Black tea fermentation process

Abstract

The invention discloses a black tea fermentation process, and relates to the technical field of tea making processes, and the black tea fermentation process specifically comprises the following steps: black tea is crushed and sieved to 150-200 meshes, and black tea fine powder is obtained; uniformly mixing black tea, glucose, xylooligosaccharide, vitamins, sodium chloride, calcium chloride, fructo-oligosaccharide and water, sterilizing to obtain a sterilized culture medium, and cooling for later use; carrying out enlarged culture on lactic acid bacteria to obtain a secondary seed solution; transferring the secondary seed solution into a sterilized culture medium, adding magnetic composite particles, and culturing and fermenting to obtain a fermentation solution; performing high-temperature treatment on the fermentation liquor, and adding a preservative to obtain black tea fermentation filtrate; by introducing a double-component system of calcium chloride and xylooligosaccharide, efficient release and stabilization of active ingredients of the black tea are realized, by introducing a magnetic composite particle and alternating magnetic field staged control technology, the fermentation efficiency and stability of the black tea are further improved, and the efficient, stable and sustainable black tea fermentation process is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of tea-making processes, and particularly to a black tea fermentation process. Background Art

[0002] Black tea is one of the basic tea categories. Belonging to the fully fermented tea category, it is made from the buds and leaves of tea trees through typical process steps such as withering, rolling, fermentation, and drying. Among them, fermentation is the key process in black tea processing. It is named because the color of its dry tea and the brewed tea soup are mainly red.

[0003] Black tea contains various nutrients and functional components such as vitamins, caffeine, amino acids, minerals, polysaccharides, and tea polyphenols. Research shows that black tea has the effects of assisting in reducing blood sugar, blood pressure, and blood lipids. The tea pigments in black tea have effects such as antioxidant, anti-tumor, anti-inflammatory and antibacterial, anti-mutation, antiviral, and deodorization.

[0004] For example, in the Chinese patent with the application number CN202211216309.5, a black tea fermentation filtrate and fermentation process, the preparation raw materials of the camellia flower fermentation liquid include: camellia flower, lactic acid bacteria, fructooligosaccharide, and water. The camellia flower fermentation filtrate prepared by this invention contains rich biological small molecule natural nutrients such as tea polyphenols and flavonoids, has strong antioxidant and anti-wrinkle properties, simple preparation raw materials, safe and environmentally friendly process, controllable process, and strong applicability. This patent improves the fermentation efficiency by optimizing the strain ratio and culture medium components, but there are still problems such as low enzyme-bacteria synergy efficiency, poor process stability caused by multi-parameter linkage, and component loss during the preparation process. Summary of the Invention

[0005] By providing a black tea fermentation process in the embodiments of the present application, the problems in the prior art during the preparation process, such as low enzyme-bacteria synergy efficiency, poor process stability caused by multi-parameter linkage, and loss of active components, are solved. By introducing a two-component system of calcium chloride and xylooligosaccharide, and utilizing the synergistic effect of calcium ions activating enzymatic hydrolysis and xylooligosaccharide regulating the metabolism of the flora, the efficient release and stabilization of the active components of black tea are achieved. In addition, by introducing magnetic composite particles and alternating magnetic field staged control technology, the fermentation efficiency and stability of black tea are further improved, the problems of low efficiency of endogenous enzymes, bacteria-enzyme competition, and low enzyme utilization rate are solved, and the high efficiency, stability, and sustainability of the black tea fermentation process are realized.

[0006] The embodiments of the present application provide a black tea fermentation process, which specifically includes the following steps:

[0007] (1) Crush black tea and sieve it to 150 - 200 meshes to obtain fine black tea powder;

[0008] (2) Mix black tea, glucose, xylo-oligosaccharide, vitamins, sodium chloride, calcium chloride, fructo-oligosaccharide, and water evenly, sterilize to obtain a sterilized culture medium, and cool for standby;

[0009] (3) Expand the culture of lactic acid bacteria to obtain a secondary seed solution;

[0010] (4) Transfer the secondary seed solution to the sterilized culture medium, add magnetic composite particles, and culture and ferment to obtain a fermentation broth;

[0011] (5) After subjecting the fermentation broth to high-temperature treatment, add a preservative to obtain a black tea fermentation filtrate.

[0012] Further, the preparation raw materials, by weight, include 6 parts of black tea, 5 parts of lactic acid bacteria, 6 parts of glucose, 0.45 - 0.55 parts of xylo-oligosaccharide, 0.7 parts of vitamins, 0.7 parts of sodium chloride, 0.04 - 0.06 parts of calcium chloride, 0.3 parts of fructo-oligosaccharide, 0.1 part of preservative, 80 parts of water, and 0.09 parts of magnetic composite particles.

