CN105494715B - A kind of technique of inocalation method production dark green tea - Google Patents
A kind of technique of inocalation method production dark green tea Download PDFInfo
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- CN105494715B CN105494715B CN201410487033.3A CN201410487033A CN105494715B CN 105494715 B CN105494715 B CN 105494715B CN 201410487033 A CN201410487033 A CN 201410487033A CN 105494715 B CN105494715 B CN 105494715B
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Abstract
The invention belongs to microorganisms and technical field of food biotechnology, relate to a kind of dark green tea production technology for being inoculated with the coronoid process dissipate capsule bacterium through breeding, it is Eurotium cristatum NHRI-BC-1.5.1 including taking classification naming, it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is the coronoid process dissipate capsule bacterium of CGMCC No.8730 as floating strain, after the spelling heap step, before steaming step, or after the metrology steps, the dark green tea production technology being inoculated with before secondary steaming step, not only dark green tea product quality height and stability are good for the dark green tea product of preparation, but also the controllability of the golden flower quantity of dark green tea product is realized substantially, further realize the standardization control of dark green tea product.
Description
Technical field
The invention belongs to microorganisms and technical field of food biotechnology, and in particular to a kind of technique of inocalation method production dark green tea.
Background technique
Dark green tea is that one of big teas of tradition six (green tea, black tea, white tea, green tea, dark green tea and yellow tea) and China are distinctive
One big teas.Common dark green tea includes Anhua dark green tea, yunnan puer tea, Guangxi Liu Bao tea, Hubei green tea etc. always.Wherein traditional Anhua
Dark green tea mainly uses the large leaf group kind in Hunan snowy peak mountain range tea area for raw material, is finished, is rubbed, pile fermentation, drying etc. four
The product for orange with dark brown glossy, the flavour alcohol of dry tea color and/or micro-puckery, soup look, slightly unique rosin that big technique is process
The Dark Green Tea of matter feature and the product general name being process using it as feed purification.It gains the name because originating from Hunan China Anhua County.
Contain the bioactive ingredients such as tea polyphenols and its metabolite, polysaccharide, a variety of amino acid, vitamin, organic acid in the dark green tea of Anhua,
And part distinctive active constituent in the dark green tea of Anhua, research shows that Anhua dark green tea may have lower hyperlipidemia, hypertension, hyperglycemia, lose weight, shield of relieving the effect of alcohol
The health efficacies such as liver, anti-oxidant, anti-radiation, its distinctive fragrance flavour, Anhua dark green tea are more and more welcomed by the people in addition.
Fu-brick tea in the dark green tea of Anhua is concerned because it contains " golden flower " bacterium, i.e. coronoid process dissipate capsule bacterium, and coronoid process dissipates capsule
Bacterium promotes the transformation of tealeaves material composition by its secondary metabolite, forms the distinctive fragrance flavour of Anhua black tea Fu-brick tea and is good for
Kang Gongxiao, coronoid process dissipate capsule bacterium are also referred to as the probiotics in dark green tea.The production technology packet of traditional Anhua black tea Fu-brick tea product
Raw material preliminary working (Dark Green Tea production: → pile-fermentation → drying is rubbed in picking → water-removing →) → preliminary working is checked and accepted → is included to sieve whole
Reason → spelling heap → decatize → pile-fermentation → metering → steam pressure system of building → floating → drying → packaging and other steps.And wherein " floating "
Step is the distinctive process of Anhua black tea Fu-brick tea, and coronoid process dissipate capsule bacterium raised growth is bred in dark green tea production process, is promoted black
The rapid conversion of material composition in tea forms the distinctive fragrance of Fu-brick tea, flavour and effect.How to allow flower to send out well, is Anhua
The key element of dark green tea Fu-brick tea production.
It is basic to use " natural floating " technology at present in industrialization Fu-brick tea production -- i.e. coronoid process dissipates in process of production
Capsule bacterium enters dark green tea naturally in the form of environmental microorganism, i.e., " seed " derives from environment.However it is above-mentioned using " natural floating "
Production technology but have the following problems: 1, " floating " quality is affected by environment big, since coronoid process dissipate capsule bacterium seed source is in ring
Border, brings dark green tea into different production links, and the quantity and activity of seed depend on environment temperature, humidity, cleannes, production frequency
The factors such as secondary and raw material, and there is place limitation, since factory extends or migrates, " floating " will fluctuate widely very
It extremely can not floating；2, " floating " parameter is unstable, and due to bringing the quantity and activity difference of the seed of production line into, identical product is not
It is had differences with batch floating quantity, floating period, is unable to reach factory there are floating quantity and requires, floating period universal mistake
The problems such as long (at least 12 days)；3, product quality is unstable, since coronoid process dissipate capsule bacterium growing state determines that (coronoid process dissipates product quality
The secondary metabolite of capsule bacterium secretion promotes the material composition transformation in tealeaves), therefore the unstable of floating leads to different batches
The fragrance flavour and taste of product have differences, and quality safety and health efficacy characteristic have differences, for example, since coronoid process dissipates capsule
The growth of bacterium can inhibit the growth of other fungies, so if originally seed vitality is weak, coronoid process dissipate capsule bacterium fails in floating early stage
Raised growth then causes other fungies to breed, pollutes.
Therefore, the prior art has gradually developed the control that tealeaves especially dark green tea product is carried out using the method for inoculating microbes
System.As disclosed a kind of dark green tea fungus growing process in Chinese patent literature CN102524442A, and in particular to by dark green tea raw material through wet
Heap steams tea, sprinkling coronoid process dissipate capsule bacterium conidia powder, then tealeaves is pressed into brick, high temperature sterilization after sealing, tealeaves floating one after cooling
Dark green tea product is made in drying after the section time, is had using this method and shortens fermentation period, and thalli growth inside and outside brick tea can be made equal
It is even luxuriant, and " golden flower " can be kept non-discolouring for a long time and the advantages of " golden flower " does not disappear.For another example Chinese patent literature
A kind of method improving quality of Liupu tea disclosed in CN101669559A dissipates cystospore suspension using addition coronoid process after first steam
It sprays the mode of inoculation and tealeaves strain is pulverized into the method that foam inoculates to increase the effect of floating after multiple steaming.But it is above-mentioned
There are drawbacks for method: first, lack strain breeding thereof step, bacterial strain uses therefor can not be during the fermentation by keeping coronoid process dissipate capsule bacterium
The growth vigors of other bacterium is achieved the purpose that antibacterial, needs are repeatedly inoculated with or are sterilized, and the scattered capsule of coronoid process could be enhanced
The growth vigor of bacterium inhibits the growths of other bacterium；Second, quality control index is lacked to bacterial strain production seed, the work to " seed "
Property, heat resistance etc. supports without data；Third lacks systematic Study to inoculation production link, proves oese without data
Section is optimal link, both can guarantee that seed was successfully accessed and has excellent activity, and had not also accounted for production convenience；4th,
Effect of inoculation is not evaluated explicitly, " floating " situation of products obtained therefrom also needs to be further increased.
