WO2018161216A1 - 一种冠突散囊菌及其应用 - Google Patents

一种冠突散囊菌及其应用 Download PDF

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WO2018161216A1
WO2018161216A1 PCT/CN2017/075759 CN2017075759W WO2018161216A1 WO 2018161216 A1 WO2018161216 A1 WO 2018161216A1 CN 2017075759 W CN2017075759 W CN 2017075759W WO 2018161216 A1 WO2018161216 A1 WO 2018161216A1
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tea
temperature
cfu
tea leaves
strain
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PCT/CN2017/075759
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English (en)
French (fr)
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王春玲
李颂
田海霞
郝彬秀
孙哲浩
黄沛
马跃
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中国茶叶有限公司
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Priority to PCT/CN2017/075759 priority Critical patent/WO2018161216A1/zh
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/08—Oxidation; Fermentation
    • A23F3/10—Fermentation with addition of microorganisms or enzymes
    • 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
    • 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
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention belongs to the field of microorganisms and food biotechnology, and in particular, the invention relates to a new plant type of the genus Coronaria and its application in the fermentation process of tea leaves.
  • Post-fermented tea that is, black tea
  • black tea belongs to one of the six traditional Chinese teas.
  • the fundamental feature distinguishing it from other teas is that microbial fermentation is involved in the processing of tea.
  • typical black tea includes Hunan Anhua Black Tea, Yunnan Pu'er Cooked Tea, Guangxi Liubao Tea, and Hubei Green Brick Tea.
  • the traditional fermentation process of various types of black tea also known as pile fermentation, refers to the time when the tea leaves are piled up under a certain natural temperature and humidity for a period of time, and the taste of the tea is sensed by experience to stop the fermentation.
  • Sclerotinia sclerotiorum is one of the many strains involved in the fermentation of black tea. It is one of the earliest fungi found in black tea of Anhui Anhua. It is called “Golden flower fungus” because of the formation of golden color closed capsule inside brick tea. ".
  • the unique "flowering" process of Anhua Black Tea Brick Tea is a process that promotes the growth of Coronaria sphaeroides, which makes the tea have a unique aroma, taste and efficacy. It is generally believed that the quality and quantity of the growth of Coriolus sphaeroides is one of the important indicators for judging the quality of Anhua black tea brick tea.
  • the number of Cochlear spores is not less than 200 ⁇ 10 3 CFU/g tea, and this requirement is increased to 300 ⁇ 10 3 CFU/g tea in local standards.
  • the flowering process has been introduced into other teas, such as the fermentation of Liubao tea, Pu'er tea, oolong tea (Dahongpao) and white tea, using the environment of the bacterium
  • the flowering is carried out at a suitable temperature (usually 25-40 ° C) and humidity (60-90%) (typically 10-15 days).
  • This process is similar to the traditional brick-and-tea flowering process, and belongs to natural fermentation.
  • the main drawbacks of natural fermentation are: with the changes of the external environment, when the content of other microbes around is high, the Coronaria can not become a dominant organism, leading to failure of flowering, mildew of the product; the microorganisms involved in the fermentation can be brought into the natural environment.
  • the production of mycotoxins causes food safety hazards; the composition of fermenting microorganisms is complex, resulting in uncontrollable product quality, unstable composition, and the sensory quality of the function cannot be guaranteed; accordingly, the production cycle is not fixed, resulting in waste of production capacity.
  • the above problems limit the widespread application of the potential high-quality microbial resources of the genus Coronaria, and also limit the promotion and export of traditional fermented foods represented by fermented tea. Therefore, the industrial upgrading from the production of traditional workshops to the production of standardized industries is extremely urgent.
  • the core technical problems that need to be solved include: First, focus on food safety, ensure that the developed strains have clear genetic background, non-toxic; Second, ensure the secretion of fermentation strains Metabolites can improve the efficacy of the product, so that the fermented tea has a stable and clear health effect; third, reduce the production cost of the product, improve production efficiency, including minimizing the inoculum of the strain, and minimizing the fermentation cycle of the product.
  • the fermentation effect, and the new strain is more stable and safer, can be applied to a wide range of tea varieties, and the tea obtained by the fermentation has more health effects.
  • Still another object of the present invention is to provide a novel strain of S. cerevisiae for use in tea fermentation.
  • Another object of the present invention is to provide a tea fermentation method using the new strain of Ascomycota, and the tea obtained by the fermentation.
  • the present invention provides a Eurotium cristatum which has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 9, 2017 (referred to as CGMCC, deposit address: Beijing No. 3, No. 1 Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC No. 13368.
  • the present invention provides Eurotium cristatum CGMCC No. 13368 (hereinafter referred to as strain 13368, which can be used interchangeably with strain CGMCC No. 13368) from the tea tea storage warehouse of the first tea factory of the tea in Hunan, Hunan province (a hundred years) Separation, purification and screening of the tea leaves stored in the product. Briefly, teas of different years were used as the original materials, and after isolation and purification of multiple strains of S. cerevisiae, strain 13368 was screened by growth rate, number of hairs and toxin production. Compared with other strains of C.
  • strain 13368 has a fast growth rate, a large number of golden flowers per unit time, and a fermentation medium and No characteristics such as common toxins were detected in the fermented tea, and the tea leaves fermented by the strain 13368 had new and significant health effects. details as follows:
  • strain 13368 is a genus of the genus Coronaria, belonging to the genus Aspergillus sp., which excludes the possibility that the strain is other species of the genus Aspergillus.
  • strain 13368 has the advantageous features not previously reported in the public report:
  • the strain can effectively improve the production efficiency: when the number of target microorganisms inoculated is 0.5 ⁇ 10 3 CFU/g tea, the number of golden flowers obtained by flowering in 5 days can exceed 1000 ⁇ 10 3 CFU/g tea (ie inoculation) The amount is only 0.5 ⁇ of the target bacteria in the final product), and the content of the tea brown pigment is higher than 3%;
  • the present invention evaluated five complete safety evaluations of genetic stability, drug resistance, virulence, toxin production and toxicology (acute toxicity and genotoxicity) of strains of the genus Coronaria, and found that strain 13368 was satisfied with fermentation. Among the required strains, the only strain that meets all five safety assessment requirements;
  • the invention provides the use of the S. cerevisiae of the invention in the fermentation of tea leaves.
  • the tea leaves include pressed tea, loose tea and broken tea.
  • the pressed tea is made of tea leaves of any form as a starting material, is pressed into bricks during the fermentation process, and then undergoes a flowering process in the form of tea bricks, including the conventional internal hair coloring and the innovative surface and interior embossing of the present invention.
  • the "loose tea” refers to a strip of tea having a mesh number of ⁇ 10 mesh
  • the "broken tea” refers to a strip of tea having a mesh number of >10 mesh.
  • the tea leaves include green tea, white tea, oolong tea, yellow tea, black tea and black tea.
  • the present invention provides a method of fermenting tea leaves, the method comprising inoculating tea leaves using the S. cerevisiae of the present invention.
  • the method can be carried out for any one or more of green tea, white tea, oolong tea, yellow tea, black tea and black tea, and the obtained tea product can be pressed flower tea, loose tea or broken tea.
  • the method comprises the step of adding the Stenotrophomonas sp. strain 13368 of the invention to the tea leaves, and performing the flowering after pressing.
  • 0.5-10 ⁇ 10 3 CFU/g tea preferably 0.5-2 ⁇ 10 3 CFU/g tea, more preferably 1 ⁇ 10 3 CFU/g tea is added to the strain of Stenotrophomonas sp. 13368;
  • the embossing comprises flowering for 4-10 days at a temperature of 20-40 ° C and a relative humidity of 50-90%.
  • the method comprises the steps of: reconstituting the tea leaves in sequence, pre-fermentation, steaming, inoculation, brick pressing, flowering, and drying.
  • the rehydrating comprises rehydrating the tea leaves using water having a temperature of 70-80 ° C, so that the water content of the tea leaves is 25%-45%;
  • the pre-fermentation comprises naturally fermenting the tea leaves at a temperature of 40-60 ° C and a humidity of 50%-70% for 48-72 hours, and stirring once every 18-30 hours;
  • the steaming comprises cooling the tea leaves to 20 ° C to 30 ° C, reducing the moisture to 15% to 20%, and steaming at a steam pressure of 0.1-0.5 MPa and a temperature of 105-115 ° C for 10-20 seconds;
  • the inoculation comprises adding 0.5-10 ⁇ 10 3 CFU/g tea, preferably 0.5-2 ⁇ 10 3 CFU/g tea, more preferably 1 ⁇ 10 3 CFU/g tea to the strain of Stenotrophomonas sp. 13368; Optionally cooling the tea leaves to below 50 °C prior to inoculation;
  • the press brick comprises a tea brick which is pressed into a cube or a rectangular parallelepiped shape, the tea brick being 200-600 g, preferably 400 g;
  • the hair buds comprise flowering for 4-10 days at a temperature of 20-40 ° C and a relative humidity of 50-90%;
  • the drying comprises drying at a temperature of 40-50 ° C for 2-4 days until the moisture content in the tea leaves is less than 12%.
  • the above fermentation method can be carried out for any one or more of green tea, white tea, oolong tea, yellow tea, black tea and black tea to obtain a pressed tea which is internally embossed.
  • the number of strains per gram of tea in the pressed tea is not less than 1000 ⁇ 10 3 CFU/g tea, and the total number of other molds and bacteria is less than 2 ⁇ 10 3 CFU/g tea, and the content of tea brown pigment is not lower than 3%.
  • the present invention also provides a tea product obtained by the above method.
  • the method comprises the step of adding to the tea leaf the strain of Stenotrophomonas sp. 13368 of the invention, which is then subjected to a filming and then two flowering.
  • 0.5-10 ⁇ 10 3 CFU/g tea preferably 0.5-2 ⁇ 10 3 CFU/g tea, more preferably 1 ⁇ 10 3 CFU/g tea is added to the strain of Stenotrophomonas sp. 13368;
  • the film comprises a composite film coated on the surface of the pressed tea brick, and the composite film comprises a hydrophilic inner film layer, a gas permeable intermediate film layer and a hydrophobic outer film layer from the side contacting the tea brick.
  • Floor a composite film coated on the surface of the pressed tea brick, and the composite film comprises a hydrophilic inner film layer, a gas permeable intermediate film layer and a hydrophobic outer film layer from the side contacting the tea brick.
  • the two florets include the first florets and the second florets, wherein the first florescence comprises 2-3 days at a temperature of 24-40 ° C and a humidity of 50-90%; the secondary florescence is included at a temperature of 30 -36 ° C, humidity 18-32% under the hair for 36-50 hours, then adjust the temperature to 36-45 ° C, continue to flower for 2-4 days.
  • the first florescence comprises 2-3 days at a temperature of 24-40 ° C and a humidity of 50-90%
  • the secondary florescence is included at a temperature of 30 -36 ° C, humidity 18-32% under the hair for 36-50 hours, then adjust the temperature to 36-45 ° C, continue to flower for 2-4 days.
  • the method comprises the steps of: re-watering, pre-fermenting, sterilizing, steaming, inoculating, pressing brick, wrapping, first-time flowering, secondary flowering, drying.
  • the rehydrating comprises rehydrating the tea leaves using water having a temperature of 70-80 ° C, so that the water content of the tea leaves is 25%-45%;
  • the pre-fermentation comprises naturally fermenting the tea leaves at a temperature of 40-60 ° C and a humidity of 50%-70% for 48-72 hours, and stirring once every 18-30 hours;
  • the sterilizing comprises cooling the tea leaves to 20 ° C to 30 ° C, the moisture is reduced to 15% -20%, and sterilizing at a temperature of 90-130 ° C for 15-30 min;
  • the steaming comprises steaming at a steam pressure of 0.3-0.5 MPa and a temperature of 95-110 ° C for 8-30 seconds;
  • the inoculation comprises adding 0.5-10 ⁇ 10 3 CFU/g tea, preferably 0.5-2 ⁇ 10 3 CFU/g tea, more preferably 1 ⁇ 10 3 CFU/g tea to the strain of Stenotrophomonas sp. 13368; Optionally cooling the tea leaves to below 50 °C prior to inoculation;
  • the press brick comprises tea bricks which are pressed into a cube or a rectangular parallelepiped shape, preferably from 20 to 400 g, more preferably from 50 to 200 g.
  • the film comprises a composite film coated on the surface of the pressed tea brick, thereby forming a sealed bag with tea bricks therein, wherein the composite film comprises a hydrophilic inner film layer from the side contacting the tea bricks in order. a gas permeable intermediate film layer and a hydrophobic outer film layer, wherein each of the composite film layers has small pores, and the pores have a pore density of 5000-7000/m 2 , and the total of the composite membrane a composite film having a basis weight of 30 to 70 g/m 2 , preferably a composite film of 50 g/m 2 ;
  • the gas permeable intermediate film layer is a PE gas permeable film layer having a pore density of 6000/m 2 and a basis weight of 5 to 10 g g/m 2 , preferably 8 g/m 2 ;
  • the inner film layer and the hydrophobic outer film layer are a hydrophilic nonwoven fabric and a hydrophobic nonwoven fabric, respectively, each having a cell density of preferably 6000/m 2 and a basis weight of 12.5 to 30 g/m 2 , preferably 20 g/ Square meters, the material of which is, for example, imported polypropylene with PP material;
  • the first flowering step comprises performing 2-3 days at a temperature of 24-40 ° C and a humidity of 50-90%;
  • the secondary florescence comprises: after checking that the surface of the tea brick in the sealed bag is covered with the golden flower visible to the naked eye, the tea brick is taken out to make the flower 30-50 at a temperature of 30-36 ° C and a humidity of 18-32%. Hours, then adjust the temperature to 36-45 ° C, continue to spend 2-4 days;
  • the drying comprises drying at a temperature of 40-50 ° C for 2-4 days until the moisture content in the tea leaves is less than 12%.
  • the above fermentation method can be carried out for any one or more of green tea, white tea, oolong tea, yellow tea, black tea and black tea to obtain a pressed tea having internal and surface hair buds.
  • the number of strains per gram of tea in the pressed tea is not less than 1000 ⁇ 10 3 CFU/g tea, and the total number of other molds and bacteria is less than 2 ⁇ 10 3 CFU/g tea, and the content of tea brown pigment is not lower than 3%.
