CN107647010A - A kind of method of mixed fermentation with various bacterium green brick tea - Google Patents
A kind of method of mixed fermentation with various bacterium green brick tea Download PDFInfo
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- CN107647010A CN107647010A CN201710990717.9A CN201710990717A CN107647010A CN 107647010 A CN107647010 A CN 107647010A CN 201710990717 A CN201710990717 A CN 201710990717A CN 107647010 A CN107647010 A CN 107647010A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/08—Oxidation; Fermentation
- A23F3/10—Fermentation with addition of microorganisms or enzymes
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Abstract
The present invention discloses a kind of method of mixed fermentation with various bacterium green brick tea, using rhizopus stolonifer bacterium and total shape branch mucor co-fermentation green brick tea, belongs to microbial fermentation technology application field.The present invention uses rhizopus stolonifer bacterium, and deposit number is CCTCC M 2017240；The preservation time is on May 5th, 2017；Total shape branch mucor, deposit number is CCTCC M 2017241；The preservation time is on May 5th, 2017；Belong to total shape branch mucor fungi；Its application is fermenting and producing green brick tea.Use the present invention mixed fungus fermentation green brick tea method for：Two kinds of fungies of liquid culture, collect the fungal spore respectively, launch in green brick tea fermented tea heap, and control temperature, humidity are fermented.The method of present invention fermentation green brick tea, compared with traditional green brick tea fermentation process, tea product mouthfeel more alcohol and silk floss are slided, and fermentation period is short, greatly improves productivity ratio.
Description
Technical field
The present invention relates to two kinds of fungies, rhizopus stolonifer bacterium and the side of the mucoraceous application of total shape branch and its fermentation green brick tea
Method, belong to microbial fermentation technology application field.
Background technology
Our province is the big province of tealeaves, and yield is big, and medium-to-high grade tea is all process by the fresh leaf of spring tea, summer and autumn tea it is fresh
Leaf can only be processed into the low large tea of added value, but large tea price continuously drops in recent years, and Tea Production structural adjustment is compeled
The eyebrows and eyelashes.
Post-fermented tea (black tea) refers to carry out holding heap before it is dried or after drying, by microbial action and hydrothermal
Polyphenols is promoted fully to carry out autoxidation, so as to the fragrance alcohol formed, the dark red dry tea of soup look.The existing Hunan of sample tea
Day point, tribute point, raw point, black brick tea, ornamental slab for paving the floor tea is special to obtain brick tea, commonly obtains brick tea, Hubei green brick tea, Guangxi Liu Bao tea, four
The South Road brick tea and West Road brick tea, the tight tea in Yunnan etc. in river, black tea finished product is various, and frying technology causes the method phase not to the utmost of type with pressure
Together, all it is slightly old through raw material, holds heap discoloration, High Temperature Gas is steamed, and pressure causes the processes such as type.
Green brick tea is one of Hubei special product, is reprocessing tea, unique flavor made of black tea is after pressing.Green brick tea is special
The formation of different flavor is mainly formed in fermentation stage, and using damp and hot with microorganism collective effect material conversion occurs for black tea fermentation,
Fermentation period is grown, and qualitative characteristics protrude.
Hubei green brick tea uses height above sea level 600-1200 rice high mountain fresh leaves of tea plant as raw material, fermented, Cheng Hua, steam pressure, drying
It is made etc. program.Blue bricks tea color green grass or young crops is brown, aroma of pure, and soup look reddish yellow, flavour is aromatic, is one of black tea to show unique characteristics.From
Research of the forefathers to the black teas such as Hunan Anhua black tea, yunnan puer tea series learns that the process of fermentation has to the quality for changing tea
Play the role of very crucial.