As it can be seen that keeping coronoid process dissipate capsule bacterium during tea leaf fermentation how by optimizing and standardizing inoculating microbes technique
The growth vigor to other bacterium, achieve the purpose that promoted dark green tea product quality, become a problem urgently to be resolved.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in dark green tea product quality in the prior art unstable, golden flower number
Uncontrollable problem is measured, and then a kind of inocalation method production dark green tea of a kind of dark green tea product with stable quality and golden flower controllable quantity is provided
Technique, i.e., realization the good strain of breeding, prepare seed, choose link, produced dark green tea.
In order to solve the above technical problems, the present invention provides a kind of techniques of inocalation method production dark green tea, including to Dark Green Tea
It carries out Raw material processing, spell heap, decatize, pile-fermentation, metering, secondary decatize, the step for pressing brick, floating, drying, in the spelling heap
After step, before steaming step or after the metrology steps, before secondary steaming step, with deposit number for CGMCC No.8730
Eurotium cristatum NHRI-BC-1.5.1 coronoid process dissipate capsule bacterium probiotic strain carry out inoculating microbes intervention.
The inoculation step is that access contains only the solid seed of the bacterial strain or obtains the bacterial strain through Liquid Culture
The liquid seeds arrived.
The solid seed obtains as follows:
Bacterial strain streak inoculation after taking activation is into on-liquid culture medium, under the conditions of 25-30 DEG C, is protected from light culture 6-8 days,
And aseptically, add water elution culture medium, and collect eluent, thalline were collected by centrifugation to get.
Preferably, the on-liquid culture of the inoculation is based under the conditions of 28 DEG C, is protected from light culture 7 days, and aseptically,
Add sterile water elution culture medium, and collect eluent, bead shake culture 30min is added.
The on-liquid culture medium includes PDA solid medium and the solid using tealeaves, tea powder, tea cream for biomass
Culture medium.
The inoculum density of the solid seed is (3-40) * 106A/kg tealeaves.Preferably, after the spelling heap step,
The density of the solid seed accessed before steaming step is 10*106A/kg tealeaves, or after the metrology steps, secondary decatize
The density of the solid seed accessed before step is 3*106A/kg tealeaves.
The liquid seeds obtain as follows:
Spore bacteria suspension is made with sterile water elution in bacterial strain after taking activation, and is seeded in suitable fluid nutrient medium,
Seed liquor is collected in shake culture 3-5 days under the conditions of 25-30 DEG C, and thalline were collected by centrifugation to obtain the final product.
Preferably, the Liquid Culture after inoculation is based under the conditions of 28 DEG C, and 125rpm shake culture 3 days, collecting seed liquor was
?.
The fluid nutrient medium includes PDA liquid medium, and suitable tea juice, millet paste.
The inoculum density of the liquid seeds is (5-60) * 106A/kg tealeaves.Preferably, after the spelling heap step,
The density of the liquid seeds accessed before steaming step is 30*106A/kg tealeaves, or after the metrology steps, secondary decatize
The density of the liquid seeds accessed before step is 6*106A/kg tealeaves.
The bacterial strain further includes the steps that activation: under aseptic condition that the strain inoculated is quiet into PDA solid medium
Set culture.
The condition for controlling first, second pressurization steaming step is independent of each other are as follows: 90-110 DEG C of temperature, pressure
0.3-0.5MPa, steaming time 8-12s.
The pile fermentation height of the pile fermentation step is 0.8-1.2m, and the pile-fermentation time is 1.5-2.5h.
In the floating step, controlled at 25-35 DEG C, relative air humidity 65-85%, constant temperature and humidity floating 9-
10 days.
It further include checking and accepting the solid seed or described before inoculation in the technique of the inocalation method production dark green tea
The step of liquid seeds:
The step of liquid seeds are checked and accepted is as follows:
The step of seed density detects specifically includes: aseptically, liquid seeds described in 1mL being taken to be diluted to dilution
Liquid, taking dilution gradient respectively is 10-4、10-5、10-6Each 1mL of dilution be added in sterile petri dish, then respectively to the training
The PDA solid medium that 15-20mL warm is added in ware is supported, is cultivated in 28 DEG C of sterile culture casees 3 days, then bacterium colony counts；
If the inoculum density is not less than 3*107/ mL culture medium, then the liquid seeds are available；If not up to above-mentioned standard, the liquid
Seed is unavailable.
The step of solid seed is checked and accepted is as follows:
The step of seed density detects specifically includes: aseptically, solid seed described in 1g is taken to be diluted to dilution,
Taking dilution gradient respectively is 10-5、10-6、10-7Each 1mL of dilution be added in sterile petri dish, then respectively to the culture
The PDA solid medium of 15-20mL warm is added in ware, is cultivated in 28 DEG C of sterile culture casees 3 days, then bacterium colony counts；If
The inoculum density is not less than 107/ gram culture medium, then the solid seed is available；If not up to above-mentioned standard, the solid seed
It is unavailable.
The present invention provides a kind of dark green teas that the technique by above-mentioned inocalation method production dark green tea is prepared.