  • the present invention also provides a tea product obtained by the above method.
  • the method comprises the steps of bottling tea leaves and then adding to the tea leaves the S. aureus strain 13368 of the invention for flowering, wherein the tea leaves are loose tea or broken tea.
  • 0.5-10 ⁇ 10 3 CFU/g tea preferably 0.5-2 ⁇ 10 3 CFU/g tea, more preferably 1 ⁇ 10 3 CFU/g tea, is added to the strain of Stenotrophomonas sp. 13368;
  • the embossing comprises flowering for 6-10 days at a temperature of 20-40 ° C and a humidity of 30-75%.
  • the method comprises the steps of: reconstituting the tea leaves in sequence, pre-fermenting, bottling, sterilizing, inoculation, flowering, drying.
  • the rehydrating comprises rehydrating the tea leaves using water having a temperature of 70-80 ° C, so that the water content of the tea leaves is 25%-45%;
  • the pre-fermentation comprises naturally fermenting the tea leaves at a temperature of 40-60 ° C and a humidity of 50%-70% for 48-72 hours, and stirring once every 18-30 hours;
  • the bottling comprises cooling the tea leaves to 20 ° C to 30 ° C, and reducing the moisture to 15% to 20%, and then loading into the charging bottle, wherein the charging bottle has a capacity of 500-2000 ml / bottle, tea leaves
  • the loading therein is 1/4 to 3/4, preferably 2/3 of the capacity;
  • the charging bottle is in the shape of an inverted trumpet, and the opening is provided with two caps each having a vent hole a breathable water absorbing film layer is further disposed between the two caps; preferably, the breathable water absorbing film layer is a sponge layer of 1-5 mm;
  • said sterilizing comprises sterilizing at a temperature of 80-125 ° C for 5-35 min;
  • said inoculation comprises first cooling the sterilized tea leaves to 30-80 ° C, then 0.5-10 x 10 3 CFU/g tea, preferably 0.5-2 x 10 3 CFU/g tea, more preferably 1 x 10 3 CFU/g tea was added to the strain of Stenotrophomonas 13368;
  • the hair buds comprise flowering for 6-10 days at a temperature of 20-40 ° C and a relative humidity of 30-75%;
  • said drying comprises removing the tea leaves from the loading bottle and then drying at a temperature of 40-50 ° C until the water content in the tea leaves is 5-12%.
  • the above fermentation method can be carried out for any one or more of green tea, white tea, oolong tea, yellow tea, black tea and black tea to obtain loose tea or broken tea.
  • the number of strains per gram of tea in the loose tea or broken tea is not less than 1000 ⁇ 10 3 CFU/g tea, and the total number of other molds and bacteria is less than 2 ⁇ 10 3 CFU/g tea, and the content of tea brown pigment is not Less than 3%.
  • the present invention also provides a tea product obtained by the above method.
  • the present invention selects a new strain of Candida albicans which can effectively ferment tightly pressed tea or even loose tea and broken tea by using the growth rate, the amount of hair and the production of toxin as screening criteria.
  • This new strain has the following advantages compared to the known bacterium:
  • the novel strain of the present invention was identified as a capsular bacterium, and the identification data covered macroscopic morphology, microscopic morphology, electron microscopic identification and gene sequencing analysis, and was the first well-identified and characterized Phytophthora sphaeroides in the field. Moreover, the strain screening and comparative experiments proved that compared with other strains of S. cerevisiae, the new strain 13368 of the present invention had no obvious change in morphological characteristics after culture to seven generations, and the gene sequence was unchanged, and the strain trait was stable.
  • the present invention is the first to complete the five complete safety of genetic stability, drug resistance, toxin production, strain culture virulence and bacterial toxicology (acute toxicity and genotoxicity) Sexual evaluation found that strain 13368 was the only strain that met the five complete safety evaluation requirements among the strains with excellent fermentation performance.
  • the invention has examined various aspects of a total of 150 strains of the genus Aspergillus sp. and the strain of Coronaria sphaeroides, and revealed to some extent that there are significant intra-species differences in the bacterium of the genus Coronaria (see the examples below). .
  • the new strain of the Schwannoma strain of the present invention has better fermentation performance. It has been confirmed that the novel strain 13368 of the present invention can even inoculate 0.05% as low as the target microorganism amount (e.g., the final product has a dendritic bacterium content of 1000 ⁇ 10 3 CFU/g, and the inoculum amount is 0.5 ⁇ 10 3 CFU / g), to achieve the number of golden flowers of 1000 ⁇ 10 3 CFU / g tea leaves in the shortest 5 days.
  • the target microorganism amount e.g., the final product has a dendritic bacterium content of 1000 ⁇ 10 3 CFU/g, and the inoculum amount is 0.5 ⁇ 10 3 CFU / g
  • the novel strain of the present invention has the advantages of reduced inoculum and shortened fermentation cycle, which can significantly reduce production costs and increase production turnover.
  • the novel strain of the bacterium of the present invention has obvious health effects.
  • the tea product fermented by the novel strain 13368 of the present invention has higher beneficial ingredients, including the theaflavin, the soluble active polysaccharide component, compared to other Ascomycota. It has also been proved that the fermentation of the new strain 13368 of the present invention can improve the blood sugar control ability of the tea, and also can help prevent and treat fatty liver, and at the same time, can enhance the activation of amylase and protease in promoting starch and protein digestion. Rate, so it can significantly promote digestion. These effects have not previously been seen in the publicly reported strains of S. cerevisiae fermented tea products.
  • the application of the new strain of the genus Coronaria in the present invention is also more extensive. It is known in the art that the genus Coronaria can participate in the black tea fermentation, and due to the growth habit of the bacterium, only the tea can be fermented and pressed, and the golden flower produced by the fermentation only exists in the interior of the pressed tea.
  • the new strain 13368 of the invention can ferment green tea, white tea, oolong tea, yellow tea, black tea and black tea, and the tea form is not limited to pressing tea, even the broken tea and loose tea can be effectively fermented, not only can be obtained internally and A pressed tea with a good fermentation/flowering effect on the surface, as well as a broken tea and loose tea with good fermentation/flowering effect. This greatly broadens the source and form of tea, making the fermentation process more flexible and convenient, and the products are more diversified to meet different market needs.
  • the present invention also provides various novel fermentation processes.
  • the present invention provides a method of pressing the hair inside and outside the tea by introducing a filming operation by two florets.
  • a composite film comprising a hydrophilic inner film layer, a gas permeable intermediate film layer and a hydrophobic outer film layer is used in the film coating operation, and each film layer is provided with small holes.
  • the inner membrane layer is in contact with the pressed tea which needs to be fermented, so that the pressed tea which is in contact with the inner membrane layer has good water retention performance, and the intermediate membrane layer can replace the gas generated during the fermentation with the outside air.
  • the outer membrane layer can discharge water vapor generated in the composite membrane.
  • the two flowering steps of the tea brick were carried out, and the first flowering made the surface of the tea brick fading, and the secondary flowering caused the growth of the golden flower to start from the surface of the tea brick and gradually extended to the inside, and changed.
  • the traditional situation in which the tea flower grows from the inside begins, and finally the pressed tea with uniform gold flower distribution on the inside and the surface is obtained.
  • This improvement not only obtained the tea production with more golden flower growth.
  • the product but also completely solved the drawbacks of the golden closed capsule of the Coriolus sphaeroides that can only be seen inside when only the tea bricks are opened.
  • the present invention also provides a method for performing the flowering of Coronaria sphaeroides on loose tea and broken tea.
  • the traditional Coriolus sp. fermented tea has only the hairy spores in the interior of the pressed tea.
  • loose tea and broken tea it is difficult to achieve effective colostomy.
  • loose tea and broken tea have the advantages of convenient carrying and convenient drinking in practical applications.
  • the present invention introduces the bottling process in the fermentation process, that is, first put loose tea or crushed tea in the charging bottle.
  • the opening of the charging bottle is provided with two caps each having a venting hole, and a breathable water absorbing film layer is disposed between the two caps so that the bottle can be realized during the embossing process.
  • the replacement of air inside and outside to ensure the oxygen supply of tea fermentation; and the breathable water absorption film layer can initially filter the air entering the bottle body at the same time, and adsorb the humid air collected at the bottle mouth to prevent the condensation of water vapor into the bottle.
  • the moisture content of the bottle mouth material is too high, and at the same time, the environmental microorganisms are isolated to prevent contamination of the bottle mouth bacteria.
  • the tea leaves are naturally fermented at a certain temperature for a certain period of time, so that the microorganisms such as Aspergillus and yeast naturally present in the tea and the fermentation environment can effectively function, thereby laying a better foundation for the pure fermentation of the subsequent strain 13368. Microenvironment and material basis.
  • the internal flowering method of the pressed tea provided by the present invention and the internal hair-spreading method and the hair-spreading method of loose tea and broken tea combine the excellent habits of the new strain 13368 of the genus Coronaria, with flexible and convenient process steps.
  • the flower yield of 1000 ⁇ 10 3 CFU/g tea leaves was obtained in the shortest 5 days, and various forms of tea products were finally obtained to meet different market demands.
  • These products have good fermentation/flowering effect, and no toxins are produced. In particular, they also have obvious health effects such as lowering blood sugar, preventing fatty liver and promoting digestion, and are significantly superior to similar products on the market.
  • Figure 1 shows the macroscopic morphology of the C. sphaeroides strain CGMCC No. 13368;
  • Figures 2A to 2E in Figure 2 show the macroscopic morphology of the C. sphaeroides strain CGMCC No. 13368, respectively;
  • Figure 3 shows the ITS rDNA sequence phylogenetic tree of the C. sphaeroides strain CGMCC No. 13368 and related species;
  • Figure 4 shows the phylogenetic tree of the ⁇ -tubulin sequence of the C. sphaeroides strain CGMCC No. 13368 and related species;
  • Figure 5 shows the results of intraperitoneal glucose tolerance test in tea obtained by fermentation of C. sphaeroides strain CGMCC No. 13368, wherein Figure 5A shows the results of the blood glucose tolerance test (compared with the control group, P ⁇ 0.05), FIG. 5B is the area under the blood glucose curve (P ⁇ 0.01 compared with the control group), and FIG. 5C is the glycated glycemic protein result (P ⁇ 0.05 compared with before fermentation).
  • Fig. 6 shows the results of a fatty liver test of tea obtained in a mouse obtained by fermentation of C. jejuni strain CGMCC No. 13368.
  • the medium components and culture conditions are as follows:
  • PDA solid medium potato dipping powder 5 g, glucose 20 g, agar 20 g, water 1000 ml, pH 5.6;
  • PDA liquid medium 5 grams of potato soaked powder, 15 grams of glucose, 10 g of peptone, 5 g of sodium chloride, 1000 ml of water, pH 5.6;
  • Preparation method of solid seed the activated strain is streaked into PDA solid medium, cultured at 25-30 ° C for 6-8 days in the dark, and the medium is added with water under aseptic conditions. Collect the eluate and collect the cells by centrifugation.
  • Preparation method of liquid seed The activated strain is eluted with sterile water to prepare a suspension of spores, and inoculated into a PDA liquid medium, and cultured at 25-30 ° C for 6-8 days under shaking, and the seed liquid is collected. That's it.
  • CYA medium sucrose 30g, yeast extract 5g, sodium nitrate 3g, potassium chloride 0.5g, magnesium sulfate 0.01g, ferrous sulfate 0.01g, copper sulfate 0.01g, dipotassium hydrogen phosphate 1g, agar 20g, distilled water 1000 ml, pH 6.3.
  • Malt extract broth medium 30 g of malt extract, add distilled water to 1000 mL, pH 6.3.
  • the mixed solution obtained in the above step is subjected to gradient dilution, and 1 mL of the bacterial-containing liquid having a dilution ratio of 10 5 is applied to the PDA solid plate medium for cultivation;
  • the coated PDA solid plate medium was placed in a constant temperature incubator at 28-30 ° C for 7 days, during which the colony identification of the colony was performed according to the morphological, physiological and biochemical characteristics of the bacterium. And numbered, the diameter of the colony was taken every 12 hours and recorded;
  • the coated PDA solid plate medium was placed in a constant temperature incubator at 28-30 ° C for 7 days, during which the colony identification of the colony was performed according to the morphological, physiological and biochemical characteristics of the bacterium. And numbered, the diameter of the colony was taken every 12 hours and recorded;
  • the obtained 150 strains were each inoculated on a PDA solid medium in an eggplant-shaped flask, cultured at 28 ° C, cultured for 8 days, and then washed with 100 mL of sterile water, and then the cells containing the cells were washed. The mixture is filtered through sterile gauze to obtain a spore suspension, which is diluted to spores. Spores in the suspension were counted.
  • the spore suspension of each strain was adjusted to the same spore concentration of 1 x 10 6 spores/mL with PDA liquid medium.
  • the black tea was steamed at a steam pressure of 0.3 MPa for 15 seconds, and then cooled to below 50 ° C, and the spore suspension of the 150 strains was added, and the initial inoculum amount was 1 ⁇ 10 3 CFU/g tea. Then, the bricks were manually pressed in accordance with the specifications of 1 kg of the mixed raw materials obtained above, and the flowers were sprayed at a temperature of 27 ° C and a relative humidity of 75%. On the 5th day of the flowering, a special sampling tool was used to take 25g of the tea sample inside the center of the tea brick, and the total number of colonies of the sclerotium and the bacteria was calculated according to the total number of colonies of GB 4789.2-94.
  • the tea sample was also dried at a temperature of 50 ° C for 4 days until the water content in the tea leaves was less than 12%. Then, according to the total number of colonies of GB4789.2-94, the total number of colonies of Coriolus sphaeroides in tea was calculated again, and the presence of mildew and the degree of golden flower in the product were analyzed.
  • the strain numbered DET-0022 is rapid in flowering, and the total number of "Golden Flower” colonies in the tea sample can reach 1000 ⁇ 10 3 CFU/g tea or more, and the amount of bacteria is less than 2 ⁇ 10 3 on the 5th day of flowering. CFU/g tea leaves; after drying, the total number of "Golden Flower” bacteria colonies can reach above 1000 ⁇ 10 3 CFU/g tea leaves; the “Golden Flower” inside the fermented tea products grows evenly and has a good taste.
