An ionic liquid-based tea fermentation process

Abstract

The present invention relates to a tea fermentation process based on ionic liquids, belonging to the field of microbial technology. The present invention provides a tea fermentation process based on ionic liquids. After mixing tea residues with ionic liquids to form a fermentation substrate, a bacterial liquid is inoculated, and anaerobic fermentation is carried out in a bioreactor to obtain a fermentation product. By combining ionic liquids with bacterial fermentation and using ionic liquids to replace water as a fermentation reagent, the present invention can synergistically improve the fermentation efficiency of tea residues, release antioxidant substances in the tea residues, and thus improve the utilization rate of tea residues. At the same time, the low oxygen content of ionic liquids is more conducive to the anaerobic fermentation of bacteria, and ionic liquids also have good recyclability. After fermentation, they can be recovered and reused through simple treatment, effectively reducing reagent consumption and fermentation costs, and bringing higher economic benefits and sustainability to the tea fermentation process.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a tea fermentation process based on ionic liquids. Background Art

[0002] After tea is brewed or processed, tea residues will be generated, and the tea residues still contain rich nutrients such as tea polyphenols. Currently, the commonly used methods for treating tea residues are landfill and incineration, which not only cause environmental pollution but also waste the rich antioxidant substances remaining in the tea residues. In addition, there are also secondary extractions of tea residues to utilize the antioxidant substances in the tea residues, but most of the existing extraction technologies are traditional extraction methods, which are difficult to fully extract the bioactive substances therein, and many effective components still remain in the tea residues.

[0003] Therefore, there is an urgent need for a method that can fully extract the antioxidant substances from tea residues. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a tea fermentation process based on ionic liquids that can fully extract the antioxidant substances from tea residues.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a tea fermentation process based on ionic liquids, in which tea residues and ionic liquids are mixed as a fermentation substrate and then inoculated with a bacterial solution, and anaerobic fermentation is carried out in a bioreactor to obtain a fermentation product;

[0007] The chemical structural formula of the ionic liquid is , where R in the formula is -CH2-CH3 or -CH2-CH2-CH2-CH3;

[0008] The bacteria include Clostridium butyricum and / or Enterococcus faecalis.

[0009] Currently, most of the traditional reagents for anaerobic fermentation are mainly water. Although water as a reagent has advantages such as low cost and easy availability, its solubility is limited (the solubility of some substrates or products in water is relatively low, which may limit the metabolic efficiency of microorganisms and the accumulation of target products), its oxygen content is high (it has an adverse effect on some anaerobic fermentation processes, thus affecting the growth and metabolic pathways of fermentation strains), and its volatility is relatively high (it may accelerate the water loss of the system under high-temperature fermentation conditions, affecting the overall reaction stability). As a single reagent, its ability to regulate the fermentation system is weak, and it is difficult to provide a stable pH and ionic strength, resulting in poor controllability of the fermentation process.

[0010] As a new type of "green reagent", ionic liquids have not been widely used in the field of assisted fermentation. However, due to their unique properties, ionic liquids exhibit obvious advantages in fermentation. Their low volatility reduces the risk of environmental pollution, and their strong solubility is conducive to the mass transfer of substrates and metabolites, promoting the growth and metabolism of microorganisms. In addition, the low dissolved oxygen content of ionic liquids provides an ideal environment for anaerobic fermentation, while reducing the oxidation risk of target products and enhancing their stability and activity. Given these characteristics, ionic liquids are not only suitable for tea residue fermentation but may also play an important role in the fermentation of other organic wastes. This invention systematically studies for the first time the effect of ionic liquids in significantly improving the fermentation efficiency of tea residues and the yield of bioactive substances under specific fermentation conditions, opening up a new technical path for the high-value utilization of tea residues.

[0011] Based on this, by combining ionic liquids with bacterial fermentation and using ionic liquids to replace water as the fermentation reagent, the present invention can synergistically improve the fermentation efficiency of tea residues, destroy the cell structure of tea residues, increase the cell membrane permeability, release the antioxidant substances inside the cells, and thus improve the utilization rate of tea residues. At the same time, the low oxygen content of ionic liquids is more conducive to the anaerobic fermentation of bacteria, and ionic liquids also have good recyclability. After fermentation, they can be recovered and reused through simple treatment, effectively reducing reagent consumption and fermentation costs, and bringing higher economic benefits and sustainability to the tea fermentation process.

