Accurate fermentation method of Fuzhuan tea

Abstract

The invention provides a precise fermentation method of Fuzhuan tea. Comprising the following steps: S1, raw material pretreatment: 1) screening and blending; 2) steaming and pile fermentation: the steaming temperature is 85-95 DEG C, and the duration time is 15-30 s; 3) adding the tea juice until the water content of the tea base is 11-12%; 4) adding a compound fermentation carrier accounting for 1-2% of the weight of the tea base, and mechanically stirring uniformly; (5) feeding into a brick press for pressing; (6) cooling and shaping, wherein the cooling time is 120-150 min; s2, three-stage fermentation: 1) germinating at the temperature of 27.5-28.5 DEG C and the humidity of 83-87% RH in the first to third days; the method comprises the following steps of (1) adding a composite fermentation carrier into a culture medium, (2) carrying out proliferation at the temperature of 29.5-30.5 DEG C and the humidity of 89-91% RH on the 4th-8th day, (3) entering a stable period at the temperature of 31.5-32.5 DEG C and the humidity of 78-82% RH on the 9th-12th day, and (S3) recovering and regenerating the carrier. According to the method, an intelligent controlled release system (an environment response type antibacterial microsphere and composite fermentation carrier-microsphere synergistic release mechanism) is adopted, so that the fermentation period is shortened to 10-12 days, and the efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Fuzhuan tea process, and particularly relates to a precise fermentation method of Fuzhuan tea. BACKGROUND

[0002] As an important category of dark tea, Fuzhuan tea mainly relies on the action of Eurotium cristatum in the fermentation process to form Eurotium cristatum in the tea base and promote the transformation of tea contents. However, the traditional fermentation of Fuzhuan tea relies on microbial communities in the natural environment, which has problems such as long fermentation period, unstable quality, high risk of contamination by miscellaneous bacteria and the like. The existing technology attempts to optimize fermentation by artificially inoculating pure culture strains, but direct use of mycelium is easy to be damaged by high temperature in processing, and simple spore inoculation also faces challenges such as asynchronous germination and insufficient metabolic activity. In addition, the traditional process lacks precise regulation of the growth environment of microorganisms, resulting in low generation efficiency of key flavor substances such as theaflavins and tea polysaccharides, which restricts the standardized production and quality improvement of Fuzhuan tea.

[0003] In recent years, although there are studies on immobilization of microorganisms using porous materials, ordinary carriers cannot meet multiple requirements such as spore protection, mycelium expansion, nutrient slow release and miscellaneous bacteria inhibition. Especially in the production environment with large fluctuations in temperature and humidity, the existing technology is difficult to achieve rapid establishment and maintenance of the dominant position of the microbial community. Therefore, developing a fermentation technology that can synergistically regulate the morphological conversion of microorganisms and dynamically release functional components has become a key problem that needs to be broken through in the Fuzhuan tea industry. SUMMARY

[0004] The technical problem to be solved: In view of the above technical problems, the purpose of the present application is to provide a precise fermentation method of Fuzhuan tea, which shortens the fermentation period to 10-12 days by using an intelligent controlled-release system (environment-responsive antibacterial microspheres and a composite fermentation carrier-microspheres synergistic release mechanism), greatly improving the efficiency. The environment-responsive antibacterial microspheres are tea polyphenol-chitosan core-shell microspheres, wherein the core layer: 0.1-0.2% tea polyphenol (mainly EGCG) provides antibacterial activity; the shell layer: sodium alginate / chitosan composite film (pH / humidity double response). The 20-50 μm large pores of the multi-level pore carrier fix the microspheres, and the 5-10 nm mesopores adjust the release kinetics. When the humidity is greater than 85%, the sodium alginate shell absorbs water and swells, the pore size expands, and the tea polyphenol is released slowly (initial antibacterial, release rate 0.8 mg / h); when the number of miscellaneous bacteria increases, the pH rises due to metabolism, and when the pH is greater than 6.0, the chitosan dissolves in weak alkaline conditions, the shell layer collapses, and the high-concentration tea polyphenol is quickly released to inhibit bacteria; when the temperature is 30℃, the difference in thermal expansion coefficient of the mesopores of the carrier causes the “nano valve” effect, and the fucose is released through the mesopores to increase the supply of carbon source.

[0005] Technical scheme: A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: raw material pretreatment 1) screening and blending: blend clean black tea with tea stems in a certain proportion; 2) steaming and pile fermentation: steaming temperature is 85-95℃, and duration is 15-30 s; 3) add tea juice, and the moisture content of tea base is 11-12%; 4) add 1-2% of composite fermentation carrier according to the weight of tea base, and mechanically mix evenly; 5) send to the brick press for pressing; 6) cooling and shaping: cooling time is 120-150 min; S2: three-stage fermentation 1) in the first 3 days, germination is carried out at a temperature of 27.5-28.5℃ and a humidity of 83-87% RH (spore germination, slow release of microspheres); 2) in the fourth to eighth days, proliferation is carried out at a temperature of 29.5-30.5℃ and a humidity of 89-91% RH (fucose promotes growth, pH triggers controlled release); 3) in the ninth to twelfth days, enter the stable period at a temperature of 31.5-32.5℃ and a humidity of 78-82% RH (metabolite conversion, carrier recovery); S3: carrier recovery and regeneration 1) after fermentation, adsorb the composite fermentation carrier by the permanent magnet roller (magnetic field strength 0.5T); 2) carrier regeneration: remove residual bacteria by ultrasonic cleaning (40kHz, 10min); 3) after drying at 80℃, reload spores and fucose.