  • strain DET-0022 the dominant strains of the above two aspects are all strains of the number DET-0022.
  • strain DET-0022 was deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee. The address of the depository is Datun Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC NO. .13368, the deposit date is January 9, 2017.
  • the strain of Stenotropha sp. 13368 was identified as follows:
  • Macroscopic morphology cultured on CYA medium for 7 days at 25 °C, colony diameter 27-30 mm, central brown, surrounded by yellow; central bulge; texture velvety; dark central brown on the reverse side, yellow around; a small amount of dark brown exudation Liquid, producing a brown soluble pigment. See Figure 1 for details.
  • Fig. 2A Microscopic morphology: hyphae yellow decorated, entangled, thinner (Fig. 2A); large closed capsule, yellow, present in the decorative mesh, diameter 110-200 ⁇ m (Fig. 2B), rupture after release to release a large number of ascus (Fig. 2C); ascospore spherical or nearly spherical, 10-16 ⁇ m in diameter, containing ascospores (Fig. 2D); ascospores oval or elliptical, 4-5 ⁇ 3-4 ⁇ m in size (Fig. 2E); Conidia structure.
  • the ITS rDNA gene sequence was amplified using the DNA sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 as forward and reverse primers, respectively, using the extracted strain 13368 genomic DNA as a template, as shown in SEQ ID NO. .
  • the ITS rDNA was sequence-aligned with the ITS rDNA of several related strains. The results are as follows:
  • SEQ ID NO. 4 and SEQ ID NO. 5 were used as forward and reverse primers, respectively, and the extracted strain 13368 genomic DNA was used as a template to amplify the ⁇ -tubulin gene sequence, which is shown in SEQ ID NO. 6.
  • the ⁇ -tubulin was sequence-matched with ⁇ -tubulin of a plurality of related strains, and the results are as follows:
  • the phylogenetic tree of strain 13368 and the related species of ⁇ -tubulin sequence were drawn by MEGA5.0 software, and the similarity was repeated for 1000 times. The results are shown in Fig. 4.
  • the invention fully satisfies the macroscopic morphology, microscopic morphology and gene sequencing analysis of the strain 13368, and is the first time for the fermentation of the Coronaria sphaeroides, and is the first disclosure of various characteristics of the genus Coronaria.
  • 16 strains were not the genus Coronaria, but other species of the genus Aspergillus. These species are less safe than C. sphaeroides, have little research on their fermentation characteristics, are not suitable for tea fermentation, and may be the cause of some toxins produced by tea.
  • Strain 13368 and 29 other strains of Candida albicans capable of rapid flowering were serially subcultured on PDA solid medium for 7 generations, and the morphological characteristics of each generation were observed for 18SrDNA/ITS and ⁇ -tubulin gene sequences. Sequencing confirmed whether the strain was stable and inherited.
  • strain 13368 had no obvious changes in its morphological characteristics after culture for seven generations, and the gene sequence did not change, and the strain traits could be stably inherited.
  • identity of the two reached 100%, which was in the same position in the ITS rDNA phylogenetic tree; and the first generation of the comparative strain 13368
  • the seventh-generation ⁇ -tubulin gene sequence was found to be 100% identical, and was also in the same position in the ⁇ -tubulin phylogenetic tree.
  • strain 13368 was sensitive to common antifungal drugs, while the remaining strains of S. cerevisiae were resistant to certain antifungal drugs.
  • step 2) The tea leaves reconstituted in step 1) are placed in a charging bottle, and the opening of the charging bottle is provided with two caps each having a vent hole, and between the two caps, Breathable water absorbing film layer;
  • the content of the theaflavin and the soluble active polysaccharide components 1, 2 in the loose tea product was measured after counting the species of the sclerotium.
  • the detection method of the tea brown pigment is carried out according to Qiuping Wang et al., Effects of enzymatic action on the formation of the abrownin during solid state fermentation of Pu-erh tea, J Sci Food Agric 2011; 91: 2412-2418; The method was carried out in accordance with Ping Xu et al., Physicochemical characterization of puerh tea polysaccharides and their antioxidant and ⁇ -glycosidase inhibition, J. of Functional Foods., V. 6, 2014. 1: 545-554.
  • Tea brown pigment can indicate the degree of fermentation of tea, which is directly related to lipid-lowering, hypoglycemic, digestive and laxative activities; soluble active polysaccharide components 1, 2 are related to hypolipidemic, hypoglycemic and digestive activities.
  • the strain 13368 fermented tea products had the largest number of crown sacs, tea The content of brown pigment and soluble active polysaccharide components 1, 2 is also much higher than that of other strains, and it can be seen that strain 13368 can be more significantly enhanced than other strains of S. The health benefits of fermented tea leaves.
  • Example 1 It is worth noting that the investigation of a total of 150 strains of the genus Aspergillus strain and the strain of Coronaria sphaeroides in Example 1 and Example 2 reveals to some extent that there are significant species in the genus Coronaria. Internal difference.
  • the colonies of the S. cerevisiae strain 13368 were transferred to the malt-impregnated broth medium, placed at about 28 ° C, and aerobically cultured for 14 days. After the culture was subjected to flowing steam for 1 hour, it was filtered, and the obtained filtrate was partially concentrated by freeze-drying 2.5 times for the experiment, and partially used for the experiment.
  • mice male and female, were randomly divided into 4 groups, 20 in each group, set as: medium blank control group, medium 2.5 times concentrated blank control group, culture stock group, culture 2.5 times concentrated group.
  • the medium, the medium 2.5 times concentrated solution, the culture stock solution, and the culture 2.5 times concentrated solution were administered to the mice at a dose of 20.0 mL/kg BW, respectively, and then continuously observed for 14 days, and the body weight of the mice during the experiment was recorded. Change, poisoning performance and death.
  • test substance was 13368 bacteria powder (referred to as "TA"), and the concentration was 0.25g/mL in sterile water.
  • the test was administered orally three times within 24 hours.
  • the actual dose was 12.5g/kg. BW.
  • mice Twenty healthy mice, half male and half female, weighing 18-22 g, were fasted for 16 hours overnight (fasting, no restriction of drinking water), and the frequency of the first two doses was 20 mL/kg BW. The final dose was 10 mL/kg BW. After 0.5 h and 2 h after administration, the observation was performed once a day, and the poisoning performance, the time of occurrence and disappearance of the symptoms, and the time of death were recorded, and the observation period was 14 days. Gross anatomy of dead or dying animals.
  • the acute oral toxicity tolerance dose is greater than 12.5g/kg BW, and its LD50 is greater than 12.5g/kg. BW.
  • the test substance was 13368 powder (referred to as "TA") of the strain of Ascomycota, and was autoclaved with the initial powder suspension (concentration: 0.25 g/mL), and then diluted with sterile water under aseptic conditions. A total of 5 dose groups of the test substance were set (actual exposures were 2.5, 0.83, 0.28, 0.09, 0.03 mg/dish); an untreated control group, a solvent (sterile water) control group, and a positive control group were set at the same time.
  • the non-metabolic activation conditions were sodium azide (1.5 ⁇ g/dish) and diclosan (50 ⁇ g/dish). Under the metabolic activation condition, the positive control was 2-acetylaminopurine (10 ⁇ g/dish), 1. 8-dihydroxyindole (50 ⁇ g/dish), cyclophosphamide (20 ⁇ g/dish).
  • 0.1 mL of the bacterium of Salmonella typhimurium, 0.1 mL of the test substance, 0.1 mL of the test substance, and 0.5 mL of the S9 mixture were added to the top agar of 45 ° C, and the mixture was thoroughly mixed and rapidly poured into the bottom medium. The colonies were counted after 48 hours of incubation at 37 ° C (72 hours in TA98 culture). The entire experiment was repeated once under the same conditions.
  • test substance was the strain 13368 powder (referred to as "TA").
  • the positive controls were all cyclophosphamide.
  • mice 50 healthy mice, weighing 25-35 g, were randomly divided into 5 groups, male and female, and were set as vehicle control group (water), TA low group, TA middle group, TA high group and positive control group.
  • vehicle control group water
  • TA low group 50 healthy mice, weighing 25-35 g
  • TA middle group 50 healthy mice, weighing 25-35 g
  • TA high group 50 healthy mice, weighing 25-35 g
  • oral gavage was performed twice at intervals of 24 hours; for positive control, two intraperitoneal injections were administered at intervals of 24 hours.
  • Six hours after the second administration the animals were sacrificed by cervical dislocation, and the femur bone marrow was taken.
  • the number of polychromatic red blood cells (PCE) in 200 red blood cells per animal was counted, and the proportion was calculated.
  • the number of observations per animal was 2000.
  • the number of micronuclei cells in the red blood cells, and the micronucleated cell rate is measured in thousands.
  • test substance was the strain 13368 powder (referred to as "TA").
  • the positive controls were all cyclophosphamide.
  • mice weighing 25-35 g were randomly divided into 5 groups, which were set as vehicle control group (water; 5), TA low group (5), TA middle group (5), and TA high. Group (10) and positive control group (5).
  • vehicle control group water; 5
  • TA low group (5) TA middle group (5)
  • TA high TA high
  • Group (10) and positive control group (5) For water or test substances, single oral gavage is given within 24 hours; for positive control, single intraperitoneal injection is given within 24 hours. Five animals in each group were sacrificed 24 hours after administration; another 5 rats in the TA high group were sacrificed 48 hours after administration, before the experimental animals were sacrificed. Hourly intraperitoneal injection of colchicine (cold colchicine is now available, dose: 5mg/kg BW, dose: 10mL/kg BW).
  • chromosome breaks csb
  • chromosome exchange cse
  • chromatid breaks ctb
  • chromatid exchange cte
  • chromosome fragments frg
  • double centromere dic
  • no centromere ring r
  • centromere ring r
  • translocation t
  • fissure csf
  • pulverization pvz
  • chromosome number change indicators such as aneuploidy ( U), polyploid (pol), intranuclear replication (end) and other types of distortion and count.
  • Chromosome aberration rate (number of distorted cells / total number of observed cells) ⁇ 100%
  • miceroscopic examination showed that under the conditions of this experiment, the mitotic index of spermatogonial cells in each dose treatment group was not lower than 50% of the solvent control.
  • the statistical results of chromosome structural distortion and other index distortion of spermatogonia in each group are shown in the table. 10 and Table 11.
  • the test results showed that compared with the solvent control, the three doses set, namely 5g/kg BW, 2.5g/kg BW, 1.25g/kg BW, did not show the chromatid cleavage of the mouse spermatogonia in the test substance.
  • **p ⁇ 0.01 **p ⁇ 0.01, with a very statistically significant difference compared to the solvent control.
  • test results show that the test substance has no mutagenic or other genotoxic effects.
  • the black tea was fermented by the fermentation method of the genus Aspergillus strain 13368, and the dried tea product fermented by the strain was obtained and named as "13368DT"; Black tea raw material, named "UDT”.
  • Blank group normal diet + distilled water
  • HFD group 60kcal% high fat diet + distilled water
  • LT group 60 kcal% high fat diet + 75 mg / kg body weight 13368 DT;
  • MT group 60kcal% high fat diet + 250mg/kg body weight 13368DT;
  • HT group 60kcal% high fat diet + 750mg/kg body weight 13368DT;
  • MUT group 60 kcal% high fat diet + 250 mg/kg body weight UDT.
  • mice Three days before the end of the experiment, 6 groups of mice were fasted overnight, and then 1.5 g/kg of glucose was intraperitoneally injected, and blood glucose was measured at 0, 30, 60, and 120 minutes after the injection using tail vein blood.
  • the results showed that after ingestion of 13368DT (medium and high doses, MT and HT), the blood glucose tolerance test (IPGTT) blood glucose level, area under the blood glucose curve (AUC) and glycated hemoglobin were higher than the control group (HFD) and unfermented tea leaves ( UDT) was significantly reduced, demonstrating that 13368 fermentation significantly increased the ability of tea to control blood sugar.
  • IPGTT blood glucose tolerance test
  • AUC area under the blood glucose curve
  • UDT unfermented tea leaves
  • Liver samples from 6 groups of mice in part (a) were embedded in optimized section temperature compounds, frozen sections, fixed with 4% paraformaldehyde in PBS, and then stained with 3% oil red O. Liver TG and TC were analyzed using a commercial kit.
  • Black tea was fermented according to the fermentation method of part (d) of Example 2, using the strains of Stem cell sp. 13368, DTE-0086 and DTE-0104, except that the flowering time of step 5 was extended. The samples were taken at regular intervals, and the amount of CFU of the strain per gram of tea was determined, and 100 ⁇ 10 3 CFU/g tea leaves fermented by strain 13368 and hair flower loose tea products of 1000 ⁇ 10 3 CFU/g tea leaves were prepared, respectively. DTE-0086 or DTE-0104 fermented 1000 ⁇ 10 3 CFU/g tea leaf hair loose tea products. Another black tea and traditional brick tea (from the first tea factory in Anhua, Hunan) were taken.
  • the prepared 6 supernatant samples were assayed for activity activation of amylase and protease.
  • the activation rate of amylase is determined by reference to http://www.sigmaaldrich.com/china-mainland/zh/technical-documents/protocols/biology/enzymatic-assay-of-protea se-casein-as-a-substrate .html is performed by the method published by Sigma; the activity activation rate of protease is determined by Lee Wah Koh et al., Evaluation of different teas against The method described by starch digestibility by mammalian glycosidases, J Agric Food Chem. 2010 Jan 13; 58(1): 148-54. The results are shown in Table 12.
  • strain 13368 on amylase and protease activity is significantly higher than that of traditional brick tea, unfermented tea, and other strains of fermented tea, and increases with the increase of the number of strains, which has significant digestive effect.
  • the following fermentation process was carried out using black tea to prepare a pressed tea product which was internally embossed by the S. cerevisiae strain 13368.
  • the following fermentation process was carried out using green tea to prepare a pressed tea product which was internally embossed by the S. cerevisiae strain 13368.
  • the following fermentation process was carried out using black tea to prepare a pressed tea product which was germinated inside and on the surface of the strain S.