[0012] As a preferred embodiment of the tea fermentation process described in the present invention, the tea residues can be replaced with solid tea products containing antioxidant substances such as fresh tea leaves, tea powder, and fermented tea leaves.

[0013] As a preferred embodiment of the tea fermentation process described in the present invention, the chemical structural formula of the ionic liquid is , and the bacterium is Clostridium butyricum.

[0014] As a preferred embodiment of the tea fermentation process described in the present invention, the chemical structural formula of the ionic liquid is , and the bacterium is Enterococcus faecalis.

[0015] As a preferred embodiment of the tea fermentation process described in the present invention, the mass percentage concentration of the ionic liquid is < 12 wt%.

[0016] As a preferred embodiment of the tea fermentation process described in the present invention, the mass percentage concentration of the ionic liquid is 1 - 11 wt%.

[0017] As a preferred embodiment of the tea fermentation process described in the present invention, the mass percentage concentration of the ionic liquid is 10 wt%.

[0018] As a preferred embodiment of the tea fermentation process of the present invention, the mass of the ionic liquid accounts for 30-60% of the total mass of the fermentation substrate.

[0019] As a preferred embodiment of the tea fermentation process of the present invention, the mass of the ionic liquid accounts for 35-55% of the total mass of the fermentation substrate.

[0020] As a preferred embodiment of the tea fermentation process of the present invention, the mass of the ionic liquid accounts for 35% of the total mass of the fermentation substrate. Through condition optimization, the present invention finds that when the mass of the ionic liquid accounts for 35% of the total mass of the fermentation substrate, the content of antioxidant substances in the fermentation product is the highest.

[0021] As a preferred embodiment of the tea fermentation process of the present invention, the conditions for anaerobic fermentation are to ferment at 25-55 °C for 4-11 days in an anaerobic environment.

[0022] As a preferred embodiment of the tea fermentation process of the present invention, the conditions for anaerobic fermentation are to ferment at 30-50 °C for 5-10 days in an anaerobic environment.

[0023] As a preferred embodiment of the tea fermentation process of the present invention, the conditions for anaerobic fermentation are to ferment at 40 °C for 10 days in an anaerobic environment. Through condition optimization, the present invention finds that when fermenting at 40 °C for 10 days, the content of antioxidant substances in the fermentation product is the highest.

[0024] As a preferred embodiment of the tea fermentation process of the present invention, the bacterial liquid is obtained by inoculating bacteria into a culture solution and shaking and culturing until OD 600 = 0.5-0.8.

[0025] As a preferred embodiment of the tea fermentation process of the present invention, the bacterial liquid is obtained by inoculating bacteria into a culture solution and shaking and culturing until OD 600 = 0.5. When OD 600 is 0.5, the bacteria are in the logarithmic growth phase, and the bacterial vitality is better at this time. When inoculated into the fermentation substrate, they can grow and reproduce better.

[0026] As a preferred embodiment of the tea fermentation process of the present invention, the ratio of the bacterial liquid to the tea residue and the ionic liquid is bacterial liquid: (tea residue + ionic liquid) = (1-5) mL: 10 g.

[0027] As a preferred embodiment of the tea fermentation process of the present invention, the ratio of the bacterial liquid to the tea residue and the ionic liquid is bacterial liquid: (tea residue + ionic liquid) = (2-4) mL: 10 g.

[0028] As a preferred embodiment of the tea fermentation process of the present invention, the ratio of the bacterial liquid to the tea residue and the ionic liquid is bacterial liquid: (tea residue + ionic liquid) = 2 mL: 10 g.

[0029] As a preferred embodiment of the tea fermentation process of the present invention, the Enterococcus faecalis is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO. M2016249.

[0030] As a preferred embodiment of the tea fermentation process of the present invention, the Clostridium butyricum is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO. M2019071.