[0013] Further, the lactic acid bacteria is a lactobacillus composition, which, by weight, includes: 1.5 parts of Lactobacillus plantarum, 4 parts of Lactobacillus paracasei, 0.5 part of Lactobacillus rhamnosus, and 0.5 part of Lactobacillus acidophilus.

[0014] Further, the magnetic composite particles include magnetic nanoparticles and cellulase. The magnetic nanoparticles are loaded with cellulase to form cellulase magnetic composite particles, and the cellulase loading amount is 30 mg / g of particles.

[0015] Further, the magnetic nanoparticles have a core-shell structure, the core layer is Fe3O4, the shell layer is SiO2, and the mass ratio of Fe3O4 to SiO2 is 1:0.7.

[0016] Further, the magnetic composite particles also include calcium ion magnetic composite particles and xylo-oligosaccharide magnetic composite particles, and the addition amounts are 0.06 parts and 0.04 parts respectively;

[0017] The calcium ion magnetic composite particles have a calcium chloride loading amount of 20 mg / g of particles, and the xylo-oligosaccharide magnetic composite particles have a xylo-oligosaccharide loading amount of 25 mg / g of particles.

[0018] Further, the calcium ion magnetic composite particles are prepared by cross-linking with a chitosan-CaCl2 mixed solution, and the mass ratio of Fe3O4, SiO2 to chitosan is 1:0.7:0.3; the xylo-oligosaccharide magnetic composite particles are prepared by encapsulating with a poly(N-isopropylacrylamide) thermosensitive hydrogel, and the mass ratio of Fe3O4, SiO2 to poly(N-isopropylacrylamide) is 1:0.7:0.2.

[0019] Further, in step (4), it is combined with phased control of an alternating magnetic field. Specifically, during the cell wall breaking period (0 - 12 h), the stirring speed is 150 r / min, and the magnetic field strength is continuously 0.1 T.

[0020] During the conversion period (12 - 24 h), the stirring speed is 100 r / min, and the magnetic field is turned off.

[0021] During the recovery period (24 - 28 h), the stirring speed is 80 r / min, the magnetic field strength is 0.1 T, the pulse frequency is 2 Hz, and the duty cycle is 50%.

[0022] Further, the frequency of the alternating magnetic field generator is 50 Hz, and the magnetic induction intensity is 0.1 T.

[0023] Further, magnetic particles are added in stages in step (4). Specifically, during the cell wall breaking period, calcium ion magnetic composite particles and xylooligosaccharide magnetic composite particles are added, and the temperature is raised to 35 °C at a rate of 0.3 °C / h.

[0024] During the conversion period, xylooligosaccharide magnetic composite particles are added and maintained at 32 ± 0.5 °C.

[0025] During the recovery period, the temperature is lowered to 25 °C at a rate of 3.5 °C / h.

[0026] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0027] Firstly, by introducing a two-component system of Ca 2+ (calcium chloride) and xylooligosaccharide (XOS), based on the synergistic effect of Ca 2+ activating enzymatic hydrolysis and xylooligosaccharide regulating the microbial metabolism, the efficient release and stabilization of the active components of black tea are realized, solving the problem in black tea fermentation that the maintenance of the dynamic balance of the strain - enzyme - substrate depends on multi-parameter regulation (such as pH buffering and staged temperature control), resulting in high process complexity and insufficient stability.

[0028] Secondly, by introducing magnetic composite particles, the immobilized enzyme technology is combined with the magnetic field dynamic regulation process, solving the problems of low efficiency of endogenous enzymes, competition between bacteria and enzymes, and low enzyme utilization rate, and further improving the fermentation efficiency and stability of black tea; the Fe3O4 core layer in the magnetic composite particles provides superparamagnetism, enabling magnetic field recovery; the SiO2 shell layer has a mesoporous structure that can protect the enzyme activity, isolate lactic acid erosion, and simultaneously load cellulase. Fe3O4 and SiO2 are bonded by chemical bonds to ensure the stability of the core - shell structure. The pore size of SiO2 is adapted to cellulase, and through the mesoporous confinement effect, the enzyme immobilization efficiency is improved.

[0029] Thirdly, by introducing pH-responsive calcium ion magnetic particles and thermosensitive xylooligosaccharide magnetic particles and combining with the magnetic field staged controlled release mechanism, the synergistic effect of enzyme activation and flora induction is achieved; the staged release of the magnetic control particles enables the particles to produce a synergistic effect. Chitosan in the calcium ion magnetic particles dissolves at pH < 5.5, releasing Ca 2+ to activate the active center of cellulase and stabilize the tertiary structure of the enzyme; poly(N-isopropylacrylamide) in the thermosensitive xylooligosaccharide magnetic particles triggers the contraction of the thermosensitive layer at 32 °C, releasing xylooligosaccharide and promoting the secretion of xylanase; the mesoporous structure of the cellulase particles immobilizes cellulase through physical adsorption and glutaraldehyde cross-linking. The magnetic field enhances the enzyme-substrate contact frequency and improves the cell wall breaking efficiency;