[0006] Further, the mass ratio of the black tea to the tea stems is (8:2) - (9:1).

[0007] Further, the tea juice is a water solution of black tea extracted by hot water at 80-90℃, and the solid content is 5-8%.

[0008] Further, the preparation method of the composite fermentation carrier is: S11: mix food-grade mesoporous silica and Fe3O4 nanoparticles at a mass ratio of 9:1, add 3% amino silane coupling agent, and react at 80℃ for 6h to obtain amino-modified mixed particles; S12: uniformly mix PMMA microspheres and amino-modified mixed particles at a mass ratio of 1:(5-7), press into shape, calcine at 550℃ for 4h to remove the template, form 20-50μm through macropores, and obtain a multi-level pore carrier; S13: immerse the hierarchical porous carrier in a dopamine-Tris buffer solution, oscillate at 22-25 DEG C for 24h to obtain a polydopamine-coated hierarchical porous carrier, and then immerse the carrier in a Eurotium cristatum spore suspension, and adsorb the spores under a vacuum negative pressure of -0.08 MPa for 30-35 min to obtain a hierarchical porous carrier loaded with Eurotium cristatum spores; S14: fill the antibacterial microspheres into the macropores of the hierarchical porous carrier loaded with Eurotium cristatum spores by vibration screening, immerse the carrier in a 4-5% fucose solution for 50-60 min, and vacuum dry the carrier at 60 DEG C until a constant weight is obtained.

[0009] Further, the food-grade mesoporous silica has a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g.

[0010] Further, the Fe3O4 nanoparticles have a particle size of 5-8 nm.

[0011] Further, the PMMA microspheres have a particle size of 30-35 μm.

[0012] Further, the dopamine-Tris buffer solution has a pH of 8.5 and a concentration of 2 mg / mL.

[0013] Further, the content of Eurotium cristatum spores in the Eurotium cristatum spore suspension in S13 is 1.0 x 10 8 -3.0 x 10 8 CFU / mL.

[0014] Further, the antibacterial microspheres are prepared by the following method in S14: Step 1: mix tea polyphenols, chitosan and a dilute acid solution to obtain a solution containing 0.1-0.2% tea polyphenols and 0.5% chitosan; Step 2: drop the solution into a 2% CaCl2 solution, solidify at a stirring rate of 500 rpm for 30 min, and filter and screen to obtain antibacterial microspheres with a particle size of 20-50 μm.

[0015] Further, the pressing pressure is 8-10 MPa.

[0016] The above method is used to prepare the brick tea. Advantages

[0017] 1、The application shortens the fermentation period to 10-12 days by adopting an intelligent controlled release system (environmentally responsive antibacterial microspheres and composite fermentation carrier-microsphere synergistic release mechanism), greatly improving efficiency. The environmentally responsive antibacterial microspheres are tea polyphenol-chitosan core-shell microspheres, wherein the core layer: 0.1-0.2% tea polyphenol (mainly EGCG) provides antibacterial activity; the shell layer: sodium alginate / chitosan composite film (pH / humidity double response). The 20-50 μm large pores of the multi-level pore carrier fix the microspheres, and the 5-10 nm mesopores adjust the release kinetics. When the humidity is >85%, the sodium alginate shell swells by absorbing water, the pore size expands, and the tea polyphenol is released slowly (initial antibacterial activity, release rate 0.8 mg / h); when the number of miscellaneous bacteria increases, the pH rises due to metabolism, and when the pH is >6.0, the chitosan dissolves in weak alkaline conditions, the shell layer collapses, and high-concentration tea polyphenol is rapidly released to inhibit bacteria; at a temperature of 30℃, the difference in the thermal expansion coefficient of the mesopores of the carrier causes a "nano valve" effect, and the fucose is accelerated to release through the mesopores, and the amount of carbon source supply is increased.

[0018] 2、The application realizes efficient colonization of Eurotium cristatum and inhibition of miscellaneous bacteria by designing a composite fermentation carrier. The multi-level pore carrier (mesopore 5-10 nm+macropore 20-50 μm) is used to load Eurotium cristatum spores, antibacterial microspheres and fucose, and the spore adhesion is enhanced by a polydopamine coating. Spore directional colonization: the macropore structure of the carrier provides a space for microbial growth, and the mesopore serves as a microenvironment regulation unit for Eurotium cristatum spores, which optimizes the growth conditions of the microbial community by adsorbing and slowly releasing metabolites; dynamic inhibition of miscellaneous bacteria: the tea polyphenol-chitosan microspheres release through humidity / pH double response, and the mold detection rate is reduced to 0.4-2.0%; precise nutrition supply: fucose release promotes the exclusive growth of Eurotium cristatum, ensuring the stable generation of core flavor substances of brick tea.

[0019] 3、The application optimizes microbial metabolism and flavor substance generation through a three-stage fermentation process, and regulates temperature and humidity in stages (germination period 28℃ / 85%RH→proliferation period 30℃ / 90%RH→stable period 32℃ / 80%RH); the first stage: efficient spore germination: a low-temperature and high-humidity environment (1-3 days) increases the spore germination rate to more than 95% (the constant temperature of 30℃ is only 78.9%); the second stage: directional conversion of tea polyphenol: the middle temperature triggers the release of antibacterial components from the microspheres, and promotes enzymatic oxidation at the same time, increasing the content of theaflavins; the third stage: efficient recovery of the carrier: the end of the period is characterized by stable product recovery, and the recovery rate of the carrier is >80%.

