Probiotic-enzymolysis synergistic fermentation functional tea beverage and preparation method thereof

Abstract

The invention relates to the technical field of preparation of fermented drinks, in particular to a probiotic-enzymolysis synergistic fermentation functional tea beverage and a preparation method thereof. Aiming at the technical problems that the content of polyphenols in tea leaves is high, the growth of probiotics is inhibited, the use of mixed enzymes lacks time sequence control, and the activity of the probiotics is reduced in the shelf life, the invention provides the following scheme: carrying out directional enzymolysis pretreatment on tea raw materials, adopting response type enzyme-controlled release capsules to assist fermentation, and constructing a three-stage fermentation control strategy; tea soup before fermentation is pretreated through a pulsed electric field, initial infectious microbe load is reduced, and low-temperature sterilization is achieved; after fermentation, the probiotics are microencapsulated, so that the survival rate of the probiotics stored at normal temperature is remarkably increased. The functional tea beverage prepared by the invention is high in viable count of probiotics, good in stability, rich and harmonious in flavor and remarkable in nutritional and health-care effects.

Description

Technical Field

[0001] This invention relates to the field of fermented beverage preparation technology, specifically to a functional tea beverage prepared by probiotic-enzymatic synergistic fermentation and its preparation method. Background Technology

[0002] Tea is rich in bioactive substances such as tea polyphenols and catechins, possessing various health benefits including antioxidant properties, lipid-lowering effects, and regulation of intestinal flora. However, the high polyphenol content also presents some processing challenges: on the one hand, high concentrations of tea polyphenols inhibit many probiotics, affecting probiotic fermentation using tea as a substrate. This is a major reason why directly adding probiotics to traditional tea beverages often fails to guarantee the activity of the bacterial strains. Therefore, how to overcome the inhibitory effect of tea polyphenols on probiotics is a pressing technical problem to be solved when developing probiotic tea beverages.

[0003] On the other hand, the stability and taste of tea beverages are also key concerns for the industry. Traditional tea beverage processing suffers from problems such as cloudy tea liquor, browning, and a strong bitter taste. Currently, various chemical or physical stabilization technologies have been developed to address sedimentation and browning, such as adding antioxidants, adjusting pH, aseptic packaging, and deoxygenation treatment; excessive bitterness is usually masked by adding sweeteners. However, these methods struggle to simultaneously improve the bioavailability of functional components in tea beverages. Especially for functional tea beverages, the goal is to retain the beneficial active substances in tea and enhance their functions, but large molecular active ingredients such as tea polyphenols are not easily absorbed by the human body and are easily lost or transformed during processing.

[0004] Fermentation technology offers new insights into improving the quality and functionality of tea beverages. Through the action of microorganisms and their enzyme systems, the biotransformation of active ingredients in tea can be achieved, improving stability and bioavailability, and imparting a unique flavor to fermented beverages. Recent developments in microbially fermented teas demonstrate that microbial fermentation reduces bitterness, increases aroma compounds, and imparts new health benefits. However, traditional fermented teas primarily rely on natural or mixed-culture fermentation, making precise control of the fermentation process difficult, especially for fermentation involving the artificial inoculation of probiotics, which still faces several technical bottlenecks. For example, existing technology CN115997833A discloses a freeze-dried functional tea drink suitable for women migrating from plains to plateau environments, belonging to the category of functional tea drink preparation; it includes 30-50 parts of Cordyceps militaris and Ganoderma lucidum fermented liquid, 10-15 parts of fresh mulberry juice, 0.1-0.5 parts of microencapsulated soy isoflavones, 0.05-0.1 parts of taurine, 0.01-0.1 parts of coenzyme Q10, 0.1-0.5 parts of selenium-enriched yeast, and 1-3 parts of probiotic powder; another method for preparation includes step 1, preparing Cordyceps militaris and Ganoderma lucidum fermented liquid; step 2, preparing soy isoflavone microcapsules; step 3, preparing mulberry juice; step 4, freeze-drying; step 5, packaging; and step 6, quality inspection; it improves the taste and benefits of the beverage through the addition of beneficial ingredients and fermentation process, but its preparation process is relatively simple, the retention of effective ingredients is questionable, and the taste is not clearly described.

[0005] In summary, existing technologies for developing probiotic fermented tea beverages suffer from several drawbacks, including ineffective elimination of the antibacterial effects of tea polyphenols, lack of coordinated control between enzymatic treatment and fermentation processes, and low probiotic survival rates. Therefore, there is an urgent need for an innovative technological solution that can coordinate the synergistic effects of enzymatic hydrolysis and probiotic fermentation, optimize fermentation conditions in stages, and protect the activity of probiotics in the finished product. This would allow for the preparation of functional tea beverages that possess both the functional components of tea and highly active probiotics, resulting in a beverage with excellent flavor. Summary of the Invention

[0006] The purpose of this invention is to provide a functional tea beverage prepared by probiotic-enzymatic co-fermentation and its preparation method. Addressing the problems of tea polyphenol inhibition of probiotics, uncontrollable enzyme action timing, and low probiotic survival rate in existing technologies, this invention proposes a comprehensive solution. By combining enzymatic pretreatment with probiotic fermentation, supplemented by process control and microencapsulation protection measures, the nutritional and functional properties of the tea beverage are enhanced, significantly improving the number of viable probiotics and storage stability.

[0007] The specific technical solution is as follows: A functional tea beverage with probiotic-enzymatic hydrolysis co-fermentation and its preparation method are disclosed. The functional tea beverage is pretreated by low-temperature plasma-assisted compound enzymatic hydrolysis, then sterilized by pulsed electric field after the addition of nutrient factors. It is then fermented by three-stage probiotic parameter regulation involving pH-responsive microcapsule controlled-release enzymes. Finally, it is packaged and sealed after probiotic microencapsulation treatment to complete the preparation.