[0031] As a preferred embodiment of the tea fermentation process of the present invention, the formula of the PY culture solution is 20 g / L peptone, 10 g / L yeast extract, 2 g / L disodium hydrogen phosphate, 0.5 g / L cysteine-HCl, and the pH is adjusted to 7.0 - 7.2.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) By combining the ionic liquid with bacterial fermentation, the present invention uses the ionic liquid to replace water as the fermentation reagent, which can synergistically improve the fermentation efficiency of the tea residue, destroy the cell structure of the tea residue, increase the cell membrane permeability, release the active substances in the cells, and thus improve the availability of the tea residue.

[0034] (2) The tea fermentation process of the present invention has low fermentation cost and simple operation. Only by controlling the key fermentation conditions (such as temperature, mass percentage of the ionic liquid, etc.), high-efficiency production can be achieved. The whole fermentation process can be flexibly adjusted according to the actual situation, which is suitable for large-scale commercial applications, helps to reduce the production cost, and improves the feasibility of industrial operation.

[0035] (3) The ionic liquid selected in the present invention has low oxygen content, which is beneficial to the anaerobic fermentation of bacteria. Moreover, the ionic liquid also has good recyclability. After the fermentation is completed, it can be recovered and reused through simple treatment, effectively reducing the reagent consumption and fermentation cost, reducing the potential environmental pollution risk, and maximizing the retention of antioxidant, anti-inflammatory and other active ingredients in the tea, bringing higher economic benefits and sustainability to the tea fermentation process. Description of the Drawings

[0036] Figure 1 It is a scanning electron micrograph of the fermentation products obtained in Example 3 (A), Example 4 (B), Comparative Example 1 (C), Comparative Example 2 (D), Comparative Example 3 (E) and Comparative Example 4 (F) in Effect Example 2 of the present invention, as well as the tea residue (G) without fermentation treatment. Detailed Embodiments

[0037] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] For other materials, reagents, etc. used in the following examples and effect examples, unless otherwise specified, they can all be obtained from commercial channels.

[0039] The experimental operations (such as bacterial culture, bacterial activation, etc.) mentioned in the following examples and effect examples are all conventional techniques in the art, and reference can be made to books such as "Experimental Course of Molecular Biology" (2011, Higher Education Press), "Molecular Biology" (2017, Science Press), "Microbiological Experiments" (2019, Science Press), etc.

[0040] The chemical structural formula of ionic liquid A is , hereinafter referred to as EMIMCL, purchased from Shanghai Macklin Biochemical Co., Ltd., with the product number E809289.

[0041] The chemical structural formula of ionic liquid B is , named BMIMCL, purchased from Shanghai Macklin Biochemical Co., Ltd., with the product number B802693.

[0042] The preparation method of the above ionic liquid is as follows: Take 1 g of ionic liquid powder and add it to 9 mL of water to prepare a 10 wt% ionic liquid, and the ionic liquid of other concentrations is prepared according to this method.

[0043] Enterococcus faecalis Y17 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO. M2016249, and was donated by Teacher Li Shengkang of Shantou University.

[0044] Clostridium butyricum CG3 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO. M2019071, and was donated by Teacher Li Shengkang of Shantou University.

[0045] The anaerobic fermentation described in the following examples, comparative examples and effect examples is as follows: After transferring the fermentation substrate into an anaerobic bottle, use nitrogen to flush the anaerobic bottle for at least 5 minutes to ensure an anaerobic environment, then seal the bottle mouth with a rubber stopper, and seal the rubber stopper and the anaerobic bottle with an aluminum cap. Bacterial inoculation is carried out by injecting into the anaerobic bottle with a sampling needle.

[0046] The specific operations for detecting epicatechin and epigallocatechin by liquid chromatography in the following examples, comparative examples and effect examples are as follows:

[0047] The extract was loaded onto a column and subjected to preparative column chromatography using an Agilent Prep 100A C18 column (50×250 mm, 10 µm) equipped with an Agilent 1290 liquid chromatography system. The chromatographic peaks were monitored in real time using gradient elution, and the fractions containing high-purity epicatechin were precisely collected. The flow rate was 0.3 mL / min, the injection volume was 20 μL, and the detection wavelength was 278 nm. High-purity epicatechin and epigallocatechin were used as standards. The eluent consisted of 0.1% (v / v) formic acid (solution A) and pure methanol (solution B). The specific program was as follows:

[0048] 0 - 2 min, 75% (v / v) A;

[0049] 2 - 9 min, 75 - 66% (v / v) A;

[0050] 9 - 9.1 min, 66 - 58% (v / v) A;

[0051] 9.1 - 30 min, 58 - 55% (v / v) A;

[0052] 30 - 40 min, 55% (v / v) A;

[0053] 40 - 40.1 min, 55% - 75% (v / v) A;

[0054] 40.1 - 60 min, 75% (v / v) A.

