CN108102953A - The application of one bacillus subtilis and its Aspergillus ochraceus A that degrades in post-fermented tea pile fermentation - Google Patents
The application of one bacillus subtilis and its Aspergillus ochraceus A that degrades in post-fermented tea pile fermentation Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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Abstract
The present invention provides a bacillus subtilis, deposit number is CCTCC NO：M2017708；It degrades the present invention also provides the bacillus subtilis in post-fermented tea pile fermentation the application of ochratoxin A；The bacillus subtilis adapts to post-fermented tea pile fermentation environment (acidity, high temperature, anaerobism) and with degradation ochratoxin A ability, and after the preservation bacterium fermentative degradation ochratoxin, the variation of fermented tea sample main components is smaller, using the ochratoxin A (OTA) generated during bacterium degradation post-fermented tea pile fermentation, to ensure the safe for drinking of post-fermented tea.
Description
Technical field
The present invention relates to microbiology and biodegradable field more particularly to bacillus subtilises and its wet in post-fermented tea
The application of degradation ochratoxin A in heap.
Background technology
Post-fermented tea, also known as black tea, including Pu'er tea, green brick tea, Fu-brick tea and six fort tea etc., main product from Yunnan, Hubei,
The provinces such as Hunan, Guangxi, be China it is traditional side pin and emigrant pin teas, be domestic northwest, west and south borderland Deng Di ethnic groups and
Newly, horse, port, the Australia even essential beverage of ground people's daily life such as Russia.Pile fermentation is Fu-brick tea, Pu'er tea (ripe tea)
Etc. post-fermented teas flavor quality formed critical process.The various microorganisms that pile fermentation process is inoculated with naturally raised growth in tealeaves
Breeding, such as aspergillus, Penicillium fungi have the ability that metabolism generates ochratoxin A.
Ochratoxin A (Ochratoxin A, OTA) be it is a kind of can be hurtful to kidney, liver and nervous system
Mycotoxin, 1993, international cancer research institution (IARC) was defined as 2B class carcinogenic substances.
OTA easily exists in post-fermented tea, in the Pu'er tea reported such as Haas etal. in 2013 ochratoxin A
For content between 0.65-94.7 μ g/kg dw, detection frequency is 11.11%.After the presence of the mycotoxins such as ochratoxin A is given
Drinking for fermented tea brings huge security risk, causes the extensive concern of society.
The chemical property of OTA is stablized, and the total amount of OTA still cannot be effectively controlled using the methods of heating, absorption, although using
The methods of oxidation, soda acid processing can effectively Degradation and Transformation OTA, but nutritional ingredient to food and functional component can be brought greatly
Destruction, therefore have only at present using microorganism in itself or its metabolite have come the biological detoxication method for the OTA that degrades it is good
Application potential.Research report display, Bacillus acidi lactici (Lactobacillus sp.), aspergillus niger (Aspergillus niger),
The degradation effect of OTA in the food such as grape wine such as hay bacillus (Bacillus subtilis) is notable, but in post-fermented tea
The biodegradable research of middle ochratoxin A is not reported so far.
The content of the invention
It is an object of the invention to overcome the defect of the prior art, a bacillus subtilis is provided and its in rear fermentation
The application of degradation ochratoxin A in tea pile fermentation, which adapts to post-fermented tea pile fermentation environment, and (acidity, high temperature are detested
Oxygen) and with degradation ochratoxin A ability, utilize the ochratoxin A generated during bacterium degradation post-fermented tea pile fermentation
(OTA), to ensure the safe for drinking of post-fermented tea.
One of the objects of the present invention is to provide a bacillus subtilis, bacillus subtilis of the invention
(Bacillus subtilis), bacterium code name be DTMo1, isolated and purified during post-fermented tea pile fermentation from pile fermentation tea sample and
Come.
The microbial characteristic of bacterial strain DTMo1 is as follows：It is one kind of bacillus.Individual cells 0.4~0.5 × 0.4
~0.8 micron, uniform coloring.Without pod membrane, peritrichous can move.Gram-positive bacteria, no gemma, heterotrophism, aerobic, thalline
In rod-short, bacterium colony rough surface is opaque, and when growing in liquid medium, wrinkle mould is commonly formed in dirty white or yellowish.
