The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea

Abstract

1 Abstract The present invention belongs to the field of beverage technology and provides a method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea, which involves adding a portion of mixed sugar to hot water and stirring until completely dissolved; adding blended black tea, steeping, and filtering to remove the tea leaves; cooling the solution to room temperature; adding the fermentation starter liquid; gently placing the starter culture (SCOBY) on the surface; covering with breathable cloth to ensure air circulation; setting the initial fermentation temperature to 28°C; and fermenting for a total of 13 days. Starting on day 2 of fermentation, sugar is gradually added and the temperature is adjusted; after 8 days, the temperature is maintained at 20°C for an additional 5 days. The end of fermentation is marked by the pH stabilizing between 3.3 and 3.5, with the fermented liquid having a rich aroma and ideal acidity. The fermented liquid is then filtered to remove microbial biomass and suspended solids, and stored at - 20°C. This invention aims to obtain fermented black tea rich in phenolic and flavonoid compounds, which has strong inhibitory effects on specific strains. Abstract The present invention belongs to the field of beverage technology and provides a method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea, which involves adding a portion of mixed sugar to hot water and stirring until completely dissolved; adding blended black tea, steeping, and filtering to remove the tea leaves; cooling the solution to room temperature; adding the fermentation starter liquid; gently placing the starter culture (SCOBY) on the surface; covering with breathable cloth to ensure air circulation; setting the initial fermentation temperature to 28°C; and fermenting for a total of 13 days. Starting on day 2 of fermentation, sugar is gradually added and the temperature is adjusted; after 8 days, the temperature is maintained at 20°C for an additional 5 days. The end of fermentation is marked by the pH stabilizing between 3.3 and 3.5, with the fermented liquid having a rich aroma and ideal acidity. The fermented liquid is then filtered to remove microbial biomass and suspended solids, and stored at - 20°C. This invention aims to obtain fermented black tea rich in phenolic and flavonoid compounds, which has strong inhibitory effects on specific strains. 1 20 24 27 82 66 10 D ec 2 02 4 1 0 D e c 2 0 2 4 2 0 2 4 2 7 8 2 6 6 1 0 D e c 2 0 2 4 2 0 2 4 2 7 8 2 6 6

Description

TECHNICAL FIELD The present invention belongs to the field of beverage technology, and particularly relates to a method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea. BACKGROUND OF THE INVENTION Kombucha is a traditional fermented acidic beverage, typically made from black tea, sugar, and water. The microbial culture of kombucha consists of a complex consortium of acetic acid bacteria, yeast, lactic acid bacteria, and other microorganisms. Under suitable conditions, raw materials such as sucrose are biotransformed through fermentation, resulting in a sweet and tangy drink. During the fermentation process of kombucha, lactic acid bacteria, yeast, and acetic acid bacteria interact with each other, regulating and promoting the formation of the flavor and quality of kombucha. CN110150420A discloses a method for preparing kombucha rich in antioxidants, where water, sucrose, and black tea leaves are mixed in a weight ratio of (125-130):(12.5-13):(1-1.5) and boiled to form the base liquid. The base liquid is then inoculated with a seed liquid consisting of Bacillus aceticus at a concentration of 10A5-10A7 CFU / mL, Acetobacter xylinum at 10A6-10A8 CFU / mL, and Saccharomyces cerevisiae at 10A5-10A7 CFU / mL in a volume ratio of 1:1:1 to form the fermentation liquid. The volume ratio of the mixed seed liquid to the base liquid is (3-3.5):(97-97.5). The fermentation liquid is placed in a container with an open end covered with gauze, and fermented at 25-30°C with a humidity of 30%-40% for 10 days to obtain kombucha. This invention shortens the fermentation cycle of kombucha, resulting in kombucha with higher total flavonoid and total phenol content and