[0055] In the following examples, comparative examples, and effect examples, the formula of the PY culture medium was 20 g / L peptone, 10 g / L yeast extract, 2 g / L disodium hydrogen phosphate, 0.5 g / L cysteine-HCl, and the pH was adjusted to 7.0 - 7.2.

[0056] Pretreatment is required for observing samples using a scanning electron microscope. The specific operation can be completed with reference to the following literature. Specifically, tea residue samples under different fermentation conditions were taken, and the samples were gold-plated to improve their surface conductivity, thus meeting the requirements for imaging with a scanning electron microscope (SEM). After sputtering, the samples were evenly dispersed on the surface of the aluminum foil for easy photography and observation. Then, a ZEISS Gemini SEM 450 scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to image the samples. During the operation, the acceleration voltage was set to 5.0 kV, the magnification was 7000 times, and the SE2 detector was used to obtain the surface morphology image of the TW samples.

[0057] References for pretreatment of scanning electron microscope samples: Jun X, Deji S, Ye L, et al. Micromechanism of ultrahigh pressure extraction of active ingredients from green tea leaves[J]. Food Control, 2011, 22(8): 1473-1476.

[0058] Example 1

[0059] To investigate the effects of ionic solution concentration on Clostridium butyricum CG3 and Enterococcus faecalis Y17, ionic solutions with different concentrations were co-cultured with CG3 or Y17. The specific scheme is as follows:

[0060] Add 100 μL of ionic liquid, 50 μL of bacterial solution, 50 μL of PY culture medium, and 50 μL of liquid paraffin to each well of a 96-well plate. After mixing, measure the OD in an enzyme-linked immunosorbent assay (ELISA) reader every half hour 600 , for 24 h. Calculate the growth of CG3 and Y17 in different ionic liquids according to the OD600 value. The results are shown in Table 1.

[0061] Table 1 Effects of different concentrations of ionic liquids on Y17 and CG3

[0062]

[0063] As shown in Table 1, as the concentration of the ionic liquid increases, the inhibitory effect on the growth of CG3 and Y17 gradually increases. Therefore, selecting an ionic liquid with a concentration < 12 wt% for fermentation with CG3 or Y17 can avoid limited bacterial growth. Considering comprehensively, 10 wt% is the optimal concentration of the ionic liquid. In addition, the combination of BMIMCL and Y17 is better than that with CG3, and the combination of EMIMCL and CG3 is better than that with Y17. Therefore, the combinations of 10 wt% BMIMCL + Y17 and 10 wt% EMIMCL + CG3 are selected in the following.

[0064] Example 2

[0065] To investigate the effects of different fermentation temperatures, fermentation times, and ionic liquid contents on the tea fermentation process, the above parameter conditions were optimized. The specific scheme is as follows:

[0066] S1. Inoculate CG3 or Y17 into PY culture medium respectively and anaerobically culture at 37 °C and 250 rpm until OD 600 = 0.5 to obtain CG3 and Y17 bacterial solutions respectively;

[0067] S2. Mix 10 wt% of ionic liquid A or 10 wt% of ionic liquid B with tea residues according to a specific ratio to obtain a fermentation substrate. Inoculate the CG3 and Y17 bacterial solutions obtained in step S1 into the fermentation substrate at an inoculation amount of 20% (v / w) respectively, and anaerobically ferment for 10 days at different temperatures to obtain a fermentation product. The fermentation parameters are set as shown in Table 2. The 20% (v / w) inoculation amount means inoculating 2 mL of the bacterial solution per 10 g of the fermentation substrate.

