CN114214211A - Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof - Google Patents
Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof Download PDFInfo
- Publication number
- CN114214211A CN114214211A CN202111621139.4A CN202111621139A CN114214211A CN 114214211 A CN114214211 A CN 114214211A CN 202111621139 A CN202111621139 A CN 202111621139A CN 114214211 A CN114214211 A CN 114214211A
- Authority
- CN
- China
- Prior art keywords
- tea
- aspergillus
- aspergillus kawachii
- golden flower
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/08—Oxidation; Fermentation
- A23F3/10—Fermentation with addition of microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Virology (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a golden flower bacterium-aspergillus kawachii strain and a preparation method and application thereof. The invention relates to a golden flower fungus-aspergillus kawachii strain, which is aspergillus kawachii JH 1; the collection name is Aspergillus kawachii JH1Aspergillus chevalier JH 1; is preserved in China center for type culture Collection, and the preservation address is Wuhan university in Wuhan, China; the preservation date is as follows: 09 months and 30 days 2021; the preservation number is CCTCC NO. 20211180. According to the invention, golden flower fungus such as Aspergillus kawachii (Aspergillus chevalieri) JH1 and the like with a health care effect is artificially inoculated into different types of tea leaves such as sun-dried green raw tea, Pu-erh ripe tea, dark raw tea, white tea, black tea loose tea and the like, so that luxuriant golden flower is promoted to be generated inside a tea brick/tea cake, the quality of the tea leaves is improved, and the health care effect of the tea leaves is improved.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a golden flower bacterium-aspergillus kawachii strain as well as a preparation method and application thereof.
Background
Fuzhuan tea is a yellow capsule-closing shell which is generated in a brick tea by promoting specific microorganisms to grow, ferment and convert in the brick tea through a special process in the processing process of black tea and is called golden flower in folk. The golden flower is universal, luxuriant, bright in color and golden yellow, does not contain sundry bacteria, and is an important standard for evaluating the quality of the Fuzhuan tea. The predominant strains in Fuzhuan tea that produce golden yellow cystosepiment were now identified as Aspergillus cristatus (Aspergillus cristatus) and the original name Eurotium cristatum (Eurotium cristatum). Modern researches show that the golden flower fungus such as aspergillus guani has the effects of preventing cardiovascular and cerebrovascular diseases, reducing blood fat, cholesterol, regulating blood sugar, blood pressure and the like, and has the effects of reducing fat and losing weight. In the process of flower growing of the Fuzhuan tea, the golden flower fungus can secrete and produce hydrolytic enzymes such as alpha-amylase, cellulase, pectinase, protease and the like to promote hydrolysis of water-insoluble polysaccharides such as cellulose, starch and the like to produce water-soluble monosaccharides, disaccharides and polysaccharides; and catalyzing the oxidation of the polyphenol compounds to generate theaflavin thearubigin theabrownin and other pigments, thereby improving the quality of the tea such as color, taste, aroma and the like. The invention utilizes a golden flower bacterium Aspergillus chevalieri JH1 strain separated and purified from golden flower Pu ' er raw tea, and completes the application of the Aspergillus chevalieri JH1 strain in the steaming and pressing of black tea bricks/tea cakes and the pile fermentation of black tea such as Pu ' er tea and the like by modern bioengineering technology and referring to the manufacturing process of the black tea bricks/tea bricks such as Pu ' er tea and the like in ancient documents.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a golden flower bacterium-aspergillus kawachii strain with high bacteriostasis rate and application thereof.
The technical scheme of the invention is as follows: the invention provides the following technical scheme: the invention relates to a golden flower fungus-aspergillus kawachii strain, which is aspergillus kawachii JH 1; the collection name is Aspergillus kawachii JH1Aspergillus chevalier JH 1; is preserved in China center for type culture Collection, and the preservation address is Wuhan university in Wuhan, China; the preservation date is as follows: 09 months and 30 days 2021; the preservation number is CCTCC NO. 20211180.
Further, the ITS base sequence of the Aspergillus kawachii Chevalieri JH1 strain is the nucleotide sequence shown in SEQ ID No. 1; the beta-tubulin base sequence of the Aspergillus kawachii Aspergillus chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 2; the Calmodulin base sequence of the Aspergillus kawachii Chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 3.
Further, the strain Aspergillus kawachii Aspergillus chevalieri JH1 has slow colony growth on the Chaudou's medium, the diameter is 9-16mm at 30 ℃ for 7 days, and the positive side: fluffy powder, yellow to golden yellow; the back is earthy yellow to yellow brown, and the golden yellow cyst closure shell is less.
The invention relates to a golden flower fungus-aspergillus kawachii strain microbial inoculum prepared from a golden flower fungus-aspergillus kawachii strain.
Further, the active ingredient is at least one of the following (a), (b) and (c):
(a) the fermentation culture of the golden flower fungus-aspergillus shewanese strain;
(b) ultrasonic cracking supernatant of the obtained eurotium cristatum-aspergillus shewanii strain cell;
(c) ultrasonic cracking precipitation of the obtained golden flower fungus-aspergillus shewanese strain cell.
The preparation method of the golden flower fungus-aspergillus kawachii strain microbial inoculum comprises the following steps: aspergillus awamori chevalieri JH1 was used for preparation of Aspergillus awamori JH1 bacterial suspension after culturing in a Chaudhuri medium or PDA medium at 30 ℃ for 7 days; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104More than CFU/mL to prepare the golden flower fungus-aspergillus kawachii strain microbial inoculum.
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in preparation of autoclaved and dried Pu' er tea, black tea, white tea and black tea bricks/cakes.