The technique of inocalation method production dark green tea of the present invention is obtained by improveing to traditional dark green tea production technology
Dark green tea product with stable quality, the outstanding and realization floating quantity obtained is substantially controllable, and the advantage of technique of the invention is specific as follows:
(1) there is the advantage for reducing environmental disturbances:
Even if raw material changes, dark green tea floating quantity change fluctuation it is small: choose different batches raw material, (picking the time and
Material supplier is different), totally 3 batches, traditional handicraft and technique production of the invention is respectively adopted, final floating quantity statistics: passes
Unite technique floating range 33*104Bacterium number/g dry tea -98*104Bacterium number/g dry tea, present invention process floating range 95*104Bacterium number/g
Dry tea -114*104Bacterium number/g dry tea, the dark green tea product floating quantity of present invention process preparation, which significantly improves and stablizes, (to be sent out
Spend detected within the 12nd day)；
Even if the temperature of the external environment of production changes, it is small that the floating quantity of dark green tea changes fluctuation: fixed raw material, choosing
25 DEG C of environment temperature, 30 DEG C, 34 DEG C of progress brick tea floating experiments are taken, respectively using traditional natural fungus growing process and work of the present invention
Skill, floating quantity statistics: traditional handicraft floating range 30*104-85*104Bacterium number/g dry tea, present invention process floating range 82*
104-101*104The dark green tea product floating quantity of bacterium number/g dry tea, present invention process preparation significantly improves and stablizes (in floating
It is detected within 12nd day)；
Even if operating environment changes, it is small that floating quantity changes fluctuation: pressing brick when carrying out simulation in Beijing and Guangdong laboratory
Experiment, for traditional fungus growing process due to bringing into almost without external source environmental microorganism, floating failure, floating quantity is lower than 104Bacterium number/g
Dry tea, present invention process floating range 61*104-66*104Bacterium number/g dry tea, the dark green tea product floating number of present invention process preparation
Amount is significant to stablize (being detected at floating the 12nd day)；
(2) with the advantage of stable floating parameter
The floating period is short, can significantly improve production efficiency, save cost: through testing, carrying out 10 500 pieces of batch altogether
Fu-brick tea experiment, in the 12-13 days floating period of traditional handicraft, the 9-10 days floating period of present invention process, (period referred to since drying chamber
Temperature control floating starts to warm up dry total time to floating is terminated, and standard is to reach golden flower to be abound with, and can be used for follow-up sales)；
Dark green tea floating quantity dramatically increases: through testing, carrying out 10 batches, 500 pieces of Fu-brick tea experiments, traditional handicraft hair altogether
Flower range 30*104-115*104Bacterium number/g dry tea, average value 52*104Bacterium number/g dry tea；Present invention process floating range 83*
104-116*104Bacterium number/g dry tea, average value 94*104The dark green tea product floating of bacterium number/g dry tea, present invention process preparation is stablized
Property and quantity are significantly higher than dark green tea floating quantity in the prior art (being detected at floating the 12nd day)；
(3) the guaranteed advantage in terms of product quality
Taste: through testing, 10 batches, 500 pieces of Fu-brick tea experiments are carried out altogether, through tasting traditional handicraft and present invention process
There is 80% and 98% product to can reach super Fu-brick tea in Hunan provincial standard " Anhua black tea Fu-brick tea " respectively and evaluates standard, soup
Color reddish yellow, arohid flavour are pure, flavour is mellow；Safety index: through testing, carrying out 10 batches, 500 pieces of Fu-brick tea experiments altogether, according to
Hunan provincial standard " Anhua black tea Fu-brick tea " moisture, total ash, tea stalk content, non-tea field trash, water extraction, safety are defended
The compliance rate of whole Indexs measures such as raw index, traditional handicraft and present invention process is more than 99%；
Living contaminants: through testing, 10 batches, 500 pieces of Fu-brick tea experiments are carried out altogether, according to new technological flow examination criteria
Carry out microorganism detection, it is respectively 6 ‰ and 0 that mildew probability, which occur, in traditional handicraft and present invention process, present invention process preparation
The mildew rate probability of dark green tea product significantly reduces.
(4) seed quality of technique preparation is high through the invention, facilitates the production of dark green tea
The successful control of seeded process is had benefited from above-mentioned (1), (2), (3) three product characteristics, i.e., is prepared into using strain
The production seed quality arrived is high, and is inoculated with link reasonable.
Technique of the invention is pure-blood ferment, and seed used is the middle tea coronoid process for being most suitable for Anhua black tea Fu-brick tea production
Bulk bacteria strain, isolation and selection obtains from the high-quality middle warehouse tea a century Mu Cang, has and grows fast, high temperature resistant, inhibits miscellaneous bacteria energy
The strong feature of power, other inoculation technique strains are mostly directly to separate from tealeaves, are purified without breeding, not the process of breeding；
Used seed density is high, and liquid strain and solid kind seed density ACCEPTABLE QUALITY LEVEL respectively reach 3*107A/
Ml culture medium and 107A/g culture medium, and carry out miscellaneous bacteria microorganism detection, it is ensured that seed density and purity form production basis；
It is selected by production link, selected inoculation link simple possible, and seed heat resistance can reach inoculation and want
It asks.Used seed heat resistance is high, due to experienced high temperature steaming twice in Fu-brick tea production process, if seed heat resistance
It is poor then can not produce, it has no and the heat resistance of external source strain is clearly identified before in the prior art, through detecting work of the present invention
The liquid strain and solid seed heat resistance of skill preparation respectively reach 10% and 30% or more, and it is close according to reckoning to calculate inoculation
Degree, all inoculum concentrations reach 1 ‰ or more end product criteria, meet microbiology and be inoculated with rule substantially.New process both can be with
Improve dark green tea product quality, at the same can be used for non-traditional dark green tea production area carry out Fu-brick tea floating and other containing coronoid process
The production of bulk bacteria product.It realizes the good strain of breeding, prepares seed, choose link, produced dark green tea.
To sum up, the dark green tea of technique preparation through the invention not only product with stable quality, excellent, and the present invention provides one
The standardized dark green tea fungus growing process of kind realizes by the way that key node control is arranged in each link and uses digitalization index
It is detected, can have quantizating index in strain improvement standard, seed preparation standard and technique criterion of acceptability etc., further
Realize the controllable of dark green tea fungus growing process, and more complete in terms of the effect assessment of the dark green tea product of present invention process preparation, from
Floating stability, floating period, floating quantity, yield rate and the aspect of product quality five simultaneously to the effect of present invention process into
It has gone evaluation, and has obtained positive findings.It is a certain from the side such as floating period, floating quantity and soup look in compared to existing technology
It is evaluated in face of dark green tea production technology, cannot be from the point of view of the floating stability to high-volume dark green tea be detected, of the invention is black
Tea production technology may be implemented in production link control, realize the controllability of dark green tea floating quantity, further improve dark green tea production
The stability of product quality.
Specific embodiment
The present invention following PDA solid plate culture medium, the test tube slant PDA culture medium and PDA eggplants as described in the examples
The culture medium of shape bottle is prepared according to conventional method in the prior art, for the following each embodiments of the present invention, the PDA solid
The culture medium composition of plating medium, the test tube slant PDA culture medium and PDA eggplant-shape bottle is respectively as follows:
PDA solid plate culture medium, test tube slant culture medium: 300 grams of potato, 20 grams of glucose, 15~20 grams of agar,
1000 milliliters of tap water, natural pH；
The culture medium of PDA eggplant-shape bottle: 300 grams of potato, 20 grams of glucose, 1000 milliliters of tap water, natural pH.
The model of following reagents as described in the examples and instrument is respectively as follows:
KOD:KOD-101Toyobo；
PCR product QIAquick Gel Extraction Kit: D6492Omega；
Centrifuge: 3K15, Sigma；
Electrophoresis tank: JY-SPFT；
Electrophoresis apparatus: JY300C, manufacturer are the east Beijing Jun Yi electrophoresis equipment Co., Ltd；
Gel imaging system: JY04S-3C, manufacturer are the east Beijing Jun Yi electrophoresis equipment Co., Ltd；
PCR instrument: Life Express, manufacturer are Bo Science and Technology Ltd..
1 bacterial strain of embodiment isolates and purifies
The purification procedures of advantage " golden flower " bacterium of the dark green tea are specific as follows:
(1) tea sample is chosen
Choose Hunan Anhua tea processing factoryIn wooden warehouse in tealeaves the Fu-brick tea product of different year and
The production of yiyang, hunan city, commercially available mainstream Fu-brick tea product, the material as original strain separation.The tea sample of above-mentioned selected product
Meet GH/T 1070 to the regulation of tea packaging, meets the regulation that GH/T1071 stores tealeaves, meet GB 2762-2012
To the limitation of pollutants in food, meets in GB2763/GB26130 to the maximum remaining limitation of pesticide, meet GB/T
9833.2-2013 to the correlation standard of " compressed tea " part；Meanwhile the tea sample of selection meets: packing without breakage, without dirt
Stain；Tealeaves shape is smooth, brick tea inside floating is uniform；The same batch products chosen, " golden flower " riotous growth, growth inside are equal
It is even；
(2) selected tea sample is numbered
To Hunan Anhua tea processing factoryIt is I that the product of wooden warehouse different year is numbered respectively, II, III,
IV, V, above-mentioned number is corresponding in turn to,, the tea sample product produced in 2013 in 2010 in 2002 in 1997 in 1985；To selection
Commercially available mainstream Fu-brick tea product number be A, B, C, D, E；
(3) tea sample is pre-processed
After tea sample is collected, aseptically, " golden flower " riotous growth, the uniform tea sample I of growth inside are chosen, taken described
The tea sample 25g that center is carried disease germs is crushed, and crosses 80-100 mesh, then the tea powder of sieving is fully transferred to the sterile water of 225mL
In, mixing fullys shake；
(4) the coating culture sample liquid of tea containing bacterium body
The mixed liquor for taking sterile water to obtain above-mentioned steps carries out gradient dilution, and taking 1mL dilution gradient is 106Contain bacterium solution
Body is coated in PDA solid plate culture medium and is cultivated；
(5) it cultivates and records bacterium colony growing state
PDA solid plate culture medium after coating is placed in constant incubator and is cultivated, cultivation temperature 28-30
DEG C, it cultivates 7 days, during the cultivation process, according to the morphology of coronoid process dissipate capsule bacterium Eurotium cristatum, physiological and biochemical property
Identification and the number of bacterium colony are carried out to the morphological feature of the bacterium colony, every 12h measures the diameter and record of bacterium colony；
(6) picking purifies single colonie
Take the single colonie for growing 5 plants of most fast bacterium in above-mentioned steps, picking colony and streak inoculation to the PDA culture medium
On, cultivated 7 days under the conditions of 28-30 DEG C, three samples of each bacterium colony picking are purified, the number of secondary culture be twice,
The fastest-rising 5 plants of bacterial strains of colony diameter are saved in final selection purifying incubation；
(7) original strain is saved
The test tube slant PDA is conventionally prepared, 5 plants of bacterium of the above-mentioned selection of picking distinguish streak inoculation to the PDA
On test tube slant, under the conditions of 28-30 DEG C, after culture 7 days, backup is freezed after test tube is sealed up for safekeeping.
Likewise, the tea sample II, III, IV, V, the strain separating of A, B, C, D, E purify referring to above-mentioned experimental implementation, obtain
Sample is alternatively educated to more plants.
The results show that the speed of its bacterium colony average diameter growth of the bacterial strain of breeding is more than 5mm/d.
Wherein, the conversion bibliography Serra J.Image analysis and of irregular colony diameter
Mathematical morphology [M] .Academic Press, Inc., 1983. is converted in relation to image procossing and diameter
Content the method.
The breeding of 2 bacterial strain of embodiment
Breeding step a. is according to " gold in characteristic containing bacterium in tealeaves when fermentation and golden flower extent of growth breeding bacterial strain tealeaves
The extent of growth breeding bacterial strain of flower "
Breeding thinking: under identical condition of culture, the same a batch of Dark Green Tea raw material for deriving from the same place of production is selected
Carry out the fermentation test of strain inoculated.If the bacterial strain can satisfy tea leaf quality to strain properties possessed by zymophyte and
Requirement of " golden flower " extent of growth to bacterial strain, then be inoculated with after the bacterial strain, then in tea product the bacterial strain can fast-growth, and
Certain amount containing bacterium number can be reached in its fermented tea within a certain period of time, to grow into dominant bacteria, inhibit miscellaneous bacteria
Growth；And its good characteristics exhibit is final tealeaves appearance and quality.Otherwise it is assumed that being inoculated with the bacterial strain is not able to satisfy tea
Leaf to zymogenic requirement, i.e., fermentation process inoculation using bacterial strain comparison cannot shoot up under identical fermentation condition for
Corresponding bacterial content is not achieved in dominant strain, and the final appearance of product cannot be guaranteed with quality or its growth in tealeaves
Excessive cycle or even heartburn, mildew.
The breeding step of advantage " golden flower " bacterium of the dark green tea is specific as follows:
1) tea sample I, II, III, IV, V, A, B, C, D, E is taken to pass through the backup strain inoculated that above-mentioned steps isolate and purify respectively
It to the PDA solid medium of eggplant-shape bottle, is cultivated under the conditions of 28 DEG C, on 100mL aseptic water washing after cultivating 5-15 days
Solid medium is stated, the mixed liquor containing thallus obtained after flushing is filtered through sterile gauze then and obtains spore suspension,
Number is stand-by；
2) wherein, 3 dark green tea samples of every part of strain inoculated, and it is not inoculated with institute according to the production of conventional dark green tea floating processing method
State control group of the Fu-brick tea of the bacterial strain of separation as experiment；
3) " golden flower " bacterium for meeting breeding condition directly inside the tealeaves of picking above-mentioned steps acquisition is in PDA solid plate
Culture is purified on culture medium, the strain for meeting breeding condition at least samples portion, and the strain number after purifying culture is A series,
The bacterial strain of selection, number A are listed in the result (part) of the cold storage of the test tube slant PDA, backup, detection.
The bacterial strain obtained by the breeding of this step is 31 plants total, and when carrying out dark green tea floating, floating is rapid, can be in floating
" golden flower " bacterium total plate count in tea sample is enabled within 5th day to reach 5 × 104CFU/g over dry tealeaves, miscellaneous bacteria content less than 1 ×
102CFU/g over dry tealeaves；And floating terminates, can " golden flower " be abound with；Finished product rate reaches 99%；Fermentation tea product appearance
" golden flower " riotous growth, growth inside uniformly, good mouthfeel；Fermentation tea product meets the bacterial strain of quality testing standard.
Breeding step b. uses hot fermentation breeding high-temperature resistant strain
Breeding thinking: the bacterial strain that high temperature resistant experiment breeding obtains enables to fermentation that can carry out at relatively high temperatures,
I.e. in tea product fermentation at relatively high temperatures, it keeps strain fermentation tealeaves in breeding step a can achieve the effect that, meets practical
The seasonal variation of temperature in production carries out floating production under the conditions of summer temperature is higher.
Firstly, isolating and purifying in PDA culture medium to strain, it is more than generation to repeat picking single colonie secondary culture 10,
Selection can stablize the bacterial strain of passage growth, preferably colony diameter growth rate fast bacterial strain under the conditions of 38 DEG C；Then, it chooses
This batch of bacterial strain repeats a step a generation, and its fermentation temperature is arranged between 30-34 DEG C, carries out according to the breeding standard of step a
The breeding of strain isolates and purifies the dominant strain B wherein grown from tealeaves, saves, and it is real that number carries out next step breeding
It tests.
The breeding step of advantage " golden flower " bacterium of the dark green tea is specific as follows:
1) PDA solid plate culture medium, the original strain bacterium colony scribing line that picking number is A are made according to aforementioned conventional method
It is seeded to the PDA solid plate culture medium, then the plate is placed in 38 DEG C of constant incubator and is cultivated, is cultivated
After 7 days, it is more than generation to repeat the above steps 10 for the bacterial strain single colonie that picking can be grown, and selection can be at a temperature of described 38 DEG C
The bacterium colony of growth freezes backup with the test tube slant PDA to the strain number B of selection；
2) bacterial strain of above-mentioned selection is repeated the above steps the dark green tea floating fermentation step in a, difference is only by the hair
Drying room temperature in flower step changes into 30-34 DEG C, then carries out the 5th day in floating, takes preparation with special sampling cutter
The tea sample 25g of brick tea central interior detected；
Wherein, 3 samples of the inoculation of every part of bacterial strain；
Control sample is the corresponding original strain A of number B, and the correlated results of detection lists the bacterial strain of selection, and number is
B。
By the strain of this step breeding, can when summer environment temperature is higher, meet tealeaves floating 30-34 DEG C it
Between the needs that produce that is, in tea product fermentation at relatively high temperatures keep strain fermentation tealeaves in breeding step a attainable
Effect meets the seasonal variation of temperature in actual production, is produced under the conditions of summer temperature is higher.
It is 17 plants total that this step obtains strain excellent.
Breeding step c, breeding can largely generate the bacterial strain of cleistothecium
Inventor has found during the experiment, when cultivating bacterial strain in eggplant type bottle PDA culture medium using spread plate method, greatly
Part bacterial strain is grown in the regular Growth and Differentiation of a small number of bacterial strains except culture medium and goes out a large amount of cleistothecium at eggplant-shape bottle edge
(ascocarp), and the presence that its cleistothecium under field conditions (factors) can be stable, therefore inventor carries out breeding by the following method
The bacterial strain of cleistothecium can largely be generated:
Whether all strains examined for taking step b acquisition (it is flat should be solid in PAD culture medium using spread plate method herein
Plate culture medium) in cultivated, the marking at away from culture medium edge 2cm, picking exceeds existing a large amount of except mark line
The bacterial strain of cleistothecium carries out second generation coating culture；The cultural method of step c first generation bacterial strain is repeated until in the 5th generation, obtain
It is 14 plants total to generate the outstanding bacterial strain of cleistothecium character, to resulting strain number C, number NHRI-BC-1.5.1-- respectively
NHRI-BC-1.5.14 freezes backup with test tube slant.Wherein the bacterial strain of number NHRI-BC-1.5.1 is the objects advantages of breeding
Bacterial strain, identified its are coronoid process dissipate capsule bacterium.
3 strain Property Identification of embodiment
To the form for the coronoid process dissipate capsule bacterium Eurotium cristatum NHRI-BC-1.5.1 that above-described embodiment 2 obtains
, physiological and biochemical property and molecular biological characteristics are identified, specific as follows:
A, morphological features
The bacterial strain that number is NHRI-BC-1.5.1 is taken, is trained strain inoculated in PDA solid plate using streak plating
It supports on base, is cultivated 15 days in 28 DEG C of constant incubators, mycelium when thalli morphology is observed on picking culture medium bacterium colony, Yu Guang
It learns and is observed under microscope, amplification factor is 10 times of object lens, 16 times of eyepiece.
Observation has the results show that thallus is filiform every in light yellow.
B, colony morphology characteristic
The bacterial strain that number is NHRI-BC-1.5.1 is taken, strain inoculated is consolidated in PDA by platysome training using point sample cultivation
It supports on base, is cultivated 15 days in 28 DEG C of constant incubators, after culture terminates, be placed in 4 DEG C of refrigerators and freeze, record the spy of bacterium colony
Sign.
Observation is the results show that the coronoid process dissipate capsule bacterium Eurotium cristatum NHRI-BC-1.5.1 bacterial strain bacterium colony is dry
Dry, fine and close, round, rough surface, protrusion or protuberance, edge roughness is in mycelia dispersed, diameter maximum up to 60mm or so,
Tone is golden yellow and brown.
There are mycelium, mycelia for the coronoid process dissipate capsule bacterium Eurotium cristatum NHRI-BC-1.5.1 bacterial strain bacterium colony
Body is elongated, is divided into substrate mycelium, aerial hyphae.Substrate mycelium is brown, aerial hyphae over time with the expansion of bacterium colony
Greatly, center aerial hyphae shows white, faint yellow, golden yellow, brown, auburn variation respectively.Cultivate (1-3 early period
It), colony edge is white or faint yellow, and center is golden yellow, diameter 2-10mm；It cultivates mid-term (2-5 days), bacterium colony
There is brown or sepia bacterium colony, diameter 5-40mm in center；Late stage of culture (after 5-9 days), bacterium colony is divided into 3-4
Layer, edge are white with faint yellow, are golden yellow inside edge, brownish coloured colonies its raw mycelia in center grows golden yellow softgel shell；
Late stage of culture is accompanied by brown diffusate, brown diffusate more obvious, brown diffusate after culture terminates to freeze simultaneously
In spot distribution on brown bacterium colony surface layer, golden yellow and faint yellow bacterium colony layer is seldom or there is no diffusates.Meanwhile it oozing out
Liquid can penetrate into culture medium, and culture medium is contaminated for brown.Its faint yellow or white colony layer and golden yellow chromatograph remain 2-
The collarium size of 10mm, brownish coloured colonies layer can become larger with the increase of bacterium colony.
C, reproduction and genital structure form
The bacterial strain that number is NHRI-BC-1.5.1 is taken, bacterial strain streak inoculation is " miscellaneous using inhibition subtractive in such as document
Type mutation produces the relevant gene [J] of spore between handing over technical appraisement to thank to Wa Shi aspergillus " (Tan Yumei, Wang Hai, Liu Yongxiang wait bacteriology
Report, 2013,32 (1): 56-63) described in hypotonic and hypertonic solid medium in, in 28 DEG C cultivate 3 days, collection mycelium,
Ascospore and conidium stained preparation observe conidium and ascocarp situation under an optical microscope, collect bacterium
Silk and ascospore save in liquid nitrogen, observe with scanning electron microscope the micromorphology of each genital structure.
Observation is the results show that the coronoid process dissipate capsule bacterium Eurotium cristatum NHRI-BC-1.5.1 bacterial strain can be low
It seeps and generates ascospore and conidium in solid medium, ascus is wrapped up and finally discharged, cleistothecium quilt by yellow cleistothecium
Thalli growth winding；Cleistothecium naked eyes as it can be seen that size be 90~170 μm, it is oval；Cleistothecium includes a large amount of ascus, bonding
Together, ascus is in irregular spherical；Ascus after release includes 8 ascospores；4-6 μm of ascospore diameter is similar to river
Freshwater mussel, lobed convex, the coarse tool wart of centre of surface, the smooth of the edge, there are obvious dent in two valve junctions；Conidium is in concatenate
Shape can be divided into spore head of concatenating, sporophore two large divisions by its structure；Wherein, multi beam spore is concatenated in spore head, conidium
There are dents for junction；Single conidium size is 4~5 μm, is in ellipsoid, surface is covered with sharp wart.
D, the routine biochemistry property of bacterial strain
The coronoid process dissipate capsule bacterium Eurotium cristatum NHRI-BC-1.5.1 bacterial strain heteroplasia oxygen consumption, tolerance arid,
It is resistant to hyperosmosis, thalli growth can be carried out using LB liquid medium, can be metabolized using glucose, sucrose, maltose,
PH tolerance is 4.5-6.5, and growth temperature is 24-38 DEG C, and suitable growth temperature when fermented tea is 26-34 DEG C.
E, molecular biological characteristics
The bacterial strain that number is Eurotium cristatum NHRI-BC-1.5.1 is taken, is trained on PDA slant medium
Health grows bacterium colony.
1), thallus DNA extracts
Picking hypha,hyphae about 10mg is added fungal DNA and extracts 300 μ l of lysate, and be vortexed precipitating.In 65 DEG C of water-baths
Isometric chloroform: isoamyl alcohol (24:1) is added in 30min, is vortexed and mixes, and 13000rpm is centrifuged 5min.Supernatant is taken, it is different that volume is added
Propyl alcohol mixes；13000rpm is centrifuged 5min.Supernatant is abandoned, 70% ethyl alcohol is added and mixes, 13000rpm is centrifuged 1min.Ethyl alcohol is abandoned,
It is inverted centrifuge tube, is dried.30 μ lddH2O, dissolving DNA is added；
2), 18S rDNA/ITS is expanded
(1) DNA for taking above-mentioned steps to obtain, dilution are used as PCR reaction template, are carried out using DNA sequence dna described in primer pair
PCR reaction amplification, the reagent in the PCR reaction system of following list 1 is sequentially added into PCR pipe；
Table 1, PCR reaction system (component is in terms of μ L):
(2) PCR pipe for taking above-mentioned steps to obtain carries out the PCR of the DNA sequence dna according to the response procedures of the PCR of the following table 2
Reaction amplification；
The response procedures of table 2, PCR
(3) above-mentioned steps after completion of the reaction, take reaction product to carry out electrophoresis detection with 1% agrose gel, and determination has
After specific amplified, takes the reaction product to carry out PCR reaction amplification again according to above-mentioned steps (1) and (2), then recycle PCR
Product carries out electrophoresis detection, the electrophoresis result are as follows: wherein M:marker2000:2000,1000,750,500,250,100bp,
Bright band is 750bp；
The method of the PCR product recycling is as follows:
The Buffer CP of 4 times of volumes is taken to be added in the 1.5mL centrifuge tube containing 100 μ L of PCR reactant volume, so
Acutely concussion afterwards, of short duration centrifugation is then placed on adsorption column in collecting pipe, then the mixture that step (3) obtains is transferred to absorption
In column (750 μ L every time once turns endless, after waiting centrifugations, outwells waste liquid, then remaining mixture is transferred to adsorption column centrifugation), in
After 13000rpm is centrifuged lower 1min, filtrate is abandoned, the eluent of 700 μ L is then added into the adsorption column, under 13000rpm
After being centrifuged 1min, filtrate is abandoned, then 500 μ L eluents are added into the adsorption column, is centrifuged 1min in 13000rpm, abandons filtrate,
It is centrifuged 2min in 13000rpm again, the ethyl alcohol on adsorption column is got rid of, the adsorption column is transferred to a new 1.5mL centrifuge tube
In, the ddH2O of 30 μ L is then added to the center of the adsorption column, the 15000rpm condition again after placing 1min under room temperature
Lower centrifugation 2min, resulting filtrate are the DNA through PCR reaction amplification.
3) sequence, is measured
The DNA of above-mentioned acquisition needs the sequence measured, includes the region 18S rDNA/ITS1/5.8S rDNA/ITS2, described
Sequencing results see attached list 1, and sequence obtained is carried out sequence B LAST comparison；
The results show that the bacterial strain of Eurotium cristatum NHRI-BC-1.5.1 belongs to coronoid process dissipate capsule bacterium, homology
It is 99%.
The bacterial strain of above-mentioned identification is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as
CGMCC), depositary institution address is city, BeiJing, China, the road great Tun, Chaoyang District, Institute of Microorganism, Academia Sinica, deposit number
For CGMCC No.8730, preservation date is on January 16th, 2014.
Embodiment 4
The present embodiment prepares the solid seed, and specific step is as follows:
(1) taking classification naming is Eurotium cristatum NHRI-BC-1.5.1, has been preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number is that the coronoid process dissipate capsule bacterium of CGMCC No.8730 is stored refrigerated, standby
With；
(2) strain in above-mentioned steps (1) is inoculated under aseptic technique on fresh PDA slant medium,
It is placed under clean environment and cultivates 2.5 days, for use；
(3) it takes the bacterial strain streak inoculation of activation into test tube PDA solid medium, under the conditions of 25 DEG C, is protected from light culture 8
It, and aseptically, the above-mentioned test tube PDA culture medium of sterile water elution is taken, and collect eluent, thalline were collected by centrifugation, i.e.,
Solid seed is obtained, it is spare.
Using above-mentioned steps preparation solid seed carry out inocalation method production dark green tea technique specific step is as follows:
(a) the dark green tea raw material of traditionally (→ pile-fermentation → drying is rubbed in picking → water-removing →) processing is taken to carry out
Preliminary working carries out an acceptance inspection the dark green tea raw material of preliminary working, carries out deciding grade and level bunch according to dark green tea standard sample, choose superfine, level-one and
The Dark Green Tea of second level carries out spelling heap；
(b) above-mentioned solid seed is accessed to Dark Green Tea after above-mentioned spelling heap, the inoculum density of the solid seed is 10*106
A/kg tealeaves, and the water content for controlling dark green tea raw material is 15%, carries out decatize, vapour pressure to above-mentioned raw materials using high-temperature steam
Power is 0.4MPa, and temperature is 102 DEG C, steaming time 9s；
(c) Dark Green Tea after above-mentioned decatize is subjected to pile-fermentation, pile fermentation height is 0.8m, and pile fermentation time 1.5h is pressed
Requirement according to product is measured；
(d) tealeaves after metering is subjected to secondary decatize, steaming condition is steam pressure 0.3MPa, temperature 110
DEG C, steaming time 8 seconds；
(e) raw material after decatize is subjected to artificial pressure brick according to the specification of 1kg；
(f) obtained Fu brick semi-finished product are transferred to drying chamber, control drying chamber is not more than 65% in nurturing period, floating phase humidity, temperature
Degree be 25 DEG C, carry out floating fermentation, floating 9 days, then be no more than 50 DEG C under the conditions of to product be dried to get.
Embodiment 5
The present embodiment prepares the solid seed, and specific step is as follows:
(1) taking classification naming is Eurotium cristatum NHRI-BC-1.5.1, has been preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number is that the coronoid process dissipate capsule bacterium of CGMCC No.8730 is stored refrigerated, standby
With；
(2) strain in above-mentioned steps (1) is inoculated under aseptic technique on fresh PDA slant medium,
It is placed under clean environment and cultivates 2.5 days, for use；
(3) by the strain streak inoculation activated in above-mentioned steps (2) into tea solids culture medium, under the conditions of 30 DEG C,
It is protected from light culture 6 days, and aseptically, takes the above-mentioned tea solids culture medium of sterile water elution, and collect eluent, centrifugation is received
Collection thallus is spare to get solid seed；
The solid seed is checked and accepted according to the following steps:
The step of seed density detects specifically includes: aseptically, solid seed described in 1g is taken to be diluted to dilution,
Taking dilution gradient respectively is 10-5、10-6、10-7Each 1mL of dilution be added in sterile petri dish, then respectively to the culture
The PDA solid medium of 15-20mL warm is added in ware, is cultivated in 28 DEG C of sterile culture casees 3 days, then bacterium colony counts；If
The inoculum density is not less than 107/ gram culture medium, then the solid seed is available；If not up to above-mentioned standard, the solid seed
It is unavailable.
Using above-mentioned steps preparation solid seed carry out inocalation method production dark green tea technique specific step is as follows:
(a) the dark green tea raw material of traditionally (→ pile-fermentation → drying is rubbed in picking → water-removing →) processing is taken to carry out
Preliminary working carries out an acceptance inspection the dark green tea raw material of preliminary working, carries out deciding grade and level bunch according to dark green tea standard sample, choose superfine, level-one and
The Dark Green Tea of second level carries out spelling heap；
(b) decatize, steam pressure 0.5MPa are carried out to the dark green tea raw material after above-mentioned spelling heap using high-temperature steam, temperature is
90 DEG C, steaming time 12s；
(c) Dark Green Tea after above-mentioned decatize is subjected to pile fermentation, pile fermentation height is 1.2m, pile fermentation time 2.5h, according to production
The requirement of product is measured；
(d) after measuring, above-mentioned solid seed is accessed in above-mentioned dark green tea raw material, the inoculum density of the solid seed is 3*
106A/kg tealeaves, and the water content for controlling dark green tea raw material is 30%, carries out secondary decatize to above-mentioned raw materials using high-temperature steam,
Steaming condition is steam pressure 0.5MPa, and temperature is 110 DEG C, steaming time 8 seconds；
(e) raw material after above-mentioned decatize is subjected to artificial pressure brick according to the specification of 1kg；
(f) Fu brick semi-finished product obtained above are transferred to drying chamber, control drying chamber is not more than in nurturing period, floating phase humidity
75%, temperature is 30 DEG C, carries out floating fermentation, floating 10 days, place then is dried to product under the conditions of being no more than 50 DEG C
Reason to get.
Embodiment 6
The present embodiment prepares the liquid seeds, and specific step is as follows:
(1) taking classification naming is Eurotium cristatum NHRI-BC-1.5.1, has been preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number is that the coronoid process dissipate capsule bacterium of CGMCC No.8730 is stored refrigerated, standby
With；
(2) strain in above-mentioned steps (1) is inoculated under aseptic technique on fresh PDA slant medium,
It is placed under clean environment and cultivates 2 days, for use；
(3) spore bacteria suspension is made with sterile water elution in the bacterial strain after taking activation, and is seeded in PDA liquid medium,
Shake culture 3 days under the conditions of 25 DEG C, collect seed liquor, thalline were collected by centrifugation to get.
Using above-mentioned steps preparation liquid seeds carry out inocalation method production dark green tea technique specific step is as follows:
(a) the dark green tea raw material of traditionally (→ pile-fermentation → drying is rubbed in picking → water-removing →) processing is taken to carry out
Preliminary working carries out an acceptance inspection the dark green tea raw material of preliminary working, carries out deciding grade and level bunch according to dark green tea standard sample, choose superfine, level-one and
The Dark Green Tea of second level carries out spelling heap；
(b) in the dark green tea raw material after aforesaid liquid seed to be accessed to above-mentioned spelling heap with tea juice, the inoculation of the liquid seeds
Density is 30*106A/kg tealeaves, and the water content for controlling dark green tea raw material is 25%, is carried out using high-temperature steam to above-mentioned raw materials
Decatize, steam pressure 0.3MPa, temperature are 100 DEG C, steaming time 10s；
(c) Dark Green Tea after above-mentioned decatize is subjected to pile fermentation, pile fermentation height is 1m, pile fermentation time 2h, according to product
It is required that being measured；
(d) tealeaves after metering being subjected to secondary decatize, steaming condition is steam pressure 0.4MPa, and temperature is 90 DEG C,
Steaming time 9 seconds；
(e) raw material after above-mentioned decatize is subjected to artificial pressure brick according to the specification of 1kg；
(f) Fu brick semi-finished product obtained above are transferred to drying chamber, control drying chamber is not more than in nurturing period, floating phase humidity
70%, temperature is 27 DEG C, carries out floating fermentation, floating 10 days, place then is dried to product under the conditions of being no more than 50 DEG C
Reason to get.
Embodiment 7
The present embodiment prepares the liquid seeds, and specific step is as follows:
(1) taking classification naming is Eurotium cristatum NHRI-BC-1.5.1, has been preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number is that the coronoid process dissipate capsule bacterium of CGMCC No.8730 is stored refrigerated, standby
With；
(2) strain in above-mentioned steps (1) is inoculated under aseptic technique on fresh PDA slant medium,
It is placed under clean environment and cultivates 2 days, for use；
(3) spore bacteria suspension is made with sterile water elution in the bacterial strain after taking activation, and is inoculated in the fluid nutrient medium of millet paste,
Shake culture 5 days under the conditions of 30 DEG C, collect seed liquor, thalline were collected by centrifugation to get；
The liquid seeds are checked and accepted according to the following steps:
The step of seed density detects specifically includes: aseptically, liquid seeds described in 1mL being taken to be diluted to dilution
Liquid, taking dilution gradient respectively is 10-4、10-5、10-6Each 1mL of dilution be added in sterile petri dish, then respectively to the training
The PDA solid medium that 15-20mL warm is added in ware is supported, is cultivated in 28 DEG C of sterile culture casees 3 days, then bacterium colony counts；
If the inoculum density is not less than 3*107/ mL culture medium, then the liquid seeds are available；If not up to above-mentioned standard, the liquid
Seed is unavailable.
Using above-mentioned steps preparation liquid seeds carry out inocalation method production dark green tea technique specific step is as follows:
(a) the dark green tea raw material of traditionally (→ pile-fermentation → drying is rubbed in picking → water-removing →) processing is taken to carry out
Preliminary working carries out an acceptance inspection the dark green tea raw material of preliminary working, carries out deciding grade and level bunch according to dark green tea standard sample, choose superfine, level-one and
The Dark Green Tea of second level carries out spelling heap；
(b) decatize, steam pressure 0.5MPa are carried out to above-mentioned raw materials using high-temperature steam, temperature is 110 DEG C, when decatize
Between be 8s；
(c) Dark Green Tea after above-mentioned decatize is subjected to pile fermentation, pile fermentation height is 1.2m, pile fermentation time 2.5h, according to production
The requirement of product is measured；
(d) after measuring, aforesaid liquid seed is accessed in dark green tea, the inoculum density of the liquid seeds is 6*106A/kg
Tealeaves, and the water content for controlling dark green tea raw material is 25%, the tealeaves after pile fermentation is carried out secondary decatize, steaming condition is steam
Pressure is 0.3MPa, and temperature is 100 DEG C, steaming time 8 seconds；
(e) raw material after above-mentioned decatize is subjected to artificial pressure brick according to the specification of 1kg；
(f) Fu brick semi-finished product obtained above are transferred to drying chamber, control drying chamber is not more than in nurturing period, floating phase humidity
85%, temperature is 35 DEG C, carries out floating fermentation, floating 10 days, place then is dried to product under the conditions of being no more than 50 DEG C
Reason to get.
Embodiment 8-11
Embodiment 8-11 is identical as the production technology of the dark green tea of embodiment 4-7 respectively, and difference is only that, does not access
The liquid or solid seed that voluntarily prepares, and access commercially available coronoid process dissipate capsule bacterium Eurotium cristatum in the prior art
Conidia powder, other production technologies are remained unchanged with parameter.The coronoid process dissipate capsule bacterium conidia powder provides biotechnology purchased from Changsha Hunan
Co., Ltd.
Embodiment 12-14
Embodiment 12-14 is identical as the production technology of the dark green tea of embodiment 4, and difference is only that the solid kind
The inoculum concentration of son is respectively 1*106A/kg tealeaves, 5*106A/kg tealeaves and 4*107A/kg tealeaves, other production technologies with
Parameter remains unchanged.
Embodiment 15-18
Embodiment 15-18 is identical as the production technology of the dark green tea of embodiment 4-7 respectively, and difference is only that, prepares
Solid/liquid culture medium used in the solid/liquid seed is respectively containing the solid medium that tealeaves is biomass, contains tea
Cream is solid medium, tea juice and the millet paste of biomass, other production technologies are remained unchanged with parameter.Wherein, it is made a living with tealeaves
The solid medium of substance refers to manufactured loose tea culture medium after processing sterilized using Dark Green Tea identical with when criticizing raw materials for production,
The same other biologicals matter culture mediums such as tea cream, tea juice and millet paste that contain also are passed through using Dark Green Tea identical with batch raw materials for production are worked as
After sterilization treatment, further finishing is made.
Effect example
The indexs such as sensory evaluation, golden flower quantity and the mildew rate of dark green tea of Statistics Implementation example 4-18 preparation, wherein sense organ
The standard of evaluation referring to the fragrance of the super Fu-brick tea sensory review standard evaluation product in " Anhua black tea Fu-brick tea ", flavour,
The statistics of the compliance rate of soup look and taste, golden flower quantity conventionally counts coronoid process dissipate capsule bacterium in the dark green tea product of preparation
Number, mildew rate conventionally detect.Resulting statistical result is as follows:
The indices (floating 12 days) of dark green tea product described in table 3
As seen from the above table, the dark green tea product that manufacturing technique method through the invention is prepared not only quality and stability
It is significantly increased, sensory evaluation compliance rate has reached 100%, and coronoid process dissipate capsule bacterium quantity is high and stablizes, multiple batches of more than 100*
104Bacterium number/gram dry tea, mildew rate are almost 0, solve and uncontrollable ask since " floating seed " derives from natural environment
Topic stabilizes product quality in product sensory quality and secure context promotion.Meanwhile by embodiment 12-14 it is found that base of the present invention
Control of this realization to the floating quantity in the dark green tea product of production, and this is can not achieve in traditional dark green tea production technology
, the present invention further realizes the standardization control of dark green tea product, obtains significant technical effect.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (12)
1. a kind of technique of inocalation method production dark green tea, including Raw material processing is carried out to Dark Green Tea, spells heap, decatize, pile-fermentation, meter
Amount, secondary decatize, the step for pressing brick, floating, drying, which is characterized in that after the spelling heap step, before steaming step, or
After the metrology steps, before secondary steaming step, the Eurotium cristatum for being CGMCC No.8730 with deposit number
NHRI-BC-1.5.1 eurotium cristatum strain carries out inoculating microbes.
2. the technique of inocalation method production dark green tea according to claim 1, which is characterized in that the inoculation step is to access only
Solid seed containing the bacterial strain or the liquid seeds for obtaining the bacterial strain through Liquid Culture.
3. the technique of inocalation method production dark green tea according to claim 2, which is characterized in that the solid seed is according to as follows
Method obtains:
Bacterial strain streak inoculation after taking activation is into on-liquid culture medium, under the conditions of 25-30 DEG C, is protected from light and cultivates 6-8 days, and
Under aseptic condition, add water elution culture medium, and collect eluent thalline were collected by centrifugation to get.
4. the technique of inocalation method according to claim 3 production dark green tea, which is characterized in that the on-liquid culture medium includes
PDA solid medium and utilization tealeaves, tea powder, the solid medium that tea cream is biomass.
5. the technique of inocalation method production dark green tea according to claim 3 or 4, which is characterized in that the solid seed connects
Kind density is (3-40) × 106A/kg tealeaves.
6. the technique of inocalation method production dark green tea according to claim 2, which is characterized in that the liquid seeds are according to as follows
Method obtains:
Spore bacteria suspension is made with sterile water elution in bacterial strain after taking activation, and is seeded in suitable fluid nutrient medium, in 25-
Shake culture 3-5 days under the conditions of 30 DEG C collects seed liquor, and thalline were collected by centrifugation to obtain the final product.
7. the technique of inocalation method according to claim 6 production dark green tea, which is characterized in that the fluid nutrient medium includes
PDA liquid medium and tea juice, millet paste.
8. the technique of inocalation method production dark green tea according to claim 6 or 7, which is characterized in that the liquid seeds connect
Kind density is (5-60) × 106A/kg tealeaves.
9. the technique of any inocalation method production dark green tea of -4 or 6-7 according to claim 1, which is characterized in that described in control
The condition of decatize, secondary steaming step are as follows: 90-110 DEG C of temperature, pressure 0.3-0.5MPa, steaming time 8-12s.
10. the technique of any inocalation method production dark green tea of -4 or 6-7 according to claim 1, which is characterized in that the pile fermentation
The pile fermentation height of fermentation step is 0.8-1.2m, and the pile-fermentation time is 1.5-2.5h.
11. the technique of any inocalation method production dark green tea of -4 or 6-7 according to claim 1, which is characterized in that the floating
In step, controlled at 25-35 DEG C, relative air humidity 65-85%, constant temperature and humidity floating 9-10 days.
12. a kind of probiotics dark green tea being prepared by any production technology of claim 1-11.
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| CN101579024A (en) * | 2009-06-18 | 2009-11-18 | 谌小丰 | Artificial inoculation production method of camellia nitidissima |
| CN102524442A (en) * | 2012-01-27 | 2012-07-04 | 向华 | Fungus growing process for black tea |
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