[0068] S3. Take 1 mL of the fermentation product obtained in step S2 and mix it with 10 mL of 70 v / v% ethanol, and extract at 70 °C for 1 h to obtain an extract.

[0069] S4. Use liquid chromatography technology to qualitatively and quantitatively analyze epicatechin and epigallocatechin in the extract obtained in step S3. The results are shown in Table 2.

[0070] Table 2 Effects of different fermentation parameters on the contents of epicatechin and epigallocatechin in the fermentation broth

[0071]

[0072] As shown in Table 2, under the conditions of the same fermentation temperature and the mass percentage of the ionic liquid, the contents of epicatechin and epigallocatechin in the fermentation broth after 10 days of fermentation are higher. For example, when fermenting at 40 °C and 35 wt% ionic liquid, the contents of epicatechin and epigallocatechin in the fermentation broth after 10 days of fermentation are increased by 7.39% and 11.96% (epicatechin) and 130.74% and 203.28% (epigallocatechin) respectively compared with the fermentation broth after 5 days of fermentation. This shows that fermenting for 10 days can release more active ingredients in the tea residues.

[0073] Under the conditions of the same fermentation time and the mass percentage of the ionic liquid, the epicatechin and epigallocatechin in the fermentation broth show a trend of first increasing and then decreasing with the increase of temperature, and the contents of epicatechin and epigallocatechin in the fermentation broth at 40 °C are higher than those in the fermentation broth at 30 °C and 50 °C. For example, when fermenting for 10 days and at 35 wt% ionic liquid, the contents of epicatechin and epigallocatechin in the fermentation broth fermented at 40 °C are increased by 102.72% and 95.08% (epicatechin) and 34.13% and 28.57% (epigallocatechin) respectively compared with the fermentation broth fermented at 30 °C, and the contents of epicatechin and epigallocatechin in the fermentation broth fermented at 40 °C are increased by 54.81% and 29.73% (epicatechin) and 21.56% and 22.98% (epigallocatechin) respectively compared with the fermentation broth fermented at 50 °C. This shows that when the fermentation temperature is 40 °C, more active ingredients in the tea residues can be released.

[0074] Under the conditions of the same fermentation time and fermentation temperature, different mass percentages of ionic liquids have different effects on the contents of epicatechin and epigallocatechin in the fermentation broth. More commonly, when the mass percentage of the ionic liquid is relatively low (35 wt%), the content of epigallocatechin is relatively high. Therefore, considering comprehensively, an ionic liquid with a mass percentage of 35% is preferably used for fermentation.

[0075] In summary, fermentation products with high antioxidant substances can be obtained when the mass percentage of the ionic liquid is 35 - 55% and the fermentation conditions are fermentation at 30 - 50 °C for 5 - 10 days. And the optimal conditions are that the mass percentage of the ionic liquid is 35% and the fermentation condition is fermentation at 40 °C for 10 days.

[0076] Example 3

[0077] This example provides an ionic liquid-based tea fermentation process, including the following steps:

[0078] A1. Inoculate Y17 into PY culture medium respectively, and anaerobically culture at 37 °C and 250 rpm until OD 600 = 0.5 to obtain Y17 bacterial solutions respectively;

[0079] A2. Mix 10 wt% BMIMCL with tea residues to obtain a fermentation substrate. At this time, the mass of BMIMCL accounts for 35% of the total mass of the fermentation substrate. Inoculate the Y17 bacterial solution obtained in step A1 into the fermentation substrate at an inoculation amount of 20% (v / w), oscillate at 160 rpm for 15 min to mix the bacterial solution and the fermentation substrate evenly, and anaerobically ferment at 40 °C for 10 days to obtain a fermentation product. The 20% (v / w) inoculation amount means inoculating 2 mL of the bacterial solution per 10 g of the fermentation substrate.