[0008] Specifically, the steps include the following: S1: The tea raw material is crushed and subjected to low-temperature plasma treatment. After treatment, it is added to purified water preheated at 90-95℃ and extracted for 5 minutes. After cooling, compound plant hydrolytic enzyme is added. After enzymatic hydrolysis for 1 hour, immobilized enzyme microbeads are added to the tea soup and the temperature is maintained for 3-4 hours. After the reaction, the tea soup is heated to 85℃ and kept warm for 5 minutes to inactivate the enzyme system. The tea soup is then filtered to obtain enzymatically hydrolyzed tea soup A. S2: Add carbon source and nutrients to enzymatically hydrolyzed tea soup A, then place it in a pulsed electric field for treatment. After treatment, cool it quickly to obtain sterile tea soup B and transfer it into a sterile fermentation tank. S3: The sterile tea soup B is fermented in three stages. The temperature is gradually reduced and the pH is adjusted in each stage. Probiotics, enzyme-controlled release capsules, growth-promoting nutrient additives and flavor-enhancing yeast are added in sequence to carry out fermentation and obtain fermented tea soup C. S4: Quickly cool the fermented tea soup C to 10℃, let it stand for 1 hour, filter it through a 200-mesh filter to clarify it, take 50% of the volume of the filtrate for probiotic microencapsulation treatment, disperse it back into the fermented tea soup, add an external antibacterial agent, and then package and seal it to obtain a functional tea beverage.

[0009] Furthermore, the low-temperature plasma treatment described in S1 includes the following processing parameters: discharge power of 200W, air as the working gas, and processing time of 60 seconds.

[0010] Further, in S1, after cooling, a compound plant hydrolytic enzyme is added, wherein the temperature after cooling is 50°C, and the compound plant hydrolytic enzyme includes a combination of cellulase and pectinase, or a combination of cellulase and xylanase; the immobilized enzyme microbeads in S1 are specifically immobilized tanninase / β-glucosidase microbeads.

[0011] Furthermore, the carbon source and nutrients described in S2 include fructooligosaccharides or prebiotic xylooligosaccharides, and the nutrients include a combination of zinc gluconate and yeast selenium, or a combination of yeast selenium and zinc sulfate; the pulsed electric field treatment described in S2 has specific operating parameters including an electric field strength of 12-18 kV / cm, a pulse frequency of 300 Hz, a pulse duration of 50 μs, and a treatment time of 2-5 ms.

[0012] Furthermore, the three-stage fermentation process described in S3 includes Stage I, which is the rapid proliferation stage of probiotics, lasting 12 hours; Stage II, which is the synthesis stage of probiotic metabolites, lasting 24 hours; and Stage III, which is the flavor enhancement stage, lasting 12 hours.

[0013] Furthermore, the gradual reduction of temperature described in S3 specifically includes reducing the temperature from 35-37°C to 30-34°C, and finally reducing it to 23-28°C; the pH regulation described in S3 includes two stages: maintaining pH 6.0 by automatically adding neutralizing solution during the probiotic proliferation period, and naturally regulating pH by utilizing the metabolism of probiotics.

[0014] Furthermore, the probiotics described in S3 include a combination of Lactobacillus plantarum, Lactobacillus thermophilus, and Lactobacillus paracasei, or Lactobacillus rhamnosus; the enzyme-controlled release capsule described in S3 contains a combination of β-glucosidase, ferulic acid esterase, and laccase; the growth-promoting nutrient additive described in S3 includes yeast extract and cysteine; the flavor-enhancing yeast described in S3 includes a combination of Saccharomyces cerevisiae SC and Hansenula polymorpha, Xylosporinus, or Kluyveromyces.

[0015] Furthermore, the microencapsulation process of probiotics described in S4 specifically includes: centrifuging to collect probiotics and a portion of yeast, then washing twice with sterile physiological saline and resuspending; mixing the bacterial suspension into a 2% w / v sodium alginate solution, stirring well, and then adding it dropwise to a CaCl2 solution to form gel beads; after solidifying for 30 minutes, transferring the gel beads to a 90% concentration chitosan solution with a 0.4% degree of deacetylation and soaking for 30 minutes to form a chitosan layer; then transferring them to a 1% concentration pectin solution and letting them stand for 30 minutes to form the outermost membrane; and finally rinsing the surface acid with sterile water.

[0016] The present invention also provides a functional tea beverage with probiotic-enzymatic co-fermentation, wherein the functional tea beverage contains active ingredients of tea, probiotic fermentation products and encapsulated probiotic bacteria.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention utilizes targeted enzymatic hydrolysis pretreatment to significantly hydrolyze ester-type catechins and complex polyphenols in tea infusion, converting them into small-molecule phenolic acids and sugars, thereby reducing the inhibitory effect of tea polyphenols on probiotics. The gallic acid and other substances produced by enzymatic hydrolysis no longer have a significant inhibitory effect on probiotics at low concentrations, but instead provide antioxidant benefits; while the released glucose directly increases the fermentable carbon source.

[0018] (2) The present invention uses an enzyme-controlled release capsule design to precisely match the action of the enzyme with the fermentation stage. Enzyme interference is avoided during the cell proliferation period, ensuring the growth rate of probiotics; enzymes are released in a timely manner during the acid production period, realizing synchronous enzymatic fermentation and improving substrate utilization and product conversion rate.

[0019] (3) This invention achieves decoupling optimization of cell growth and metabolite generation through three-stage temperature and pH regulation. The high-temperature pH control strategy in stage I increases cell biomass; the low-temperature pH conditions in stage II, combined with the action of enzymes, increase lactic acid production; and the low-temperature aroma production in stage III increases the concentration of volatile esters and preserves the aroma more fully.