Further, the preparation method of the Pu' er tea, the black tea, the white tea and the black tea brick/cake comprises the following steps: (1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is transplanted on a Chao's medium or a PDA medium and cultured for 7 days at the temperature of 30 ℃ for preparing a suspension of Aspergillus kawachii JH1 bacteria;extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more;
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 4min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, inoculating the suspension of the eurotium cristatum-Aspergillus kawachii JH1 bacteria, placing in a constant-temperature incubator with the temperature of 30 ℃ and the humidity of less than 75% to perform pure culture of Aspergillus kawachii Aspergillus chevalieri JH1, culturing for 7 days, and drying in a natural state;
(3) preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing eurotium cristatum obtained by pure culture with tea such as sun-dried raw tea as matrix with Yunnan big leaf sun-dried green tea according to inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3-5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 18 ℃; when the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity; the number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g; the pollution of mixed bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process; drying to obtain a large amount of golden flower fungus Aspergillus chevalieri JH1 strain, which can be used for making golden flower Pu 'er tea brick/tea cake and natural pile fermentation of ripe golden flower Pu' er tea loose tea;
(4) steaming and pressing tea brick/tea cake such as Pu' er tea and the like:
the production of the golden flower fungus requires that the tea cake has certain water content and proper temperature and humidity are required; mixing the eurotium cristatum strain-Aspergillus kawachii Chevalieri JH1 obtained by enlarged culture with the sun-dried green tea of the Yunnan big leaf according to the mass ratio of 1-3%, adding a proper amount of drinking water for humidifying, and steaming under normal pressure and high temperature steam after humidifying for 30 min-1 h; the steam temperature is between 100 ℃; steaming for 2-3 min;
(5) and (3) drying the grown flower of the Pu' er tea brick/tea cake: the step of flowering refers to a process of mass propagation of golden flower fungus under a certain hydrothermal action, hydrolysis, oxidation and polymerization are carried out on quality components such as tea polyphenol, amino acid and the like in tea leaves through corresponding extracellular enzymes, and autoclaved tea cakes or tea bricks are arranged in a drying room according to a certain sequence; and when the flowers bloom for 7 days, entering a drying stage, gradually increasing the temperature of a drying room to 40 ℃, and drying for 3 days to obtain the Pu' er tea, the black tea, the white tea and the black tea brick/cake.
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in finished products of golden flower Pu-er ripe tea loose tea.
Further, the preparation method of the finished product of the golden flower Pu-erh ripe loose tea comprises the following steps: (1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is cultured on a Chaudhuri culture medium or a PDA culture medium at 30 ℃ for 7 days, and then is used for preparing a suspension of Aspergillus kawachii JH 1; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more;
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing the golden flower fungus-aspergillus kawachii JH1 bacterial suspension and Yunnan large-leaf sun-dried raw tea according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 2min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus-aspergillus kawachii JH1 with the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, and inoculating suspension of eurotium cristatum-aspergillus shepheri JH 1; performing pure culture in a constant-temperature incubator at 30 ℃ and humidity of less than 75%, taking out after 7 days of culture, and drying in a natural state;
(3) preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture by taking tea leaves such as sun-dried raw tea and the like as a matrix with a large-leaf sun-dried green tea of Yunnan according to an inoculation amount of 5% by mass, steaming for 5min under normal pressure and high temperature steam after humidifying for 30min to 1 h, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation; when the fermentation is carried out for 5 days, the golden flower begins to appear in the tea, and when the solid state fermentation is carried out for 14 days, the number of aspergillus kawachii communities reaches 1.5 multiplied by 107CFU/g, drying to obtain a large amount of golden flower fungus-Aspergillus chevalieri JH1 strain, which can be used for making golden flower Pu 'er tea brick/tea cake and natural pile fermentation of golden flower Pu' er tea loose tea;
(4) exogenous inoculation natural pile fermentation of Pu-Er ripe tea:
mixing the golden flower fungus-aspergillus kawachii JH1 strain with the Yunnan big-leaf green-dried raw tea according to the inoculation amount of 1-3% by mass percent, adding a proper amount of drinking water, piling the green-dried raw tea by over 2000kg, enabling the pile height to be 70-80 cm, and covering the hemp cloth after steam sterilization treatment;
(5) and after the pile fermentation is finished, natural drying, storage aging, screening and picking are carried out to prepare the finished product of the golden flower Pu-erh ripe tea loose tea.
Has the advantages that: the golden flower fungus with the health care effect of Aspergillus kawachii (Aspergillus chevalieri) JH1 and the like is artificially inoculated into different types of tea leaves such as sun-cured raw tea, Pu-er ripe tea, dark raw tea, white tea, black tea loose tea and the like, autoclaved into tea bricks/tea bricks with a certain specification, and the flourishing golden flower is promoted to be generated inside the tea bricks/tea cakes under the specific temperature and humidity control condition, so that the production of the golden flower tea bricks/tea cakes is completed, the quality of the tea leaves is improved, and the health care effect of the tea leaves is promoted.
Detailed Description
The present invention will be further specifically described below by way of specific examples, but it should not be construed as limiting the scope of the present invention.
Example 1
The invention relates to a golden flower fungus-aspergillus kawachii strain, which is aspergillus kawachii JH 1; the collection name is Aspergillus kawachii JH1Aspergillus chevalier JH 1; is preserved in China center for type culture Collection, and the preservation address is Wuhan university in Wuhan, China; the preservation date is as follows: 09 months and 30 days 2021; the preservation number is CCTCC NO. 20211180.
The ITS base sequence of the Aspergillus kawachii Chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 1; the beta-tubulin base sequence of the Aspergillus kawachii Aspergillus chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 2; the Calmodulin base sequence of the Aspergillus kawachii Chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 3.
The bacterial strain Aspergillus kawachii Chevalieri JH1 of the invention has slow colony growth on a Chaudou culture medium, the diameter is 12mm at 30 ℃ for 7 days, and the front side: fluffy powder, yellow to golden yellow; the back is earthy yellow to yellow brown, and the golden yellow cyst closure shell is less.
The invention relates to a golden flower fungus-aspergillus kawachii strain microbial inoculum prepared from a golden flower fungus-aspergillus kawachii strain. The active component is at least one of the following components (a), (b) and (c):
(a) the fermentation culture of the golden flower fungus-aspergillus shewanese strain;
(b) ultrasonic cracking supernatant of the obtained eurotium cristatum-aspergillus shewanii strain cell;
(c) ultrasonic cracking precipitation of the obtained golden flower fungus-aspergillus shewanese strain cell.
The preparation method of the golden flower fungus-aspergillus kawachii strain microbial inoculum comprises the following steps: aspergillus awamori chevalieri JH1 was used for preparation of Aspergillus awamori JH1 bacterial suspension after culturing in a Chaudhuri medium or PDA medium at 30 ℃ for 7 days; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104More than CFU/mL to prepare the golden flower fungus-aspergillus kawachii strain microbial inoculum.
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in preparation of autoclaved and dried Pu' er tea, black tea, white tea and black tea bricks/cakes.
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in finished products of golden flower Pu-er ripe tea loose tea.
Example 2
The bacterial strain Aspergillus kawachii Chevalieri JH1 of the invention has slow colony growth on a Chaudou culture medium, the diameter is 9mm at 30 ℃ for 7 days, and the front side: fluffy powder, yellow to golden yellow; the back is earthy yellow to yellow brown, and the golden yellow cyst closure shell is less.