  • the tea leaves obtained in the step 3) are steamed at a steam pressure of 0.3 MPa and a temperature of 95 ° C for 30 seconds;
  • the gas permeable intermediate film layer in the composite film is a PE gas permeable film layer, and the pore density thereof is 6000 / square meter, its weight is 8 grams / square meter; hydrophilic inner membrane layer and hydrophobic outer membrane layer are hydrophilic non-woven fabric and hydrophobic non-woven fabric, respectively, the pore density is 6000 / Square meter, its weight is 20g / square meter, its material is PP imported polypropylene;
  • the tea bricks are taken out and allowed to bloom for 40 hours at a temperature of 32 ° C and a humidity of 20%, and then the temperature is adjusted to 38 ° C, and the flowering is continued. day;
  • the following fermentation process was carried out using white tea to prepare a pressed tea product which was flowered inside and on the surface of the strain S.
  • the tea is naturally fermented at a temperature of 60 ° C and a humidity of 70% for 72 hours, and stirred once every 24 hours;
  • the tea leaves obtained in the step 3) are steamed at a steam pressure of 0.5 MPa and a temperature of 110 ° C for 15 seconds;
  • the gas permeable intermediate film layer in the composite film is a PE gas permeable film layer having a pore density of 6000/m 2 and a gram weight of 8 g/m 2 ; the hydrophilic inner film layer and the hydrophobic outer film layer respectively.
  • the hydrophilic type non-woven fabric and the hydrophobic type non-woven fabric have a pore density of 6,000/m 2 and a basis weight of 20 g/m 2 , and the material is PP imported polypropylene;
  • the tea bricks are taken out and allowed to bloom for 50 hours at a temperature of 34 ° C and a humidity of 18%, and then the temperature is adjusted to 40 ° C, and the flowering is continued. Day
  • the following fermentation process was carried out using black tea ( ⁇ 10 mesh) to prepare a loose tea product which was produced by the strain S.
  • the tea is naturally fermented at a temperature of 60 ° C and a humidity of 70% for 72 hours, and stirred once every 24 hours;
  • the tea leaves are cooled to 20 ° C and the moisture is reduced to 20%, they are charged into a charging bottle, wherein the capacity of the charging bottle is 1000 ml / bottle, and the amount of the tea leaves therein is 2 / of the capacity.
  • the opening of the charging bottle is provided with two caps each having a venting hole, and a breathable water absorbing film layer is disposed between the two caps, which is a sponge layer of 5 mm;
  • the following fermentation process was carried out using black tea (less than 10 mesh) to prepare a loose tea product which was hair-stained by S. sphaeroides strain 13368.
  • the tea leaves are cooled to 30 ° C and the moisture is reduced to 15%, they are loaded into a charging bottle, wherein the capacity of the charging bottle is 2000 ml / bottle, and the amount of the tea leaves therein is 2 / 2 of the capacity.
  • the opening of the charging bottle is provided with two caps each having a vent hole, which is a 3 mm sponge layer;
  • the oolong tea (>10 mesh) was used as a raw material to carry out the fermentation according to the procedure of Example 9, and 100 parts of the broken tea product which was produced by the S. sphaeroides strain 13368 was prepared.
  • the white tea (>10 mesh) was used as a raw material to carry out the fermentation according to the procedure of Example 10, and 100 parts of the broken tea product which was produced by the S. sphaeroides strain 13368 was prepared.
  • Example 13 Pre-fermentation, preparation of fermented tea of Stenotrophomonas sp. 13368 strain
  • the pre-fermentation step is not carried out, that is, after the tea leaves are rehydrated, optionally cooling, and then steaming or bottling according to a specific process, 100 parts are also prepared. Corresponding tea products.
  • the total number of colonies of strain 13368 and the total number of colonies of other molds and bacteria in each of the fermented tea products of Examples 5-12 were calculated according to the total number of colonies of GB4789.2-94. Among them, the distinction between strain 13368 and other molds and bacteria is described in the present application regarding the characteristics of strain 13368. At the same time, the content of the tea brown pigment in each fermented tea product was examined.
  • the fermented tea product prepared in Comparative Example 13 without pre-fermentation was compared with each of the fermented tea products of Examples 5 to 12, and it was found that the natural fermentation process not only accelerated the subsequent flowering of the strain 13368, It can further improve the color, aroma, taste and health effects of the final tea product, and combine with the flowering effect of strain 13368 to obtain a tea with improved effect. product.
  • the specific performance is that the soup color is more red and transparent, the taste is more mellow, the number of the sclerotium is increased, and the product "bacteria" is more intense, and the regulation and promotion of blood sugar are more obvious. The results are shown in Table 13.

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Abstract

提供了一种冠突散囊菌菌株，其保藏编号为CGMCC No.13368。还提供了该冠突散囊菌菌株在茶叶发酵中的应用，以及利用该菌株发酵茶叶的方法。

Description

一种冠突散囊菌及其应用 技术领域
本发明属于微生物和食品生物技术领域，具体而言，本发明涉及冠突散囊菌的一种新株型及其在茶叶发酵过程中的应用。
背景技术
后发酵茶，即黑茶，属于中国传统六大茶类之一，其区别于其他茶叶的根本特征在于有微生物发酵参与茶叶的加工过程。根据原料和生产地区的不同，典型的黑茶包括湖南安化黑茶、云南普洱熟茶、广西六堡茶、湖北青砖茶等。各类黑茶的传统发酵过程又称渥堆发酵，是指将茶叶堆放在一定自然温湿度下一段时间，凭经验感知茶叶口感后，停止发酵。
冠突散囊菌是参与黑茶发酵的众多菌种之一，是最早于湖南安化黑茶茯砖茶中发现的一种真菌，因在砖茶内部形成金花色闭囊壳又称“金花菌”。安化黑茶茯砖茶特有的“发花”工序，就是促使冠突散囊菌大量生长，使得茶叶具有独特的香气、滋味和功效的过程。一般认为冠突散囊菌生长的质量和数量是评判安化黑茶茯砖茶品质的重要指标之一。研究结论认为，冠突散囊菌进一步增强了茯砖茶的健康功效(茯砖茶内具有茯茶素A、B等特有活性物质，这些物质首次在冠突散囊菌发酵的茯砖茶中被鉴定出来)，并促使茶叶产生了独特的“菌花香”。在安化黑茶茯砖茶国家标准中要求冠突散囊菌数量不低于200×103CFU/g茶，地方标准中这一要求提高到300×103CFU/g茶。
除去安化黑茶茯砖茶外，近年来发花工序已被引入其他茶，例如六堡茶、普洱茶、乌龙茶(大红袍)和白茶的发酵中，利用环境中的冠突散囊菌为菌种来源，在适宜温度(通常25-40℃)、湿度(60-90％)下进行发花(一般周期10-15天)，该工序与传统茯砖茶发花工序类似，均属于自然发酵。自然发酵的主要弊端为：随外界环境变化，当周围其他微生物含量高时，冠突散囊菌无法成为优势生物，导致发花失败，产品发生霉变；自然环境中带入参与发酵的微生物可产生真菌毒素，造成食品安全隐患；发酵微生物组成复杂，造成产品品质不可控，成分不稳定，功效感官品质无法得到保障；相应地，生产周期等不固定，造成产能浪费。上述问题限制了冠突散囊菌这一潜在优质微生物资源的广泛应用，也限制了以发酵茶为代表的传统发酵食品的推广和出口。因此，从传统作坊生产向标准化工业生产的产业升级迫在眉睫。
为解决上述问题，已有研究开始转向冠突散囊菌菌株的分离和应用，即将特定的冠突散囊菌发酵菌株引入茶叶的发酵加工中，以提高产品品质。但是，这种技术对分离纯化的冠突散囊菌提出了极高的要求，特别是目前对冠突散囊菌的科学研究较之其他已被广泛应用的发酵用菌还较少。如果试图应用到发酵中的冠突散囊菌菌株存在鉴定不准确、遗传性状不稳定、具有耐药性等问题，甚至自身有毒(例如产毒素或具有其他毒性)，反而会带来较传统发酵更严重的风险；如果应用的菌株生长速度慢，环境适应性差，则前期菌株培养会造成大量资源浪费，生产周期延长，生产成本增加；而如果菌株的代谢特征存在“缺陷”，就会导致发酵产品风味不佳或不具有健康功效，导致产品失败。