[0030] Fourthly, during the cell wall breaking stage, exogenous enzymes and endogenous enzymes synergistically degrade cellulose, and the magnetic field directionally controls the particle dispersion; during the conversion stage, XOS-induced flora dominates the metabolism, the magnetic field is turned off to reduce interference, and the flora focuses on polyphenol conversion and by-product inhibition. Through the spatio-temporal division of labor in different periods of each stage, a dynamic synergistic network of "enzyme cell wall breaking - bacteria conversion" is formed. Specific Embodiments

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs; the terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments, and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0032] Example 1: A black tea fermentation process specifically includes the following steps:

[0033] (1) Crush black tea and sieve it to 150 - 200 mesh to obtain fine black tea powder;

[0034] (2) Mix black tea, glucose, xylooligosaccharide, vitamins, sodium chloride, calcium chloride, fructooligosaccharide, and water evenly, maintain at 85 °C and 103.4 kPa for 10 minutes for sterilization, then raise the temperature to 95 °C for 5 minutes for sterilization to obtain a sterilized culture medium, and cool for later use;

[0035] (3) Expand the culture of lactic acid bacteria to obtain a secondary seed liquid;

[0036] (4) Transfer the secondary seed liquid to the sterilized culture medium, and carry out constant temperature culture and fermentation at 32 ± 0.5 °C, and shake culture at a stirring speed of 80 - 120 r / min for 24 - 36 hours to obtain a fermentation broth;

[0037] (5) After high-temperature treatment of the fermentation broth, add a preservative and mix evenly to obtain a black tea fermentation filtrate;

[0038] The raw materials for the preparation include, by weight, 6 parts of black tea, 5 parts of lactic acid bacteria, 6 parts of glucose, 0.45-0.55 parts of xylo-oligosaccharides (XOS), 0.7 parts of vitamins, 0.7 parts of sodium chloride, 0.04-0.06 parts of calcium chloride (CaCl2·2H2O), 0.3 parts of oligofructose, 0.1 parts of preservatives and 80 parts of water;

[0039] The inoculation amount of lactic acid bacteria is 0.83 times that of black tea; the lactic acid bacteria is lactobacillus; the composition of lactobacillus, by weight, includes: 1.5 parts of Lactobacillus plantarum, 4 parts of Lactobacillus paracasei, 0.5 parts of Lactobacillus rhamnosus, and 0.5 parts of Lactobacillus acidophilus;

[0040] The specific process of step (3) is as follows: transferring the lactic acid bacteria to an MRS liquid culture medium, culturing at 32±0.5°C to obtain a primary seed solution, and then expanding the primary seed solution in an MRS liquid culture medium, culturing at 32±0.5°C for 12-18 hours to obtain a secondary seed solution;

[0041] The specific process of step (5) is as follows: the fermentation liquid is subjected to a first high-temperature treatment at a temperature of 65°C for 30 minutes, and then ultrasonicated after cooling to 25°C to obtain a treated fermentation liquid, and then filtered through a 10 μm pore filter plate to remove residues to obtain a clarified liquid, and the clarified liquid is subjected to a second high-temperature treatment at a temperature of 85°C for 15 minutes, and then cooled to 25°C and a preservative is added to obtain a black tea fermentation filtrate.

[0042] Experiments were conducted for the technical solution of this embodiment, wherein the amounts of xylo-oligosaccharides and calcium chloride added in each experiment are shown in Table 1 below:

[0043] Table 1

[0044] Group Xylooligosaccharide addition (parts) Calcium chloride addition (parts) Control 0 0 Experiment 1 0.45 0.04 Experiment 2 0.50 0.05 Experiment 3 0.55 0.06

[0045] The samples prepared in the comparative example and experiments 1 to 3 were subjected to performance tests, and the test indicators and methods are shown in Table 2 below:

[0046] Table 2

[0047]

[0048] The test results are shown in Table 3 below;

[0049] Table 3

[0050] Group Polyphenol conversion rate (%) Total biogenic amine content (mg / kg) Fermentation period (h) Polyphenol retention rate after 30 days (%) Control 67.2±1.8 38.5±2.7 40±2 72.1±1.5 Experiment 1 82.5±1.2 15.3±1.4 34±1 85.6±1.8 Experiment 2 89.4±0.9 9.2±0.8 30±1 90.3±0.7 Experiment 3 86.7±1.5 12.5±1.1 32±1 87.9±1.2

[0051] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0052] By introducing Ca 2+A binary system of calcium chloride (CaCl₂) and xylooligosaccharides (XOS), based on Ca 2+ activates the synergistic effect of enzymatic hydrolysis and xylooligosaccharide-regulated microbial metabolism to achieve the efficient release and stabilization of active components in black tea, and solves the problem in black tea fermentation that the maintenance of the dynamic balance of strain-enzyme-substrate depends on multi-parameter regulation (such as pH buffering and staged temperature control), resulting in high process complexity and insufficient stability.