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in preparation of autoclaved and dried Pu' er tea, black tea, white tea and black tea bricks/cakes.
1. Material
The Yunnan big leaf sun-dried green tea which meets the national geographical sign product requirements of GB/T22111-2008 Pu' er tea is taken as the raw material. Aspergillus kawachii has a preservation number of CCTCC NO: M20211180 in Aspergillus chevalieri JH 1. And a Chachi medium.
2. Testing instrument
JR-1003 electronic balance (Shanghai Xinnuo Instrument plant); HH-S28S model digital display constant temperature water bath (environmental protection instrument factory of gold jar); model DFZ-O1 electrothermal vacuum drying oven (hengfeng medical instruments ltd, yellow stone); JH-752 ultraviolet-visible spectrophotometer (Shanghai cyanine scientific instrument Co., Ltd.); agilent model 1200 high performance liquid chromatograph (Agilent, usa); a low temperature refrigerator (SANYO corporation, japan); an FZ-102 type micro plant sample pulverizer (Guangming medical instruments, Beijing); labonce-150TH constant temperature and humidity test chamber (Beijing Lanbeishi constant temperature technology, Inc.); SFG type solid state fermentor (Zhenjiang river engineering and bioengineering devices).
The preparation method of the brick/cake comprises the following steps:
(1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is transplanted on a Chao's medium or a PDA medium and cultured for 7 days at the temperature of 30 ℃ for preparing a suspension of Aspergillus kawachii JH1 bacteria; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more.
(2) Pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 4min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, inoculating the suspension of the eurotium cristatum-Aspergillus kawachii JH1 bacteria, and placing in a constant-temperature incubator at the temperature of 30 ℃ and the humidity of less than 75% to perform pure culture of Aspergillus kawachii Aspergillus chevalieri JH 1. After 7 days of culture, drying was carried out in the natural state. And observing the growth condition of colonies of the golden flower fungus, and measuring the quantity of aspergillus kawachii according to GB 4789.15-2010 national food safety standard 'measuring method in food microbiology inspection mould and yeast counting'.
The morphological observation shows that after the tea is purely cultured for 7 days, the tea has a luxuriant, compact and bright golden yellow cyst closure shell, namely golden flower, and has no mixed bacteria colonies such as black mold, white mold, blue mold, red mold and the like. The growth vigor of the golden flower fungus in the fermentation tank is better than that of the golden flower fungus in the culture tank. Based on the high temperature resistance of aspergillus kawachii, the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be achieved within 5min of high-temperature steaming under normal pressure, and simultaneously, higher biological activity of aspergillus kawachii is kept. At 30 deg.C, Aspergillus kawachii JH1 in tea is propagated in large amount, and the number is 1 × 10 after 7 days6CFU/g is higher than the standard.
(3) Preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3-5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 18 ℃. When the tea is fermented for 5 days, the interior of the tea begins to appear 'golden flower', and the golden flower is propagated in large quantities. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, and the pollution of other bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process. After drying, a large amount of golden flower fungus Aspergillus chevalieri JH1 strains can be obtained, and the strains can be used for manufacturing golden flower Pu 'er tea bricks/tea cakes and natural pile fermentation of the golden flower Pu' er tea ripe tea loose tea.
(4) Steaming and pressing tea brick/tea cake such as Pu' er tea and the like:
the production of the golden flower fungus requires that the tea cake (brick tea) has certain water content and proper temperature and humidity. Mixing the eurotium cristatum strain-Aspergillus kawachii Chevalieri JH1 obtained by enlarged culture with the sun-dried green tea of the Yunnan big leaf according to the mass ratio of 1-3%, adding a proper amount of drinking water for humidifying, and steaming under normal pressure and high temperature steam after humidifying for 30 min-1 h. The steam temperature is between 100 ℃. Steaming for 2-3 min. If a high-pressure steam device is adopted, the steaming time is properly shortened, and the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be fulfilled on the premise of keeping part of the activity of aspergillus kawachii JH 1. According to the existing Pu' er tea steaming and pressing process, the maximum stress is 100-150 Mpa. The thickness of the center of the tea cake is 2.5cm, and the tea cake can be used for pressing 357 g, 400 g, 500 g, 1000g and other tea cakes with different specifications according to the difference of the moulds. The thickness of the brick tea is 4-4.5cm, and the brick tea can be used for pressing four brick tea with different specifications, namely 500 g, 800 g, 1000g and 2000 g according to the difference of moulds. After a plurality of tests, the initial water content of the tea cake is preferably 30%; the initial water content of the brick tea is preferably 28%.
(5) And (3) drying the grown flower of the Pu' er tea brick/tea cake:
the flowering refers to a process of mass propagation of golden flower fungus under a certain hydrothermal action, and the hydrolysis, oxidation, polymerization and other actions are generated on quality components such as tea polyphenol, amino acid and the like in tea leaves through corresponding extracellular enzymes, so that the original color, taste and aroma of the tea leaves are improved. Arranging the autoclaved tea cakes or tea bricks in a drying room with the size of 4m multiplied by 4m according to a certain sequence. The interval between two rows, must not be less than 5cm to facilitate the circulation of air. In the stage of flowering, the drying room is warmThe temperature is controlled between 26 ℃ and 30 ℃ and is gradually increased; the humidity is controlled below 75%, and when the humidity exceeds 75%, ventilation and moisture removal are required. And (3) after the tea cake or the tea brick grows for 7 days, entering a drying stage, gradually increasing the temperature of the drying room to 40 ℃, and after drying for 3 days, finishing the drying when the water content of the tea cake or the tea brick is lower than 13%. When the lowest temperature of the air temperature is above 16 ℃, the highest temperature is 32 ℃ and the air humidity is below 65% during the flowering and drying, the flowering and drying of the Puer tea cake or the tea brick can be realized without the help of the temperature control of a drying room. Sensory evaluation shows that finished products of golden flower Pu' er raw tea cakes or tea bricks are dark green and have slight color, flourishing flowers, obvious dry smell and fungus fragrance, obvious nectar fragrance of tea soup, orange yellow and transparent bright soup color, mellow and pure taste, obvious and lasting salivation promoting effect and no bitter taste. The number of Aspergillus kawachii in the tea cake is 20 × 104CFU/g, the number of Aspergillus kawachii in the golden camellia brick is 50 multiplied by 104CFU/g。
Example 3
Example 3 differs from example 2 in that: the bacterial colony of the strain Aspergillus kawachii Aspergillus chevalieri JH1 grows slowly on a Chaudou culture medium, the diameter is 16mm in 7 days at 30 ℃, and the front side: fluffy powder, yellow to golden yellow; the back is earthy yellow to yellow brown, and the golden yellow cyst closure shell is less.