因此现阶段开发冠突散囊菌菌株及配套标准化发酵工艺，需要解决的核心技术问题包括：第一、重点关注食品安全，确保开发的菌株遗传背景清晰，无毒；第二、保证发酵菌株分泌代谢产物后能提高产品功效，让发酵茶具有稳定、明确的健康功效；第三、降低产品生产成本，提高生产效率，包括尽量降低菌株接种量，最大化缩短产品发酵周期等。
发明内容
针对上述问题，本发明的目的是提供一种冠突散囊菌的新菌株，相比于已有的冠突散囊菌，该新菌株能够在更短时间内以更低的接种量实现快速发酵效果，并且该新菌株更稳定、更安全，能够应用于广范围的茶叶种类，并且经其发酵得到的茶叶具有更多的健康功效。
本发明的又一个目的是，提供冠突散囊菌的新菌株在茶叶发酵中的应用。
本发明的另一个目的是，提供一种采用该冠突散囊菌新菌株的茶叶发酵方法以及由此发酵得到的茶叶。
本发明的目的是通过以下技术方案来实现的。
一方面，本发明提供一种冠突散囊菌(Eurotium cristatum)，该菌株已于2017年1月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏编号为CGMCC No.13368。
本发明提供的冠突散囊菌(Eurotium cristatum)CGMCC No.13368(下文简称为菌株13368，与菌株CGMCC No.13368可互换使用)从中茶湖南安 化第一茶厂木质茶叶存储仓库(百年
Figure PCTCN2017075759-appb-000001
)中储存的茶叶中分离、纯化和筛选获得。简言之，将不同年份的茶叶作为原始材料，从中分离、纯化出多个冠突散囊菌菌株之后，以生长速度、发花数量和毒素产生等为筛选标准，筛选获得菌株13368。与该过程中分离、筛选、鉴定的其他冠突散囊菌菌株相比以及与其他已知菌株相比，菌株13368具有生长速度快、单位时间发花得到的金花数量多、发酵培养基与发酵茶中均未检测到常见毒素等特征，并且经由菌株13368发酵的茶叶具有新且显著的健康功效。具体如下：
首先，经宏观形态、微观形态和基因测序分析，菌株13368为冠突散囊菌，属散囊菌目发菌科散囊菌属，排除了该菌株为散囊菌属其他种的可能。
第二，菌株13368具有之前公开报道中不具有的优势特征：
1.该菌株能够有效提高生产效率：当接种的目标微生物数量为0.5×103CFU/g茶时，5天发花得到的金花数量即可超过1000×103CFU/g茶(即接种量只有最终产品中含目标菌量的0.5‰)、茶褐素含量高于3％；
2.本发明首次对冠突散囊菌菌株进行了遗传稳定性、耐药性、毒力、产毒素和毒理学(急性毒性和遗传毒性)五项完整安全性评价，发现菌株13368是满足发酵要求的菌株中，唯一一个符合该五项全部安全性评价要求的菌株；
3.功效表征结果表明，菌株13368发酵的茶叶具有缓解脂肪肝、降低餐后血糖、促进蛋白脂肪消化、调节肝脂酶等糖脂代谢通路的活性等功效，该功效之前未见于公开报道的单独冠突散囊菌菌株发酵茶叶。
另一方面，本发明提供本发明的冠突散囊菌在茶叶发酵中的应用。
就茶叶形式而言，所述茶叶包括紧压茶、散茶和碎茶。所述紧压茶以任何形式的茶叶为起始原料，在发酵过程中进行压砖，然后以茶砖形式经历发花工序，包括传统的内部发花和本发明创新的表面与内部依次发花。所述“散茶”是指目数≤10目的条索茶，所述“碎茶”是指目数>10目的条索茶。在散茶和碎茶的发酵过程中，保持其起始形态，实现冠突散囊菌在茶叶中的发花。
就茶叶种类而言，所述茶叶包括绿茶、白茶、乌龙茶、黄茶、红茶及黑茶。
又一方面，本发明提供一种发酵茶叶的方法，所述方法包括采用本发明的冠突散囊菌对茶叶进行接种。所述方法可以针对绿茶、白茶、乌龙茶、黄茶、红茶及黑茶中任一种或多种进行，得到的茶叶产品可以是经发花的紧压茶、散茶或碎茶。
根据本发明的实施方式，所述方法包括向茶叶中加入本发明的冠突散囊菌菌株13368，压制后进行发花的步骤。
其中优选地，以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；
优选地，所述发花包括在温度20-40℃、相对湿度50-90％下发花4-10天。
根据本发明的具体实施方式，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、汽蒸、接种、压砖、发花、干燥。
优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
优选地，所述汽蒸包括将茶叶冷却至20℃-30℃、水分降至15％-20％后，在蒸汽压力0.1-0.5Mpa、温度105-115℃下汽蒸10-20秒；
优选地，所述接种包括以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；接种之前可选地将茶叶冷却至50℃以下；
优选地，所述压砖包括将茶叶压制成立方体或长方体形状的茶砖，所述茶砖为200-600g、优选400g；
优选地，所述发花包括在温度20-40℃、相对湿度50-90％下发花4-10天；
优选地，所述干燥包括在温度40-50℃下干燥2-4天，直至茶叶中的含水率低于12％。
上述发酵方法可以针对绿茶、白茶、乌龙茶、黄茶、红茶及黑茶中任一种或多种进行，以获得经内部发花的紧压茶。经检测可知，该紧压茶中每克茶的菌株数量不低于1000×103CFU/g茶，其他霉菌、细菌总数均小于2×103CFU/g茶，茶褐素含量不低于3％。基于此，本发明还提供由上述方法发酵得到的茶叶产品。
根据本发明的实施方式，所述方法包括向茶叶中加入本发明的冠突散囊菌菌株13368，压制后进行裹膜、然后两次发花的步骤。
其中优选地，以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；
优选地，所述裹膜包括在压制得到的茶砖表面裹上复合膜，所述复合膜自接触茶砖的一侧向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层；
优选地，两次发花包括首次发花与二次发花，其中首次发花包括在温度24-40℃、湿度50-90％下发花2-3天；二次发花包括在温度30-36℃、湿度18-32％下发花36-50小时，再将温度调整为36-45℃，继续发花2-4天。
根据本发明的具体实施方式，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、灭菌、汽蒸、接种、压砖、裹膜、首次发花、二次发花、干燥。
优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
优选地，所述灭菌包括将茶叶冷却至20℃-30℃、水分降至15％-20％后，在温度90-130℃下灭菌15-30min；
优选地，所述汽蒸包括在蒸汽压力0.3-0.5Mpa、温度95-110℃下汽蒸8-30秒；
优选地，所述接种包括以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；接种之前可选地将茶叶冷却至50℃以下；
优选地，所述压砖包括将茶叶压制成立方体或长方体形状的茶砖，所述茶砖优选为20-400g，更优选为50-200g。
优选地，所述裹膜包括在压制得到的茶砖表面裹上复合膜，由此形成内有茶砖的密封袋，其中所述复合膜自接触茶砖的一侧向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层，其中所述复合膜中的各个膜层具有小孔，所述小孔的孔密度为5000-7000个/平方米的膜层，复合膜的总克重为30～70克/平方米的复合膜，优选50克/平方米的复合膜；
更优选地，所述透气型中间膜层为PE透气膜层，其孔密度为6000个/平方米，其克重5～10g克/平方米，优选8克/平方米；所述亲水型内膜层和疏水型外膜层分别为亲水型无纺布和疏水型无纺布，其孔密度均优选为6000个/平方米，其克重为12.5～30g/平方米，优选20g/平方米，其材质例如采用PP材质的进口丙纶；
优选地，所述首次发花步骤包括在温度24-40℃、湿度50-90％下发花2-3天；
优选地，所述二次发花包括在检查到密封袋内的茶砖表面布满肉眼可见金花后，取出茶砖，使其在温度30-36℃、湿度18-32％下发花36-50小时，再将温度调整为36-45℃，继续发花2-4天；
优选地，所述干燥包括在温度40-50℃下干燥2-4天，直至茶叶中的含水率低于12％。
上述发酵方法可以针对绿茶、白茶、乌龙茶、黄茶、红茶及黑茶中任一种或多种进行，以获得内部和表面均发花的紧压茶。经检测可知，该紧压茶中每克茶的菌株数量不低于1000×103CFU/g茶，其他霉菌、细菌总数均小于2×103CFU/g茶，茶褐素含量不低于3％。基于此，本发明还提供由上述方法发酵得到的茶叶产品。
根据本发明的实施方式，所述方法包括先将茶叶装瓶，然后向茶叶中加入本发明的冠突散囊菌菌株13368进行发花的步骤，其中所述茶叶为散茶或碎茶。
其中优选地，以茶叶重量的0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；
优选地，所述发花包括在温度20-40℃、湿度30-75％下发花6-10天。
根据本发明的具体实施方式，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、装瓶、灭菌、接种、发花、干燥。
优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
优选地，所述装瓶包括将茶叶冷却至20℃-30℃、水分降至15％-20％后装入装料瓶中，其中所述装料瓶的容量为500-2000ml/瓶，茶叶在其内的装量为所述容量的1/4-3/4、优选2/3；优选地，所述装料瓶为倒喇叭状，开口处设置有各自具有通气孔的两个瓶盖，在所述两个瓶盖之间还设置有透气吸水膜层；优选地，所述透气吸水膜层为1-5mm的海绵层；
优选地，所述灭菌包括在温度80-125℃下灭菌5-35min；
优选地，所述接种包括先将经灭菌的茶叶冷却至30-80℃，然后以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入 冠突散囊菌菌株13368；
优选地，所述发花包括在温度20-40℃、相对湿度30-75％下发花6-10天；
优选地，所述干燥包括将茶叶从装料瓶中取出，然后在温度40-50℃下干燥，直至茶叶中的含水率为5-12％。
上述发酵方法可以针对绿茶、白茶、乌龙茶、黄茶、红茶及黑茶中任一种或多种进行，以获得发花的散茶或碎茶。经检测可知，该散茶或碎茶中每克茶的菌株数量不低于1000×103CFU/g茶，其他霉菌、细菌总数均小于2×103CFU/g茶，茶褐素含量不低于3％。基于此，本发明还提供由上述方法发酵得到的茶叶产品。
以下是本发明的详细说明。
一方面，本发明通过以生长速度、发花数量和毒素产生等为筛选标准，从茯砖茶产品中筛选出一株能有效发酵紧压茶甚至散茶与碎茶的冠突散囊菌新菌株。与已知冠突散囊菌相比，该新菌株具有以下优点：
第一，本发明的新菌株鉴定为冠突散囊菌，鉴定数据涵盖宏观形态、微观形态、电子显微镜鉴定和基因测序分析，是本领域首个得到充分鉴定与表征的冠突散囊菌。并且，菌株筛选与对比实验证明，相比于其他冠突散囊菌菌株，本发明的新菌株13368在培养至七代后，其形态学特征无明显变化，基因序列无变化，菌株性状可稳定遗传；菌本身对常见抗真菌药物更敏感，同时完全无毒素产生；而其余发酵性能相对优良的冠突散囊菌在培养七代后，在宏观形态、微观形态和基因测序分析方面各自具有一定变化。特别是就毒素产生，本发明首次对冠突散囊菌菌株进行了遗传稳定性、耐药性、产毒素、菌株培养物毒力和菌体毒理学(急性毒性和遗传毒性)五项完整安全性评价，发现菌株13368在发酵性能优良的菌株中，是唯一一个符合该五项完整安全性评价要求的菌株。本发明对总共150株散囊菌属菌株和冠突散囊菌菌株进行的各方面考察，还在一定程度上揭示了冠突散囊菌存在显著的种内差异(详见下文的实施例)。
此外，发酵及食品生产领域公知，本发明的冠突散囊菌新菌株这种稳定遗传、对环境药物敏感且不产生毒素的特征使其成为更适宜的茶叶发酵菌株，甚至是茶叶产品本身的组成成分之一。
第二，本发明的冠突散囊菌新菌株的发酵性能更好。已证明，本发明的新菌株13368甚至可以以低至目标微生物量0.05％的接种量(如终产品冠突 散囊菌含量为1000×103CFU/g，接种量为0.5×103CFU/g)，实现最短5天得到1000×103CFU/g茶叶的金花数量。相比之下，常规自然发酵的茶叶中金花数量无法在如此短期之内达到1000×103CFU/g，甚至需要在大接种量的情况下在8天以上才能有较好的发酵/发花效果，多数传统发酵产品无法达到1000×103CFU/g这一技术指标。因此，本发明的新菌株具有接种量降低，发酵周期缩短的优势，能够显著减少生产成本，提高生产周转率。
第三，本发明的冠突散囊菌新菌株具有明显的健康功效。相比于其他冠突散囊菌，经本发明的新菌株13368发酵的茶叶产品具有更高的有益成分，包括茶褐素、可溶性活性多糖组分。并且还已证明，经本发明的新菌株13368发酵，能够提高茶叶的血糖控制能力，还使其能辅助防治脂肪肝，同时在促淀粉与蛋白消化方面，还能提高淀粉酶、蛋白酶的活性激活率，因此能显著促消化。这些功效之前未见于公开报道的冠突散囊菌菌株发酵茶叶产品。
第四，本发明的冠突散囊菌新菌株的应用也更广泛。本领域已知冠突散囊菌可以参与黑茶发酵，且由于该菌的生长习性，仅能发酵紧压茶，发酵产生的金花仅存在于紧压茶的内部。本发明的新菌株13368可以对绿茶、白茶、乌龙茶、黄茶、红茶及黑茶进行发酵，并且茶叶形式不限于紧压茶，即使是碎茶与散茶也可有效发酵，不仅可以获得内部与表面均具有良好发酵/发花效果的紧压茶，也可获得良好发酵/发花效果的碎茶与散茶。由此极大地拓宽了茶叶来源与形式，使得发酵工艺更灵活方便，产品更多样化，适应不同市场需求。
另一方面，在本发明的冠突散囊菌新菌株的基础上，本发明还提供了各种新型发酵工艺。
首先，为了改进紧压茶发花效果，除了内部发花方法之外，本发明还通过引入裹膜操作，通过两次发花提供了一种紧压茶内外发花的方法。其中在裹膜操作中采用了包括亲水型内膜层、透气型中间膜层和疏水型外膜层的复合膜，且各个膜层设置有小孔。该内膜层与需要发酵的紧压茶接触，可以使与内膜层相接触的紧压茶具有很好的保水性能，同时中间膜层可以将发酵过程中产生的气体与外界的空气进行置换，外膜层可以将复合膜内产生的水汽排出。在用这种复合构造的膜包裹后，进行茶砖的两次发花步骤，首次发花使茶砖表面发花，二次发花使金花的生长从茶砖表面开始，逐渐延伸至内部，改变了传统紧压茶发花从内部开始生长的常规情况，最终获得内部、表面均有均匀金花分布的紧压茶。这种改进不仅获得了金花生长更为充分的茶叶产 品，而且还彻底解决了之前只有打开茶砖才能看到内部存在的冠突散囊菌金色闭囊壳的弊端。
其次，本发明还首创地提供了在散茶和碎茶上进行冠突散囊菌发花的方法。传统冠突散囊菌发酵茶仅在紧压茶内部具有冠突散囊菌发花，对于散茶和碎茶，难以实现有效的冠突散囊菌发花。与此相对的是，散茶和碎茶在实际应用中却具有携带便利、饮用方便的优势。为了能充分利用散茶和碎茶，拓宽茶叶来源与形式，使得产品更多样化，本发明通过在发酵过程中引入装瓶操作，即先将散茶或碎茶置于装料瓶中再进行发花，实现了散茶或碎茶安全、可控、标准同时高效的发花效果。在装瓶操作中，装料瓶的开口处设置有各自具有通气孔的两个瓶盖，在所述两个瓶盖之间还设置有透气吸水膜层，使得在发花过程中可以实现瓶体内外空气的置换，保证茶发酵的供氧量；并且透气吸水膜层可对外界进入瓶体的空气进行初步过滤，同时对聚集在瓶口的湿空气进行吸附，防止水蒸气冷凝回流入瓶中，使瓶口物料水分过高，同时隔离环境微生物，防止瓶口杂菌污染。