stronger antioxidant properties. However, the traditional kombucha fermentation process has a long cycle 2024278266   10 Dec 2024 and is sensitive to factors such as raw materials, microbial strains, and fermentation conditions. Additionally, the carbon sources, nitrogen sources, and nutrients available for microbial growth in the tea are relatively low. Furthermore, tea polyphenols and caffeine possess antibacterial properties. As a result, the traditional fermentation process of kombucha yields a lower tea concentration and acidity, leading to a lower content of phenolic and flavonoid compounds, which is not conducive to the wider application of kombucha. Currently, there is an urgent need to develop a kombucha production method that has high antioxidant activity and antibacterial properties, while being simple in process. STATEMENT OF INVENTION To address the issues in the background technology, the present invention provides a method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea. The resulting kombucha product has a high content of polyphenols and flavonoids, with significantly enhanced antioxidant and antibacterial properties. The specific technical solution is as follows: The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea includes the following steps: S1. Prepare the sugar tea solution; Add a portion of the mixed sugar to hot water and stir until completely dissolved; Add the blended black tea, steep, and filter to remove the tea leaves; Furthermore, in step S1, add 50% of the total mass of mixed sugar, with the water temperature at 80°C, resulting in a sugar concentration of 47.2 g / L. Add mixed sugar, with the following mass percentages: Glucose: 46%, which can be rapidly utilized by microorganisms to promote growth. Fructose: 24%, used to enhance the flavor of the fermentation liquid and support yeast metabolism. Sucrose: 13%, used for continuous energy supply to support the growth of acetic acid bacteria. Maltose: 17%, used to promote enzyme production in molds and increase the release of phenolic compounds. Add blended black tea at a concentration of 13.8 g / L, steep at 80°C for 10 minutes, 2024278266   10 Dec 2024 then turn off the heat and continue steeping for an additional 12 minutes. Add blended black tea in the following mass ratio: Darjeeling tea: 62%; Keemun tea: 38%. S2. Cooling and inoculation; Cool the solution to room temperature; add the fermentation starter liquid; Gently place the starter culture (SCOBY) on the surface; Furthermore, the starter culture includes: Lactobacillus plantarum ATCC 14917, for efficient biotransformation of phenolic compounds to enhance antioxidant properties. Komagataeibacterxylinus ATCC 53524, used for the production of acetic acid and glucuronic acid, enhancing antibacterial properties. Saccharomyces cerevisiae ATCC 9763, used to promote fermentation, produce secondary metabolites, and enhance flavor. Aspergillus oryzae ATCC 42149, used to produce p-glucosidase, promoting the release of phenolic compounds. Mix the above-mentioned strains in the following mass ratio to prepare the fermentation starter liquid: Lactobacillus plantarum: 35%; Komagataeibacterxylinus: 32%; Saccharomyces cerevisiae:24%; Aspergillus oryzae:9%. S2 The fermentation starter liquid is added at a concentration of 200 mL / L, with the starter culture concentration being 11.8 g / L. S3. Fermentation; Cover with breathable cloth to ensure air circulation; The initial fermentation temperature is 28°C; The total fermentation time is 13 days; Starting on day 2 of fermentation, gradually add sugar and adjust the temperature; After 8 days, maintain the temperature at 20°C and continue fermentation for an additional 5 days. On days 2, 4, 6, and 8 of fermentation, add 22%, 15%, 9%, and 4% of the remaining sugar mass, respectively; When adding the sugar, dissolve it in a small amount of hot water, then cool it and 2024278266   10 Dec 2024 add it to the fermentation liquid. On days 2, 4, 6, and 8 of fermentation, the temperatures are as follows: 28°C, 24°C, 22°C, and 20°C; After 8 days, maintain the temperature at 20°C and continue fermentation for an additional 5 days. The initial pH of fermentation is 4.5, and the target pH is maintained between 3.3 and 