In step (3), preparation of the eurotium cristatum-Aspergillus kawachii strain Aspergillus chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3-5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 18 ℃. When the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, and the pollution of other bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process. Drying to obtain a large amount of Aspergillus chevalieri JH1 strain, which can be used for preparing golden flower Pu' er tea brick/tea cakeAnd (3) performing natural pile fermentation on the bulk tea of the golden flower Pu-er tea.
In the step (4), steaming and pressing the tea brick/tea cake of Pu' er tea and the like:
the production of the golden flower fungus requires that the tea cake (brick tea) has certain water content and proper temperature and humidity. Mixing the eurotium cristatum strain-Aspergillus kawachii Chevalieri JH1 obtained by enlarged culture with the sun-dried green tea of the Yunnan big leaf according to the mass ratio of 1-3%, adding a proper amount of drinking water for humidifying, and steaming under normal pressure and high temperature steam after humidifying for 30 min-1 h. The steam temperature is between 100 ℃ and 121 ℃. Steaming time is 3 min. If a high-pressure steam device is adopted, the steaming time is properly shortened, and the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be fulfilled on the premise of keeping part of the activity of aspergillus kawachii JH 1. According to the existing Pu' er tea steaming and pressing process, the maximum stress is 100-150 Mpa. The center thickness of the tea cake is 2.5-3.5cm, and the tea cake can be used for pressing tea cakes with different specifications such as 357 g, 400 g, 500 g, 1000g and the like according to the difference of moulds. The brick tea thickness is 4cm, can be used for pressing four different specification brick tea of 500 grams, 800 grams, 1000 grams, 2000 grams according to the mould difference. After a plurality of tests, the initial water content of the tea cake is preferably 27-30%; the initial water content of the brick tea is preferably 26-28%.
In the step (5), the flower formation and drying of the Pu' er tea brick/tea cake:
the flowering refers to a process of mass propagation of golden flower fungus under a certain hydrothermal action, and the hydrolysis, oxidation, polymerization and other actions are generated on quality components such as tea polyphenol, amino acid and the like in tea leaves through corresponding extracellular enzymes, so that the original color, taste and aroma of the tea leaves are improved. Arranging the autoclaved tea cakes or tea bricks in a drying room with the size of 4m multiplied by 4m according to a certain sequence. The interval between two rows, must not be less than 5cm to facilitate the circulation of air. In the flowering stage, the temperature of the drying room is controlled between 26 ℃ and 30 ℃, and the temperature is gradually increased; the humidity is controlled below 75%, and when the humidity exceeds 75%, ventilation and moisture removal are required. After the tea cake or the tea brick grows for 7 days, the drying stage is carried out, the temperature of the drying room is gradually increased to 40 ℃, and after the drying for 3 days, the tea cake or the tea brick is dried when the water content is lower than 13 percentAnd (4) bundling. When the lowest temperature of the air temperature is above 16 ℃, the highest temperature is 28-32 ℃ and the air humidity is below 65% during the flowering and drying, the flowering and drying of the Puer tea cake or the tea brick can be realized without the help of the temperature control of a drying room. Sensory evaluation shows that finished products of golden flower Pu' er raw tea cakes or tea bricks are dark green and have slight color, flourishing flowers, obvious dry smell and fungus fragrance, obvious nectar fragrance of tea soup, orange yellow and transparent bright soup color, mellow and pure taste, obvious and lasting salivation promoting effect and no bitter taste. The number of Aspergillus kawachii in the tea cake is 20 × 104CFU/g, the number of Aspergillus kawachii in the golden camellia brick is 50 multiplied by 104CFU/g。
Example 4
Example 4 differs from example 2 in that: in step (3), preparation of the eurotium cristatum-Aspergillus kawachii strain Aspergillus chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3-5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be between 30 ℃. When the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, and the pollution of other bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process. After drying, a large amount of golden flower fungus Aspergillus chevalieri JH1 strains can be obtained, and the strains can be used for manufacturing golden flower Pu 'er tea bricks/tea cakes and natural pile fermentation of the golden flower Pu' er tea ripe tea loose tea.
In the step (4), steaming and pressing the tea brick/tea cake of Pu' er tea and the like:
the production of the golden flower fungus requires that the tea cake (brick tea) has certain water content and proper temperature and humidity. Mixing the eurotium cristatum strain-Aspergillus kawachii Chevalieri JH1 obtained by enlarged culture with the sun-dried green tea of the Yunnan big leaf according to the mass ratio of 1-3%, adding a proper amount of drinking water for humidifying, and steaming under normal pressure and high temperature steam after humidifying for 30 min-1 h. The steam temperature is between 100 ℃ and 121 ℃. Steaming for 2-3 min. If a high-pressure steam device is adopted, the steaming time is properly shortened, and the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be fulfilled on the premise of keeping part of the activity of aspergillus kawachii JH 1. According to the existing Pu' er tea steaming and pressing process, the maximum stress is 100-150 Mpa. The center thickness of the tea cake is 2.5-3.5cm, and the tea cake can be used for pressing tea cakes with different specifications such as 357 g, 400 g, 500 g, 1000g and the like according to the difference of moulds. The brick tea thickness is 4.5cm, can be used for pressing four different specification brick tea of 500 grams, 800 grams, 1000 grams, 2000 grams according to the mould difference. After a plurality of tests, the initial water content of the tea cake is preferably 27-30%; the initial water content of the brick tea is preferably 26-28%.