[0020] 4、The intelligent controlled release system and the three-stage fermentation form a space-time coupling in the application: the first stage (1-3 days) of slow-release of the microspheres inhibits miscellaneous bacteria, creating an advantageous environment for spore germination; the second stage (4-8 days) of rapid release triggered by pH responds to the peak of miscellaneous bacteria competition; the third stage (9-12 days) of continuous release of metabolic substrates through mesopores stabilizes the quality.

[0021] 5、The present application guarantees the process safety and the carrier combination strength through the steaming-pressing synergistic pretreatment, softens the tea base at 90℃ for 15-30s, and forms the dense brick body through pressing at 8-10MPa. The steaming reduces the mold load of the raw material, achieves the initial inactivation of miscellaneous bacteria, and the carrier-tea base is tightly combined after pressing.

[0022] 6、The mesopore of the multi-level pore carrier in the present application can also load the tea polyphenol degradation product, enhance the synergistic effect of antibiosis, and limit the too fast diffusion of the tea polyphenol, so that the tea polyphenol and chitosan form the gradient release in the microspheres.

[0023] 7、The present application significantly reduces the production cost through the carrier recycling regeneration technology. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The picture of the dispersed Fuzhuan tea prepared in Example 16; Figure 2 The microscope picture of the mature ascocarps in the Fuzhuan tea prepared in Example 16; Figure 3 The microscope picture of the ascocarps and mycelium in the Fuzhuan tea prepared in Example 16. DETAILED DESCRIPTION

[0025] The present application proposes a precise fermentation method of Fuzhuan tea. In order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application will be further described in detail below in cooperation with the examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. Example 1

[0026] The preparation method of the antibacterial microspheres is as follows: Step 1: mix tea polyphenol, chitosan and 1% acetic acid solution to obtain a solution with a tea polyphenol content of 0.1% and a chitosan content of 0.5%; Step 2: drop into 2% CaCl2 solution, solidify at a stirring rate of 500rpm for 30min, filter and screen to obtain antibacterial microspheres with a particle size of 20-50μm. Example 2

[0027] The preparation method of the antibacterial microspheres is as follows: Step 1: mix tea polyphenol, chitosan and 1% acetic acid solution to obtain a solution with a tea polyphenol content of 0.2% and a chitosan content of 0.5%; Step 2: drop into 2% CaCl2 solution, solidify at a stirring rate of 500rpm for 30min, filter and screen to obtain antibacterial microspheres with a particle size of 20-50μm.

[0028] Table 1

[0029] The antibacterial microspheres prepared in Example 2 were selected for subsequent experiments. Example 3

[0030] The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm were mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane was added, and the mixture was reacted at 80°C for 6 h to obtain aminated mixed particles; S12: PMMA microspheres with a particle size of 30-35 μm were uniformly mixed with the aminated mixed particles at a mass ratio of 1:5, and then compression molded and calcined at 550°C for 4 h to remove the template and form 20-50 μm through macropores to obtain a hierarchical pore carrier; S13: The hierarchical pore carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and oscillated at 25°C for 24 h to obtain a polydopamine-coated hierarchical pore carrier, which was then immersed in a sporangium suspension of Eurotium cristatum with a content of 2.0 x 10 8 CFU / mL, and adsorbed for 33 min under vacuum negative pressure of-0.08 MPa to obtain a hierarchical pore carrier loaded with spores of Eurotium cristatum; S14: The antibacterial microspheres were filled into the macropores of the hierarchical pore carrier loaded with spores of Eurotium cristatum by vibration screening, and then immersed in a 5% fucose solution for 55 min and dried at 60°C to constant weight. Example 4

[0031] The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm were mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane was added, and the mixture was reacted at 80°C for 6 h to obtain aminated mixed particles; S12: PMMA microspheres with a particle size of 30-35 μm were uniformly mixed with the aminated mixed particles at a mass ratio of 1:6, and then compression molded and calcined at 550°C for 4 h to remove the template and form 20-50 μm through macropores to obtain a hierarchical pore carrier; S13: The hierarchical pore carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and oscillated at 25°C for 24 h to obtain a polydopamine-coated hierarchical pore carrier, which was then immersed in a sporangium suspension of Eurotium cristatum with a content of 2.0 x 10 8A multi-level pore carrier loaded with Eurotium cristatum spores is obtained by adsorbing CFU / mL of Eurotium cristatum spore suspension under a vacuum negative pressure of -0.08 MPa for 33 min; S14: Fill the antibacterial microspheres into the macropores of the multi-level pore carrier loaded with Eurotium cristatum spores by vibration screening, immerse in a 5% fucose solution for 55 min, and vacuum dry at 60°C to constant weight. Example 5

[0032] The preparation method of the composite fermentation carrier is as follows: S11: Take food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm are mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane is added, and the reaction is carried out at 80°C for 6 h to obtain amin-modified mixed particles; S12: Mix PMMA microspheres with a particle size of 30-35 μm and amin-modified mixed particles uniformly at a mass ratio of 1:7, press into shape, and calcine at 550°C for 4 h to remove the template to form 20-50 μm through macropores, thereby obtaining a multi-level pore carrier; S13: Immerse the multi-level pore carrier in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, oscillate at 25°C for 24 h to obtain a polydopamine-coated multi-level pore carrier, and then immerse it in a solution containing 2.0×10 8 A multi-level pore carrier loaded with Eurotium cristatum spores is obtained by adsorbing CFU / mL of Eurotium cristatum spore suspension under a vacuum negative pressure of -0.08 MPa for 33 min; S14: Fill the antibacterial microspheres into the macropores of the multi-level pore carrier loaded with Eurotium cristatum spores by vibration screening, immerse in a 5% fucose solution for 55 min, and vacuum dry at 60°C to constant weight. Example 6