Table one
The physicochemical characteristics of bacterial strain DTMo1 are as follows：Aerobic bacteria, available protein, a variety of sugar and starch, decomposes tryptophan shape
Into indoles.It is heterotrophism, aerobic.Glucose, sucrose, maltose, mannose ferment are the positive, and lactose fermentation is feminine gender.
Table two
The 16S rRNA sequences such as SEQ ID NO of bacterial strain DTMo1:Shown in 1, primer 2 7F (5'-AGAGTT are utilized
) and the 16S of 1492R (5'-TAC GGCTACCTTGTTACGACTT-3') through the PCR amplification bacterial strain TGATCCTGGCTCAG-3'
RRNA genes.By 16S rRNA sequences in GenBank sequence analysis and tetraploid rice, GenBank accession
Number is MG256170, by the phase for comparing bacterial strain DTMo1 and Bacillus subtilis strain NBRC 13719
It is 99% like property.In short, by the Morphological Identification of bacterial strain, bio-chemical characteristics and 16S rRNA sequence analyses and homology
Compare, it is thus determined that Strains B. subtilis DTMo1 is bacillus subtilis.
Based on information above, bacterial strain DTMo1 is accredited as bacillus subtilis (Bacillus subtilis).The bacterium shows
It has been preserved in China typical culture collection center (CCTCC), address：No. 299 Wuhan of Wuhan City, Hubei Province Wuchang District Bayi Road
In institution of higher education, Wuhan University's collection, postcode：430072, deposit number CCTCCNO：M2017708, preservation date 2017
November 22.
The second object of the present invention is malicious in providing bacillus subtilis Aspergillus ochraceus of degrade in post-fermented tea pile fermentation
The application of plain A.
In the specific embodiment of the present invention, the application comprises the following steps：
Bacteria suspension will be made after the activated processing of bacillus subtilis strain in step 1；
Step 2, by the bacterial suspension inoculation activated in step 1 to LB fluid nutrient mediums expand it is numerous, zymotic fluid centrifugation removal on
Clear liquid, distilled water rinse to obtain fermenting microbe repeatedly, are diluted with distilled water bacillus subtilis strain being configured to unit volume
The bacteria suspension of the bacterial population containing different number is inoculated with for follow-up tealeaves pile-fermentation；
Step 3, the tea sample for taking post-fermented tea pile fermentation add ochratoxin A solution；
Step 4, the bacillus subtilis bacteria suspension that various concentration is inoculated with into the tea sample in post-fermented tea pile fermentation later stage, mixing
Uniformly, ferment 3-7 days in 28-35 DEG C, after everfermentation, tea sample is directly dried for standby.
Preferably, the concentration of bacteria suspension is 10 in the step 16~1010CFU/ml。
Preferably, the tea sample in post-fermented tea pile fermentation later stage is taken in the step 3, the ochratoxin A for adding 10 μ g/ml is molten
Liquid makes in tea sample ochratoxin A content be 5 μ g/ml, and spraying distilled water makes water content >=40%, adjust tea sample pH value for≤
6。
Preferably, inoculum concentration is 10 in the step 46~108CFU/g, temperature are fermented 3-7 days between 28 DEG C~35 DEG C
Afterwards, the degradation rate of ochratoxin A reaches 88.61% in pile fermentation tea sample, and after bacillus subtilis detoxification fermentation, fermented tea
The variation of sample main components is small.
The invention has the advantages that：
Bacillus subtilis provided by the invention adapts to post-fermented tea pile fermentation environment (acidity, high temperature, anaerobism) and has
Degradation ochratoxin A ability, using the ochratoxin A (OTA) generated during bacterium degradation post-fermented tea pile fermentation, to protect
Hinder the safe for drinking of post-fermented tea, the degradation rate of ochratoxin A is up to 88.61% in pile fermentation tea sample, and is sent out through the preservation bacterium
After ferment degradation ochratoxin A, the variation of fermented tea sample main components is smaller.