[0020] (4) This invention reduces the number of contaminants in the fermentation liquid by introducing a pulsed electric field heatless sterilization pretreatment, so that the final product can meet commercial sterility requirements without high-temperature sterilization. Combined with innovative probiotic multilayer microencapsulation protection technology, the shelf life of the product is significantly extended. Attached Figure Description

[0021] Figure 1 This is a flowchart of the functional tea beverage prepared by probiotic-enzymatic hydrolysis co-fermentation according to the present invention; Figure 2 This is the standard curve used for the determination of total polyphenol content in Experimental Example 1 of the present invention; Figure 3 This is a comparison chart showing the determination of substance content at different stages during the preparation process of Example 1 in Experimental Example 1 of the present invention; Figure 4 This is a comparison chart of the test results for Experiment Example 2 of the present invention. Detailed Implementation

[0022] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0023] This invention proposes a functional tea beverage with probiotic-enzymatic hydrolysis co-fermentation and its preparation method, as shown in the appendix. Figure 1 The diagram shown is a flowchart of the functional tea beverage prepared by probiotic-enzymatic hydrolysis co-fermentation according to the present invention. The specific technical solution is as follows: 1. Targeted enzymatic hydrolysis pretreatment of tea raw materials Low-temperature plasma is used to modify the surface of tea leaves. At a lower temperature, the active particles generated by the plasma etch the surface of the tea leaves, forming micron-sized pores and cracks, thus increasing the specific surface area. This treatment helps with the subsequent penetration of enzymes and the leaching of tea components. At the same time, the plasma also has a bactericidal effect, which can partially reduce the microbial load on the surface of the tea leaves.

[0024] After plasma treatment, a complex plant hydrolytic enzyme system, preferably containing cellulase, pectinase, and amylase, is added to the tea leaves to decompose polysaccharides and macromolecules in the tea cell walls, thereby releasing soluble components such as tea polyphenols and tea polysaccharides. Next, a co-immobilized tanninase and β-glucosidase system is added. Tanninase specifically hydrolyzes ester-type polyphenols, such as removing the galloyl group from ester-type catechins like epigallocatechin gallate (EGCG), generating free epicatechin and gallic acid. This not only reduces the antibacterial activity of tea polyphenols, weakening their inhibitory effect on probiotics, but also produces gallic acid and glucose as potential carbon sources or metabolic promoters for probiotics. β-glucosidase can act on certain glycoside components in the tea leaves, releasing glucose and corresponding aglycones, increasing the content of fermentable sugars and releasing potential aroma compounds from the tea.

[0025] 2. Optimization of culture medium nutrients In the enzymatically hydrolyzed tea liquor base, specific nutrient factors are added to optimize the fermentation substrate for probiotics. A certain amount of functional oligosaccharides is preferably added as prebiotics to promote probiotic proliferation. Simultaneously, trace amounts of zinc and selenium are supplemented to enhance the activity of bacterial enzymes and their antioxidant capacity. Zinc is a cofactor for many enzymes, and appropriate amounts of zinc ions can promote the growth and metabolism of probiotics. Selenium can be accumulated by probiotics to form selenoamino acids, giving the product additional nutritional value. Through nutrient premixing technology, oligosaccharides and organic trace elements are pre-dissolved uniformly and then added to the enzymatically hydrolyzed tea liquor, resulting in a more balanced carbon-to-nitrogen ratio in the fermentation medium. This satisfies the various factors required for probiotic growth, facilitating the acquisition of high viable cell counts and high acid production capacity.

[0026] 3. Responsive enzyme-controlled release capsules to assist fermentation To address the adverse effects of simultaneously adding multiple enzymes on early-stage fermentation strains, this invention employs a pH-responsive enzyme-controlled release capsule containing a combination of enzymes: β-glucosidase, ferulic acid esterase, and laccase. Ferulic acid esterase hydrolyzes ferulic acid ester bonds in plant cell walls, releasing phenolic acids such as ferulic acid; laccase oxidizes polyphenolic compounds, promoting the conversion and polymerization of flavor compounds. Considering that laccase generally exhibits high activity under neutral to alkaline conditions, while the fermentation environment in this invention is acidic in the later stages, the laccase used is a modified adaptive enzyme with its optimal pH adjusted from approximately 7.0 to 5.5. The aforementioned combination of enzymes is covalently or physically immobilized within the core matrix of the microcapsule, ensuring that the enzymes are not lost or prematurely involved in the reaction before release. The capsule shell is made of pH-sensitive biomaterials, preferably chitosan-algin composite gel or a special phenolic resin coating, characterized by stability and insolubility near neutral conditions, and swelling or dissolution when the ambient pH drops below 5.0.

[0027] The enzyme capsules are added to the culture medium at the start of fermentation. Initially, the capsules remain intact, preventing enzyme release and thus avoiding interference with probiotic growth. As the probiotics grow and enter the acid-producing phase, the pH gradually decreases to approximately 5.0. At this point, the capsule shell rapidly disintegrates, releasing the internal enzyme components instantaneously into the system. This occurs during the peak acid-producing stage of fermentation, a crucial period for the flavor transformation of substances like tea polyphenols. The enzyme release at this time allows β-glucosidase to further release the potential aroma of the tea leaves; ferulic acid esterase releases ferulic acid, which can subsequently be metabolized by yeast into aroma compounds such as 4-ethylguaiacol; and laccase, under acidic conditions, moderately oxidizes and polymerizes some phenols, reducing bitterness and generating substances that contribute to color and flavor. This responsive release design avoids the potential cell inhibition caused by premature enzyme action in the early stages of fermentation while fully utilizing the acidic environment in the later stages to trigger enzyme participation, enhancing the flavor complexity and quality of the final product.

[0028] 4. Three-stage fermentation control strategy To address the varying needs at different stages of probiotic fermentation, this invention divides the fermentation process into three stages and controls key parameters in each stage to optimize the results: (1) Stage I, cell proliferation period, 0-12 hours, the focus is on rapidly increasing the cell number. The fermentation temperature is set at 35-37℃, the initial pH of the culture medium is adjusted to 6.0 and maintained; sterile air or oxygen is moderately introduced to maintain a dissolved oxygen level of 30-35%; at the same time, growth promoters are added to further shorten the lag period and increase the maximum specific growth rate; at the end of this stage, the cell concentration is greatly increased, the target is OD 600 A pH >8.0 corresponds to a viable cell count of over 10^9 CFU / mL, laying the foundation for efficient acid production in the next stage. It is important to note that pH control at this stage is crucial to prevent premature acidification of the culture medium, which promotes cell accumulation.