[0033] The preparation method of the composite fermentation carrier is as follows: S11: Take food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm are mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane is added, and the reaction is carried out at 80°C for 6 h to obtain amin-modified mixed particles; S12: Mix PMMA microspheres with a particle size of 30-35 μm and amin-modified mixed particles uniformly at a mass ratio of 1:6, press into shape, and calcine at 550°C for 4 h to remove the template to form 20-50 μm through macropores, thereby obtaining a multi-level pore carrier; S13: the multi-level porous carrier is immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, oscillated at 25°C for 24 hours, and a polydopamine-coated multi-level porous carrier is obtained; after being taken out, the multi-level porous carrier is immersed in a fucose solution with a content of 1.0 x 10 8 CFU / mL of Eurotium cristatum spore suspension, adsorbed for 33 min under a vacuum negative pressure of -0.08 MPa, to obtain a multi-level porous carrier loaded with Eurotium cristatum spores; S14: the antibacterial microspheres are filled into the macropores of the multi-level porous carrier loaded with Eurotium cristatum spores through vibration screening, immersed in a 5% fucose solution for 55 min, and dried at 60°C to a constant weight, and the composite fermentation carrier is obtained. Example 7

[0034] The preparation method of the composite fermentation carrier is as follows: S11: food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm are mixed at a mass ratio of 9:1, 3% γ-aminopropyltriethoxysilane is added, and reaction is carried out at 80°C for 6 h to obtain amin-modified mixed particles; S12: PMMA microspheres with a particle size of 30-35 μm and amin-modified mixed particles are uniformly mixed at a mass ratio of 1:6, compression molded, and calcined at 550°C for 4 h to remove the template to form 20-50 μm through macropores, and a multi-level porous carrier is obtained; S13: the multi-level porous carrier is immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, oscillated at 25°C for 24 hours, and a polydopamine-coated multi-level porous carrier is obtained; after being taken out, the multi-level porous carrier is immersed in a fucose solution with a content of 3.0 x 10 8 CFU / mL of Eurotium cristatum spore suspension, adsorbed for 33 min under a vacuum negative pressure of -0.08 MPa, to obtain a multi-level porous carrier loaded with Eurotium cristatum spores; S14: the antibacterial microspheres are filled into the macropores of the multi-level porous carrier loaded with Eurotium cristatum spores through vibration screening, immersed in a 5% fucose solution for 55 min, and dried at 60°C to a constant weight, and the composite fermentation carrier is obtained. Example 8

[0035] The preparation method of the composite fermentation carrier is as follows: S11: food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g and Fe3O4 nanoparticles with a particle size of 5-8 nm are mixed at a mass ratio of 9:1, 3% γ-aminopropyltriethoxysilane is added, and reaction is carried out at 80°C for 6 h to obtain amin-modified mixed particles; S12: The PMMA microspheres with a particle size of 30-35 pm were mixed with the amino-modified mixed particles at a mass ratio of 1:6, uniformly mixed, and compression molded, and then calcined at 550°C for 4h to remove the template to form 20-50 pm through macropores, thereby obtaining a hierarchical porous carrier; S13: The hierarchical porous carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and oscillated at 25°C for 24h to obtain a polydopamine-coated hierarchical porous carrier. After being taken out, the hierarchical porous carrier was immersed in a solution containing 2.0 x 10 8 CFU / mL of the Eurotium cristatum spore suspension, and adsorbed under a vacuum negative pressure of -0.08 MPa for 30 min to obtain a hierarchical porous carrier loaded with Eurotium cristatum spores; S14: The antibacterial microspheres were filled into the macropores of the hierarchical porous carrier loaded with Eurotium cristatum spores by vibration screening, and then immersed in a 5% fucose solution for 55 min and vacuum dried at 60°C to constant weight. Example 9

[0036] The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g of Fe3O4 nanoparticles with a particle size of 5-8 nm were mixed at a mass ratio of 9:1, 3% γ-aminopropyltriethoxysilane was added, and the mixture was reacted at 80°C for 6h to obtain amino-modified mixed particles; S12: The PMMA microspheres with a particle size of 30-35 pm were mixed with the amino-modified mixed particles at a mass ratio of 1:6, uniformly mixed, and compression molded, and then calcined at 550°C for 4h to remove the template to form 20-50 pm through macropores, thereby obtaining a hierarchical porous carrier; S13: The hierarchical porous carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and oscillated at 25°C for 24h to obtain a polydopamine-coated hierarchical porous carrier. After being taken out, the hierarchical porous carrier was immersed in a solution containing 2.0 x 10 8 CFU / mL of the Eurotium cristatum spore suspension, and adsorbed under a vacuum negative pressure of -0.08 MPa for 30 min to obtain a hierarchical porous carrier loaded with Eurotium cristatum spores; S14: The antibacterial microspheres were filled into the macropores of the hierarchical porous carrier loaded with Eurotium cristatum spores by vibration screening, and then immersed in a 5% fucose solution for 55 min and vacuum dried at 60°C to constant weight. Example 10