Description of the drawings
Fig. 1 is forms of the bacterial strain DTMo1 provided in an embodiment of the present invention on LB culture mediums；
The optical microscopic morphology that Fig. 2 is bacterial strain DTMo1 provided in an embodiment of the present invention is observed；
Fig. 3 is that the Liquid Culture fermentation that the embodiment of the present invention 1 provides starts OTA liquid chromatographic detection figures；
Fig. 4 is OTA liquid chromatographic detection figures in zymotic fluid after the culture that provides of the embodiment of the present invention 13 days；
Fig. 5 is that the Liquid Culture fermentation that the embodiment of the present invention 4 provides starts OTA liquid chromatographic detection figures；
Fig. 6 is OTA liquid chromatographic detection figures in zymotic fluid after the culture that provides of the embodiment of the present invention 43 days.
Specific embodiment
The screening of 1 bacterial strain DTMo1 of embodiment
(1) by the bacterial strain DTMo1 isolated and purified during post-fermented tea pile fermentation from pile fermentation tea sample it is activated from
Bacteria suspension is made after reason, concentration is about 106CFU/ml。
(2) prepared LB fluid nutrient mediums are dispensed after high-temperature sterilization into the triangular flask of 200ml, is accumulated per bottle
50ml, then the OTA solution of 10 μ g/ml of 20ml and the bacteria suspension of 100 μ L are added into triangular flask, it is sealed in 28-35 DEG C of shaking table
Middle shaken cultivation, shaking speed 200r/min.Control group is set, and blank control system does not add OTA, adds isometric distillation
Water, other conditions are identical.
(3) after bacterium solution culture 3d, the OTA in 2 days extraction 5mL culture mediums, HPLC measure the content of OTA, utilize
HPLC detects the ability of strains for degrading OTA.
(4) OTA extractions and HPLC detections
5ml culture solutions are taken, add in 900 μ L methanol-formic acid (25：1) extract, is shaken after 90min, and with 0.45 μm of filter
Membrane filtration, filtrate are dissolved in after being dried up with nitrogen evaporator in 0.5mL methanol, are housed in 4 DEG C and are detected for HPLC, until chromatography point
Analysis, each concentration is in triplicate.
HPLC testing conditions：Splitter Agilent HC-C18 chromatographic columns, 5 μm, 4.6 × 250mm, fluorescence detector swashs
Send out wavelength 330nm, launch wavelength 460nm；Constant gradient program, mobile phase are acetonitrile：Water：Acetic acid (57%, 41%, 2%), flow velocity
1mL/min。
It screening and obtains the bacterial strain DTMo1 best to OTA degradation capabilities, the changes of contents of OTA is shown in Fig. 3-4 in culture medium,
The fermentation of middle Fig. 3 Liquid Cultures starts OTA liquid chromatographic detection figures, OTA liquid chromatographic detection figures in zymotic fluid after Fig. 4 cultures 3 days.
OTA degradation rates (%)=(C-C0)/C × 100% (1)
(1) in formula：C is the initial concentration (μ g/ml) of OTA in zymotic fluid；Concentration (the μ that C0 is OTA in zymotic fluid after 3 days
g/ml)。
OTA appearance times are 30.30min or so, and OTA recovery of standard addition is 85% in fluid nutrient medium, according to formula (1)
It is 91.3% to the degradation rate of OTA to draw bacterial strain DTMo1.
The identification of embodiment 2, bacterial strain DTMo1
The 16S rRNA sequences such as SEQ ID NO of bacterial strain DTMo1:Shown in 1, primer 2 7F (5'-AGAGTT are utilized
) and the 16S of 1492R (5'-TAC GGCTACCTTGTTACGACTT-3') through the PCR amplification bacterial strain TGATCCTGGCTCAG-3'
RRNA genes.By 16S rRNA sequences in GenBank sequence analysis and tetraploid rice, GenBank accession
Number is MG256170, by the phase for comparing bacterial strain DTMo1 and Bacillus subtilis strain NBRC 13719
It is 99% like property.Bacterial strain DTMo1, by the Morphological Identification of bacterial strain, bio-chemical characteristics and 16S rRNA sequence analyses and
Tetraploid rice is by the similitude for comparing bacterial strain DTMo1 and Bacillus subtilis strain NBRC 13719
99%, it is thus determined that Strains B. subtilis DTMo1 is bacillus subtilis.
