 | 2019-02-01 | 中科沣文（天津）新能源科技有限公司 | A kind of preparation method of the coronoid process dissipate capsule bacterium bacterium powder for dark green tea fermentation |
| CN109892420A (en) * | 2019-04-10 | 2019-06-18 | 福建师范大学 | A kind of method of fermenting twice double cure method preparation coronoid process dissipate capsule bacterium oolong tea |
| CN109943438A (en) * | 2019-04-10 | 2019-06-28 | 福建师范大学 | A kind of health red rice method for preparing medicated wine of addition coronoid process dissipate capsule bacterium fermentation |
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| CN104738256A (en) * | 2013-12-31 | 2015-07-01 | 深圳华大基因科技有限公司 | Fu tea and preparation method and use thereof |
| CN104739888A (en) * | 2013-12-31 | 2015-07-01 | 深圳华大基因科技有限公司 | Eurotium cristatum powder as well as preparation method and application thereof |
| CN103865810A (en) * | 2014-03-26 | 2014-06-18 | 湖南城市学院 | Method for culturing golden flower fungus powder by utilizing tea leftovers |
| CN103865811A (en) * | 2014-03-26 | 2014-06-18 | 湖南城市学院 | Process for cyclically preparing eurotium cristarum by adding nutrient solution into tea residues |
| CN105831311A (en) * | 2016-04-05 | 2016-08-10 | 咸阳泾渭茯茶有限公司 | Processing method of Fuzhuan tea using summer and autumn raw tea |
| CN106318877A (en) * | 2016-08-29 | 2017-01-11 | 刘翔 | Method for separating and purifying eurotium cristatum from fu-brick tea and liquid culture method of eurotium cristatum |
| CN106358765A (en) * | 2016-11-16 | 2017-02-01 | 陕西北极宫茶叶有限公司 | Method of cultivating eurotium cristatum on solid-state medium |
| CN107862697A (en) * | 2017-10-27 | 2018-03-30 | 陕西科技大学 | A kind of golden flower bacterium method of counting for Fu tea quality testings |
| CN107862697B (en) * | 2017-10-27 | 2021-03-23 | 陕西科技大学 | Golden flower fungus counting method for Fuzhuan tea quality detection |
| CN108142984A (en) * | 2017-12-12 | 2018-06-12 | 湖北中烟工业有限责任公司 | A kind of method for preparing tobacco leaf clout fragrance using the golden flower bacterium fermentation of tea source |
| CN109294929A (en) * | 2018-10-30 | 2019-02-01 | 中科沣文（天津）新能源科技有限公司 | A kind of preparation method of the coronoid process dissipate capsule bacterium bacterium powder for dark green tea fermentation |
| CN109892420A (en) * | 2019-04-10 | 2019-06-18 | 福建师范大学 | A kind of method of fermenting twice double cure method preparation coronoid process dissipate capsule bacterium oolong tea |
| CN109943438A (en) * | 2019-04-10 | 2019-06-28 | 福建师范大学 | A kind of health red rice method for preparing medicated wine of addition coronoid process dissipate capsule bacterium fermentation |
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Application publication date: 20130313 |