3.5; Measure the pH daily. If the pH drops too quickly, reduce the sugar addition or extend the addition interval. S4. End of fermentation and storage The end of fermentation is indicated by the pH stabilizing between 3.3 and 3.5, with the fermented liquid exhibiting a rich aroma and ideal acidity. Filter the fermented liquid to remove microbial biomass and suspended solids. Store the fermented liquid at-20°C. The present invention aims to obtain fermented black tea rich in phenolic and flavonoid compounds, which exhibits strong inhibitory effects on specific strains, including Staphylococcus aureus MCTIC 4163, Escherichia coli NCTIC 8196, Pseudomonas aeruginosa MUA26, and Bacillus cereus MU-A44. DETAILED DESCRIPTION OF THE INVENTION The embodiments of the present invention are intended to be illustrative, and not to limit the scope of the present invention. Any slight difference in nutrient composition due to the difference in milk source should not be used as a reason for the inconsistency between the embodiment of the present invention and the specification, so the present invention aims to protect the purpose of adding nutrients, rather than to make the ratio particularly strict. The ground is limited to a specific value. The present invention also provides the following embodiments: The materials used in the method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea are as follows: (1) Blended black tea The selected black tea comes from high-altitude regions due to its high content of phenolic and flavonoid compounds, including: 2024278266   10 Dec 2024 Darjeeling Tea: Produced in the Himalayan region of India, it has a fresh flavor and a high phenolic content. Keemun Tea: Produced in Qimen County, Anhui Province, China, it has a distinctive floral and fruity aroma and is rich in flavonoids. The blending ratio is: Darjeeling Tea 62%; Keemun Tea 38% o This combination maximizes the content of phenolic and flavonoid compounds in the tea. (2 ) Fermentation starter liquid rich in specific microorganisms The microbial strains include: Lactobacillus plantarum ATCC 14917, for efficient biotransformation of phenolic compounds and enhancement of antioxidant properties. Komagataeibacterxylinus ATCC 53524, for efficient biotransformation of phenolic compounds and enhancement of antioxidant properties. Saccharomyces cerevisiae ATCC 9763,used to promote fermentation, produce secondary metabolites, and enhance flavor. Aspergillus oryzae ATCC 42149, used to produce P-glucosidase and promote the release of phenolic compounds. Mix the above-mentioned microbial strains in the following mass ratio to prepare the fermentation starter liquid: Lactobacillus plantarum: 35%; Komagataeibacterxylinus: 32%; Saccharomyces cerevisiae:24%; Aspergillus oryzae:9%o (3)Selection of Mixed Sugars The sugar combination, according to mass percentage, includes: Glucose: 46%,rapidly utilized by microorganisms, promoting growth. Fructose: 24%,used to enhance the flavor of the fermentation liquid and support yeast metabolism. Sucrose: 13%,provides continuous energy to support the growth of acetic acid bacteria. Maltose: 17%,provides continuous energy to support the growth of acetic acid bacteria. 2024278266   10 Dec 2024 The method for preparing antibacterial kombucha liquid through the fermentation of highland blended black tea and mixed sugars includes the following steps: S1. Prepare the sugar tea solution; Add 50% of the total amount of mixed sugar to water at 80°C, stirring until completely dissolved, resulting in a concentration of 47.2 g / L. Add the blended black tea at a concentration of 13.8 g / L, steep at 80°C for 10 minutes, then turn off the heat and continue steeping for an additional 12 minutes; afterward, filter to remove the tea leaves. S2. Cooling and Inoculation; Cool the solution to room temperature; add the fermentation starter liquid at a concentration of 200 mL / L. Gently place the starter culture (SCOBY) at a concentration of 11.8 g / L on the surface of the solution. S3. Fermentation; Cover with breathable cloth to ensure proper air circulation; The initial fermentation temperature is 28°C; The total fermentation time is 13 days; Starting on day 2 of fermentation, gradually add sugar and adjust the