[0037] The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2food-grade mesoporous silica with a particle size of 5-8 nm and Fe3O4 nanoparticles with a particle size of 5-8 nm were mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane was added, and reaction was carried out at 80°C for 6h to obtain amin-modified mixed particles; S12: PMMA microspheres with a particle size of 30-35 μm were mixed with amin-modified mixed particles at a mass ratio of 1:6, and were uniformly mixed and pressed into a shape, and were calcined at 550°C for 4h to remove the template to form 20-50 μm through macropores, and a hierarchical pore carrier was obtained; S13: The hierarchical pore carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and was oscillated at 25°C for 24h to obtain a polydopamine-coated hierarchical pore carrier, and after being taken out, was immersed in a solution containing 2.0×10 8 CFU / mL of Eurotium cristatum spore suspension, adsorbed under vacuum negative pressure-0.08 MPa for 33 min to obtain a hierarchical pore carrier loaded with Eurotium cristatum spores; S14: The antibacterial microspheres were filled into the macropores of the hierarchical pore carrier loaded with Eurotium cristatum spores by vibration screening, and were immersed in a 4% fucose solution for 55 min, and were vacuum dried at 60°C to constant weight. Comparative Example 1

[0038] The difference between this comparative example and Example 4 is that PMMA microspheres are not added, and the specific process is as follows: The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g of food-grade mesoporous silica and Fe3O4 nanoparticles with a particle size of 5-8 nm were mixed at a mass ratio of 9:1, 3% γ-aminopropyl triethoxysilane was added, and reaction was carried out at 80°C for 6h to obtain amin-modified mixed particles; S12: The mixed particles were pressed into a shape, and were calcined at 550°C for 4h to obtain a carrier; S13: The hierarchical pore carrier was immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2 mg / mL, and was oscillated at 25°C for 24h to obtain a polydopamine-coated hierarchical pore carrier, and after being taken out, was immersed in a solution containing 2.0×10 8 CFU / mL of Eurotium cristatum spore suspension, adsorbed under vacuum negative pressure-0.08 MPa for 33 min to obtain a hierarchical pore carrier loaded with Eurotium cristatum spores; S14: Immersed in a 5% fucose solution for 55 min, and vacuum dried at 60°C to constant weight. Comparative Example 2

[0039] The difference between this comparative example and Example 4 is that there is no polydopamine coating, and the specific process is as follows: The preparation method of the composite fermentation carrier is as follows: S11: Take food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g, 3% γ-aminopropyl triethoxysilane is added, and reaction is carried out at 80°C for 6h to obtain aminated mixed particles; S12: The PMMA microspheres with a particle size of 30-35μm are uniformly mixed with the aminated mixed particles at a mass ratio of 1:6, and are pressed and formed, and are calcined at 550°C for 4h to remove the template, to form 20-50μm through macropores, and a multi-level pore carrier is obtained; S13: The multi-level pore carrier is immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2mg / mL, and is oscillated at 25°C for 24h to obtain a multi-level pore carrier coated with polydopamine, and then is immersed in a Eurotium cristatum spore suspension with a content of 2.0×10 8 CFU / mL, and is adsorbed under a vacuum negative pressure of-0.08MPa for 33min to obtain a multi-level pore carrier loaded with Eurotium cristatum spores; S14: The antibacterial microspheres are filled into the macropores of the multi-level pore carrier loaded with Eurotium cristatum spores through vibration screening, and are immersed in a 5% fucose solution for 55min, and are vacuum dried at 60°C until constant weight. Comparative Example 3

[0040] The difference between this comparative example and Example 4 is that there is no Fe3O4 nanoparticles, and the specific process is as follows: The preparation method of the composite fermentation carrier is as follows: S11: Take food-grade mesoporous silica with a pore size of 5-10 nm and an average specific surface area of 807 m 2 / g, 3% γ-aminopropyl triethoxysilane is added, and reaction is carried out at 80°C for 6h to obtain aminated mixed particles; S12: The PMMA microspheres with a particle size of 30-35μm are uniformly mixed with the aminated mixed particles at a mass ratio of 1:6, and are pressed and formed, and are calcined at 550°C for 4h to remove the template, to form 20-50μm through macropores, and a multi-level pore carrier is obtained; S13: The multi-level pore carrier is immersed in a dopamine-Tris buffer solution with a pH of 8.5 and a concentration of 2mg / mL, and is oscillated at 25°C for 24h to obtain a multi-level pore carrier coated with polydopamine, and then is immersed in a Eurotium cristatum spore suspension with a content of 2.0×10 8 CFU / mL, and is adsorbed under a vacuum negative pressure of-0.08MPa for 33min to obtain a multi-level pore carrier loaded with Eurotium cristatum spores; S14: The antibacterial microspheres were filled into the macropores of the multi-level porous carrier loaded with Eurotium cristatum spores by vibration screening, immersed in a 5% fucose solution for 55 min, and vacuum dried at 60°C to constant weight.