Pile fermentation technique is that black tea quality forms key, its essence is tea base occur under damp and hot and microorganism collective effect with
Polyphenols is that a series of biochemical reaction occurs for main chemical composition, so as to form the distinctive flavor quality of black tea.It is domestic at present
The research to black tea quality focuses mostly on outside is characterised by during pile fermentation in Fu-brick tea and Pu'er tea etc., Pu'er tea fragrance component
A large amount of aromatic compounds are formd, such as 1,2,3- trimethoxy-benzenes, 1,2- dimethoxy benzenes etc..And " arohid flavour " of Fu-brick tea
Also mainly formed in the case where " growing dim " period is by microbial actions such as coronoid process dissipate capsule bacteriums, wherein linalool and its oxide are to perfume (or spice)
Gas contributes the larger element of the first species.In addition, the air-dried ageing after pile fermentation also has a significant impact to the fragrance of black tea.Processingization above
Learn research to lay a good foundation to verify black tea flavor quality formation mechenism, to the optimization of black tea processing technology and the lifting of product quality
With positive effect.
The microorganism participated in black tea fermentation mainly has fungi, yeast, bacterium, actinomyces etc., and wherein filamentous fungi is excellent
Gesture bacterial strain, plays an important role during the fermentation.Compared with other black teas such as Fu-brick tea, Pu'er tea, green brick tea processing quality
Chemical research is less, and on green brick tea fermentation process, change is even more that there is not been reported during first system, pile fermentation, from preparatory treating unfired tea
The changing rule of green brick tea volatile ingredient and Crack cause are still not clear in the whole dynamic process terminated to pile fermentation.
The content of the invention
Present invention aims at provide two kinds of fungal bacterial strains that can be used in fermentation green brick tea；And mixed with two kinds of fungies
The method for closing fermentation green brick tea.
The fungi that the present invention utilizes isolates and purifies from green brick tea fermented tea heap and obtained；A kind of bacterial strain is total shape branch Mucor
Bacterium (Mucor racemosus Fres), is named as total shape branch mucor xingding-2；Another bacterial strain is rhizopus stolonifer bacterium
Strain (Rhizopus stolonifer), is named as rhizopus stolonifer bacterium xingding-3；Chinese Typical Representative culture is preserved in contain
Center；Deposit number is respectively CCTCC M 2017241, CCTCC M 2017240；The preservation time is on May 5th, 2017.
Use the present invention two kinds of fungi fermentations production green brick tea method for：Liquid two kinds of fungies of culture respectively, enrichment
Its spore is made spore suspension and counted.The ratio that 200,000 fungal spores are launched according to solarization Mao Qingcha per jin is inoculated with.
It is specifically as follows using the method for two kinds of fungi fermentation green brick teas：
(1) inclined-plane culture：Rhizopus stolonifer bacterium xingding-3 and total shape branch mucor xingding-2 are inoculated in tiltedly respectively
Cultivated on the culture medium of face, obtain inclined-plane bacterial strain；
(2) fermented bacterium expands culture：Inclined-plane inoculation is taken to obtain seed culture into sterilized seed culture medium
Liquid, culture are completed to centrifuge 10min abandoning supernatants after 3000r/min, obtain strain spore；
(3) tea leaf fermentation：It is 20-50% to choose green gross tea and sprinkle water to water capacity, the strain spore of harvest in inoculation step (2)
Fermented after son, yeasting is dustless pollution-free constant incubator pile fermentation, and it is 25-30 DEG C to control temperature, humidity 85-
95%, after fermenting 3-4 days, when tea heap temperature is raised to 50-60 DEG C, turning is carried out, tea heap may proceed to growth white after turning
Mycelium, and rise with temperature, when temperature is raised again to 50-55 DEG C, tealeaves color is completely converted into crineous, gives out ferment
When fragrant, stop；
(4) type is made in tea steaming：The tealeaves decatize that will be fermented, the tealeaves after decatize are put into shaped device shaping, shaping
Naturally cool afterwards to put, cool to put the time be 35-45 hours, completes the making of green brick tea loose tea.