In the step (5), the flower formation and drying of the Pu' er tea brick/tea cake:
the flowering refers to a process of mass propagation of golden flower fungus under a certain hydrothermal action, and the hydrolysis, oxidation, polymerization and other actions are generated on quality components such as tea polyphenol, amino acid and the like in tea leaves through corresponding extracellular enzymes, so that the original color, taste and aroma of the tea leaves are improved. Arranging the autoclaved tea cakes or tea bricks in a drying room with the size of 4m multiplied by 4m according to a certain sequence. The interval between two rows, must not be less than 5cm to facilitate the circulation of air. In the flowering stage, the temperature of the drying room is controlled between 26 ℃ and 30 ℃, and the temperature is gradually increased; the humidity is controlled below 75%, and when the humidity exceeds 75%, ventilation and moisture removal are required. And (3) after the tea cake or the tea brick grows for 7 days, entering a drying stage, gradually increasing the temperature of the drying room to 40 ℃, and after drying for 3 days, finishing the drying when the water content of the tea cake or the tea brick is lower than 13%. When the lowest temperature of the air temperature is above 16 ℃, the highest temperature is 28-32 ℃ and the air humidity is below 65% during the flowering and drying, the flowering and drying of the Puer tea cake or the tea brick can be realized without the help of the temperature control of a drying room. Sensory evaluation shows that finished products of golden flower Pu' er raw tea cakes or tea bricks are dark green and have slight color, flourishing flowers, obvious dry smell and fungus fragrance, obvious nectar fragrance of tea soup, orange yellow and transparent bright soup color, mellow and pure taste, obvious and lasting salivation promoting effect and no bitter taste. The number of Aspergillus kawachii in the tea cake is 20 × 104CFU/g, Chinese thank-leaf of golden camellia brickThe number of Aspergillus awamori is 50 × 104CFU/g。
Example 5
The invention relates to application of a golden flower fungus-aspergillus kawachii strain in finished products of golden flower Pu-er ripe tea loose tea.
The preparation method of the golden flower fungus-aspergillus kawachii strain in the finished product of the golden flower Pu-er ripe tea loose tea comprises the following steps:
(1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is cultured on a Chaudhuri culture medium or a PDA culture medium at 30 ℃ for 7 days, and then is used for preparing a suspension of Aspergillus kawachii JH 1; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more.
(2) Pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 2min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus-aspergillus kawachii JH1 taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, and inoculating the suspension of eurotium cristatum-aspergillus shepheri JH 1. The pure culture is carried out in a constant temperature incubator with the temperature of 30 ℃ and the humidity of less than 75 percent. After 7 days of culture, the cells were removed and dried in the natural state. And observing the growth condition of colonies of the golden flower fungus, and measuring the quantity of aspergillus kawachii according to GB 4789.15-2010 national food safety standard 'measuring method in food microbiology inspection mould and yeast counting'.
The morphological observation shows that after the tea is purely cultured for 7 days, the tea has a luxuriant, compact and bright golden yellow cyst closure shell, namely golden flower, and has no mixed bacteria colonies such as black mold, white mold, blue mold, red mold and the like. The growth vigor of the golden flower fungus in the fermentation tank is better than that of the golden flower fungus in the culture tank. Based on the high temperature resistance of aspergillus kawachii, the aspergillus niger in the tea can be achieved within 5min of high temperature steaming under normal pressureAnd the sterilization of other bacteria such as aspergillus fumigatus, and the like, and simultaneously keeps higher biological activity of aspergillus kawachii. At 30 deg.C, Aspergillus kawachii JH1 in tea is propagated in large amount, and the number is 1 × 10 after 7 days6CFU/g is higher than the standard.
(3) Preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 35 ℃. When the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, and the pollution of other bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process. After drying, a large amount of golden flower fungus Aspergillus chevalieri JH1 strains can be obtained, and the strains can be used for manufacturing golden flower Pu 'er tea bricks/tea cakes and natural pile fermentation of the golden flower Pu' er tea ripe tea loose tea.
(4) Exogenous inoculation natural pile fermentation of Pu-Er ripe tea:
mixing the golden flower fungus-aspergillus kawachii JH1 strain with the Yunnan big-leaf sun-dried green raw tea according to the inoculation amount of 1-3% (w/w), and adding a proper amount of drinking water to keep the initial moisture content (water content) of the tea between 37-48%. The green-sunning raw tea used for pile fermentation is more than 2000kg, the pile fermentation height is 70 cm to 80 cm, and the linen after steam sterilization treatment is covered on the pile fermentation height.
The reactor core temperature is controlled between 35 ℃ and 60 ℃ in the pile fermentation process, and the whole pile fermentation time is 35 days. Turning the pile once every 5 days, and deblocking at the first or second turning. Sampling and observing the growth condition of the golden flower fungus during turning. The number of aspergillus kawachii and the phenomenon of golden flower in the process of natural pile fermentation by exogenous inoculation of Pu-erh ripe tea are shown in table 1.
TABLE 1
And after the pile fermentation, carrying out natural drying, storage aging, screening, picking and other processing procedures to finish the processing of the loose Pu-Er ripe tea finished product. Obvious flowering phenomenon appears in the pile fermentation process and continues until the pile fermentation is finished. After the Pu-Er ripe tea fermented by natural pile fermentation inoculated by the external source of the eurotium cristatum-Aspergillus kawachii is autoclaved into bricks/cakes, the high biological activity of the Aspergillus kawachii is still kept, and the phenomenon of continuous flowering can occur in the storage and aging process.
Example 6
Example 6 differs from example 5 in that:
the invention relates to a preparation method of a finished product of golden flower Pu-er ripe tea loose tea by using a golden flower fungus-aspergillus kawachii strain, which comprises the following steps:
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 3min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus-aspergillus kawachii JH1 taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, and inoculating the suspension of eurotium cristatum-aspergillus shepheri JH 1. The pure culture is carried out in a constant temperature incubator with the temperature of 30 ℃ and the humidity of less than 75 percent. After 7 days of culture, the cells were removed and dried in the natural state. And observing the growth condition of colonies of the golden flower fungus, and measuring the quantity of aspergillus kawachii according to GB 4789.15-2010 national food safety standard 'measuring method in food microbiology inspection mould and yeast counting'.
The shape observation shows that after the pure culture for 7 days, the tea leaves have splendid, compact and bright golden yellow cyst closure shells, namely the tea leavesThe golden flower has no mixed bacteria colony such as black mold, white mold, blue mold, red mold and the like. The growth vigor of the golden flower fungus in the fermentation tank is better than that of the golden flower fungus in the culture tank. Based on the high temperature resistance of aspergillus kawachii, the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be achieved within 5min of high-temperature steaming under normal pressure, and meanwhile, higher biological activity of aspergillus kawachii is reserved. At 30 deg.C, Aspergillus kawachii JH1 in tea is propagated in large amount, and the number is 1 × 10 after 7 days6CFU/g is higher than the standard.
(3) Preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 4min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be between 33 ℃. When the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, and the pollution of other bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process. After drying, a large amount of golden flower fungus Aspergillus chevalieri JH1 strains can be obtained, and the strains can be used for manufacturing golden flower Pu 'er tea bricks/tea cakes and natural pile fermentation of the golden flower Pu' er tea ripe tea loose tea.