[0080] Example 4

[0081] This example provides an ionic liquid-based tea fermentation process, including the following steps:

[0082] B1. Inoculate CG3 into PY culture medium respectively, and anaerobically culture at 37 °C and 250 rpm until OD 600 = 0.5 to obtain CG3 bacterial solutions respectively;

[0083] B2. Mix 10 wt% EMIMCL with tea residues to obtain a fermentation substrate. At this time, the mass of EMIMCL accounts for 35% of the total mass of the fermentation substrate. Inoculate the CG3 bacterial solution obtained in step B1 into the fermentation substrate at an inoculation amount of 20% (v / w), and anaerobically ferment at 40 °C for 10 days to obtain a fermentation product. The 20% (v / w) inoculation amount means inoculating 2 mL of the bacterial solution per 10 g of the fermentation substrate.

[0084] Comparative Example 1

[0085] This comparative example provides a tea fermentation process based on ionic liquid. Its steps are similar to those of Example 3, with the difference that BMIMCL in step A2 is replaced by water, and the remaining steps and parameters remain unchanged.

[0086] Comparative Example 2

[0087] This comparative example provides a tea fermentation process based on ionic liquid. Its steps are similar to those of Example 4, with the difference that EMIMCL in step B2 is replaced by water, and the remaining steps and parameters remain unchanged.

[0088] Comparative Example 3

[0089] This comparative example provides a tea fermentation process based on ionic liquid. Its steps are similar to those of Example 3, with the difference that the Y17 bacterial solution in step A2 is replaced by PY culture solution, and the remaining steps and parameters remain unchanged.

[0090] Comparative Example 4

[0091] This comparative example provides a tea fermentation process based on ionic liquid. Its steps are similar to those of Example 4, with the difference that the CG3 bacterial solution in step B2 is replaced by PY culture solution, and the remaining steps and parameters remain unchanged.

[0092] Effect Example 1

[0093] Quantify the contents of epicatechin and epigallocatechin in the fermentation substrates obtained in Examples 3 - 4 and Comparative Examples 1 - 4 according to the operations in steps S3 - S4 of Example 2. The results are shown in Table 3.

[0094] Table 3 Determination results of the contents of epicatechin and epigallocatechin in the fermentation substrates obtained in different groups

[0095]

[0096] As shown in Table 3, overall, the contents of epicatechin and epigallocatechin in the fermentation products obtained in Examples 3 and 4 are higher than those in the comparative examples. The sum of epicatechin (4.17 mg / g) and the sum of epigallocatechin (19.51 mg / g) in Comparative Example 1 and Comparative Example 3 are both lower than those in Example 3 (5.96 mg / g, 39.18 mg / g). The epicatechin content in Example 3 is increased by 42.92% compared with the sum of Comparative Example 1 and Comparative Example 3, and the epigallocatechin content in Example 3 is increased by 100.82% compared with that of Comparative Example 1 + 3. The sum of epicatechin (3.27 mg / g) and the sum of epigallocatechin (30.09 mg / g) in Comparative Example 2 and Comparative Example 4 are both lower than those in Example 4 (40.73 mg / g). The epicatechin content in Example 4 is increased by 57.49% compared with the sum of Comparative Example 2 and Comparative Example 4, and the epigallocatechin content in Example 4 is increased by 35.36% compared with the sum of Comparative Example 2 and Comparative Example 4, indicating that combining ionic liquid with CG3 or Y17 for anaerobic fermentation can synergistically increase the content of antioxidant substances in the fermentation product, thereby improving the utilization rate of tea residue.

[0097] Effect Example 2

[0098] The tea residues in the fermentation products obtained in Examples 3 - 4 and Comparative Examples 1 - 4 were observed by scanning electron microscopy, and the results are shown in Figure 1 .

[0099] Under anaerobic conditions, butyric acid bacteria can produce metabolic products such as butyric acid, acetic acid, hydrogen, and carbon dioxide through the glycolysis and butyric acid fermentation pathways. Butyric acid is a short-chain fatty acid that can reduce the local environmental pH and has strong cell membrane penetration ability, which helps to destroy the structural stability of the plant cell wall.

[0100] As a facultative anaerobic lactic acid bacterium, Enterococcus faecalis can also produce organic acids (such as lactic acid and acetic acid) and certain extracellular enzymes. Its metabolic activity not only enhances the acidic environment in the system but also further destroys the integrity of the cell wall and improves the cell membrane permeability. In particular, the lactic acid produced by it can, to a certain extent, assist butyric acid bacteria in forming a more stable anaerobic microenvironment, promoting the accumulation of butyric acid and strengthening the enzymatic reaction process.