[0029] (2) Stage II, product synthesis period, 12-36 hours, focusing on acid production and enzymatic conversion. After the cell density reaches a high level, the external alkali control is stopped, allowing the pH of the fermentation broth to drop freely. When entering Stage II, the temperature is lowered to about 30-34℃. Appropriately lowering the temperature slows down the further rapid proliferation of the cells, and the milder temperature is conducive to the preservation of flavor substances and the synergistic effect of enzymes. At the same time, the pH is triggered to switch to 5.0. The metabolism of lactic acid bacteria will lower the environmental pH from 6 to about 5. At this time, the aforementioned pH response capsules dissolve, releasing the three enzymes contained within, and starting synchronous enzymatic hydrolysis-fermentation. During this stage, lactic acid bacteria metabolize a large amount of sugars in the culture medium into organic acids, especially lactic acid, maintaining the pH at a level of 5.0. Due to acid inhibition, its growth slows down but its metabolism is active, while the released enzymes work synergistically. During this period, the acid production rate and the enzyme reaction rate are matched, achieving further conversion of raw materials and flavor enhancement.

[0030] (3) Stage III, flavor maturation period, 36-48 hours, focuses on aroma and flavor harmony. With the primary fermentation basically complete and sufficient organic acid production, the temperature is further lowered to 23-28℃. This lower temperature helps retain and generate aromatic substances. At the beginning of Stage III, specific aroma-producing strains are introduced into the fermentation system, preferably a small number of yeast strains or aroma-producing lactic acid bacteria for co-fermentation. At this time, the residual sugar in the culture medium is not high, but there is a certain amount of ethanol and a large amount of organic acids. Acidophilic microorganisms such as yeast can still be active at low pH. They will utilize the residual sugar and a small amount of ethanol, and undergo esterification reactions with organic acids to generate various ester aroma substances. Some yeasts can also secrete aroma enzymes to further modify the aroma components of the fermentation broth. Stage III is not aimed at acid production, but rather at allowing the flavors to fully react and blend.

[0031] 5. Sterilization and preservation of probiotic activity The resulting tea beverage, due to its low pH, possesses a certain degree of antibacterial activity. However, to ensure product safety and extend shelf life, appropriate sterilization treatment is still necessary. Considering the need to preserve probiotic activity, this invention does not employ traditional high-temperature pasteurization but instead uses a gentle, non-thermal sterilization method—pulsed electric field (PEF) treatment—on the tea base before fermentation. Before fermentation and inoculation with probiotics, the enzymatically hydrolyzed tea infusion with added nutrients is first treated with PEF (Probiotic-Enzyme-Fuel). The tea infusion is placed in a PEF device and a high-voltage pulsed electric field is applied for tens of microseconds to milliseconds. This strong electric field can destroy the cell membrane of microorganisms and kill most of the live bacteria within microseconds. However, due to the extremely short treatment time, the temperature of the tea infusion hardly rises, and the quality of tea polyphenols, amino acids, etc., is not damaged. This pretreatment greatly reduces the amount of contaminating bacteria in the material, creating a near-non-competitive environment for pure probiotic fermentation. After fermentation, the tea beverage is free from serious contamination by contaminating bacteria throughout the process and can be directly packaged without further high-temperature sterilization. To further ensure the safety of the ready-to-drink process and maintain the survival rate of probiotics, this invention combines probiotic microencapsulation protection technology before filling. The probiotic cells in the fermentation liquid are separated and concentrated, resuspended in sterile water, and then made into calcium alginate gel beads by drop addition. The probiotics are encapsulated in these beads, and then multi-layered encapsulation is used to form storage-resistant microcapsules. The preferred three-layer structure is as follows: the first layer is sodium alginate cross-linked with calcium ions to form a gel framework; the second layer is a positively charged chitosan that forms a polyelectrolyte composite film on the surface of the alginate gel; and the third layer is further coated and neutralized with negatively charged polysaccharides such as pectin or naringenin, improving the density and strength of the microcapsules. Ultimately, the microcapsules are barely visible to the naked eye and do not affect the taste. A small amount of natural antibacterial agents, preferably tea polyphenol extracts, nisin, or cinnamon essential oil, can also be adsorbed or bound to the outer layer of the microcapsules to provide protection against spoilage bacteria in the beverage system. Through the above treatment, probiotics are physically isolated within the microcapsules, isolating them from the influence of adverse external environments. Simultaneously, the gel properties of the microcapsules themselves provide a moist microenvironment, delaying the death of probiotics.

[0032] Example 1 A functional tea beverage produced through probiotic-enzymatic hydrolysis synergistic fermentation and its preparation method are as follows: Core raw material preparation: 100g of high-quality dried green tea leaves, 2 liters of purified water; compound plant hydrolytic enzymes, cellulase and pectinase, with enzyme activities ≥100,000 U / g and ≥80,000 U / g, respectively; immobilized tanninase / β-glucosidase microbeads, with a total tanninase activity ≥500U and a total β-glucosidase activity ≥300U per microbead, and approximately 10mg of enzyme protein per milliliter of microbead suspension; functional fructooligosaccharide powder with an average degree of polymerization of 4; organic zinc fortifier, zinc gluconate; organic selenium fortifier, yeast selenium; lactic acid bacteria inoculum, plant... Lactobacillus LP, cultured at a concentration of 10^9 CFU / mL; yeast inoculum, Hansenula polymorpha and Saccharomyces cerevisiae SC, 10^8 CFU / mL; enzyme-controlled release capsules, each containing ≥1000 U of total β-glucosidase activity, ≥500 U of total ferulic acid esterase activity, and ≥200 U of total laccase activity, with an optimal pH of laccase approximately 5.5; the capsule shell material is a chitosan-alginic acid composite membrane, which completely dissolves within 15 minutes at pH ≤ 5.0; growth-promoting nutrient additives, yeast extract 5g, L-cysteine ​​0.1g.