The microbial characteristic of bacterial strain DTMo1 is as follows：It is one kind of bacillus.Individual cells 0.7~0.8 × 2~
3 microns, uniform coloring.Without pod membrane, peritrichous can move.Gram-positive bacteria, no gemma, heterotrophism, aerobic, thalline is in short
It is rod-shaped,.Bacterium colony rough surface is opaque, and when growing in liquid medium, wrinkle mould is commonly formed in dirty white or yellowish.
The physicochemical characteristics of bacterial strain DTMo1 are as follows：Aerobic bacteria, available protein, a variety of sugar and starch, decomposes tryptophan shape
Into indoles.It is heterotrophism, aerobic.Glucose, sucrose, maltose, mannose ferment are the positive, and lactose fermentation is feminine gender.
Fig. 1 is forms of the bacterial strain DTMo1 on LB culture mediums, the form observed under the optical microphotograph of Fig. 2 bacterial strains DTMo1.
Embodiment 3, the culture of bacillus subtilis
Bacillus subtilis strain DTMo1 is inoculated in LB fluid nutrient mediums, shaking speed 200r/min, temperature 28-
35 DEG C, up to the zymotic fluid of bacillus subtilis DTMo1 after 72h cultures.Zymotic fluid is centrifuged into removal supernatant, precipitation is steamed
Distilled water rinses to obtain fermenting microbe repeatedly.Bacillus subtilis DTMo1 is configured to unit volume containing difference with distilled water dilution
The bacteria suspension of number of bacteria number is inoculated with for follow-up tealeaves pile-fermentation.
The formula of the LB culture mediums is：Tryptone 10g, dusty yeast 5g, (solid medium adds 1.5% to sodium chloride 10g
Agar), add distilled water to 1L, 121 DEG C of sterilizing 20min.
Embodiment 4, bacterial strain DTMo1 are to the degradation of OTA during post-fermented tea pile fermentation
Bacteria suspension will be made after the activated processing of bacterial strain DTMo1, concentration is about 106CFU/ml。
The tea sample in post-fermented tea pile fermentation later stage is taken, the OTA solution of 10 μ g/ml of addition makes OTA contents in tea sample be 5 μ g/ml,
Spraying distilled water makes water content be maintained at 40%, and it is respectively 5,6 to adjust tea sample pH value, and then inoculum density is 10 respectively4、105、
106Bacteria suspension is uniformly mixed, and is fermented 3-7 days in 28-35 DEG C, the ability of analysis bacterial strain DTMo1 degradations OTA.
The tea sample after everfermentation of learning from else's experience is dry, crushes, and 5g crushes sample in 250mL triangular flasks, and it is 1% to add 100mL concentration
(w/v) sodium bicarbonate solution vibrates 30min on the oscillator, and quantitative filter paper (Whatman No.1) mistake is used after standing 10min
Filter.10mL sample extracting solutions is taken to add in 10 mL phosphate buffers in a teat glass, mix.
Purification：Select SPE-C18 solid phase extraction columns, adsorbent capacity 500mg.Successively with 10mL phosphate buffers and
5mL distilled water activates pillar, then by 20mL extracting solutions by SPE-C18 solid phase extraction columns, flow control 60 drops/point.
It is eluted again with 3mL methanol, collects eluent.Nitrogen dries up at 40 DEG C, and mobile phase solution is settled to 0.4mL, through 0.22 μm of micropore
It is detected after membrane filtration for high performance liquid chromatograph.
HPLC testing conditions：Splitter be Agilent HC-C18 chromatographic columns, 5 μm, 4.6 × 250mm, fluorescence detector,
Excitation wavelength 330nm, launch wavelength 460nm；Mobile phase is acetonitrile：Methanol：0.15mol/L phosphoric acid (1/1/1, v/v/v), stream
Fast 0.8mL/min, 20 μ L of sample size.