[0053] The binary system acts together to promote the activation of biological enzymes. Among them, Ca 2+ acts as a metal coenzyme. Calcium ions (Ca 2+ ) are the key cofactors at the active center of cellulase (Cel7A). By forming coordination bonds (binding constant Kd = 1.8 μM) with the Glu212 / Asp214 sites of the enzyme protein, the tertiary structure of the enzyme is stabilized, the activity of cellulase is increased by 1.6 times, and the degradation of cellulose in the black tea cell wall is accelerated; xylooligosaccharides produce an induction effect. Xylooligosaccharides (β-1,4 xyloside bonds), as the degradation products of hemicellulose, induce the secretion of xylanase (XynA) (activity reaches 154.7 U / mL) by activating the xynR gene of Lactobacillus paracasei (the expression level is increased by 3.2 times), and target the decomposition of arabinoxylan in the cell wall;

[0054] The binary system acts together to achieve the interaction between substrate and microflora. Among them, Ca 2+ regulates the intracellular pH (stabilized at 5.2 - 5.5) through the Ca 2 + -ATPase pump on the bacterial membrane to achieve transmembrane regulation, promote the proton motive force (PMF) of lactic acid bacteria, and significantly improve the metabolic efficiency; xylooligosaccharides are preferentially metabolized by Lactobacillus paracasei, activate the carbon metabolic pathway through the phosphotransferase system (PTS), inhibit the growth of competitive miscellaneous bacteria (such as Escherichia coli), and the purity of the microflora is increased by 85%;

[0055] In addition, the binary system produces molecular complex protection. Among them, Ca 2+ forms a stable complex (binding energy ΔG = -28.6 kJ / mol) with the catechol group of black tea polyphenols (such as EGCG), inhibits the activity of polyphenol oxidase (PPO), reduces the generation of quinone substances, the browning index decreases, and the β-glycosidic bond of xylooligosaccharides forms a hydrogen bond network with theaflavins to delay photooxidative degradation, and the retention rate of polyphenols is increased in the 30-day accelerated test;

[0056] By introducing Ca 2+In synergistic action with xylo-oligosaccharide, cellulase and the introduced xylanase, the cell wall porosity is increased, improving the cell wall breaking efficiency; the polyphenol conversion rate is increased, the theaflavin content is increased, promoting the release of active ingredients; the total amount of biogenic amines is reduced, the tyramine synthase activity is inhibited, thus inhibiting metabolic by-products; the fermentation time is shortened, the bacterial proliferation rate is increased, thus optimizing the fermentation cycle; calcium-polyphenol chelates are formed, the oxidation induction period is prolonged, significantly enhancing the stability of the product;

[0057] For black tea fermentation, through the synergistic cell wall breaking by two enzymes (cellulase + xylanase), the dissolution rate of tea polyphenols is increased, and the extraction amount of flavonoids is increased; in addition, during the fermentation process, the bound polyphenols (such as tea polyphenol glycosides) are converted into free forms, and the antioxidant value is significantly increased;

[0058] Xylo-oligosaccharide selectively promotes the proliferation of Lactobacillus paracasei and inhibits the growth of amine-producing bacteria (such as Enterococcus), reducing the biogenic amine risk and shortening the fermentation cycle, solving the problems of low extraction rate, many by-products, poor stability, etc. in traditional fermentation, improving the fermentation efficiency while reducing the loss of active ingredients and inhibiting the production of by-products.

[0059] Example 2: In the above Example 1, by introducing Ca 2+ A two-component system in synergy with xylo-oligosaccharide, based on Ca 2+ Activating the synergistic action of enzymatic hydrolysis and xylo-oligosaccharide-regulated microbial metabolism, realizing the efficient release and stabilization of the active ingredients of black tea, improving the fermentation efficiency while reducing the loss of active ingredients and inhibiting the production of by-products, and further improving on the basis of Example 1 for further enhancing the fermentation efficiency and stability of black tea.

[0060] In step (4), magnetic composite particles are also added. The magnetic composite particles include magnetic nanoparticles and cellulase. The magnetic nanoparticles are loaded with cellulase to form cellulase magnetic composite particles, and the cellulase loading is 30 mg / g of the particles; the addition amount of the magnetic composite particles is 0.09 parts by weight;

[0061] Among them, the magnetic nanoparticles have a core-shell structure, the core layer is Fe3O4, the shell layer is SiO2, and the mass ratio of Fe3O4 to SiO2 is 1:0.7; the particle size of the magnetic nanoparticles is 80 - 100 nm;

[0062] The preparation of the magnetic composite particles is specifically as follows;

[0063] The magnetic nanoparticles are impregnated in 0.1 M phosphate buffer (pH 6.5) and ultrasonically dispersed for 20 min;

[0064] Cellulase is added according to an enzyme loading of 30 mg / g of the particles, and shaken and adsorbed at 4°C for 12 h;

[0065] Add 0.5% glutaraldehyde crosslinking agent and fix at room temperature for 2 h;

[0066] After magnetic separation, freeze-dry to obtain magnetic composite particles (the enzyme activity retention rate is ≥85%).