[0041] Performance test: Specific surface area: determined according to the BET nitrogen adsorption method (GB / T 19587-2017); Spore load: determined according to the plate count method (GB 4789.15-2016); Fucose load: determined according to the sulfuric acid-phenol method (GB 5009.8-2016); Microsphere filling rate: determined according to SEM image analysis (ISO 21363:2020); Magnetic response: determined according to the vibrating sample magnetometer (VSM) test; The results are shown in Table 2 below: Table 2

[0042] The specific surface area reflects the degree of development of the carrier pore structure, and the higher the value, the stronger the adsorption capacity. As can be seen from the above table, the specific surface area of the examples is slightly lower than that of Comparative Example 1 (805 m 2 / g) due to the introduction of macroporous structure, but it is more suitable for multifunctional requirements. The spore load directly reflects the ability of the carrier to immobilize Eurotium cristatum spores, and the polydopamine coating (Examples 3-10) significantly increases the load compared to Comparative Example 2 without coating. Each example can stably load fucose, indicating that the adsorption capacity of the carrier mesoporous structure to sugars is consistent. After comprehensive consideration, the performance of the composite fermentation carrier of Example 7 is better, which is used for subsequent tests. Example 11

[0043] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: raw material pretreatment 1) screening and blending: clean black tea and tea stems are blended at a mass ratio of 8:2; 2) steaming and heap fermentation: the steaming temperature is 90°C, and the duration is 25 s; 3) add tea juice, the moisture content of the tea base is 12%; the tea juice is the aqueous solution of black tea extracted by hot water at 90°C, and the solid content is 7%; 4) add 1.5% of the composite fermentation carrier prepared in Example 7 based on the weight of the tea base, and mechanically mix evenly; 5) send to the brick press for pressing, the pressing pressure is 9 MPa; 6) cooling and shaping: the cooling time is 150 min; S2: three-stage fermentation 1) Germination was carried out on days 1-3 at a temperature of 28℃ and a humidity of 85%RH; 2) Propagation was carried out on days 4-8 at a temperature of 30℃ and a humidity of 90%RH; 3) The system enters a stable period on days 9-12 at a temperature of 32℃ and a humidity of 80%RH; S3: Carrier Recycling and Regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: Ultrasonic cleaning (40kHz, 10min) removes residual bacteria; 3) After drying at 80℃, the spores and fucose are reloaded. Example 12

[0044] A precise fermentation method for Fu brick tea includes the following steps: S1: Raw material pretreatment 1) Screening and blending: The clean black tea leaves and tea stems are blended at a mass ratio of 9:1; 2) Steam fermentation: The steam temperature is 90℃, and the duration is 25 seconds; 3) Add tea juice until the moisture content of the tea leaves is 12%; the tea juice is an aqueous solution of black tea extracted with 90℃ hot water, with a solid content of 7%; 4) Add the composite fermentation carrier prepared in Example 7 at 1.5% of the weight of the tea leaves, and mechanically mix evenly; 5) The bricks are fed into a brick press for pressing at a pressure of 9 MPa. 6) Cooling and shaping: Cooling time is 150 min; S2: Three-stage fermentation 1) Germination was carried out on days 1-3 at a temperature of 28℃ and a humidity of 85%RH; 2) Propagation was carried out on days 4-8 at a temperature of 30℃ and a humidity of 90%RH; 3) The system enters a stable period on days 9-12 at a temperature of 32℃ and a humidity of 80%RH; S3: Carrier Recycling and Regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: Ultrasonic cleaning (40kHz, 10min) removes residual bacteria; 3) After drying at 80℃, the spores and fucose are reloaded. Example 13

[0045] A precise fermentation method for Fu brick tea includes the following steps: S1: Raw material pretreatment 1) Sieving and blending: clean black tea and tea stem were blended at a mass ratio of 8:2; 2) Steaming and pile fermentation: steaming temperature was 90°C, and the duration was 15 s; 3) Adding tea juice to the tea base to a moisture content of 12%; the tea juice was an aqueous solution of black tea extracted by hot water at 90°C, and the solid content was 7%; 4) Adding 1.5% of the composite fermentation carrier prepared in Example 7 by weight of the tea base, and mechanically stirring evenly; 5) Sending into a brick press for pressing, and the pressing pressure was 9 MPa; 6) Cooling and setting: the cooling time was 150 min; S2: Three-stage fermentation 1) Germination at 28°C and 85% RH for 1-3 days; 2) Proliferation at 30°C and 90% RH for 4-8 days; 3) Entering the stable period at 32°C and 80% RH for 9-12 days; S3: Carrier recovery and regeneration 1) After the fermentation was completed, the composite fermentation carrier was adsorbed by a permanent magnet roller (magnetic field strength 0.5 T); 2) Carrier regeneration: removing residual bacterial bodies by ultrasonic cleaning (40 kHz, 10 min); 3) After drying at 80°C, spores and fucose were reloaded. Example 14

[0046] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: Raw material pretreatment 1) Sieving and blending: clean black tea and tea stem were blended at a mass ratio of 8:2; 2) Steaming and pile fermentation: steaming temperature was 90°C, and the duration was 30 s; 3) Adding tea juice to the tea base to a moisture content of 12%; the tea juice was an aqueous solution of black tea extracted by hot water at 90°C, and the solid content was 7%; 4) Adding 1.5% of the composite fermentation carrier prepared in Example 7 by weight of the tea base, and mechanically stirring evenly; 5) Sending into a brick press for pressing, and the pressing pressure was 9 MPa; 6) Cooling and setting: the cooling time was 150 min; S2: Three-stage fermentation 1) Germination at 28°C and 85% RH for 1-3 days; 2) Proliferation at 30°C and 90% RH for 4-8 days; 3) On the 9th-12th day, enter the stable period at 32℃ and 80% RH; S3: Carrier recovery and regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: ultrasonic cleaning (40kHz, 10min) to remove residual bacteria; 3) After drying at 80℃, reload spores and fucose. Example 15