temperature; On days 2, 4, 6, and 8 of fermentation, add 22%, 15%, 9%, and 4% of the remaining sugar mass, respectively; When adding the sugar, dissolve it in a small amount of hot water, then cool it and add it to the fermentation liquid. On days 2, 4, 6, and 8 of fermentation, the temperatures are as follows: 28°C, 24°C, 22°C, and 20°C; After 8 days, maintain the temperature at 20°C and continue fermentation for an additional 5 days. This gradual addition of sugar and temperature reduction process helps ensure that the fermentation products are produced more steadily and stably, making it easier to control and allowing the flavor to evolve gradually. The initial pH of fermentation is 4.5, and the target pH is maintained between 3.3 and 3.5;Measure the pH daily. If the pH drops too quickly, reduce the sugar addition or extend the intervals between sugar additions. S4. End of Fermentation and Storage 2024278266   10 Dec 2024 The end of fermentation is marked by the pH stabilizing between 3.3 and 3.5, and the fermented liquid having a rich aroma and ideal acidity. The fermented liquid is then filtered to remove microbial biomass and suspended solids. It is stored at -20°C for future use. Mechanisms for Enhancing Antibacterial and Antioxidant Properties of the Invention: 1. Enrichment of Phenolic and Flavonoid Compounds; Tea selection: Highland black tea is rich in phenolic compounds. Microbial action: The enzymes produced by Aspergillus oryzae promote the release of tea polyphenols. Lactobacillus plantarum biotransforms phenolic compounds, producing more biologically active forms. 2. Enhanced Antibacterial Properties; High concentrations of phenolic compounds have a disruptive effect on cell membranes, inhibiting bacterial growth. Organic acids such as acetic acid and lactic acid lower the pH, inhibiting pathogenic bacteria. Experiments show that high concentrations of phenolic compounds and organic acids have a significant inhibitory effect on S. aureus, E. coli, P. aeruginosa, and B. cereus. 3. Enhanced Antioxidant Properties; Secondary metabolites generated during fermentation, such as phenolic acids and flavonoids, exhibit strong antioxidant activity. Microbial metabolism produces enzymes that promote the accumulation of antioxidants. As shown in Figure 1, the experiments (K1-K17) compared 17 groups, where K15 (the technology of this embodiment) achieved the highest values for both phenolic compounds and flavonoids. The 17 groups tested different fermentation parameters, including fermentation time, temperature, starter liquid, and process variations. Among these, K15 (the technology of this embodiment) achieved the best overall antibacterial properties. Table 1: Antibacterial Test Results Antibacterial Activity Analysis Results of Kombucha Liquid (mm) Sample S. aureus MCTIC 4163 E. coli NCTIC 8196 P. aeruginosa MUA26 B. cereus MU-A44 K1 6.00±0.00c 7.00±0.00g 6.00±0.00 6.00±0.00 K2 6.00±0.00c 6.00±0.00g 6.00±0.00 6.00±0.00 2024278266   10 Dec 2024 K3 6.00±0.00c 9.00±0.00f 6.60±0.18 6.00±0.00 K4 7.00±0.00c 13.33±1.15bc 7.00±0.20 8.00±0.00 K5 8.00±0.00c 12.67±0.58bcd 6.00±0.00 8.67±0.58 K6 6.00±0.00c 11.00±0.00de 6.00±0.00 6.00±0.00 K7 6.30±0.00c 12.00±0.00cd 6.00±0.00 8.67±0.58 K8 6.00±0.00c 6.00±0.00g 6.50±0.17 6.00±0.00 K9 6.00±0.00c 6.00±0.00g 6.00±0.00 6.00±0.00 K10 6.00±0.00c 6.00±0.00g 6.00±0.00 6.00±0.00 K11 11.00±0.00a 14.00±0.00cd 6.00±0.00 12.00±0.00 K12 6.00±0.00c 11.67±0.58cde 6.00±0.00 8.67±0.58 K13 6.00±0.00c 11.67±0.58cde 6.00±0.00 8.67±0.58 K14 6.00±0.00c 10.00±0.00ef 6.00±0.00 6.00±0.00 K15 9.43±0.57b 17.10±0.00b 8.30±0.34 14.33±0.87 K16 6.00±0.00c 12.00±0.00cd 6.00±0.00 8.33±0.58 K17 6.00±0.00c 12.67±2.08bcd 6.00±0.00 9.20±0.68 Positive control 52.00 25.00 16.00 20.00 It should be noted that the above embodiments are provided to illustrate the technical solutions of the present invention, not to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will understand that they can still modify the technical solutions described in these embodiments, or make equivalent substitutions to some or all of the technical features. These modifications or substitutions do not depart from the scope of the technical solutions of the present invention as described in the embodiments.