OTA appearance times are 30.41min or so in tea sample, and OTA recovery of standard addition is 82.6% in tea sample,
OTA degradation rates (%)=(C-C0)/C × 100% (1)
(1) in formula：C is the initial concentration (μ g/ml) of OTA in zymotic fluid；Concentration (the μ that C0 is OTA in zymotic fluid after 3 days
g/ml)。
Show that bacterial strain DTMo1 in inoculum concentration is 106CFU/g according to formula (1), tea sample water content 40%, during 28 DEG C of temperature,
Ferment 7d, and the degradation rate of OTA is up to 88.61%.
5 different fermentations time of embodiment, influences of the pH value bacterial strain DTMo1 to the degradation rate of OTA during tealeaves pile fermentation
Experimental method as described in Example 4, wherein：
Processing 1：PH value is 5, inoculum concentration 104CFU/ml, fermentation time 3 days, degradation rate 68.10%；
Processing 2：PH value is 5, inoculum concentration 104CFU/ml, fermentation time 7 days, degradation rate 72.35%；
Processing 3：PH value is 5, inoculum concentration 105CFU/ml, fermentation time 3 days, degradation rate 70.09%；
Processing 4：PH value is 5, inoculum concentration 105CFU/ml, fermentation time 7 days, degradation rate 73.61%；
Processing 5：PH value is 5, inoculum concentration 106CFU/ml, fermentation time 3 days, degradation rate 79.48%；
Processing 6：PH value is 5, inoculum concentration 106CFU/ml, fermentation time 7 days, degradation rate 80.26%；
Processing 7：PH value is 6, inoculum concentration 104CFU/ml, fermentation time 3 days, degradation rate 69.11%；
Processing 8：PH value is 6, inoculum concentration 104CFU/ml, fermentation time 7 days, degradation rate 83.40%；
Processing 9：PH value is 6, inoculum concentration 105CFU/ml, fermentation time 3 days, degradation rate 69.73%；
Processing 10：PH value is 6, inoculum concentration 105CFU/ml, fermentation time 7 days, degradation rate 83.79%；
Processing 11：PH value is 6, inoculum concentration 106CFU/ml, fermentation time 3 days, degradation rate 71.30%；
Processing 12：PH value is 6, inoculum concentration 106CFU/ml, fermentation time 7 days, degradation rate 88.61%；
As known from the above, 12 groups of degradation rate highest is handled, i.e. pH value is 6, inoculum concentration 106CFU/ml, fermentation time 7
My god, degradation rate 88.61%；
Table three
Embodiment 6：After the preservation bacterium fermentative degradation ochratoxin, the variation of fermented tea sample main components is smaller.
Inoculating strain DTM01 (presses 106CFU/g tea sample) the variation such as following table of tealeaves main component after different time ferments
Table four
As seen from the above table, through bacterial strain DTM01 to tea sample carry out later stage fermentation processing, tea sample main component amplitude of variation compared with
It is small, illustrate to add appropriate bacterial strain DTM01 during tealeaves pile fermentation, while OTA contents are significantly reduced, to pile fermentation tealeaves product
Matter influences little.
Summary the experimental results showed that, bacillus subtilis provided by the invention adapts to post-fermented tea pile fermentation environment
(acidity, high temperature, anaerobism) and with degradation ochratoxin A ability, generation during bacterium degradation post-fermented tea pile fermentation is utilized
Ochratoxin A (OTA), to ensure the safe for drinking of post-fermented tea, the degradation rate of ochratoxin A is up in pile fermentation tea sample
88.61%, and after the preservation bacterium fermentative degradation ochratoxin, the variation of fermented tea sample main components is smaller.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention god.