[0067] In step (4), culture is carried out in combination with an alternating magnetic field generator, and staged control is implemented;

[0068] Among them, the frequency of the alternating magnetic field generator is 50 Hz, and the magnetic induction intensity is 0.1 T;

[0069] The staged control is specifically as follows: during the cell wall breaking period, 0 - 12 h, the stirring speed is 150 r / min, and the magnetic field intensity is a continuous 0.1 T;

[0070] During the conversion period, 12 - 24 h, the stirring speed is 100 r / min, and the magnetic field is turned off;

[0071] During the recovery period, 24 - 28 h, the stirring speed is 80 r / min, the magnetic field intensity is 0.1 T, the pulse frequency is 2 Hz, and the duty cycle is 50%;

[0072] After the fermentation in step (5) is completed, turn on the permanent magnet recovery device (the surface magnetic field intensity is 0.5 T); perform magnetic absorption for 10 min for particle recovery; wash the recovered particles 3 times with pH 6.0 phosphate buffer solution and store them at 4 °C for recycling.

[0073] Experiments were carried out on the technical solution of this embodiment based on Experiment 2 of Example 1. As Experiment 4, the difference between Experiment 4 of this embodiment and Experiment 2 is that magnetic composite particles are also added in step (2) of the technical solution of this embodiment, and culture is carried out in combination with an alternating magnetic field generator in step (4), and staged control is implemented; performance tests were carried out on the samples prepared in Experiment 4 of this embodiment, and the test results are shown in Table 4 below;

[0074] Table 4

[0075] Group Polyphenol conversion rate (%) Total biogenic amine content (mg / kg) Fermentation period (h) Polyphenol retention rate after 30 days (%) Experiment 4 92.8±0.7 6.1±0.6 26±1 93.1±0.5

[0076] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0077] By introducing magnetic composite particles, the immobilized enzyme technology is combined with the magnetic field dynamic regulation process, solving the problems of low efficiency of endogenous enzymes, competition between bacteria and enzymes, and low enzyme utilization rate, and further improving the fermentation efficiency and stability of black tea;

[0078] The Fe3O4 core layer in the magnetic composite particles provides superparamagnetism (saturation magnetization ≥ 60 emu / g) and enables magnetic field recovery; the SiO2 shell layer has a mesoporous structure (pore size 5 - 8 nm), which can protect the enzyme activity, isolate lactic acid erosion, and simultaneously load cellulase (enzyme loading 30 mg / g). Fe3O4 and SiO2 are bonded by chemical bonds to ensure the stability of the core-shell structure. The SiO2 pore size is adapted to cellulase (molecular size ≈ 4.5 nm), and through the mesoporous confinement effect, the enzyme immobilization efficiency is improved;

[0079] By controlling the alternating magnetic field in stages, the fermentation efficiency and stability of black tea are further improved. During the cell wall breaking period (0 - 12 h), a continuous magnetic field of 0.1 T is used to enhance the enzyme-substrate contact and improve the cellulase activity; during the conversion period (12 - 24 h), the magnetic field is turned off to reduce the interference of the magnetic field on the flora, enabling lactic acid bacteria to focus on metabolism and increasing the polyphenol conversion rate; during the recovery period (24 - 28 h), a 2 Hz pulsed magnetic field (0.1 T) is used to achieve efficient enrichment of particles under low-speed stirring (80 r / min), improving the recovery rate. By dynamically regulating, the temporal and spatial division of labor between enzymatic cell wall breaking and flora metabolism is balanced, shortening the fermentation cycle and simultaneously inhibiting the generation of biogenic amines;

[0080] By introducing magnetic composite particles and staged control of alternating magnetic fields, the following effects are produced on black tea fermentation;

[0081] Efficient cell wall breaking and active ingredient release: The magnetic particle-immobilized enzyme (1700 U / mL) degrades the cell wall directionally with the assistance of a magnetic field, significantly increasing the porosity, further increasing the polyphenol conversion rate, and significantly increasing the release amount of active ingredients such as theaflavins;

[0082] Precise metabolic regulation and safety control: The SiO2 shell physically isolates the enzyme and the flora, reducing lactic acid inhibition and by-product generation. At the same time, the staged control of the dynamic magnetic field (cell wall breaking - conversion - recovery) optimizes the division of labor between bacteria and enzymes, improving the fermentation stability and product consistency;

[0083] High stability and recycling: The magnetic recovery system realizes the recycling of enzyme resources. Combined with ultrafiltration technology and calcium-polyphenol chelation protection, the polyphenol retention rate is increased in 30 days, improving the fermentation efficiency and stability of black tea.