[0033] S1: Pulverize dried green tea leaves to approximately 60 mesh size. Place 100g of the pulverized tea leaves in a low-temperature plasma treatment chamber and treat for 60 seconds under a discharge power of 200W and air as the working gas to create visible micropores on the surface of the tea leaves. Immediately after treatment, add the tea leaves to 2 L of preheated purified water at 90℃ and extract for 5 minutes, then cool to 50℃. Add 5g of compound plant hydrolytic enzyme and slowly stir for 1 hour for enzymatic hydrolysis. During enzymatic hydrolysis, the viscosity of the tea soup decreases slightly, and the color changes from dark green to brownish-green. Then, add 20mL of immobilized tanninase / β-glucosidase microbeads to the tea soup and maintain a gentle shaking reaction at 50℃ for 3 hours. After the reaction, stop the enzymatic hydrolysis, heat the tea soup to 85℃ and keep it warm for 5 minutes to inactivate the enzyme system. Then filter with double-layer gauze to remove tea residue and enzyme microbeads, obtaining 1.8L of clear enzymatically hydrolyzed tea soup base.

[0034] S2: Add 20g of functional fructooligosaccharides, 0.5g of zinc gluconate, and 0.2g of yeast selenium powder to the above enzymatically hydrolyzed tea infusion. After stirring and dissolving, the resulting culture medium has a total sugar content of 12g / L and a C / N ratio of 20:1. Then, place the culture medium into a pulsed electric field processor, set the field strength to 15kV / cm, the pulse frequency to 300Hz, each pulse to last 50μs, and the treatment time to 2ms. After treatment, quickly cool the tea infusion and transfer it to a sterile fermenter for later use.

[0035] S3: Stage I, adjust the fermenter temperature to 37℃, introduce sterile air to adjust dissolved oxygen to 30% saturation level, add 90mL of Lactobacillus plantarum inoculum to the fermenter aseptically, and add 5g of enzyme-controlled release capsules and mix well to suspend; adjust the fermenter pH control system to set the value to 6.0, and maintain the pH by automatically adding neutralizing solution; add 5g of growth-promoting nutrient additive yeast extract and 0.1g of cysteine, and maintain 37℃ and 30% oxygen solubility for stirring and incubation for 12 hours. Entering stage II, the temperature inside the tank is lowered to 32℃, and the automatic pH control is stopped. The metabolism of lactic acid bacteria causes the pH to drop from 6.0 to about 5.0. When the pH is approximately 5.0, the capsule shells placed in the tank begin to dissolve, indicating that the enzyme components have been released. After that, fermentation is continued at 32℃ in a sealed micro-anaerobic state for 24 hours. In stage III, the temperature of the fermenter is further reduced to 25°C, and sterile air with a volume fraction of 5% is introduced into the fermenter for stirring to prevent the generation of reducing odors under anaerobic conditions, while not significantly affecting the already slow lactic acid fermentation. 20 mL of Saccharomyces cerevisiae SC inoculum and 10 mL of Hansenula polymorpha inoculum are inoculated into the fermentation broth, and 0.5 wt% glucose of the mixture is added. Fermentation continues at 25°C for 12 hours.

[0036] S4: Quickly cool the fermented mixture to 10°C, let it stand for 1 hour to allow a small amount of precipitate to form, filter it through a 200-mesh filter to clarify it, and take 50% of the filtrate for probiotic microencapsulation. The probiotic fraction, including Lactobacillus plantarum and some yeast, was collected by centrifugation. The centrifugation parameters included a centrifugation rate of 5000 rpm and a centrifugation time of 10 min. The microbial suspension was then washed twice with sterile physiological saline and resuspended. The bacterial suspension was mixed into 100 mL of 2% (w / v) sodium alginate solution, stirred well, and then added dropwise to 100 mmol / L CaCl2 solution using a syringe pump to form gel beads. After solidification for 30 minutes, the gel beads were transferred to a 90% concentration chitosan solution with a 0.4% degree of deacetylation (containing 0.1 mol / L acetic acid) and soaked for 30 minutes to form a chitosan layer. Then, the microbeads were transferred to a 1% concentration pectin solution and allowed to stand for 30 minutes to form the outermost membrane, thus obtaining a three-layer probiotic microcapsule. After rinsing the surface acid with sterile water, the microcapsules were dispersed back into the fermented tea liquid.

[0037] Finally, 0.2g of tea polyphenol extract per liter was added to the mixture as an external antibacterial agent. The treated product was then dispensed into containers under ultra-clean conditions, sealed, and the final product, a probiotic functional green tea beverage, was obtained.

[0038] Example 2 The preparation method is the same as in Example 1, except that: Raw materials and enzyme system: Take 100g of oolong tea leaves and grind them into coarse powder. The probiotics selected are Lactobacillus thermophilus liquid and Lactobacillus paracasei liquid, each with a concentration of 10^9 CFU / mL; the flavor strain Xylosomalidase is used for aroma production, with a concentration of 10^8 CFU / mL; the enzyme ratio in the enzyme capsules is similar to that in Example 1, but the total activity of laccase is ≥300U; S1: After plasma treatment, oolong tea powder was extracted in water at 95℃ for 5 minutes; 20 mL of immobilized tanninase / β-glucosidase was gently stirred at 50℃ for 4 hours. S2: 0.3g of yeast selenium powder and 0.2g of zinc sulfate; subjected to pulsed electric field treatment with a field strength of 18kV / cm and a treatment time of 3ms; S3: Inoculate with Lactobacillus thermophilus and Lactobacillus paracasei; maintain pH 6.2 in stage I, cool to 34°C in stage II, cool to 28°C in stage III, and inoculate with 20 mL of xylosomal yeast inoculum; All other steps are the same.