[0101] In addition, both butyric acid bacteria and Enterococcus faecalis can secrete a variety of hydrolases, such as cellulase, hemicellulase, and protease, etc. These enzymes can synergistically decompose the main structural polysaccharides and cell wall proteins in tea residue, promoting the release of cell contents.

[0102] Meanwhile, the introduction of ionic liquid significantly changes the physicochemical properties of the tea residue surface. As a green solvent, ionic liquid has a low surface tension and strong polarity, and can physically or chemically interact with lignin, cellulose, etc. in the tea residue cell wall, reducing the cell wall rigidity and enhancing the swelling property of the material and the accessibility of enzymes. This pretreatment effect of structural loosening provides a good foundation for subsequent microbial metabolic activities, facilitating the infiltration of bacterial metabolites and the rapid release of active ingredients.

[0103] Based on this, compared with the tea residues of Figure 1 D, 1F, and 1G, Figure 1 a large number of bacteria are colonized on the surface of the tea residues of Figure 1 A, 1B. The bacteria utilize the nutrients on the tea residue surface for growth and metabolism, and even form holes ( Figure 1 the direction indicated by the red arrow in A) and depressions ( Figure 1 the dotted part in B) on the tea residue surface. This is because under the action of the ionic liquid, the cell wall of the tea residue and its rigidity are damaged, and the surface tension and solubility are changed, promoting the release of cell contents, enabling the bacteria to colonize well on the tea residue. Supplemented by various enzymes, organic acids, etc. secreted by the bacteria, the tea residue cell wall is further broken, and the cell membrane permeability is enhanced, so that more active ingredients (epicatechin, epigallocatechin, etc.) in the tea residue are released, thereby synergistically improving the utilization rate of the tea residue. If only the ionic liquid is used for fermentation, the ionic liquid can only damage the structure of the tea residue cell wall, but cannot cause a large amount of release of the tea residue cell contents and cannot further increase the content of active ingredients ( Figure 1 C, 1E); when the bacteria ferment in an environment without ionic liquid, since the tea residue cell wall, surface tension, and solubility of the tea residue are not damaged by the ionic liquid, it is difficult for the bacteria to colonize on the tea residue surface in large numbers (

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An ionic liquid-based tea fermentation process, characterized in that, Mix tea dregs with an ionic liquid to form a fermentation substrate, inoculate with a bacterial liquid, and perform anaerobic fermentation in a bioreactor to obtain a fermentation product; The chemical structural formula of the ionic liquid is , where R is -CH2-CH3 or -CH2-CH2-CH2-CH3; The bacterium is Clostridium butyricum and / or Enterococcus faecalis; the Clostridium butyricum is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO. M2019071, and the Enterococcus faecalis is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO. M2019071; The mass percentage concentration of the ionic liquid is 1-11 wt%; The ratio of the bacterial liquid to the tea dregs and the ionic liquid is bacterial liquid: (tea dregs + ionic liquid) = (1-5) mL: 10 g.

2. The tea fermentation process according to claim 1, characterized in that, The mass of the ionic liquid accounts for 30-60% of the total mass of the fermentation substrate.

3. The tea fermentation process according to claim 2, characterized in that, The mass of the ionic liquid accounts for 35-55% of the total mass of the fermentation substrate.

4. The tea fermentation process according to claim 1, wherein The conditions for the anaerobic fermentation are to ferment at 25-55 °C for 4-11 days in an anaerobic environment.

5. The tea fermentation process according to claim 4, characterized in that, The conditions for the anaerobic fermentation are to ferment at 30-50 °C for 5-10 days in an anaerobic environment.

6. The tea fermentation process according to claim 1, characterized in that, The bacterial liquid is obtained by inoculating bacteria into a culture medium and culturing them with shaking until OD 600 = 0.5 - 0.

8.

7. The tea fermentation process according to claim 1, characterized in that, The ratio of the bacterial liquid to the tea dregs and the ionic liquid is bacterial liquid: (tea dregs + ionic liquid) = (2-4) mL: 10 g.

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