[0084] Example 3: In Example 2 above, by introducing magnetic composite particles to realize the combination of immobilized enzyme technology and dynamic magnetic field regulation, the problems of low efficiency of endogenous enzymes, competition between bacteria and enzymes, and low enzyme utilization rate are solved, and effects such as improving the fermentation efficiency and stability of black tea and shortening the fermentation cycle are produced. To further improve the fermentation efficiency and stability of black tea, it is further improved on the basis of Example 2.

[0085] The magnetic composite particles further include calcium ion magnetic composite particles and xylo-oligosaccharide magnetic composite particles; the addition amounts of the calcium ion magnetic composite particles and xylo-oligosaccharide magnetic composite particles are 0.06 parts and 0.04 parts by weight, respectively;

[0086] The magnetic nanoparticles are loaded with CaCl2 to form calcium ion magnetic composite particles, and the CaCl2 loading amount is 20 mg / g of particles; the xylo-oligosaccharide loading amount is 25 mg / g of particles;

[0087] The preparation of the calcium ion magnetic composite particles is specifically as follows: the magnetic nanoparticles are impregnated in a 5% chitosan-CaCl2 mixed solution (chitosan:CaCl2 = 1:2), cross-linked and cured at pH 6.0, and then freeze-dried to obtain;

[0088] Among them, the mass ratio of Fe3O4, SiO2 and chitosan is 1:0.7:0.3;

[0089] The preparation of the xylo-oligosaccharide magnetic composite particles is specifically as follows: the magnetic nanoparticles are impregnated in a 10% XOS solution, wrapped with a poly N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel (LCST = 32 °C), and then vacuum-dried to obtain;

[0090] Among them, the mass ratio of Fe3O4, SiO2 and PNIPAM is 1:0.7:0.2;

[0091] In step (4), different magnetic nanoparticles are added at different stages, specifically;

[0092] During the cell wall breaking period, calcium ion magnetic composite particles and xylo-oligosaccharide magnetic composite particles are added, and the temperature is gradually increased from 32 to 35 °C at a heating rate of 0.3 °C / h;

[0093] During the conversion period, xylo-oligosaccharide magnetic composite particles with a weight of 0.04 are added, and the temperature is 32 ± 0.5 °C;

[0094] During the recovery period, the temperature is decreased from 32 °C to 25 °C at a rate of 3.5 °C / h.

[0095] Based on the technical solution of this example, an experiment is carried out on the basis of Experiment 4 in Example 3. As Experiment 5, the difference between Experiment 5 in this example and Experiment 4 is that in this example, the magnetic nanoparticles further include calcium ion magnetic composite particles and xylo-oligosaccharide magnetic composite particles, and different magnetic nanoparticles are added at different stages in step (4); the performance of the sample prepared in Experiment 5 of this example is tested, and the test results are shown in Table 5 below;

[0096] Table 5

[0097] Group Polyphenol conversion rate (%) Total biogenic amine content (mg / kg) Fermentation period (h) Polyphenol retention rate after 30 days (%) Experiment 5 94.5±0.6 4.2±0.4 22±1 94.8±0.4

[0098] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0099] By introducing pH-responsive calcium ion magnetic particles and thermosensitive xylooligosaccharide magnetic particles, and combining with the magnetic field staged controlled release mechanism, the synergistic effect of enzyme activation and flora induction is realized;

[0100] The staged release of the magnetically controlled particles enables the particles to produce a synergistic effect. Chitosan in the calcium ion magnetic particles dissolves at pH < 5.5, releasing Ca 2+ activates the active center (Glu212 / Asp214 site) of cellulase (Cel7A), with a binding constant Kd = 1.8 μM, and stabilizes the tertiary structure of the enzyme; poly(N-isopropylacrylamide) (PNIPAM) in the thermosensitive xylooligosaccharide magnetic particles triggers the contraction of the thermosensitive layer at 32 °C (LCST), releasing xylooligosaccharide (XOS), inducing the expression of the xynR gene of Lactobacillus paracasei (increased by 3.5 times), and promoting the secretion of xylanase (XynA); the mesoporous structure (pore size 5-8 nm) of the cellulase particles immobilizes cellulase through physical adsorption and glutaraldehyde cross-linking, and the magnetic field (0.1 T) enhances the enzyme-substrate contact frequency and improves the cell wall breaking efficiency;

[0101] During the cell wall breaking stage, exogenous enzymes (cellulase particles) and endogenous enzymes (Ca 2+ activation) synergistically degrade cellulose, and the magnetic field directionally controls the particle dispersion; during the conversion stage, XOS induces the dominant metabolism of the flora, turns off the magnetic field to reduce interference, and the flora focuses on polyphenol conversion and by-product inhibition. Through the spatio-temporal division of labor in different periods of each stage, a "enzyme cell wall breaking - bacteria conversion" dynamic synergistic network is formed;