(4) Exogenous inoculation natural pile fermentation of Pu-Er ripe tea:
mixing the golden flower fungus-aspergillus kawachii JH1 strain with the Yunnan big-leaf sun-dried green raw tea according to the inoculation amount of 1-3% (w/w), and adding a proper amount of drinking water to keep the initial moisture content (water content) of the tea between 37-48%. The green-sunning raw tea used for pile fermentation is more than 2000kg, the pile fermentation height is 70 cm to 80 cm, and the linen after steam sterilization treatment is covered on the pile fermentation height.
Example 7
Example 7 differs from example 5 in that:
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 4min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus-aspergillus kawachii JH1 taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, and inoculating the suspension of eurotium cristatum-aspergillus shepheri JH 1. The pure culture is carried out in a constant temperature incubator with the temperature of 30 ℃ and the humidity of less than 75 percent. After 7 days of culture, the cells were removed and dried in the natural state. And observing the growth condition of colonies of the golden flower fungus, and measuring the quantity of aspergillus kawachii according to GB 4789.15-2010 national food safety standard 'measuring method in food microbiology inspection mould and yeast counting'.
The morphological observation shows that after the tea is purely cultured for 7 days, the tea has a luxuriant, compact and bright golden yellow cyst closure shell, namely golden flower, and has no mixed bacteria colonies such as black mold, white mold, blue mold, red mold and the like. The growth vigor of the golden flower fungus in the fermentation tank is better than that of the golden flower fungus in the culture tank. Based on the high temperature resistance of aspergillus kawachii, the aim of sterilizing the mixed bacteria such as aspergillus niger and aspergillus fumigatus in the tea can be achieved within 5min of high-temperature steaming under normal pressure, and meanwhile, higher biological activity of aspergillus kawachii is reserved. At 30 deg.C, Aspergillus kawachii JH1 in tea is propagated in large amount, and the number is 1 × 10 after 7 days6CFU/g is higher than the standard.
(3) Preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture with tea leaves such as sun-dried raw tea and the like as a matrix with the sun-dried raw tea of the Yunnan large leaf according to the inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 18 ℃. When the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity. The number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g, avoiding mixed bacteria such as black mold, blue mold, white mold and red mold during solid state fermentation and dryingAnd (4) pollution. After drying, a large amount of golden flower fungus Aspergillus chevalieri JH1 strains can be obtained, and the strains can be used for manufacturing golden flower Pu 'er tea bricks/tea cakes and natural pile fermentation of the golden flower Pu' er tea ripe tea loose tea.
(4) Exogenous inoculation natural pile fermentation of Pu-Er ripe tea:
mixing the golden flower fungus-aspergillus kawachii JH1 strain with the Yunnan big-leaf sun-dried green raw tea according to the inoculation amount of 1-3% (w/w), and adding a proper amount of drinking water to keep the initial moisture content (water content) of the tea between 37-48%. The green-sunning raw tea used for pile fermentation is more than 2000kg, the pile fermentation height is 70 cm to 80 cm, and the linen after steam sterilization treatment is covered on the pile fermentation height.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Sequence listing
<110> Marchang of Yunnan agricultural university
<120> golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof
<130> 2021
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 482
<212> DNA
<213> Artificial sequence (ITS base sequence of Aspergillus chevalieri JH1 Strain, Aspergillus kawachii)
<400> 1
cctggttaaa aagattggtt gcgaggctag ctgccagctg gacctacggg agcgggtgac 60
aaagccccat acgctcgagg accagacatg gtgccgccac tgccttttgg gcccgtcccc 120
gttgccaggg acggaagccc aacacacaag ccgtgcttga gggcagcaat gacgctcgga 180
caggcatgcc ccccggaata ccagggggcg caatgtgcgt tcaaagactc gatgattcac 240
tgaattctgc aattcacatt aattatcgca tttcgctgcg ttcttcatcg atgccggaac 300
caagagatcc gttgttgaaa gttttaacga ttgtttaact aaaaactcag actgcaaact 360
tcagacagcg ttcaaatgtt agtctccggc gggccgtggc cacgccgaag caacagggta 420
cagatagaca cggatgggag gttggaccca gagggcccgc actcggtaat gatccttccg 480
ca 482
<210> 2
<211> 423
<212> DNA
<213> Artificial sequence (beta-tubulin base sequence)
<400> 2
ccagttgtta ccggcaccgg actgaccgaa aacgaagtta tcgggacgga agagctggcc 60
gaagggaccg gcacggacgg cgtccatggt accgggctca aggtcgacga ggacggcacg 120
ggggacatat ttgttgttgg aggcctgtca ctgtcagaac tgtttcccac acagacacga 180
atgaattagg caaacctcgt tgaagtagac gttcatacgc tccaactgga ggtcggagga 240
gccattgtag ctgcaatcca tttagatatc catatccgat acgcgtccat ctcggagacc 300
cgactgtact tacacaccag agccgtcgag accgtgctcg ccggagatag tctgcctgtg 360
atactgttag tatatcaccc aatctggact ctgattataa tgcggacata ccagaaagca 420
gca 423
<210> 3
<211> 741
<212> DNA
<213> Artificial sequence (Calmodulin base sequence)
<400> 3
gagcaagtct ccgagtacaa ggaggcgttc tccctcttcg taagtgcgct ggccatctcc 60
atgtggcatt ttccccgtcg gcgttgaggg cagtaaactg accacgattt ctgcatttct 120
acaggacaag gatggcgatg gttagtgatc ccgctctact ctgcatcaaa tgcctctttt 180
actccaccat tcgattggtt ccgaccgtga tatcatattc gctgttgaaa taatatcttt 240
ccggttaacg acataatact gatggatctc tgcgattaca ggccagatca ccaccaagga 300
gctgggtacc gttatgcgct cgctgggcca gaacccctcc gagtcggagt tgcaggacat 360
gatcaacgag gttgacgctg acaacaacgg caccattgat ttccctggta tgcgatcgtc 420
ccgacatgaa aggtcacgta acaagatagg cgattctgac catactagaa ttccttacca 480
tgatggcacg gaagatgaag gacaccgatt ccgaggaaga gatccgggaa gctttcaagg 540
tcttcgatcg cgacaacaac ggtttcattt ccgctgcgga gctgcgccac gttatgacct 600
ccattggcga gaagcttacc gatgacgaag ttgacgagat gattcgtgag gctgaccagg 660
acggtgatgg ccgtattgac tgtatgtgaa cctttttctc ggtcgtatgt gaagaagcta 720
atcgttacca gacaacgaat t 741
Claims (10)
1. A golden flower fungus-aspergillus shewanii strain is characterized in that: the strain is aspergillus kawachii JH 1; the collection name is Aspergillus kawachii JH1Aspergillus chevalier JH 1; is preserved in China center for type culture Collection, and the preservation address is Wuhan university in Wuhan, China; the preservation date is as follows: 09 months and 30 days 2021; the preservation number is CCTCC NO. 20211180.