[0047] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: Raw material pretreatment 1) Sieving and blending: blend clean black tea and tea stems at a mass ratio of 8:2; 2) Steaming and pile fermentation: steaming temperature is 90℃, and the duration is 25s; 3) Add tea juice to the tea base with a moisture content of 12%; the tea juice is a water solution of black tea extracted by hot water at 90℃, with a solid content of 7%; 4) Add 1.0% of the composite fermentation carrier prepared in Example 7 by weight of the tea base, and mechanically mix evenly; 5) Send to the brick press for pressing, with a pressing pressure of 9MPa; 6) Cooling and shaping: cooling time is 150min; S2: Three-stage fermentation 1) On the 1st-3rd day, germination is carried out at 28℃ and 85% RH; 2) On the 4th-8th day, proliferation is carried out at 30℃ and 90% RH; 3) On the 9th-12th day, enter the stable period at 32℃ and 80% RH; S3: Carrier recovery and regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: ultrasonic cleaning (40kHz, 10min) to remove residual bacteria; 3) After drying at 80℃, reload spores and fucose. Example 16

[0048] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: Raw material pretreatment 1) Sieving and blending: blend clean black tea and tea stems at a mass ratio of 8:2; 2) Steaming and pile fermentation: steaming temperature is 90℃, and the duration is 25s; 3) Add tea juice to the tea dregs until the moisture content is 12%; the tea juice is a water solution of black tea extracted by hot water at 90℃, with a solid content of 7%; 4) Add the composite fermentation carrier prepared in Example 7 at 2.0% by weight of the tea dregs, and mechanically mix evenly; 5) Send into the brick press for pressing, with a pressing pressure of 9 MPa; 6) Cooling and shaping: cooling time is 150 min; S2: Three-stage fermentation 1) Germination at 28℃ and 85% RH for the first 3 days; 2) Proliferation at 30℃ and 90% RH for the 4th to 8th days; 3) Enter the stable period at 32℃ and 80% RH for the 9th to 12th days; S3: Carrier recovery and regeneration 1) After fermentation, adsorb the composite fermentation carrier by the permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: remove residual bacteria by ultrasonic cleaning (40 kHz, 10 min); 3) After drying at 80℃, reload spores and fucose. Example 17

[0049] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: Raw material pretreatment 1) Sieving and blending: blend clean black tea and tea stems at a mass ratio of 8:2; 2) Steaming and heap fermentation: steaming temperature is 90℃, and the duration is 25 s; 3) Add tea juice to the tea dregs until the moisture content is 12%; the tea juice is a water solution of black tea extracted by hot water at 90℃, with a solid content of 7%; 4) Add the composite fermentation carrier prepared in Example 7 at 1.5% by weight of the tea dregs, and mechanically mix evenly; 5) Send into the brick press for pressing, with a pressing pressure of 8 MPa; 6) Cooling and shaping: cooling time is 150 min; S2: Three-stage fermentation 1) Germination at 28℃ and 85% RH for the first 3 days; 2) Proliferation at 30℃ and 90% RH for the 4th to 8th days; 3) Enter the stable period at 32℃ and 80% RH for the 9th to 12th days; S3: Carrier recovery and regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: ultrasonic cleaning (40kHz, 10min) to remove residual bacteria; 3) After drying at 80℃, reload spores and fucose. Example 18

[0050] A precise fermentation method of Fuzhuan tea, comprising the following steps: S1: raw material pretreatment 1) Sieving and blending: blend clean black tea and tea stems at a mass ratio of 8:2; 2) steaming and heap fermentation: steaming temperature is 90℃, and the duration is 25s; 3) add tea juice, the moisture content of tea base is 12%; the tea juice is the aqueous solution of black tea extracted by hot water at 90℃, and the solid content is 7%; 4) add 1.5% of the composite fermentation carrier prepared in Example 7 according to the weight of tea base, and mechanically mix evenly; 5) send to the brick press for pressing, the pressing pressure is 10MPa; 6) cooling and shaping: cooling time is 150min; S2: three-stage fermentation 1) germination at 28℃ and 85%RH for 1-3 days; 2) proliferation at 30℃ and 90%RH for 4-8 days; 3) enter the stable period at 32℃ and 80%RH for 9-12 days; S3: carrier recovery and regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller (magnetic field strength 0.5T); 2) Carrier regeneration: ultrasonic cleaning (40kHz, 10min) to remove residual bacteria; 3) After drying at 80℃, reload spores and fucose. Comparative Example 4

[0051] The difference between this comparative example and Example 11 is that no composite fermentation carrier is added, and an equal amount of Eurotium cristatum spores is directly added. Comparative Example 5

[0052] The difference between this comparative example and Example 11 is that there is no three-stage temperature control, but constant temperature at 30℃ is used. Comparative Example 6

[0053] The difference between this comparative example and Example 11 is that there is no steaming and heap fermentation step, and the rest is the same.

[0054] Performance test: E. terricolis amount and E. terricolis colonization rate: according to GB / T 32719.5 and GB 4789.15-2016, combined with fluorescence in situ hybridization technology for determination; Theaflavin content: according to GB / T 30483-2013 for determination; Carrier recovery rate: method: weighing calculation after magnetic separation (cycle 5 times to take the average), Calculation formula: recovery rate (%) = (recovered carrier dry weight / initial carrier dry weight) x 100; Mold contamination detection: according to GB 4789.15-2016 "National food safety standard Food microbiological examination Mold and yeast count" for determination.

[0055] Table 3

[0056] From table 3, it can be seen that the examples 11-18 of the present application all show excellent fermentation performance, and the E. terricolis colonization rate is generally higher than 90% (88.6%-97.8%), which is significantly better than the comparative examples 4-6 (62.4%-85.2%); the theaflavin content reaches 1.65-2.24 mg / g, which is increased by 62%-120% compared with the comparative examples, the carrier recovery rate is stable at 90.3%-94.6%, the maximum residual rate is 9.7%, which meets the safety standard; the mold detection rate is controlled at 0.4%-2.0%, which is much lower than the comparative examples 4-6 (8.1%-18.1%), and no pathogenic mold is detected. The optimal example is example 16: the E. terricolis colonization rate is the highest (97.8±0.9%), the theaflavin content is the highest (2.24±0.14 mg / g), the mold contamination rate is the lowest (0.4±0.1%), and the carrier recovery rate is higher (84.6%).

[0057] The E. terricolis in the brick tea prepared from example 16 was observed under a microscope, as shown in Figure 2 and 3 .

[0058] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A precise fermentation method for Fu brick tea, characterized in that, Includes the following steps: S1: Raw material pretreatment 1) Sieving and blending: Blending clean black tea leaves with tea stems in a certain proportion; 2) Steam fermentation: The steam temperature is 85-95℃, and the duration is 15-30 seconds; 3) Add tea juice until the moisture content of the tea leaves is 11-12%; 4) Add 1-2% of the compound fermentation carrier by weight of the tea leaves and mechanically mix evenly; 5) Feed it into the brick press for pressing; 6) Cooling and shaping: Cooling time is 120-150 min; S2: Three-stage fermentation 1) Germination occurs on days 1-3 at a temperature of 27.5-28.5℃ and a humidity of 83-87%RH; 2) Propagation was carried out on days 4-8 at a temperature of 29.5-30.5℃ and a humidity of 89-91%RH; 3) The temperature enters a stable period on days 9-12 at 31.5-32.5℃ and 78-82%RH. S3: Carrier Recycling and Regeneration 1) After fermentation, the composite fermentation carrier is adsorbed by a permanent magnet roller; 2) Carrier regeneration: ultrasonic cleaning; 3) After drying at 80℃, the spores and fucose are reloaded.

2. The precise fermentation method for Fu brick tea according to claim 1, characterized in that, The mass ratio of the black tea to the tea stems is (8:2)-(9:1).

3. The precise fermentation method for Fu brick tea according to claim 1, characterized in that, The tea juice is an aqueous solution obtained by steeping black tea in hot water at 80-90℃, with a solid content of 5-8%.

4. The precise fermentation method for Fu brick tea according to claim 1, characterized in that, The preparation method of the composite fermentation carrier is as follows: S11: Food-grade mesoporous silica and Fe3O4 nanoparticles were mixed at a mass ratio of 9:1, 3% aminosilane coupling agent was added, and the mixture was reacted at 80℃ for 6 hours to obtain amination-modified mixed particles. S12: PMMA microspheres and aminated mixed particles are mixed evenly at a mass ratio of 1:(5-7), pressed into shape, and calcined at 550℃ for 4h to remove the template, forming 20-50μm through-pores to obtain a multi-level porous carrier. S13: Immerse the multi-porous carrier in dopamine-Tris buffer and shake at 22-25℃ for 24h to obtain a polydopamine-coated multi-porous carrier. After taking it out, immerse it in a suspension of Aspergillus cristatus spores and adsorb it under vacuum negative pressure of -0.08MPa for 30-35min to obtain a multi-porous carrier loaded with Aspergillus cristatus spores. S14: The antibacterial microspheres are filled into the macropores of a multi-level porous carrier loaded with Aspergillus cristatus spores by vibrating sieve, immersed in 4-5% fucose solution for 50-60 min, and vacuum dried at 60℃ to constant weight to obtain the product.

5. The precise fermentation method for Fu brick tea according to claim 4, characterized in that, The food-grade mesoporous silica has a pore size of 5-10 nm and an average specific surface area of ​​807 m². 2 / g.

6. The precise fermentation method for Fu brick tea according to claim 4, characterized in that, The PMMA microspheres have a particle size of 30-35 μm.

7. The precise fermentation method for Fu brick tea according to claim 4, characterized in that, The dopamine-Tris buffer solution has a pH of 8.5 and a concentration of 2 mg / mL.

8. The precise fermentation method for Fu brick tea according to claim 4, characterized in that, The content of *Aspergillus cristatus* spores in the *S13* spore suspension was 1.0 × 10⁻⁶. 8 -3.0×10 8 CFU / mL.

9. The precise fermentation method for Fu brick tea according to claim 4, characterized in that, The method for preparing the antibacterial microspheres in S14 is as follows: Step 1: Take tea polyphenols, chitosan and dilute acid solution to obtain a solution with tea polyphenol content of 0.1-0.2% and chitosan content of 0.5%; Step 2: Add dropwise to 2% CaCl2 solution, stir at 500 rpm for 30 min to solidify, filter and sieve to obtain antibacterial microspheres with a particle size of 20-50 μm.

10. Fu brick tea prepared by the method according to any one of claims 1-9.

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