[0102] By introducing pH-responsive calcium ion magnetic particles and thermosensitive xylooligosaccharide magnetic particles, and combining with the magnetic field staged controlled release mechanism, the fermentation efficiency is further improved, and the polyphenol conversion rate is further increased. Due to the synergistic degradation of cellulose and hemicellulose by exogenous and endogenous enzymes (degradation efficiency +12%), at the same time, the xylanase activity is increased; the total amount of biogenic amines is reduced, which is achieved through the staged action of bacteria and enzymes (enzyme-dominated during the cell wall breaking stage and bacteria-dominated during the conversion stage) and flora optimization; due to the gradient heating (32 to 35 °C, 0.3 °C / h) accelerating the enzyme reaction rate, and the XOS inducing the logarithmic phase of the flora to advance, the fermentation cycle is further shortened. In addition, relying on the calcium-polyphenol chelation antioxidant and the physical barrier of the thermosensitive layer, the polyphenol retention rate after 30 days is further increased and the stability is enhanced.

[0103] Example 4: Based on Example 3, to further improve the efficiency and stability of black tea fermentation, the above Example 3 introduced calcium ion and xylooligosaccharide magnetic composite particles, added them in stages, and used magnetic fields to control the release in stages, solving the problems of low endogenous enzyme efficiency, competition between bacteria and enzymes, and low enzyme utilization rate, and realizing the synergy of enzyme activation and flora induction, and achieving efficient active release in black tea fermentation.

[0104] The cell wall breaking stage is divided into the early cell wall breaking stage (0 - 4h) and the late cell wall breaking stage (4 - 12h);

[0105] In the early cell wall breaking stage (0 - 4h), the magnetic field is a weak magnetic field with a magnetic field intensity of 0.05T, and low-speed stirring is carried out at a stirring speed of 80r / min to promote moderate aggregation of particles and slow down the release of Ca 2+ and enzymes;

[0106] In the late cell wall breaking stage (4 - 12h), the magnetic field is enhanced to a magnetic field intensity of 0.1T, and high-speed stirring is carried out at a stirring speed of 150r / min to force the particles to disperse and accelerate the release to make up for the deficiency in the initial stage;

[0107] Based on Experiment 5 of Example 4, experiments were carried out on the technical solution of this example. As Experiment 6, the difference between Experiment 6 of this example and Experiment 5 is that in this example, the cell wall breaking stage is divided into the early cell wall breaking stage (0 - 4h) and the late cell wall breaking stage (4 - 12h), and the magnetic field intensity and stirring speed are different in the early and late cell wall breaking stages; the performance of the samples prepared in Experiment 6 of this example was tested, and the test results are shown in Table 6 below;

[0108] Table 6

[0109] Group Polyphenol conversion rate (%) Total biogenic amine content (mg / kg) Fermentation period (h) Polyphenol retention rate after 30 days (%) Experiment 6 95.8±0.5 3.5±0.3 20±1 95.2±0.3

[0110] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0111] By regulating the magnetic field intensity and stirring speed in stages, the control of the cell wall breaking stage is further refined, the release rhythm of particles is optimized, and the balance of particle aggregation and dispersion is actively controlled by dividing the cell wall breaking stage into the early stage (0 - 4h) and the late stage (4 - 12h). In the early cell wall breaking stage (0 - 4h), the weak magnetic field (0.05T) and low-speed stirring (80r / min) promote moderate aggregation of particles and slow down the release of Ca 2+ and cellulase (the release amount is controlled at 40%), avoiding substrate competition in the initial stage; in the late cell wall breaking stage (4 - 12h), it is switched to a strong magnetic field (0.1T) and high-speed stirring (150r / min), forcing the particles to disperse and concentrating the release of the remaining 60% of the enzymes and Ca 2+ , activating the peak value of endogenous enzyme activity (1750U / mL) and increasing the cellulose degradation rate (+10%). This staged regulation matches the metabolic rhythm of the flora and realizes the optimized distribution of enzymes;

[0112] By regulating the magnetic field intensity and stirring speed in stages, the black tea fermentation efficiency is further improved;

[0113] The polyphenol conversion rate is increased. Due to the slow release in the initial stage of cell wall breaking, the substrate competition is reduced, and more cellulose is reserved for efficient degradation in the later stage. In the later stage of cell wall breaking, Ca 2+ and enzymes are released concentratedly. The peak value of cellulase activity reaches 1750 U / mL (compared with 1700 U / mL in Comparative Experiment 5), and the porosity is increased;

[0114] The total amount of biogenic amines is further reduced, and the spatial and temporal division of labor between bacteria and enzymes is more clear: in the initial stage of cell wall breaking, the enzyme activity is controlled, and the interference of the bacterial community metabolism is reduced (the inhibition rate of tyramine synthase activity is increased from 60% to 65%), and the proportion of Lactobacillus paracasei is increased from 70% to 72%;

[0115] The fermentation cycle is shortened. In the later stage of cell wall breaking, the enzyme activity is released efficiently, the cellulose degradation rate is increased by 10%, the logarithmic phase of the XOS-induced bacterial community is advanced, and the time for the cell density (OD600) to reach the plateau is reduced.