[0039] Example 3 The preparation method is the same as in Example 1, except that: Raw materials and enzyme system: 100g of dark tea raw material (ripe Pu-erh tea); the compound enzyme uses a combination of cellulase and xylanase, with a total enzyme activity of 120,000 U; 15g / L of prebiotic xylooligosaccharide (XOS) is added; the probiotic is Lactobacillus rhamnosus, and the flavor bacteria is Kluyveromyces lactis. S1: After plasma treatment of tea leaves, water is extracted at 95℃ for 5 minutes, and 3g each of cellulase and xylanase are added at 50℃ for 1 hour of enzymatic hydrolysis. Then, an immobilized tannin enzyme system is added for 4 hours of enzymatic hydrolysis. S2: Add 15g of XOS per liter of tea infusion, add glucose until the total sugar reaches 10g / L, and perform pulsed electric field treatment with a field strength of 12kV / cm and a treatment time of 5ms. S3: Inoculate with Lactobacillus rhamnosus, starting at 10^7 CFU / mL; Stage I, 35℃, pH 6.2; Stage II, reduce to 30℃; Stage III, 23℃, inoculate with 20 mL of Kluyveromyces inoculum. All other steps are the same.

[0040] Comparative Example 1 The preparation method is the same as in Example 1, except that: The plasma treatment and enzymatic hydrolysis steps are omitted, and lactic acid fermentation is carried out directly using untreated green tea infusion. 100g of the same green tea as in Example 1 is taken and steeped in hot water at 90℃ for 10 minutes to obtain tea infusion. All other steps are the same.

[0041] Comparative Example 2 The preparation method is the same as in Example 1, except that: At the start of fermentation, the same amount of enzyme (a mixture of β-glucosidase, ferulic acid esterase, and modified laccase) was added directly to the culture medium without encapsulation. The fermentation process still used three-stage temperature control, but since the enzymes had already been released, there was no pH-triggered release phase. All other steps were the same.

[0042] Comparative Example 3 The preparation method is the same as in Example 1, except that: S4 does not undergo probiotic microencapsulation; it is directly bottled and sealed. All other steps are the same.

[0043] Experimental Example 1 The viable cell count, pH and acidity, total polyphenol content, and different types of catechin components in the tea infusion mixture during each stage of pretreatment and fermentation in the preparation process of Example 1 were tested. Each stage included: tea infusion after extraction (Stage A), tea infusion after enzymatic pretreatment (Stage B), tea infusion after fermentation stage I (Stage C), tea infusion after fermentation stage II (Stage D), tea infusion after fermentation stage III (Stage E), and tea infusion after microencapsulation / sterilization treatment (Stage F). The specific test conditions are as follows: (1) Viable bacteria count (CFU count) Methods: Plate count method, referring to standard GB 4789.35-2023 "Food Microbiology Examination - Lactic Acid Bacteria Examination"; Culture medium: selective medium for lactic acid bacteria, MRS agar (containing 0.5 g / L L-cysteine, pH 5.4); Yeast inhibition: Adding 0.2 g / L of actinomycete ketone inhibits the growth of residual yeast; Assay procedure: Take 1 mL of sample and place it in 9 mL of sterile physiological saline (0.85% NaCl), then serially dilute to 10 mL. -7 ; Spread 100 μL of the diluted solution onto an MRS plate and culture it anaerobically at 37°C for 48 h; Select plates with 30–300 CFU for counting, and calculate the unit: CFU / mL.

[0044] (2) pH and acidity determination pH measurement: The calibrated pH meter (accuracy ±0.01) was directly inserted into the sample, and the reading was taken after equilibration at 25°C; Titrated total acid (calculated as lactic acid): Refer to GB / T 12456-2021 "National Food Safety Standard - Determination of Total Acid in Food". The reagents used are 0.1 mol / L NaOH and phenolphthalein indicator (1% ethanol solution). For the determination, take 10 mL of sample, dilute with 20 mL of distilled water, and titrate to the endpoint. The formula for calculating total acid is: Total acid (%) = ,in C This is the NaOH concentration (mol / L). VIt is the titration volume (mL). m The value is the sample mass (g), and 0.09 represents the molar mass of lactic acid (g / mmol).

[0045] (3) Determination of total polyphenol content According to ISO 14502-1:2005 (Determination of polyphenols in tea), the Folin-Schowal reagent method was used for determination. The sample was centrifuged and the supernatant was collected (10,000 rpm, 10 min). After dilution, 0.5 mL of the sample was mixed with 2.5 mL of Folin reagent (10-fold dilution) and reacted in the dark for 5 minutes. Then, 2 mL of 20% Na2CO3 was added, and the volume was adjusted to 10 mL with distilled water. Measure the absorbance (OD) at 765 nm wavelength, and compare with the standard curve (see attached). Figure 2 Calculate the total polyphenol concentration (in gallic acid equivalents).

[0046] (4) Determination of different types of catechin components The determination was performed using high-performance liquid chromatography (HPLC) with an ultraviolet detector. The chromatographic column was a C18 reversed-phase column (5 μm, 250 × 4.6 mm). The mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile. The time concentration gradient of the mobile phase B was 0 min–5%, 15 min–20%, 30 min–25%, and 35 min–5%. The flow rate was 1.0 mL / min. The detection wavelengths were gallic acid 280 nm and EGCG 274 nm. The column temperature was 30°C. The sample was centrifuged (12000 rpm, 15 min) and then filtered through a 0.22 μm filter membrane.

[0047] The test results are shown in Table 1 and Appendix. Figure 3 As shown.

[0048] Table 1 Test Results of Experiment Example 1

[0049] Note: "↑ / ↓" indicates a significant increase / decrease compared to the previous stage; "*" indicates that stage I maintains pH=6.0* by automatically adding neutralizing solution.