The 16S rRNA sequences such as SEQ ID NO of bacterial strain DTMo1:1
Atataatgcaatcgagcggacagatgtgagcttgctctctgatgttagcggcggacaggtgagtaacacgtgcctaa
cctgcctgtaag actggcataactccgggaaaccggggctaataccgaatggttgtttgaaccgcatggttcaaac
ataaaaggtggcttcggctaccacttacaga tggacccgcggcgcattagctagttggtgaggtaacggctcacca
aggcaacgatgcgtagccgacctgagagggtgatcagccacactgcg actgagacacggcccagactcctacggaa
ggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtga
tgaaggtgttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggta
cctaaccagaaagccac ggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaatta
ttgggcgtaaagggctcgcaggcggtttcttaagt ctgatgtgaaagcccccggctcaaccggggagggtcattgg
aaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcg gtgaaatgcgtagagatgtggagga
acaccagtggcgttggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaa
caggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctg
cagctaacgcattaagca ctccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgc
acaagcggtggagcatgtggtttaattcgaagca acgcgaagaaccttaccaggtcttgacatcctctgacaatcc
tagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgt cgtcagctcgtgtcgtgagatgtt
gggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgact
gccggtgacaaaccggaggaaggtggctatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgcta
caatggtcagaacaaa gggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcag
tctgcaactcgactgcgtgaagctggaatcgcta gtaatcgcggatcagcatgccgcggtgaatacgttcccgggc
cttgtacacaccgcccgtcacaccacgagagtttgtaacacccgcagtcgg 。
Claims (6)
- A 1. bacillus subtilis, which is characterized in that its deposit number is CCTCCNO：M2017708.
- The application of ochratoxin A 2. bacillus subtilis according to claim 1 is degraded in post-fermented tea pile fermentation.
- The application of ochratoxin A 3. bacillus subtilis as claimed in claim 2 is degraded in post-fermented tea pile fermentation, it is special Sign is, comprises the following steps：Bacteria suspension will be made after the activated processing of bacillus subtilis strain isolated and purified out from pile fermentation tea sample in step 1；The bacterial suspension inoculation activated in step 1 to LB fluid nutrient mediums is expanded numerous, zymotic fluid centrifugation removal supernatant by step 2, Distilled water rinses to obtain fermenting microbe repeatedly；Step 3, the tea sample for taking pile fermentation add ochratoxin A solution；Step 4, the bacillus subtilis bacteria suspension that various concentration is inoculated with into the tea sample in post-fermented tea pile fermentation later stage, mixing are equal It is even, it ferments 3-7 days in 28-35 DEG C, after everfermentation, tea sample is directly dried.
- The application of ochratoxin A 4. bacillus subtilis as claimed in claim 3 is degraded in tea pile fermentation, feature exist In the concentration of bacteria suspension is 10 in the step 16~1010CFU/ml。
- The application of ochratoxin A 5. bacillus subtilis as claimed in claim 3 is degraded in post-fermented tea pile fermentation, it is special Sign is, the tea sample in post-fermented tea pile fermentation later stage is taken in the step 3, and the ochratoxin A solution of 10 μ g/ml of addition makes tea sample Middle ochratoxin A content is 5 μ g/ml, and spraying distilled water makes water content >=40%, and it is≤6 to adjust tea sample pH value.
- The application of ochratoxin A 6. bacillus subtilis as claimed in claim 3 is degraded in tea pile fermentation, feature exist In inoculum concentration is 10 in the step 46~108CFU/g, temperature is between 28 DEG C~35 DEG C, after fermenting 3-7 days, pile fermentation tea sample The degradation rate of middle ochratoxin A reaches 88.61%, and after bacillus subtilis detoxification fermentation, fermented tea sample main quality Composition transfer is small.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| 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 |
| CN110563499A (en) * | 2019-07-09 | 2019-12-13 | 湖北新保得生物科技有限公司 | Method for producing amino acid foliar fertilizer by taking yellow wine lees as matrix |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998393A (en) * | 2018-07-13 | 2018-12-14 | 云南中茶茶业有限公司 | One bacillus subtilis and its preparation have the Pu'er tea method for adjusting function of intestinal canal |
| 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 |
| 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 |
| CN109055273B (en) * | 2018-09-10 | 2022-03-04 | 安徽农业大学 | Green brick tea pile fermentation strain composition and application thereof |
| CN110563499A (en) * | 2019-07-09 | 2019-12-13 | 湖北新保得生物科技有限公司 | Method for producing amino acid foliar fertilizer by taking yellow wine lees as matrix |
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