[0116] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A black tea fermentation process, characterized in that, Specifically, it includes the following steps: (1) Crush black tea and sieve it to 150 - 200 mesh to obtain fine black tea powder; (2) Mix black tea, glucose, xylo - oligosaccharide, vitamins, sodium chloride, calcium chloride, fructo - oligosaccharide, and water evenly, sterilize to obtain a sterilized culture medium, and cool for standby; (3) Expand the culture of lactic acid bacteria to obtain a secondary seed liquid; (4) Transfer the secondary seed liquid to the sterilized culture medium, add magnetic composite particles, culture and ferment to obtain a fermentation broth; (5) After high - temperature treatment of the fermentation broth, add a preservative to obtain a black tea fermentation filtrate.

2. The black tea fermentation process according to claim 1, wherein, The preparation raw materials are calculated by weight, including 6 parts of black tea, 5 parts of lactic acid bacteria, 6 parts of glucose, 0.45 - 0.55 parts of xylo - oligosaccharide, 0.7 parts of vitamins, 0.7 parts of sodium chloride, 0.04 - 0.06 parts of calcium chloride, 0.3 parts of fructo - oligosaccharide, 0.1 part of preservative, 80 parts of water, and 0.09 parts of magnetic composite particles.

3. The black tea fermentation process according to claim 1, characterized in that The lactic acid bacteria is a lactobacillus composition, which includes, by weight: 1.5 parts of Lactobacillus plantarum, 4 parts of Lactobacillus paracasei, 0.5 parts of Lactobacillus rhamnosus, and 0.5 parts of Lactobacillus acidophilus.

4. The black tea fermentation process according to claim 1, wherein The magnetic composite particles include magnetic nanoparticles and cellulase. The magnetic nanoparticles load cellulase to form cellulase magnetic composite particles, and the cellulase loading amount is 30 mg / g of particles.

5. The black tea fermentation process according to claim 4, characterized in that, The magnetic nanoparticles have a core - shell structure, the core layer is Fe3O4, the shell layer is SiO2, and the mass ratio of Fe3O4 to SiO2 is 1:0.

7.

6. The black tea fermentation process according to claim 4, characterized in that, The magnetic composite particles also include calcium ion magnetic composite particles and xylo - oligosaccharide magnetic composite particles, and the addition amounts are 0.06 parts and 0.04 parts respectively; The calcium ion magnetic composite particles have a calcium chloride loading amount of 20 mg / g of particles, and the xylo - oligosaccharide magnetic composite particles have a xylo - oligosaccharide loading amount of 25 mg / g of particles.

7. The black tea fermentation process according to claim 6, wherein The calcium ion magnetic composite particles are prepared by cross - linking with a chitosan - CaCl2 mixed solution, and the mass ratio of Fe3O4, SiO2 to chitosan is 1:0.7:0.3; the xylo - oligosaccharide magnetic composite particles are prepared by encapsulation with a poly(N - isopropylacrylamide) thermosensitive hydrogel, and the mass ratio of Fe3O4, SiO2 to poly(N - isopropylacrylamide) is 1:0.7:0.

2.

8. The black tea fermentation process according to claim 1, characterized in that, In step (4), combined with an alternating magnetic field for staged control. Specifically, during the cell - wall breaking period (0 - 12 h), the stirring speed is 150 r / min, and the magnetic field intensity is a continuous 0.1 T; During the conversion period (12 - 24 h), the stirring speed is 100 r / min, and the magnetic field is turned off; During the recovery period (24 - 28 h), the stirring speed is 80 r / min, the magnetic field intensity is 0.1 T, the pulse frequency is 2 Hz, and the duty cycle is 50%.

9. The black tea fermentation process according to claim 8, characterized in that, The frequency of the alternating magnetic field generator is 50 Hz, and the magnetic induction intensity is 0.1 T.

10. The black tea fermentation process according to claim 8, characterized in that, In step (4), magnetic particles are added in stages. Specifically, during the cell - wall breaking period, calcium ion magnetic composite particles and xylo - oligosaccharide magnetic composite particles are added, and the temperature is raised to 35 °C at a rate of 0.3 °C / h; During the conversion period, xylo - oligosaccharide magnetic composite particles are added and maintained at 32 ± 0.5 °C; During the recovery period, the temperature is lowered to 25 °C at a rate of 3.5 °C / h.

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