In described step (1), rhizopus stolonifer bacterium xingding-3 and total shape branch mucor xingding-2 are inoculated with respectively
In on slant medium, cultivated 48 hours at 28 DEG C, obtain inclined-plane bacterial strain；Slant medium used, the composition often contained in 1L
For：Glucose 20.0g, potato 200.0g, agar 15g, distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
In described step (2), inclined-plane bacterial strain is taken to be inoculated into respectively in sterilized seed culture medium, in 175rpm, 28
Isothermal vibration culture 48h under conditions of DEG C, obtains seed culture fluid, and culture is completed to discard after 3000r/min centrifugations 10min
Clear liquid, obtain strain spore；Seed culture medium used, the composition often contained in 1L are：Glucose 20.0g, potato 200.0g,
Distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
In described step (3), it is 30% that green gross tea, which is sprinkled water to water capacity,；The strain spore harvested in inoculation step 2, is pressed
The ratio that 200,000 fungal spores are launched according to tea per jin is fermented；Yeasting is dustless pollution-free constant incubator pile fermentation,
It is 25 DEG C to control temperature, humidity 90%.
In described step (4), the tealeaves decatize that will ferment, steam temperature is 100 DEG C, steaming time 3min.
Beneficial effect：
1st, the fungal bacterial strain rhizopus stolonifer bacterium xingding-3 that can be used in green brick tea fermenting and producing is separately cultured out first；
2nd, the present invention is substantially reduced green brick tea fermentation period, improved using mixed fungus fermentation method production green brick tea
Production efficiency；
3rd, the method for the additional mixed fungus bacterial strain of present invention application, makes fermentation process quickly form specific advantages flora, should
For fermenting and producing green brick tea, the mode of production has new meaning.
Preservation explanation
Depositary institution：China typical culture collection center
Address：Wuhan City, Hubei Province Hongshan District Bayi Road, Wuhan University
Preservation date：On May 5th, 2017
Deposit number:CCTCC M 2017240 and CCTCC M 2017241
Classification And Nomenclature：Rhizopus stolonifer Rhizopus stolonifer (xingding-3) and total shape branch mucor Mucor
racemosusFres(xingding-2)
Brief description of the drawings
Fig. 1：The morphological features under the microscope of rhizopus stolonifer bacterium xingding-3 used in the present invention.
Fig. 2：The morphological features under the microscope of mucor xingding-2 used in the present invention.
Fig. 3：Rhizopus stolonifer bacterium xingding-3 positive forms on PDA plate of the present invention.
Fig. 4：The form of rhizopus stolonifer bacterium xingding-3 reverse side on PDA plate of the present invention.
Fig. 5：Mucor xingding-2 of the present invention positive forms on PDA plate.
Fig. 6：The form of mucor xingding-2 reverse side on PDA plate of the present invention.
Fig. 7：Add rhizopus stolonifer bacterium xinding-3 fermentation green brick tea tea heap samples.
Fig. 8：Add rhizopus stolonifer bacterium xingding-3 and total shape branch mucor xingding-2 fermentation green brick tea tea heap samples.
Embodiment
It is method commonly used in the art unless otherwise specified in following embodiments；Agents useful for same lives instrument to indicate production
Manufacturer person, being can be by the conventional products of acquisition purchased in market.
The rhizopus stolonifer bacteria strain xingding-3 of embodiment 1 Morphological Identification；
Bacterial strain xingding-3 is isolated from green brick tea fermented tea heap, and its form of micro- sem observation is as shown in Figure 1.Looking into respectively
Cultivate, 28 DEG C, incubation time 2-5d of cultivation temperature, observe on the culture matrixes such as family name's agar, Cha Shi yeast agars, wort agar
Form.