[0050] Combined with appendix Figure 3 The analysis of the above results shows that, in terms of viable cell count dynamics, *Lactobacillus plantarum* rapidly proliferated under optimized conditions of 37℃ / pH 6.0 in Stage I, reaching the end of the logarithmic phase after 12 hours; in Stage II, the pH dropping to 5.0 triggered the release of enzyme capsules, and β-glucosidase hydrolyzed glycosidic bonds to release glucose, promoting a viable cell count increase to 1.1 × 10⁻⁶. 9 CFU / mL; in stage III, low temperature (25℃) and yeast competition inhibited the growth of lactic acid bacteria, resulting in a slight decrease in the number of viable bacteria; after microencapsulation, some free viable bacteria were lost during the centrifugation / embedding process, but a large number of active bacteria were retained in the microcapsule core.

[0051] Regarding the transformation of polyphenols, during the enzymatic hydrolysis stage, immobilized enzyme beads cause a large amount of EGCG to hydrolyze, and gallic acid increases significantly as a hydrolysis product. The total polyphenols decrease slightly because of catechin polymerization and enzymatic hydrolysis losses. During the fermentation stage, the enzyme capsules in stage II continue to act, further degrading EGCG. Gallic acid is utilized by microorganisms to synthesize γ-aminobutyric acid (GABA), and the increase slows down.

[0052] Regarding acidity and pH regulation, in stage I, the acid produced by lactic acid bacteria increases the total acidity, but the pH remains at 6.0 due to the maintenance of the neutralization system; in stage II, the pH drops sharply to 3.8 after neutralization stops, and the acidity increases significantly; in stage III, yeast metabolism consumes some organic acids, and the pH drops slightly to 3.6.

[0053] As can be seen from the above process, the enzymatic pretreatment causes a sharp drop in EGCG, ensuring the survival rate of probiotics; in stage II, pH triggers the release of enzyme capsules, which increases gallic acid during fermentation and provides a carbon source for the microbial community; although the number of viable bacteria decreases after microencapsulation, the encapsulation system can achieve a longer shelf life at room temperature.

[0054] Experimental Example 2 The functional tea beverages finally prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to comparative tests on viable bacterial count, pH and acidity, total polyphenol content, and different types of catechin components. Specific testing conditions were the same as in Experiment 1, except that the viable bacterial count was measured after 6 months of storage at room temperature, simulating shelf-life conditions. The test results are shown in Table 2 and Appendix. Figure 4 As shown.

[0055] Table 2 Comparison of Test Results for Experiment Example 2

[0056] Note: "↑ / ↓" indicates a significant increase / decrease compared to Example 1.

[0057] Combined with appendix Figure 4 As can be seen from the above comparison results, in terms of the difference in viable count, the thermophilic lactobacillus in Example 2 has a weaker acid resistance and a slightly lower viable count; xylooligosaccharide (XOS) in Example 3 promotes the proliferation of lactobacillus rhamnosus and increases the viable count; undegraded EGCG in Comparative Example 1 strongly inhibits probiotics and significantly reduces the viable count; and the viable bacteria in Comparative Example 3 without microencapsulation protection are completely inactivated during the shelf life.

[0058] Regarding the conversion of polyphenols, Example 2 showed that prolonged enzymatic hydrolysis resulted in greater degradation of EGCG in oolong tea; Example 3 showed that the fermentation of black tea itself could degrade polyphenols, resulting in a lower total polyphenol content; Comparative Example 1 showed that the lack of enzymatic hydrolysis led to a high residual amount of EGCG; and Comparative Example 2 showed that the enzyme released in advance was hydrolyzed by protease, resulting in a lower EGCG conversion rate.

[0059] Regarding the relationship between acidity and flavor, in Example 2, *Xylosomalemia* produced esters such as ethyl acetate, resulting in lower total acidity and a milder flavor; in Example 3, *Lactobacillus rhamnosus* had weak acid production capacity and lower acidity; in Comparative Example 2, the enzyme inhibited cell growth too early, leading to a significant reduction in lactic acid production and affecting flavor.

[0060] The above analysis shows that Comparative Example 1 demonstrates the necessity of targeted enzymatic hydrolysis and pretreatment; without pretreatment, the EGCG conversion rate is very low, and the number of viable bacteria decreases significantly. Comparative Example 2 demonstrates the advantages of enzyme timing control, as early enzyme release leads to a decrease in conversion rate. Comparative Example 3 verifies the role of the microencapsulated core, whose viable bacteria are completely inactivated during the shelf life, highlighting the protective efficacy of the sodium alginate-chitosan-pectin triple-layer encapsulation for viable bacteria. Examples 2 and 3 show that the present invention can be extended to different types of tea raw materials.

[0061] Experimental Example 3 The tea beverages prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to sensory evaluation of tea infusion quality. The sensory evaluation team consisted of 10 professional tea tasters. Referring to the national standard GB / T 23776-2018 "Sensory Evaluation Method for Tea," the scoring items based on the product characteristics of this invention included color, aroma, taste, mouthfeel, and off-flavor of live bacteria. All samples were uniformly refrigerated (4℃) and equilibrated to 10℃ (optimal drinking temperature) before consumption. The blind testing procedure included: the first round of color / aroma evaluation, where the sample was poured into a transparent cup (50mL), the color was observed, and the aroma was smelled (after standing for 30 seconds, followed by three short inhalations); the second round of taste / mouthfeel evaluation, where 10mL of tea infusion was held in the mouth, held on the tongue for 5 seconds, and then swallowed, recording the instantaneous taste and aftertaste. Each sample was rinsed with water after a 3-minute interval. Data recording used linear scaling to quantify each indicator, and a weighted total score was calculated.

[0062] Specific scoring criteria (out of 100): Table 3. Sensory evaluation method for tea infusion and weighting of each quality factor.

[0063] The specific scoring results are as follows: Table 4 Comparison of Experimental Results for Example 3

[0064] As can be seen from the results in the table above, Example 1 has no significant defects in overall sensory evaluation, Example 2 has slightly low acidity, and the aged aroma of Example 3 masks part of the fermentation aroma; Comparative Example 1 is severely bitter and has a poor taste; Comparative Example 2 has a thin flavor due to premature enzyme inactivation; Comparative Example 3 has a noticeable yeast off-flavor, which affects the taste; In summary, the sensory evaluation of the tea soup basically conforms to the design scheme of this invention.