并且，对于紧压茶、散茶和碎茶，除了本发明菌株13368的发花操作之外，在本发明的发酵工艺中、在菌株13368接种之前，还包括了预发酵操作，即在不加入本发明菌株13368的情况下，先使茶叶在一定温度下自然发酵一段时间，使得茶叶及发酵环境中天然存在的曲霉、酵母等微生物有效发挥作用，从而为后续菌株13368的纯种发酵奠定更好的微环境与物质基础。实验发现，该自然发酵过程不仅能将加快菌株13368的后续发花，对提高最终茶叶产品的色、香、味以及健康功效也有贡献，从而与菌株13368的发花作用相结合，获得效果更为改善的茶叶产品。
总之，本发明提供的紧压茶的内部发花方法与表面内部发花方法以及散茶与碎茶的发花方法结合了冠突散囊菌新菌株13368的优良习性，以灵活方便的工艺步骤，以低至目标微生物量0.05％的接种量，实现了最短5天得到1000×103CFU/g茶叶的发花量，最终获得了各种形式的多样化茶叶产品，适应不同市场需求。这些产品具有良好发酵/发花效果，同时无毒素产生，特别是还具有降血糖、防治脂肪肝、促消化等明显的健康功效，显著优于市场上同类产品。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了冠突散囊菌菌株CGMCC No.13368的宏观形态；
图2中的图2A至2E分别示出了冠突散囊菌菌株CGMCC No.13368的宏观形态；
图3示出了冠突散囊菌菌株CGMCC No.13368与相关种的ITS rDNA序列系统发育树；
图4示出了冠突散囊菌菌株CGMCC No.13368与相关种的β-tubulin序列系统发育树；
图5示出了采用冠突散囊菌菌株CGMCC No.13368发酵后获得的茶叶在小鼠中的腹腔内葡萄糖耐受试验结果，其中图5A为血糖耐受试验结果(与对照组相比，P<0.05)，图5B为血糖曲线下面积结果(与对照组相比，P<0.01)，图5C为糖化血糖蛋白结果(与发酵前相比，P<0.05)。
图6示出了采用冠突散囊菌菌株CGMCC No.13368发酵后获得的茶叶在小鼠中的脂肪肝试验结果。
实施发明的最佳方式
根据下述实施例可以更好地理解本发明，然而实施例中所描述的具体的物料配比，工艺条件及结果仅用于说明本发明，而不应当对本发明进行限制。
以下各实施例中，培养基成分和培养条件如下：
PDA固体培养基：马铃薯浸粉5克、葡萄糖20克、琼脂20克、水1000毫升，pH 5.6；
PDA液体培养基：马铃薯浸粉5克、葡萄糖15克、蛋白胨10g，氯化钠5g，水1000毫升，pH 5.6；
固体种子的制备方法：取活化后的菌株划线接种至PDA固体培养基中，于25-30℃条件下，避光培养6-8天，并在无菌条件下，加水洗脱培养基，并收集洗脱液离心收集菌体，即得。
液体种子的制备方法：取活化后的菌株以无菌水洗脱制得孢子菌悬液，并接种至PDA液体培养基中，于25-30℃条件下震荡培养6-8天，收集种子液，即得。
CYA培养基：蔗糖30g，酵母膏5g，硝酸钠3g，氯化钾0.5g，硫酸镁0.01g，硫酸亚铁0.01g，硫酸铜0.01g，磷酸氢二钾1g，琼脂20g，蒸馏水1000毫升，pH 6.3。
麦芽浸粉肉汤培养基：麦芽浸膏30g，加蒸馏水至1000mL，pH 6.3。
实施例1冠突散囊菌菌株13368的获得、筛选与鉴定
(一)获得冠突散囊菌新菌株：
(1)选取茶样
选取湖南安化茶厂不同年份生产的包装完好、品质优秀的茶样40块(肉眼可见“金花”)，作为原始菌株分离的材料。
(2)预处理茶样
在无菌条件下，取不同茶样各25g，粉碎，过60-100目筛，然后将过筛的茶粉全部转移至225mL的无菌水中，充分震荡混合；
(3)涂布培养含菌茶样液体
取无菌水将上述步骤获得的混合液进行梯度稀释，取1mL稀释倍数为105的含菌液体涂布于PDA固体平板培养基中进行培养；
(4)培养并记录菌落生长情况
将涂布后的PDA固体平板培养基置于28-30℃的恒温培养箱中，培养7天，期间按照冠突散囊菌的形态学、生理生化特征对所述菌落的形态特征进行菌落识别并编号，每12h量取菌落的直径并记录；
(4)培养并记录菌落生长情况
将涂布后的PDA固体平板培养基置于28-30℃的恒温培养箱中，培养7天，期间按照冠突散囊菌的形态学、生理生化特征对所述菌落的形态特征进行菌落识别并编号，每12h量取菌落的直径并记录；
(5)挑取纯化单菌落
取上述步骤中生长最快的单菌落，挑取菌落并划线接种至所述PDA培养基上，于28-30℃下培养7天，传代培养两次，最终选取纯化培养过程中菌落直径增长最快的150株菌株。
根据形态学(微观和宏观)、生理生化特征等初步鉴定，发现这150株菌株均为散囊菌属，分别命名为DTE-0001至DTE-0150。
(二)筛选：
对于获得的150株菌株，就生长速度和金花数量进行以下筛选实验：
1)生长速度
取获得的150株菌株各自接种至茄形瓶中的PDA固体培养基上，于28℃条件下培养，培养8天后用100mL无菌水冲洗该固体培养基，然后将冲洗后获得的含有菌体的混合液经无菌纱布过滤获得孢子悬浮液，稀释后对孢子 悬浮液中的孢子进行计数。
结果表明，编号为DET-0022的菌株在培养的第2天可产生大量菌丝体，是150株菌株中生长最快的菌株之一。相比之下，其余多数菌株在第3-4天可以产生大量肉眼可见菌丝体。
用PDA液体培养基将各个菌株的孢子悬浮液调整成相同的孢子浓度1×106孢子/mL。
2)发花效果
将黑毛茶在蒸汽压力0.3Mpa下汽蒸15秒，然后冷却至50℃以下，加入该150株菌株的孢子悬浮液，初始接种量1×103CFU/g茶。然后按照每块含有上述得到的混合原料1kg的规格进行人工压砖，在温度27℃、相对湿度75％下发花。在发花进行到第5天时，用特制的取样刀具，取用茶砖中心内部的茶样25g，并按照GB 4789.2-94菌落总数测定，计算冠突散囊菌以及杂菌的菌落总数。
还将所述茶样在温度50℃下干燥4天，直至茶叶中的含水率低于12％。然后按照GB4789.2-94菌落总数测定，再次计算茶叶中冠突散囊菌菌落总数含量，并分析产品中是否存在霉变以及金花茂盛程度。
DET-0022与其他部分菌株的结果见表1。DET-0022与其他部分菌株的结果见表1。
表1部分散囊菌属菌株的发花效果比较
Figure PCTCN2017075759-appb-000002
Figure PCTCN2017075759-appb-000003
(表1中“-”表示未检测到霉变。)
发现有45株菌可以实现快速发花效果，即第五天金花肉眼可见。其中编号为DET-0022的菌株在发花迅速，在发花第5天使得茶样中“金花”菌菌落总数能够达到1000×103CFU/g茶叶以上，杂菌含量小于2×103CFU/g茶叶；干燥后，“金花”菌菌落总数能够达到1000×103CFU/g茶叶以上；发酵茶产品内部“金花”生长均匀、口感良好。
因此，上述两个方面的优势菌株均为编号DET-0022的菌株。为了与其他菌株相区分，将菌株DET-0022保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏单位地址为中国北京市朝阳区大屯路，中国科学院微生物研究所，其保藏编号为CGMCC NO.13368，保藏日期是2017年1月9日。
(三)冠突散囊菌菌株13368鉴定性质：
冠突散囊菌菌株13368鉴定如下：
宏观形态：在CYA培养基上，25℃培养7天，菌落直径27-30mm，中央棕褐色，周围黄色；中心凸起；质地丝绒状；反面中央深褐色，周围黄色；产生少量黑褐色渗出液，产生棕色可溶性色素。具体见图1。
微观形态：菌丝黄色具饰，缠绕，较细(图2A)；闭囊壳大量，黄色，存在于具饰菌丝网中，直径110-200μm(图2B)，成熟后破裂释放出大量子囊(图2C)；子囊球形或近球形，直径10-16μm，内含子囊孢子(图2D)；子囊孢子卵圆形或椭圆形，大小为4-5×3-4μm(图2E)；未见分生孢子结构。
DNA序列分析：
ITS rDNA基因：
以示于SEQ ID NO.1和SEQ ID NO.2的DNA序列分别作为正向和反向引物，以提取的菌株13368基因组DNA作为模板，扩增ITS rDNA基因序列，示于SEQ ID NO.3。将其ITS rDNA与多个相关种菌株的ITS rDNA进行序列比对，结果见下：
Figure PCTCN2017075759-appb-000004
采用MEGA5.0软件，邻位连接法绘制菌株13368与相关种的ITS rDNA序列系统发育树，进行1000次的相似度重复计算，结果见图3。
β-tubulin基因：
以示于SEQ ID NO.4和SEQ ID NO.5的DNA序列分别作为正向和反向引物，以提取的菌株13368基因组DNA作为模板，扩增β-tubulin基因序列，示于SEQ ID NO.6。将其β-tubulin与多个相关种菌株的β-tubulin进行序列比对，结果见下：
Figure PCTCN2017075759-appb-000005
采用MEGA5.0软件，邻位连接法绘制菌株13368与相关种的β-tubulin序列系统发育树，进行1000次的相似度重复计算，结果见图4。
本发明对菌株13368进行了宏观形态、微观形态和基因测序分析各方面充分鉴定，对于应用于发酵的冠突散囊菌来说尚属首次，是冠突散囊菌各方面特征的首次披露。与此相对，经过形态学和分子生物学鉴定，发现在45株能够实现快速发花的菌株中，有16株不是冠突散囊菌，而是散囊菌属其他种。这些种较冠突散囊菌安全性低，对其发酵特性研究少，不适宜茶叶发酵，并且有可能是部分茶叶产生毒素的原因。
实施例2菌株13368与其他冠突散囊菌菌株的比较
(一)遗传稳定性
将菌株13368与其他29株能够实现快速发花的冠突散囊菌菌株在PDA固体培养基上连续传代培养至7代，观察每一代的形态学特征，进行18SrDNA/ITS和β-tubulin基因序列测序，确认菌株是否能稳定遗传。
结果确定，菌株13368在培养至七代后，其形态学特征无明显变化，基因序列无变化，菌株性状可稳定遗传。特别是，比较菌株13368第一代与第七代的ITS rDNA基因序列，发现二者的同一性达到100％，在ITS rDNA系统发育树中处于完全相同的位置；而比较菌株13368第一代与第七代的β-tubulin基因序列，发现二者的同一性同样为100％，在β-tubulin系统发育树中也处于完全相同的位置。
相比之下，部分其他冠突散囊菌菌株几乎均无法实现上述遗传稳定性，在宏观形态、微观形态和基因测序分析方面各自具有一定变化。部分其他菌株与菌株13368的遗传稳定性结果见表2。
表2部分冠突散囊菌菌株的遗传稳定性比较
菌株编号 宏观形态 微观形态 18S rDNA/ITS β-tubulin
13368 无变化 无变化 无变化 无变化
DTE-0007 无变化 发生变化 无变化 无变化
DTE-0012 无变化 无变化 无变化 出现碱基突变
DTE-0019 无变化 无变化 无变化 无变化
DTE-0023 生长速度减缓 发生变化 出现碱基突变 无变化
DTE-0024 无变化 无变化 无变化 无变化
DTE-0030 无变化 发生变化 无变化 无变化
DTE-0036 生长速度减缓 发生变化 无变化 无变化
DTE-0039 无变化 发生变化 出现碱基突变 无变化
DTE-0045 生长速度减缓 发生变化 无变化 出现碱基突变
DTE-0051 无变化 无变化 无变化 无变化
DTE-0054 生长速度减缓 无变化 无变化 无变化
DTE-0055 无变化 无变化 无变化 无变化
DTE-0062 生长速度减缓 发生变化 无变化 无变化
DTE-0067 无变化 无变化 无变化 无变化
DTE-0075 生长速度减缓 无变化 出现碱基突变 出现碱基突变
DTE-0086 无变化 发生变化 无变化 无变化
DTE-0104 生长速度减缓 无变化 无变化 出现碱基突变
DTE-0111 生长速度减缓 发生变化 无变化 无变化
DTE-0141 生长速度减缓 发生变化 无变化 无变化
(二)抗真菌药物敏感实验
制备菌株13368与其他29株冠突散囊菌菌株在PDA液体培养基中的孢子悬液，取浓度为0.4-5×104CFU/mL的菌株孢子悬液0.1mL接种于含有2倍梯度稀释的6种抗真菌药物的PDA固体培养基平板上，28℃下培养7天，观察生长情况，读取最低抑菌浓度值。用标准菌株近平假丝酵母ATCC22019作为质控菌。部分结果见表3。
表3最低抑菌浓度(MIC)值(单位：μg/mL)
  伊曲康唑 酮康唑 伏立康唑 5-氟胞嘧啶 氟康唑 环吡酮胺
13368 2 4 4 64 128 4
DTE-0019 2 4 4 64 128  
DTE-0024 8 16 8 32 128  
DTE-0030 4 16 8 抗性 抗性  
DTE-0039 2 16 8 抗性 128 2
DTE-0051 2 抗性 4 64 抗性  
DTE-0055 2 16 8 64 抗性  
DTE-0067 4 4 4 32 抗性 4
DTE-0086 8 16 8 64 128 4
DTE-0104 4 16 8 64 128 2
结果发现，菌株13368对常见抗真菌药物均敏感，而其余部分冠突散囊菌菌株对某些抗真菌药物呈抗性。
(三)培养基中常见毒素检测
将菌株13368与其他29株冠突散囊菌菌株在CYA培养基中25℃培养10天，参考相应标准或采用商购试剂盒，检测培养物中是否存在常见毒素。部分结果见表4。
表4培养基中常见毒素检测
Figure PCTCN2017075759-appb-000006
(四)发酵茶中常见毒素检测
采用菌株13368与其他29株冠突散囊菌菌株，如下发酵作为散茶的黑 茶，制得经菌株发酵的散茶产品：
1)使用温度为70℃的水将茶叶复水，使茶叶的含水率为25％；
2)将步骤1)中复水的茶叶装入装料瓶中，所述装料瓶的开口处设置有各自具有通气孔的两个瓶盖，在所述两个瓶盖之间还设置有透气吸水膜层；
3)在温度100℃下灭菌30min；
4)经灭菌的茶叶冷却至50℃，然后以2×103CFU/g茶加入各冠突散囊菌菌株；
5)在温度30℃、相对湿度45％下发花6天；
6)将茶叶从装料瓶中取出，然后在温度45℃下干燥，直至茶叶中的含水率为10％。
获得20份散茶产品，参考相应标准，检测散茶产品中是否存在常见毒素。结果发现，部分冠突散囊菌菌株发酵的散茶产品中检出了一定量的伏马毒素和赫曲霉素A。部分结果见表5。
表5散茶产品中常见毒素检测
Figure PCTCN2017075759-appb-000007
Figure PCTCN2017075759-appb-000008
(五)发酵茶中有益成分表征
对于上文第(四)部分中获得的20份散茶产品，在对产品中冠突散囊菌进行计数后，检测散茶产品中茶褐素和可溶性活性多糖组分1、2的含量。其中茶褐素的检测方法参照Qiuping Wang等人，Effects of enzymatic action on the formation of theabrownin during solid state fermentation of Pu-erh tea，J Sci Food Agric 2011；91:2412-2418进行；多糖组分的检测方法参照Ping Xu等人，Physicochemical characterization of puerh tea polysaccharides and their antioxidant andα-glycosidase inhibition，J.of Functional Foods.,V.6,2014.1:545-554进行。
菌株13368与其余部分菌株的结果见表6。
表6散茶产品中菌数量和有益成分(以质量占茶叶干重的百分比计)
Figure PCTCN2017075759-appb-000009
茶褐素能够指示茶叶发酵程度，与降脂、降血糖、促消化和通便活性直接相关；可溶性活性多糖组分1、2与降血脂、降血糖、促消化活性相关。在所有散茶产品中，菌株13368发酵的茶叶产品中冠突散囊菌数量最多，茶 褐素和可溶性活性多糖组分1、2的含量也远远高于其他菌株发酵得到的散茶产品，由此可知，相比于其他冠突散囊菌菌株，菌株13368能更为显著地增强经发酵的茶叶的健康功效。
值得注意的是，实施例1和实施例2中对总共150株散囊菌属菌株和冠突散囊菌菌株进行的各方面考察，在一定程度上揭示了冠突散囊菌存在显著的种内差异。
实施例3冠突散囊菌菌株13368的动物毒性检验
(一)菌株培养物毒力检验
将冠突散囊菌菌株13368的菌落转接到麦芽浸粉肉汤培养基中，置于28℃左右，好氧培养14天。培养物经流动蒸汽1小时后，过滤，所得滤液部分通过冷冻干燥浓缩2.5倍供实验用，部分直接供实验用。
ICR小鼠80只，雌雄各半，分别随机分为4组，每组20只，设置为：培养基空白对照组，培养基2.5倍浓缩空白对照组，培养物原液组，培养物2.5倍浓缩组。将培养基、培养基2.5倍浓缩液、培养物原液、培养物2.5倍浓缩液分别以20.0mL/kgBW的剂量给小鼠一次性灌胃后，连续观察14天，记录实验过程中小鼠的体重变化，中毒表现及死亡情况。
使用SPSS18.0软件进行数据分析，培养物组与对照组定量资料的比较用两样本t检验。
结果表明，无论是雄性小鼠还是雌性小鼠，其初始体重和终体重在培养物组与其相应的对照组间比较，均无显著性差异(p>0.05)，即在实验期间冠突散囊菌菌株13368培养物对小鼠体重无影响。
同时，未观察到受试小鼠有毒性反应或死亡。
(二)菌粉的急性经口毒性试验
受试物为冠突散囊菌菌株13368菌粉(简称“TA”)，浓度为无菌水中0.25g/mL，试验采用24小时内三次经口灌胃，实际给药剂量为12.5g/kg BW。
选用20只健康小鼠，雌雄各半，体重18-22g，给受试物前隔夜禁食16小时(禁食、不限制饮水)，给药频次为前两次给药体积为20mL/kg BW，末次给药体积为10mL/kg BW。给药后0.5h、2h观察，其后，每天观察1次，记录动物的中毒表现、症状出现和消失的时间及死亡时间，观察期为14天。对死亡或濒死动物进行大体解剖检查。
的急性经口毒性耐受剂量大于12.5g/kg BW，其LD50大于12.5g/kg  BW。
表7急性毒性试验动物体重变化
Figure PCTCN2017075759-appb-000010
Figure PCTCN2017075759-appb-000011
(三)AMES试验
受试物为冠突散囊菌菌株13368菌粉(简称“TA”)，以初始菌粉悬液(浓度为0.25g/mL)高压灭菌后，无菌条件下，以无菌水稀释。共设置受试物的5个剂量组(实际暴露量为2.5、0.83、0.28、0.09、0.03mg/皿)；同时设置未处理对照组、溶剂(无菌水)对照组及阳性物对照组，其中非代谢激活条件下阳性对照品为叠氮钠(1.5μg/皿)、敌克松(50μg/皿)，代谢激活条件下阳性对照品为2-乙酰氨基芴(10μg/皿)、1,8-二羟基蒽醌(50μg/皿)、环磷酰胺(20μg/皿)。
采用平板掺入法：即45℃顶层琼脂2mL中依次加入鼠伤寒沙门氏菌的菌液0.1mL、受试物0.1mL、活化需加入S9混合液0.5mL，充分混匀后迅速倾入底层培养基上，37℃培养48小时(TA98培养72小时)后计数菌落。整个实验在相同条件下重复检测一次。
各剂量组平板背景菌苔未见异常；各菌株阳性对照处理均成立，未处理对照(自发回变)菌落数在正常范围，受试物各剂量组回变菌落数均未超过未处理对照组菌落数2倍，不同剂量组间无剂量-效应关系。在加和不加S9条件下，均未见受试物对鼠伤寒沙门氏菌TA97a、TA98、TA100、TA102、TA1535五菌株有致突变效应。表8为AMES试验第1次结果。
Figure PCTCN2017075759-appb-000012
(四)骨髓红细胞微核试验
受试物为冠突散囊菌菌株13368菌粉(简称“TA”)。阳性对照品均为环磷酰胺。
选用50只健康小鼠，体重25-35g，随机分为5组，雌雄各半，分别设置为溶媒对照组(水)、TA低组、TA中组、TA高组、阳性对照组。对于水或受试物，间隔24小时两次经口灌胃；对于阳性对照品，间隔24小时两次腹腔注射给药。第二次给予后6小时，脱颈处死动物，取股骨骨髓，计数每只动物200个红细胞中的嗜多染红细胞数(PCE)，计算其所占比例；每只动物观察计数2000个嗜多染红细胞中微核细胞数，微核细胞率以千分率计。
发生率与溶剂对照组相比，均无统计学差异(P>0.05)，提示该受试物对小鼠骨髓细胞无致微核作用。
表9受试物对小鼠骨髓红细胞微核细胞率的影响
Figure PCTCN2017075759-appb-000013
Figure PCTCN2017075759-appb-000014
**：与阴性对照组相比，P<0.01。
(五)小鼠精原细胞染色体畸变试验
受试物为冠突散囊菌菌株13368菌粉(简称“TA”)。阳性对照品均为环磷酰胺。
选用30只健康雄性小鼠，体重25-35g，随机分为5组，分别设置为溶媒对照组(水；5只)、TA低组(5只)、TA中组(5只)、TA高组(10只)、阳性对照组(5只)。对于水或受试物，采用24小时内单次经口灌胃 给予；对于阳性对照品，24小时内单次腹腔注射给药。给药后24小时处死各组动物5只；给药后48小时处死TA高组另5只，实验动物处死之前
Figure PCTCN2017075759-appb-000015
小时腹腔注射秋水仙素(秋水仙素现用现配，给药剂量：5mg/kg BW，给药体积：10mL/kg BW)。动物安乐死后，迅速取两侧睾丸，按照标准方法制作精原细胞Giemsa染色标本后，显微镜检，每只动物观察100个中期分裂相细胞(染色体数2n±2)，每个剂量组至少观察500个中期分裂相细胞用于染色体分析。显微镜下观察并分析染色体结构改变的指标，如染色体断裂(csb)、染色体交换(cse)、染色单体断裂(ctb)、染色单体交换(cte)、染色体碎片(frg)、双着丝点(dic)、无着丝点环(r)、着丝点环(r)、易位(t)、裂隙(csf)、粉碎化(pvz)等，染色体数目改变指标，如非整倍体(U)、多倍体(pol)、核内复制(end)等畸变类型并计数。同一细胞内的染色体有一种或多种畸变类型均记作一个畸变细胞。染色体裂隙、单价体应分别记录和报告，一般不作为畸变率统计。根据以下公式计算每个处理的畸变率：
染色体畸变率＝(畸变细胞的数量/观察细胞总数)×100％
镜检结果显示，本试验条件下，受试物各剂量处理组精原细胞有丝分裂指数均不低于溶剂对照的50％，各组精原细胞染色体结构畸变和其他指标畸变的统计结果详见表10和表11。试验结果显示，与溶剂对照相比，设置的三个剂量，即5g/kg BW、2.5g/kg BW、1.25g/kg BW，均未见受试物对小鼠精原细胞染色单体断裂(ctb)、染色体断裂(csb)、染色单体交换(cte)、染色体交换(cse)、染色体易位(t)、微小体(c)、着丝点异常(r，dic)等染色体结构畸变方面的指标有影响(表10)。对各组中细胞染色体或染色单体裂隙、多倍体的统计分析发现，与溶剂对照组相比，受试物各剂量组均无显著性(P>0.05)(表11)。以上结果提示该受试物对ICR小鼠无致精原细胞染色体畸变作用。
表10受试物对ICR小鼠精原细胞染色体畸变的影响
Figure PCTCN2017075759-appb-000016
Figure PCTCN2017075759-appb-000017
**P<0.01，与溶剂对照相比具有极显著的统计学差异。
表11受试物对ICR小鼠精原细胞染色体其他指标的影响
Figure PCTCN2017075759-appb-000018
**p<0.01，与溶剂对照相比具有极显著的统计学差异。
上述AMES试验、小鼠骨髓红细胞微核试验、小鼠精原细胞染色体畸变试验结果显示，该受试物无致突变或其他遗传毒性效应。
实施例4冠突散囊菌菌株13368发酵茶的功效表征
采用冠突散囊菌菌株13368，按照实施例2中第(四)部分的发酵方法，发酵黑茶，制得经菌株发酵的散茶产品，命名为“13368DT”；取未经所述发酵的黑茶原料，命名为“UDT”。
将60只6周龄的SD大鼠随机分成6组，经胃如下饲喂，总共喂养9周：
Blank组：正常饮食+蒸馏水；
HFD组：60kcal％高脂饮食+蒸馏水；
LT组：60kcal％高脂饮食+75mg/kg体重的13368DT；
MT组：60kcal％高脂饮食+250mg/kg体重的13368DT；
HT组：60kcal％高脂饮食+750mg/kg体重的13368DT；
MUT组：60kcal％高脂饮食+250mg/kg体重的UDT。
(一)腹腔内葡萄糖耐受试验
实验结束前3天，将6组小鼠禁食过夜，然后腹腔内注射1.5g/kg葡萄糖，使用尾静脉血液测定注射后0、30、60和120分钟的血糖。结果发现，摄入13368DT(中剂量及高剂量，MT和HT)后血糖耐受实验(IPGTT)血糖值、血糖曲线下面积(AUC)和糖化血红蛋白均较对照组(HFD)和未发酵茶叶(UDT)显著降低，证明13368发酵显著提高茶叶控制血糖能力。结果分别见图5中的5A至5C。
(二)脂肪肝试验
将来自第(一)部分中6组小鼠的肝样本包埋于优化的切片温度化合物中，冷冻切片，用PBS中的4％多聚甲醛固定，然后用3％油红O染色。用商购试剂盒分析肝TG和TC。
结果发现，在摄入13368发酵茶叶后(中剂量及高剂量，MT和HT)脂肪肝脏累积较对照组(HFD)和未发酵茶叶(UDT)显著降低，结果见图6。表明菌株13368发酵的茶叶具有辅助防治脂肪肝的功效。
(三)蛋白酶和淀粉酶活性试验
采用冠突散囊菌菌株13368、DTE-0086和DTE-0104，基本上按照实施例2中第(四)部分的发酵方法，发酵黑茶，不同之处在于延长步骤5的发花时间，期间按照一定时间间隔抽取样本，测定每克茶叶中菌株的CFU数量，分别制备经菌株13368发酵的100×103CFU/g茶叶和1000×103CFU/g茶叶的发花散茶产品，以及经DTE-0086或DTE-0104发酵的1000×103CFU/g茶叶的发花散茶产品。另取未经上述发酵的黑茶和传统茯砖茶(来自湖南安化第一茶厂)。对于总共6种茶叶样品，各自称取0.200g茶叶打碎后于50mL离心管中，加入30mL沸水，混匀后于沸水浴中浸提45min，每隔10min震荡一次，提取后在冰水浴中冷却至室温，3000rpm离心5min后取上清液适当稀释备用。
将制得的6种上清液样品进行淀粉酶和蛋白酶的活性激活率测定。其中淀粉酶的活性激活率测定参考网址http://www.sigmaaldrich.com/china-mainland/zh/technical-documents/protocols/biology/enzymatic-assay-of-protea se-casein-as-a-substrate.html由Sigma公司公布的方法进行；蛋白酶的活性激活率测定参考Lee Wah Koh等人，Evaluation of different teas against  starch digestibility by mammalian glycosidases,J Agric Food Chem.2010Jan13；58(1):148-54页描述的方法进行。结果见表12。
表12蛋白酶和淀粉酶活性试验
Figure PCTCN2017075759-appb-000019
上述实验证明菌株13368发酵茶叶对淀粉酶、蛋白酶活性促进作用显著高于传统茯砖茶、未发酵茶、和其他菌株发酵茶，且随着菌株数量增加而增加，具有显著促消化功效。
实施例5冠突散囊菌菌株13368发酵茶的制备
采用黑茶进行下述发酵过程，制备经冠突散囊菌菌株13368内部发花的紧压茶产品。
1)使用温度为70℃的水将茶叶复水，使茶叶的含水率为45％；
2)将茶叶在温度40℃、湿度70％下自然发酵48小时，每24h翻动搅拌一次；
3)将茶叶冷却至30℃、水分降至20％后，在蒸汽压力0.3Mpa、温度110℃下汽蒸20秒；
4)将茶叶冷却至50℃以下，然后以1×103CFU/g茶加入冠突散囊菌菌株13368；
5)将茶叶压制成长方体形状的400g茶砖；
6)在温度28℃、相对湿度70％下发花5天；
7)在温度45℃下干燥2天，直至茶叶中的含水率低于12％。
按照上述过程制备100份紧压茶产品。
实施例6冠突散囊菌菌株13368发酵茶的制备
采用绿茶进行下述发酵过程，制备经冠突散囊菌菌株13368内部发花的紧压茶产品。
1)包括使用温度为80℃的水将茶叶复水，使茶叶的含水率为25％；
2)将茶叶在温度60℃、湿度50％下自然发酵72小时，每24h翻动搅拌一次；
3)茶叶冷却至30℃、水分降至15％，在蒸汽压力0.5Mpa、温度105℃下汽蒸15秒；
4)将茶叶冷却至50℃以下，然后以1×103CFU/g茶加入冠突散囊菌菌株13368；
5)将茶叶压制成正方体形状的400g茶砖；
6)在温度30℃、相对湿度80％下发花5天；
7)在温度40℃下干燥4天，直至茶叶中的含水率低于12％。
按照上述过程制备100份紧压茶产品。
实施例7冠突散囊菌菌株13368发酵茶的制备
采用黑茶进行下述发酵过程，制备经冠突散囊菌菌株13368内部和表面均发花的紧压茶产品。
1)使用温度为70℃的水将茶叶复水，使茶叶的含水率为25％；
2)将茶叶在温度50℃、湿度60％下自然发酵60小时，每24h翻动搅拌一次；
3)将茶叶冷却至30℃、水分降至15％后，在温度120℃下灭菌30min；
4)将步骤3)得到的茶叶在蒸汽压力0.3Mpa、温度95℃下汽蒸30秒；
5)将茶叶冷却至50℃以下，然后以10×103CFU/g茶加入冠突散囊菌菌株13368；
6)将茶叶压制成正方体形状的200g茶砖；
7)在压制得到的茶叶表面裹上复合膜，由此形成内有茶砖的密封袋，其中所述复合膜自接触茶叶的表面向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层；
其中所述复合膜中的透气型中间膜层为PE透气膜层，其孔密度为 6000个/平方米，其克重8克/平方米；亲水型内膜层和疏水型外膜层分别为亲水型无纺布和疏水型无纺布，其孔密度均为6000个/平方米，其克重为20g/平方米，其材质为PP材质的进口丙纶；
8)在温度28℃、湿度60％下发花2天；
9)在检查到密封发酵袋内茶叶表面布满肉眼可见金花后，取出茶砖，使其在温度32℃、湿度20％下发花40小时，再将温度调整为38℃，继续发花3天；
10)在温度45℃下干燥3天，直至茶叶中的含水率低于12％。
按照上述过程制备100份紧压茶产品。
实施例8冠突散囊菌菌株13368发酵茶的制备
采用白茶进行下述发酵过程，制备经冠突散囊菌菌株13368内部和表面均发花的紧压茶产品。
1)使用温度为80℃的水将茶叶复水，使茶叶的含水率为45％；
2)将茶叶在温度60℃、湿度70％下自然发酵72小时，每24h翻动搅拌一次；
3)将茶叶冷却至20℃、水分降至20％后，在温度130℃下灭菌30min；
4)将步骤3)得到的茶叶在蒸汽压力0.5Mpa、温度110℃下汽蒸15秒；
5)将茶叶冷却至50℃以下，然后以5×103CFU/g茶加入冠突散囊菌菌株13368；
6)将茶叶压制成长方体形状的200g茶砖；
7)在压制得到的茶叶表面裹上复合膜，由此形成内有茶叶的密封袋，其中所述复合膜自接触茶叶的表面向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层；
其中所述复合膜中的透气型中间膜层为PE透气膜层，其孔密度为6000个/平方米，其克重8克/平方米；亲水型内膜层和疏水型外膜层分别为亲水型无纺布和疏水型无纺布，其孔密度均为6000个/平方米，其克重为20g/平方米，其材质为PP材质的进口丙纶；
8)在温度38℃、湿度65％下发花1.5天；
9)在检查到密封发酵袋内茶叶表面布满肉眼可见金花后，取出茶砖，使其在温度34℃、湿度18％下发花50小时，再将温度调整为40℃，继续发花3天；
10)在温度50℃下干燥2天，直至茶叶中的含水率低于12％。
按照上述过程制备100份紧压茶产品。
实施例9冠突散囊菌菌株13368发酵茶的制备
采用黑茶(≤10目)进行下述发酵过程，制备经冠突散囊菌菌株13368发花的散茶产品。
1)使用温度为80℃的水将茶叶复水，使茶叶的含水率为45％；
2)将茶叶在温度60℃、湿度70％下自然发酵72小时，每24h翻动搅拌一次；
3)将茶叶冷却至20℃、水分降至20％后，装入装料瓶中，其中所述装料瓶的容量为1000ml/瓶，茶叶在其内的装量为所述容量的2/3，装料瓶的开口处设置有各自具有通气孔的两个瓶盖，在所述两个瓶盖之间还设置有透气吸水膜层，为5mm的海绵层；
4)在温度121℃下灭菌30min；
5)经灭菌的茶叶冷却至50℃，然后以1×103CFU/g茶加入冠突散囊菌菌株13368；
6)在温度25℃、相对湿度45％下发花6天；
7)将茶叶从装料瓶中取出，然后在温度40℃下干燥，直至茶叶中的含水率为10％。
按照上述过程制备100份散茶产品。
实施例10冠突散囊菌菌株13368发酵茶的制备
采用红茶(小于10目)进行下述发酵过程，制备经冠突散囊菌菌株13368发花的散茶产品。
1)使用温度为70℃的水将茶叶复水，使茶叶的含水率为25％；
2)将茶叶在温度50℃、湿度60％下自然发酵60小时，每24h翻动搅拌一次；
3)将茶叶冷却至30℃、水分降至15％后，装入装料瓶中，其中所述装料瓶的容量为2000ml/瓶，茶叶在其内的装量为所述容量的2/3，装料瓶的开口处设置有各自具有通气孔的两个瓶盖，为3mm的海绵层；
4)在温度100℃下灭菌30min；
5)经灭菌的茶叶冷却至50℃，然后以1×103CFU/g茶加入冠突散囊菌 菌株13368；
6)在温度30℃、相对湿度40％下发花6天；
7)将茶叶从装料瓶中取出，然后在温度35℃下干燥，直至茶叶中的含水率为8％。
按照上述过程制备100份散茶产品。
实施例11冠突散囊菌菌株13368发酵茶的制备
采用乌龙茶(>10目)为原料按照实施例9的过程进行发酵，制备经冠突散囊菌菌株13368发花的100份碎茶产品。
实施例12冠突散囊菌菌株13368发酵茶的制备
采用白茶(>10目)为原料按照实施例10的过程进行发酵，制备经冠突散囊菌菌株13368发花的100份碎茶产品。
实施例13不进行预发酵，冠突散囊菌菌株13368发酵茶的制备
对于实施例5-12中的发酵过程，不进行其中的预发酵步骤，即在茶叶复水后可选地进行冷却，然后根据具体过程进行之后的汽蒸或装瓶等操作，同样制备100份相应的茶叶产品。
实施例14发酵茶产品的检测和对比
按照GB4789.2-94菌落总数测定，计算实施例5-12中的各发酵茶产品中菌株13368菌落总数以及其他霉菌、细菌的菌落总数。其中，菌株13368与其他霉菌、细菌的区分参见本申请中关于菌株13368特征的描述。同时检测各发酵茶产品中的茶褐素含量。
结果发现，在实施例5-12中的各发酵茶产品中，每克茶的菌株数量均不低于1000×103CFU/g茶，其他霉菌、细菌总数均小于2×103CFU/g茶，茶褐素含量均不低于3％，其中以黑茶为原料(实施例5-10)制备得到的茶叶茶褐素含量不低于7％。
此外，比较实施例13中不进行预发酵而制备的发酵茶产品，与实施例5-12中的各发酵茶产品进行比较，发现：该自然发酵过程不仅能将加快菌株13368的后续发花，还能进一步改善最终茶叶产品的色、香、味以及健康功效，从而与菌株13368的发花作用相结合，获得效果更为改善的茶叶 产品。具体表现为汤色更为红透明亮，味道更为醇厚，冠突散囊菌数量提升且产品“菌花”香更为浓重，对血糖的调节和促消化作用更为明显。结果见表13。
表13有无预发酵的产品效果比较
Figure PCTCN2017075759-appb-000020
Figure PCTCN2017075759-appb-000021

Claims (11)

  1. 一种冠突散囊菌(Eurotium cristatum)，其保藏编号为CGMCC No.13368。
  2. 权利要求1所述的冠突散囊菌在茶叶发酵中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述茶叶的形式包括紧压茶、散茶和碎茶；
    优选地，所述茶叶的种类包括绿茶、白茶、乌龙茶、黄茶、红茶及黑茶。
  4. 一种发酵茶叶的方法，其特征在于，所述方法包括采用权利要求1所述的冠突散囊菌对茶叶进行接种。
  5. 根据权利要求4所述的方法，其特征在于，所述方法包括向茶叶中加入权利要求1所述的冠突散囊菌，压制后进行发花的步骤；
    其中优选地，以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入所述冠突散囊菌菌株；
    优选地，所述发花包括在温度20-40℃、相对湿度50-90％下发花4-10天。
  6. 根据权利要求4或5所述的方法，其特征在于，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、汽蒸、接种、压砖、发花、干燥；
    优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
    优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
    优选地，所述汽蒸包括将茶叶冷却至20℃-30℃、水分降至15％-20％后，在蒸汽压力0.1-0.5Mpa、温度105-115℃下汽蒸10-20秒；
    优选地，所述接种包括以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入所述冠突散囊菌菌株；接种之前可选地将茶叶冷却至50℃以下；
    优选地，所述压砖包括将茶叶压制成立方体或长方体形状的茶砖，所 述茶砖为200-600g、优选400g；
    优选地，所述发花包括在温度20-40℃、相对湿度50-90％下发花4-10天；
    优选地，所述干燥包括在温度40-50℃下干燥2-4天，直至茶叶中的含水率低于12％。
  7. 根据权利要求4所述的方法，其特征在于，所述方法包括向茶叶中加入权利要求1所述的冠突散囊菌，压制后进行裹膜、然后两次发花的步骤；
    其中优选地，以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入所述冠突散囊菌菌株；
    优选地，所述裹膜包括在压制得到的茶砖表面裹上复合膜，所述复合膜自接触茶砖的一侧向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层；
    优选地，两次发花包括首次发花与二次发花，其中首次发花包括在温度24-40℃、湿度50-90％下发花2-3天；二次发花包括在温度30-36℃、湿度18-32％下发花36-50小时，再将温度调整为36-45℃，继续发花2-4天。
  8. 根据权利要求4或7所述的方法，其特征在于，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、灭菌、汽蒸、接种、压砖、裹膜、首次发花、二次发花、干燥；
    优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
    优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
    优选地，所述灭菌包括将茶叶冷却至20℃-30℃、水分降至15％-20％后，在温度90-130℃下灭菌15-30min；
    优选地，所述汽蒸包括在蒸汽压力0.3-0.5Mpa、温度95-110℃下汽蒸8-30秒；
    优选地，所述接种包括以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；接种之前可选地将茶叶冷却至50℃以下；
    优选地，所述压砖包括将茶叶压制成立方体或长方体形状的茶砖，所述茶砖优选为20-400g，更优选为50-200g；
    优选地，所述裹膜包括在压制得到的茶砖表面裹上复合膜，由此形成内有茶砖的密封袋，其中所述复合膜自接触茶砖的一侧向外依次包括亲水型内膜层、透气型中间膜层和疏水型外膜层，其中所述复合膜中的各个膜层具有小孔，所述小孔的孔密度为5000-7000个/平方米的膜层，复合膜的总克重为30～70克/平方米的复合膜，优选50克/平方米的复合膜；
    更优选地，所述透气型中间膜层为PE透气膜层，其孔密度为6000个/平方米，其克重5～10g克/平方米，优选8克/平方米；所述亲水型内膜层和疏水型外膜层分别为亲水型无纺布和疏水型无纺布，其孔密度均优选为6000个/平方米，其克重为12.5～30g/平方米，优选20g/平方米，其材质例如采用PP材质的进口丙纶；
    优选地，所述首次发花步骤包括在温度24-40℃、湿度50-90％下发花2-3天；
    优选地，所述二次发花包括在检查到密封袋内的茶砖表面布满肉眼可见金花后，取出茶砖，使其在温度30-36℃、湿度18-32％下发花36-50小时，再将温度调整为36-45℃，继续发花2-4天；
    优选地，所述干燥包括在温度40-50℃下干燥2-4天，直至茶叶中的含水率低于12％。
  9. 根据权利要求4所述的方法，其特征在于，所述方法包括向茶叶中加入权利要求1所述的冠突散囊菌进行发花的步骤，其中所述茶叶为散茶或碎茶；
    其中优选地，以茶叶重量的0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；
    优选地，所述发花包括在温度20-40℃、湿度30-75％下发花6-10天。
  10. 根据权利要求4或9所述的方法，其特征在于，所述方法包括以下步骤：将茶叶依次进行复水、预发酵、装瓶、灭菌、接种、发花、干燥；
    优选地，所述复水包括使用温度为70-80℃的水将茶叶复水，使茶叶的含水率为25％-45％；
    优选地，所述预发酵包括将茶叶在温度40-60℃、湿度50％-70％下自然发酵48-72小时，每18-30h翻动搅拌一次；
    优选地，所述装瓶包括将茶叶冷却至20℃-30℃、水分降至15％-20％后装入装料瓶中，其中所述装料瓶的容量为500-2000ml/瓶，茶叶在其内的装量为所述容量的1/4-3/4、优选2/3；优选地，所述装料瓶为倒喇叭状，开口处设置有各自具有通气孔的两个瓶盖，在所述两个瓶盖之间还设置有透气吸水膜层；优选地，所述透气吸水膜层为1-5mm的海绵层；
    优选地，所述灭菌包括在温度80-125℃下灭菌5-35min；
    优选地，所述接种包括先将经灭菌的茶叶冷却至30-80℃，然后以0.5-10×103CFU/g茶、优选0.5-2×103CFU/g茶、更优选1×103CFU/g茶加入冠突散囊菌菌株13368；
    优选地，所述发花包括在温度20-40℃、相对湿度30-75％下发花6-10天；
    优选地，所述干燥包括将茶叶从装料瓶中取出，然后在温度40-50℃下干燥，直至茶叶中的含水率为5-12％。
  11. 通过权利要求1至10中任一项所述的方法发酵得到的茶叶产品。
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CN110629630A (zh) * 2019-09-23 2019-12-31 台晨茜 一种海绵城市建设用路面透水砖
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CN114304600A (zh) * 2022-01-12 2022-04-12 中华全国供销合作总社济南果品研究院 一种冠突散囊菌发酵小米粉的制备方法
CN114766547A (zh) * 2022-04-15 2022-07-22 贵州大学 一种重悬液延缓百香果腐败的方法及其专用菌
CN117801967A (zh) * 2024-03-01 2024-04-02 烟台市供销社茶业有限公司 一种培养冠突散囊菌的方法及应用

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CN110178919A (zh) * 2019-05-08 2019-08-30 西咸新区茯茶镇茶业有限公司 一种银针茶叶散茶发花工艺
CN110591925A (zh) * 2019-09-06 2019-12-20 青岛中科星熠高新技术研究院有限公司 一种冠突散囊菌生产发酵方法
CN110629630A (zh) * 2019-09-23 2019-12-31 台晨茜 一种海绵城市建设用路面透水砖
CN111235038A (zh) * 2020-02-28 2020-06-05 深圳市清生元生物技术股份有限公司 一种冠突散囊菌株及其发酵液提取物的制备方法
CN112602795A (zh) * 2021-01-11 2021-04-06 湖南平嘉鑫茶业有限公司 一种利用茶树鲜叶添加冠突散囊菌制备金花黑茶的方法
CN113331274A (zh) * 2021-06-07 2021-09-03 陕西科技大学 一种基于自然发花的散茯茶及其制备方法
CN113331274B (zh) * 2021-06-07 2022-11-25 通化唐亦舒食品有限公司 一种基于自然发花的散茯茶及其制备方法
CN113249233A (zh) * 2021-06-29 2021-08-13 北京百奥茵诺生物科技有限公司 冠突散囊菌和菌剂及它们的应用
CN113249233B (zh) * 2021-06-29 2021-11-09 北京百奥茵诺生物科技有限公司 冠突散囊菌和菌剂及它们的应用
CN114304600A (zh) * 2022-01-12 2022-04-12 中华全国供销合作总社济南果品研究院 一种冠突散囊菌发酵小米粉的制备方法
CN114304600B (zh) * 2022-01-12 2023-06-30 中华全国供销合作总社济南果品研究院 一种冠突散囊菌发酵小米粉的制备方法
CN114766547A (zh) * 2022-04-15 2022-07-22 贵州大学 一种重悬液延缓百香果腐败的方法及其专用菌
CN114766547B (zh) * 2022-04-15 2023-05-12 贵州大学 一种重悬液延缓百香果腐败的方法及其专用菌
CN117801967A (zh) * 2024-03-01 2024-04-02 烟台市供销社茶业有限公司 一种培养冠突散囊菌的方法及应用
CN117801967B (zh) * 2024-03-01 2024-04-30 烟台市供销社茶业有限公司 一种培养冠突散囊菌的方法及应用

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