Cha Shi agar colonies are divided into two for white, mycelium, and vegetative hyphae growth is presented by mediad surrounding relatively advises
Then circular, aerial hyphae is a large amount of, and colony growth is very fast.Bacterium colony is white on Cha Shi yeast agars, colonial morphology is relatively regular circle
Shape, aerial hyphae is more, and compared with Cha Shi agar, growth is faster.Bacterium colony is white on wort agar, and aerial hyphae is more,
Growth is fast compared with Cha Shi agar and Cha Shi yeast agars.Reference《Fungal identification handbook》Morphology ratio is carried out to xingding-3 bacterial strains
It is right, determine that xingding-3 bacterial strains belong to bacterium for Rhizopusstolonifer.
The bacterial strain xingding-3 Molecular Identifications of embodiment 2：
Strain gene group DNA is extracted, polymerase chain reaction is carried out to the genomic DNA using ITS1 and ITS4 universal primers
(Polymerase Chain Reaction, PCR) is answered to breed specific dna sequence, and utilizes American National Biotechnology Information
Center (National Center for Biotechnology Information, NCBI) database carries out bacterial strain comparison.
(1) 18S rDNA sequence analyses：
ITS1rDNA primer sequences：5'-TCCGTAGGTGAACCTGCGG-3'
ITS4rDNA primer sequences：5'-TCCTCCGCTTATTGATATGC-3'
Amplification obtains 18SrDNA sequences, sequence 617bp.By 18SrDNA sequences obtained by the amplification and GeneBank numbers
It is compared according to the gene order of the related strain in storehouse, the bacterium higher with bacterial strain xingding-3 homologys is found from NCBI
Strain is Rhizopus stolonifer (AM933546.1), and homology reaches 99%.
(2) LSU rDNA sequence analyses：
NL1rDNA primer sequences：5'-GCATATCAATAAGCGGAGGAAAAG-3'
NL4rDNA primer sequences：5'-GGTCCGTGTTTCAAGACGG-3'
Amplification obtains 28SrDNA sequences, sequence 683bp.By 28SrDNA sequences obtained by the amplification and GeneBank numbers
It is compared according to the gene order of the related strain in storehouse, the bacterial strain for finding higher with bacterial strain xingding-3 homologys is
Rhizopus stoloniferstrain DAOM 225707 (JN938904.1), homology reaches 99%
Therefore it can be determined that xingding-3 is rhizopus stolonifer bacterial strain.Sequence has been committed to GeneBank data on NCBI
Storehouse (Serial No. KY373221).Rhizopus stolonifer bacterium xingding-3 was preserved in China typical culture collection center in 2017
(Wuhan University)；Deposit number is CCTCC M 2017240；This biomaterial has been survived experiment test and by the experiment.
Embodiment 3 carries out green brick tea fermenting experiment using rhizopus stolonifer bacterial strain：
(1) inclined-plane culture：Rhizopus stolonifer bacterium xingding-3 is inoculated on slant medium, and it is small that 48 are cultivated at 28 DEG C
When, obtain inclined-plane bacterial strain.Slant medium used, the composition often contained in 1L are：Glucose 20.0g, potato 200.0g, agar
15g, distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
(2) fermented bacterium expands culture：The ring of inclined-plane bacterial strain one is taken to be inoculated into sterilized seed culture medium,
175rpm, isothermal vibration culture 48h under conditions of 28 DEG C, seed culture fluid is obtained, and discarded in 3000r/min centrifugations 10min
Supernatant, obtain strain spore.Seed culture medium used, the composition often contained in 1L are：Glucose 20.0g, potato
200.0g, distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
(3) tea leaf fermentation：It is 30% to choose the green gross tea of three-level and sprinkle water to water capacity.The strain spore harvested in inoculation step 2
Son, the ratio that 100,000 fungal spores are launched according to tea per jin are fermented.Yeasting is that dustless pollution-free constant incubator is wet
Heap, it is 30 DEG C to control temperature, humidity 90%.After fermentation 3 to 4 days, fermented fungal grows in tea heap surface colonization, tealeaves leaf
Visible white mycelium on piece, simultaneous tea heap temperature rises, and when tea heap temperature is raised to 45 DEG C, carries out turning, turning
Tea heap may proceed to rise afterwards, treat that temperature is raised to 40 DEG C or so, tealeaves color is completely converted into crineous, when giving out ferment perfume (or spice), stops
Only.
(4) type is made in tea steaming：The tealeaves decatize that will be fermented, steam temperature are 100 DEG C, steaming time 3min, are obtained
Tealeaves after to decatize is put into shaped device shaping, naturally cool after shaping to put, cool to put the time as 40 hours, completes green brick tea loose tea
Making.
Embodiment 4 is tested using two kinds of fungi mixed fermentation green brick teas
(1) inclined-plane culture：Rhizopus stolonifer bacterium xingding-3 and total shape branch mucor xingding-2 are inoculated in tiltedly respectively
On the culture medium of face, cultivated 48 hours at 28 DEG C, obtain inclined-plane bacterial strain.Slant medium used, the composition often contained in 1L are：Portugal
Grape sugar 20.0g, potato 200.0g, agar 15g, distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
(2) fermented bacterium expands culture：The ring of inclined-plane bacterial strain one is respectively taken to be inoculated into respectively in sterilized seed culture medium,
175rpm, isothermal vibration culture 48h under conditions of 28 DEG C, obtain seed culture fluid, are discarded in 3000r/min centrifugations 10min
Clear liquid, obtain strain spore.Seed culture medium used, the composition often contained in 1L are：Glucose 20.0g, potato 200.0g,
Distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
(3) tea leaf fermentation：It is 30% to choose the green gross tea of three-level and sprinkle water to water capacity.The strain spore harvested in inoculation step 2
Son, the ratio that fungal spore each 100,000 is launched according to tea per jin are fermented.Yeasting is dustless pollution-free constant incubator
Pile fermentation, it is 28 DEG C to control temperature, humidity 90%.After fermentation 3 to 4 days, fermented fungal grows in tea heap surface colonization, tealeaves
Visible white mycelium on blade, simultaneous tea heap temperature rises, and when tea heap temperature is raised to 50 DEG C, carries out turning, turns over
Tea heap may proceed to rise after heap, treat that temperature is raised to 45 DEG C or so, and tealeaves color is completely converted into crineous, when giving out ferment perfume (or spice),
Stop.
(4) type is made in tea steaming：The tealeaves decatize that will be fermented, steam temperature are 100 DEG C, steaming time 3min, are obtained
Tealeaves after to decatize is put into shaped device shaping, naturally cool after shaping to put, cool to put the time as 40 hours, completes green brick tea loose tea
Making.
The fermentation green brick tea of embodiment 5 is evaluated：
Reference is evaluated to fermented tea《Tealeaves organoleptic evaluation method》, from profile, soup look, fragrance, flavour etc. to this
The green brick tea that the tealeaves of experiment fermenting and producing produces with traditional mode of production mode is contrasted, as a result such as table 1.As a result show with this
Too big difference is had no in the tea of method fermenting and producing and the green brick tea quality of produced in conventional processes, with independent rhizopus stolonifer fermentation phase
And, soup look is redder, and fragrance is slightly poor than, flavour more alcohol.Illustrate that this method can be used for the production of green brick tea, and this method used time
Shorter, production efficiency is higher.
The present invention production green brick tea of table 1 evaluates contrast with traditional green brick tea
Claims (9)
1. one plant of rhizopus stolonifer bacteria strainRhizopus stolonifer Xingding-3, on May 5th, 2017 is preserved in
State's Type Tissue Collection, deposit number are CCTCC M 2017240, and preservation address is Hubei, Wuhan, Wuhan University.
2. one plant of Mucor bacteria strainMucor racemosusFresXingding-2, Chinese allusion quotation is preserved on May 5th, 2017
Type culture collection, deposit number are CCTCC M 2017241, and preservation address is Hubei, Wuhan, Wuhan University.
3. the strain isolation described in claim 1 or 2 is produced in pile fermentation technique from green brick tea is in the wet of pile-fermentation mid-term
Heap, the tealeaves for having hypha,hyphae hair tonic.
4. application of a kind of hybrid bacterial strain on fermentation green brick tea, it is characterised in that the hybrid bacterial strain is included described in claim 1
Rhizopus stolonifer bacteria strainRhizopus stolonifer Xingding-3, and the Mucor bacteria strain described in claim 2Mucor racemosusFresxingding-2。
5. the application described in claim 4, it is characterised in that comprise the following steps：
（1）Inclined-plane culture：Total shape branch mucor xingding-2 and rhizopus stolonifer bacterium xingding-3 is inoculated in slant medium
Upper culture, obtain inclined-plane bacterial strain；
（2）Fermented bacterium expands culture：Inclined-plane inoculation is taken to obtain seed culture fluid into sterilized seed culture medium,
After the completion of culture, 10min abandoning supernatants are centrifuged in 3000r/min, harvest strain spore；
（3）Tea leaf fermentation：It is 20-50% to choose green gross tea and sprinkle water to water capacity, inoculation step（2）After the strain spore of middle harvest
Fermented, yeasting is dustless pollution-free constant incubator pile fermentation, and it is 25-30 DEG C, humidity 85-95% to control temperature, hair
After ferment 3-4 days, when tea heap temperature is raised to 50-60 DEG C, turning is carried out, tea heap may proceed to grow white hypha body after turning,
And rise with temperature, when temperature is raised again to 50-55 DEG C, tealeaves color is completely converted into crineous, when giving out ferment perfume (or spice), stops
Only；
（4）Type is made in tea steaming：The tealeaves decatize that will be fermented, the tealeaves after decatize are put into shaped device shaping, after shaping from
So cool to put, cool to put the time be 35-45 hours, the making of completion green brick tea loose tea.
6. the application described in claim 5, it is characterised in that step（1）In, total shape branch Mucor xingding-2, rhizopus stolonifer
Bacterium xingding-3 is inoculated on slant medium, is cultivated 48 hours at 28 DEG C, is obtained inclined-plane bacterial strain；Slant medium used,
It is per the composition contained in 1L：The .0g of glucose 20, potato 200.0g, agar 15g, distilled water surplus, natural pH, 121 DEG C
Sterilize 20 min.
7. the application described in claim 5, it is characterised in that step（2）In, take inclined-plane inoculation to be trained to sterilized seed
Support in base, the isothermal vibration culture 48h under conditions of 175 rpm, 28 DEG C obtains seed culture fluid, culture complete after
3000r/min centrifuges 10min abandoning supernatants, obtains strain spore；Seed culture medium used, the composition often contained in 1L are：
The .0g of glucose 20, potato 200.0g, distilled water surplus, natural pH, 121 DEG C of sterilizing 20min.
8. the application described in claim 5, it is characterised in that step（3）In, it is 30% that green gross tea, which is sprinkled water to water capacity,；Inoculation step
Suddenly（2）The strain spore of middle harvest, the ratio that 100,000 fungal spores are launched according to tea per jin are fermented；Yeasting is dustless
Pollution-free constant incubator pile fermentation, it is 25 DEG C to control temperature, humidity 90%.
9. the application described in claim 5, it is characterised in that step（4）In, the tealeaves decatize that will ferment, steam temperature is
100 DEG C, steaming time 3min.
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| CN109055273A (en) * | 2018-09-10 | 2018-12-21 | 安徽农业大学 | A kind of green brick tea pile-fermentation strain composition and application |
| CN109136100A (en) * | 2018-08-21 | 2019-01-04 | 鑫鼎生物科技有限公司 | One plant of Tabin aspergillus bacterial strain and the application on fermentation green brick tea |
| CN109287784A (en) * | 2018-09-10 | 2019-02-01 | 安徽农业大学 | A kind of method of the quick pile-fermentation of green brick tea |
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