Claims

1. A method for preparing a functional tea beverage using probiotic-enzymatic synergistic fermentation, characterized in that, The functional tea beverage undergoes low-temperature plasma-assisted enzymatic hydrolysis pretreatment, followed by the addition of nutritional factors and pulsed electric field sterilization. It then undergoes three-stage probiotic parameter-controlled fermentation involving pH-responsive microcapsule-controlled-release enzymes, and is finally packaged and sealed after probiotic microencapsulation.

2. The preparation method of the functional tea beverage with probiotic-enzymatic synergistic fermentation as described in claim 1, characterized in that, Includes the following steps: S1: The tea raw material is crushed and subjected to low-temperature plasma treatment. After treatment, it is added to purified water preheated at 90-95℃ and extracted for 5 minutes. After cooling, compound plant hydrolytic enzyme is added. After enzymatic hydrolysis for 1 hour, immobilized enzyme microbeads are added to the tea soup and the temperature is maintained for 3-4 hours. After the reaction, the tea soup is heated to 85℃ and kept warm for 5 minutes to inactivate the enzyme system. The tea soup is then filtered to obtain enzymatically hydrolyzed tea soup A. S2: Add carbon source and nutrients to enzymatically hydrolyzed tea soup A, then place it in a pulsed electric field for treatment. After treatment, quickly cool it to obtain sterile tea soup B and transfer it into a sterile fermentation tank. S3: The sterile tea soup B is fermented in three stages. The temperature is gradually reduced and the pH is adjusted in each stage. Probiotics, enzyme-controlled release capsules, growth-promoting nutrient additives and flavor-enhancing yeast are added in sequence to carry out fermentation and obtain fermented tea soup C. S4: Quickly cool the fermented tea soup C to 10℃, let it stand for 1 hour, filter it through a 200-mesh filter to clarify it, take 50% of the volume of the filtrate for probiotic microencapsulation treatment, disperse it back into the fermented tea soup, add an external antibacterial agent, and then package and seal it to obtain a functional tea beverage.

3. The preparation method of the functional tea beverage with probiotic-enzymatic hydrolysis synergistic fermentation as described in claim 2, characterized in that, The low-temperature plasma treatment described in S1 has the following parameters: discharge power of 200W, air as working gas, and treatment duration of 60 seconds.

4. The preparation method of the functional tea beverage with probiotic-enzymatic hydrolysis synergistic fermentation as described in claim 2, characterized in that, The addition of the compound plant hydrolytic enzyme after cooling as described in S1, wherein the temperature after cooling is 50°C, and the compound plant hydrolytic enzyme includes a combination of cellulase and pectinase, or a combination of cellulase and xylanase; the immobilized enzyme microbeads described in S1 are specifically immobilized tanninase / β-glucosidase microbeads.

5. The preparation method of the functional tea beverage with probiotic-enzymatic hydrolysis synergistic fermentation as described in claim 2, characterized in that, The carbon source and nutrients described in S2 include fructooligosaccharides or prebiotic xylooligosaccharides, and the nutrients include a combination of zinc gluconate and yeast selenium, or a combination of yeast selenium and zinc sulfate; the pulsed electric field treatment described in S2 has specific operating parameters including an electric field strength of 12-18 kV / cm, a pulse frequency of 300 Hz, a pulse duration of 50 μs, and a treatment time of 2-5 ms.

6. The method for preparing a functional tea beverage with probiotic-enzymatic synergistic fermentation as described in claim 2, characterized in that, The three-stage fermentation process described in S3 includes Stage I, which is the rapid proliferation stage of probiotics, lasting 12 hours; and Stage II, which is the synthesis stage of probiotic metabolites, lasting 24 hours. Phase III is the flavor enhancement phase, which lasts for 12 hours.

7. The method for preparing a functional tea beverage with probiotic-enzymatic synergistic fermentation as described in claim 2, characterized in that, The gradual temperature reduction described in S3 specifically includes reducing the temperature from 35-37℃ to 30-34℃, and finally to 23-28℃. The pH regulation described in S3 includes two stages: maintaining pH 6.0 by automatically adding neutralizing solution during the probiotic proliferation period, and naturally regulating pH by utilizing the metabolism of probiotics.

8. The method for preparing a functional tea beverage with probiotic-enzymatic synergistic fermentation as described in claim 2, characterized in that, The probiotics described in S3 include a combination of Lactobacillus plantarum, Lactobacillus thermophilus, and Lactobacillus paracasei, or Lactobacillus rhamnosus; the enzyme-controlled release capsule described in S3 contains a combination of β-glucosidase, ferulic acid esterase, and laccase; the growth-promoting nutrient additive described in S3 includes yeast extract and cysteine; the flavor-enhancing yeast described in S3 includes a combination of Saccharomyces cerevisiae SC and Hansenula polymorpha, Xylosporinus, or Kluyveromyces.

9. The method for preparing a functional tea beverage with probiotic-enzymatic hydrolysis synergistic fermentation as described in claim 2, characterized in that, The microencapsulation process of probiotics described in S4 specifically includes: centrifuging to collect probiotics and a portion of yeast, then washing twice with sterile physiological saline and resuspending; mixing the bacterial suspension into a 2% w / v sodium alginate solution, stirring well, and then adding it dropwise to a CaCl2 solution to form gel beads; after solidifying for 30 minutes, transferring the gel beads to a 90% concentration chitosan solution with a 0.4% degree of deacetylation and soaking for 30 minutes to form a chitosan layer; then transferring them to a 1% concentration pectin solution and letting them stand for 30 minutes to form the outermost membrane; and finally rinsing the surface acid with sterile water.

10. The probiotic-enzymatic co-fermentation functional tea beverage prepared by the preparation method according to any one of claims 1-9, characterized in that, The functional tea beverage contains active ingredients from tea leaves, probiotic fermentation products, and encapsulated probiotic bacteria.

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