2. The A.chrysantha-Aspergillus shewanii strain of claim 1, wherein: the ITS base sequence of the Aspergillus kawachii Chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 1; the beta-tubulin base sequence of the Aspergillus kawachii Aspergillus chevalieriJH1 strain is a nucleotide sequence shown in SEQ ID No. 2; the Calmodulin base sequence of the Aspergillus kawachii Chevalieri JH1 strain is a nucleotide sequence shown in SEQ ID No. 3.
3. The A.chrysantha-Aspergillus shewanii strain of claim 2, wherein: the bacterial colony of the strain Aspergillus kawachii Aspergillus chevalieri JH1 grows slowly on a Chaudou culture medium, the diameter is 9-16mm in 7 days at the temperature of 30 ℃, and the front side: fluffy powder, yellow to golden yellow; the back is earthy yellow to yellow brown, and the golden yellow cyst closure shell is less.
4. The A. chrysantha-A. chevalieri strain microbial inoculum prepared from the A. chrysantha-A. chevalieri strain of claim 1.
5. The golden flower fungus-aspergillus kawachii strain microbial inoculum according to claim 4, wherein the active ingredients are at least one of the following (a), (b) and (c):
(a) a fermentation culture of the eurotium cristatum-aspergillus kawachii strain of claim 1;
(b) an ultrasonic lysis supernatant of the cells of the A.nitida-A.shewanensis strain obtained in claim 1;
(c) the ultrasonic lysis of the cells of the A.nitidissima-A.shewanensis strain obtained according to claim 1.
6. The method for preparing the golden flower fungus-aspergillus kawachii strain microbial inoculum of claim 1, which is characterized by comprising the following steps: aspergillus awamori chevalieri JH1 was used for preparation of Aspergillus awamori JH1 bacterial suspension after culturing in a Chaudhuri medium or PDA medium at 30 ℃ for 7 days; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104More than CFU/mL to prepare the golden flower fungus-aspergillus kawachii strain microbial inoculum.
7. The use of the golden flower fungus-aspergillus kawachii strain of any one of claims 1 to 6 in the preparation of Pu' er tea, black tea, white tea and black tea brick/cake autoclaved drying.
8. Use according to claim 7, characterized in that: the preparation method of the brick/cake comprises the following steps: (1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is transplanted on a Chao's medium or a PDA medium and cultured for 7 days at the temperature of 30 ℃ for preparing a suspension of Aspergillus kawachii JH1 bacteria; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more;
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing a golden flower fungus-aspergillus kawachii JH1 bacterial suspension (250g) and Yunnan big-leaf sun-dried raw tea (1000g) according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 4min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus taking the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, inoculating the suspension of the eurotium cristatum-Aspergillus kawachii JH1 bacteria, placing in a constant-temperature incubator with the temperature of 30 ℃ and the humidity of less than 75% to perform pure culture of Aspergillus kawachii Aspergillus chevalieri JH1, culturing for 7 days, and drying in a natural state;
(3) preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing eurotium cristatum obtained by pure culture with tea such as sun-dried raw tea as matrix with Yunnan big leaf sun-dried green tea according to inoculation amount of 5% (w/w). And (3) after 30min to 1 h of humidification, steaming for 3-5min under normal pressure and high temperature steam, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation. No water is added in the whole fermentation process, and the temperature of the fermentation environment is required to be 18 ℃; when the tea is fermented for 5 days, golden flowers begin to appear in the tea and propagate in a large quantity; the number of Aspergillus kawachii communities reaches 1.5 multiplied by 10 when the solid state fermentation is carried out for 14 days7CFU/g; the pollution of mixed bacteria such as black mold, blue mold, white mold and red mold should be avoided in the solid fermentation and drying process; drying to obtain a large amount of strains of Camellia Chysantha Aspergillus chevalieri JH1, which can be used for making brick/cake of Camellia sinensis and natural loose tea of Camellia sinensisPile fermentation;
(4) steaming and pressing tea brick/tea cake such as Pu' er tea and the like:
the production of the golden flower fungus requires that the tea cake has certain water content and proper temperature and humidity are required; mixing the eurotium cristatum strain-Aspergillus kawachii Chevalieri JH1 obtained by enlarged culture with the sun-dried green tea of the Yunnan big leaf according to the mass ratio of 1-3%, adding a proper amount of drinking water for humidifying, and steaming under normal pressure and high temperature steam after humidifying for 30 min-1 h; the steam temperature is between 100 ℃; steaming for 2-3 min;
(5) and (3) drying the grown flower of the Pu' er tea brick/tea cake: the step of flowering refers to a process of mass propagation of golden flower fungus under a certain hydrothermal action, hydrolysis, oxidation and polymerization are carried out on quality components such as tea polyphenol, amino acid and the like in tea leaves through corresponding extracellular enzymes, and autoclaved tea cakes or tea bricks are arranged in a drying room according to a certain sequence; and when the flowers bloom for 7 days, entering a drying stage, gradually increasing the temperature of a drying room to 40 ℃, and drying for 3 days to obtain the Pu' er tea, the black tea, the white tea and the black tea brick/cake.
9. The use of the golden flower fungus-aspergillus kawachii strain as claimed in any one of claims 1 to 6 in finished products of loose golden flower Pu' er ripe tea.