Claims

2024278266   10 Dec 2024WHAT WE CLAIM IS1 > The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea is characterized by the following steps:S1. Prepare the sugar tea solution;Add part of the mixed sugar to hot water and stir until completely dissolved;Add the blended black tea, steep, and filter to remove the tea leaves;S2. Cooling and inoculation;Cool the solution to room temperature; add the fermentation starter liquid;Gently place the starter culture (SCOBY) on the surface;S3. Fermentation;Cover with breathable cloth to ensure air circulation;The initial fermentation temperature is 28°C;The total fermentation time is 13 days;Starting on day 2 of fermentation, gradually add sugar and adjust the temperature; After 8 days, maintain the temperature at 20°C and continue fermentation for an additional 5 days;S4. End of fermentation and storageThe end of fermentation is indicated by the pH stabilizing between 3.3 and 3.5, with the fermented liquid exhibiting a rich aroma and ideal acidity;Filter the fermented liquid to remove the microbial biomass and suspended solids; store the fermented liquid at -20°C.2^ The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S1, 50% of the total mass of mixed sugar is added, the water temperature is 80°C, and the sugar concentration is 47.2 g / L.3 > The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S1, mixed sugar is added, comprising the following mass percentages:Glucose: 46%, which can be rapidly utilized by microorganisms to promote growth; Fructose: 24%, used to enhance the flavor of the fermentation liquid and support yeast metabolism;Sucrose: 13%, used for continuous energy supply to support the growth of acetic acid bacteria;Maltose: 17%, used to promote the enzyme production of molds and increase the release of phenolic compounds.4^ The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S1, blended black tea is added at a concentration of 13.8 g / L, steeped at 80°C for 10 minutes, after which the heating is turned off and the tea is allowed to steep for an additional 12 minutes.5^ The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step2024278266   10 Dec 2024S1, the blended black tea is added in the following mass ratio: Darjeeling tea: 62%; Keemun tea: 38%.6 > The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S2, the concentration of the fermentation starter liquid added is 200 mL / L, and the starter culture concentration is 11.8 g / L.7> The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S2, the starter culture added includes:Lactic acid bacteria Lactobacillus plantarum ATCC 14917, for efficient biotransformation of phenolic compounds and enhancing antioxidant properties;Acetic acid bacteria Komagataeibacter xylinus ATCC 53524, used for producing acetic acid and glucuronic acid, enhancing antibacterial properties;Saccharomyces cerevisiae ATCC 9763, used to promote fermentation, produce secondary metabolites, and enhance flavor;Aspergillus oryzae ATCC 42149, used to produce P-glucosidase and promote the release of phenolic compounds;Mix the above-mentioned strains in the following mass ratio to prepare the fermentation starter liquid:Lactobacillus plantarum: 35%;Komagataeibacterxylinus: 32%;Saccharomyces cerevisiae:24%;Aspergillus oryzae: 9%o7^ The method for preparing high-antibacterial and high-antioxidant kombucha liquid using highland blended black tea according to claim 1, is characterized in that in step S3,On days 2, 4, 6, and 8 of fermentation, add 22%, 15%, 9%, and 4% of the remaining sugar mass, respectively;When adding the sugar, dissolve it in a small amount of hot water, then cool it and add it to the fermentation liquid;On days 2, 4, 6, and 8 of fermentation, the temperatures are as follows: 28°C, 24°C, 22°C, and 20°C, respectively;After 8 days, maintain the temperature at 20°C and continue fermentation for an additional 5 days;The initial pH of fermentation is 4.5, and the target pH during fermentation is maintained between 3.3 and 3.

5. The pH value is measured daily, and if the pH decreases too quickly, reduce the amount of sugar added or extend the addition interval.