10. Use according to claim 9, characterized in that: the preparation method of the finished product of the golden flower Pu-erh ripe loose tea comprises the following steps: (1) preparation of a eurotium cristatum-aspergillus shewanese JH1 bacterial suspension:
aspergillus kawachii Aspergillus chevalieri JH1 is cultured on a Chaudhuri culture medium or a PDA culture medium at 30 ℃ for 7 days, and then is used for preparing a suspension of Aspergillus kawachii JH 1; extracting Aspergillus kawachii JH1 community and mixing with sterile water under sterile environment, and adjusting the number of Aspergillus kawachii JH1 in bacterial suspension to 1 × 104CFU/mL or more;
(2) pure culture of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing the golden flower fungus-aspergillus kawachii JH1 bacterial suspension and Yunnan large-leaf sun-dried raw tea according to the mass ratio of the tea to the golden flower fungus suspension of 100:25, steaming for 2min under normal pressure and high temperature steam after 30min, and putting into a sterilized fermentation tank for pure culture of the golden flower fungus-aspergillus kawachii JH1 with the sun-dried raw tea as a matrix. Or sterilizing sun-dried green tea at high temperature and high pressure, and inoculating suspension of eurotium cristatum-aspergillus shepheri JH 1; performing pure culture in a constant-temperature incubator at 30 ℃ and humidity of less than 75%, taking out after 7 days of culture, and drying in a natural state;
(3) preparation of a strain of eurotium cristatum-Aspergillus kawachii chevalieri JH 1:
mixing golden flower fungus obtained by pure culture by taking tea leaves such as sun-dried raw tea and the like as a matrix with a large-leaf sun-dried green tea of Yunnan according to an inoculation amount of 5% by mass, steaming for 5min under normal pressure and high temperature steam after humidifying for 30min to 1 h, and transferring to a sterilized fermentation tank for semi-anaerobic solid state fermentation; when the fermentation is carried out for 5 days, the golden flower begins to appear in the tea, and when the solid state fermentation is carried out for 14 days, the number of aspergillus kawachii communities reaches 1.5 multiplied by 107CFU/g, drying to obtain a large amount of golden flower fungus-Aspergillus chevalieri JH1 strain, which can be used for making golden flower Pu 'er tea brick/tea cake and natural pile fermentation of golden flower Pu' er tea loose tea;
(4) exogenous inoculation natural pile fermentation of Pu-Er ripe tea:
mixing the golden flower fungus-aspergillus kawachii JH1 strain with the Yunnan big-leaf green-dried raw tea according to the inoculation amount of 1-3% by mass percent, adding a proper amount of drinking water, piling the green-dried raw tea by over 2000kg, enabling the pile height to be 70-80 cm, and covering the hemp cloth after steam sterilization treatment;
(5) and after the pile fermentation is finished, natural drying, storage aging, screening and picking are carried out to prepare the finished product of the golden flower Pu-erh ripe tea loose tea.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111621139.4A CN114214211A (en) | 2021-12-28 | 2021-12-28 | Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111621139.4A CN114214211A (en) | 2021-12-28 | 2021-12-28 | Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114214211A true CN114214211A (en) | 2022-03-22 |
Family
ID=80706303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111621139.4A Pending CN114214211A (en) | 2021-12-28 | 2021-12-28 | Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114214211A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115251203A (en) * | 2022-07-27 | 2022-11-01 | 中华全国供销合作总社济南果品研究所 | Application and method of Xie Washi aspergillus in fermentation preparation of peony petal tea |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170358A1 (en) * | 2002-05-17 | 2005-08-04 | Morrison Christine J. | Molecular identification of aspergillus species |
| CN109679855A (en) * | 2018-11-30 | 2019-04-26 | 中国食品发酵工业研究院有限公司 | One plant of production tea flavor flavor thanks watt bulk bacteria and its application in white wine yeast |
| CN112841356A (en) * | 2021-03-19 | 2021-05-28 | 厦门惠尔康食品有限公司 | Process method for preparing golden flower fungus fermented tea drink by liquid state fermentation of aspergillus kawachii |
-
2021
- 2021-12-28 CN CN202111621139.4A patent/CN114214211A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170358A1 (en) * | 2002-05-17 | 2005-08-04 | Morrison Christine J. | Molecular identification of aspergillus species |
| CN109679855A (en) * | 2018-11-30 | 2019-04-26 | 中国食品发酵工业研究院有限公司 | One plant of production tea flavor flavor thanks watt bulk bacteria and its application in white wine yeast |
| CN112841356A (en) * | 2021-03-19 | 2021-05-28 | 厦门惠尔康食品有限公司 | Process method for preparing golden flower fungus fermented tea drink by liquid state fermentation of aspergillus kawachii |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115251203A (en) * | 2022-07-27 | 2022-11-01 | 中华全国供销合作总社济南果品研究所 | Application and method of Xie Washi aspergillus in fermentation preparation of peony petal tea |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102524442B (en) | Fungus growing process for black tea | |
| CN107080008A (en) | A kind of natural pile fermentation of golden flower Pu'er cooked teas loose tea is grown dim method | |
| CN103141599B (en) | Golden flower loose tea dark tea and processing method of golden flower loose tea dark tea | |
| CN107197967A (en) | A kind of preparation method of golden flower white teas tea cake | |
| CN102187920A (en) | Method for preparing gold-flower Puer tea by inoculating fungus microorganisms | |
| CN107699506B (en) | Saccharomyces cerevisiae in black tea fungus and application thereof | |
| CN103103061A (en) | Preparation method of pure rice-koji added with Chinese medical herbs | |
| CN109055235A (en) | A kind of preparation of the inoculating microbes strain for Pu'er tea pile fermentation and its application method | |
| CN107041436A (en) | A kind of manufacture craft of golden flower Pu'er raw teas tea cake or brick tea | |
| CN112481134A (en) | Method for extracting mulberry leaf polysaccharide by using microbial fermentation method | |
| CN102550723B (en) | Preparation of conventional Pu'er tea microbial colony seeds and application of microbial colony seeds in Pu'er tea fermentation | |
| CN107114500A (en) | A kind of the natural of Pu'er tea compressed tea is aged method of growing dim | |
| CN112175845B (en) | Saccharomyces cerevisiae suitable for brewing tea wine and application thereof | |
| CN108934785B (en) | Liquid strain culture method and cultivation method of boletus nigricans | |
| CN105494715A (en) | Process for producing dark tea through inoculation method | |
| CN108283310A (en) | A kind of preparation method and enzyme stoste of tawny daylily ferment | |
| CN107801938B (en) | Biological softening method of betel nuts | |
| CN114214211A (en) | Golden flower bacterium-aspergillus kawachii strain and preparation method and application thereof | |
| CN105368736A (en) | Bacillus aceticus and application of bacillus aceticus in coffee cherry peel and pulp fermentation vinegar | |
| CN110317734A (en) | A kind of monascus and its isolated culture method and the application of high-yield glucoamylase, Esterified Enzyme and protease | |
| CN109699766A (en) | The method for preparing fermented tea, the fermented tea prepared with this method and its application | |
| CN105230827A (en) | Eurotium cristatum tea automatic fermentation process | |
| CN115252525B (en) | Preparation method of narcissus polysaccharide fermentation liquid and application of narcissus polysaccharide fermentation liquid in cosmetics | |
| CN110699265B (en) | Method for preparing wolfberry leaf tea by fermentation of eurotium cristatum and special strain | |
| CN102604840B (en) | Rice wine yeast, rhizopus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |