A method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology

Abstract

1 Abstract This invention belongs to the field of beverage technology and provides a method for preparing high-antibacterial kombucha liquid using blended black tea, mixed sugar, and micro-oxygen technology. The process involves heating water, adding part of the mixed sugar, and infusing with highland blended black tea; natural cooling and continued steeping; removing the tea leaves; after the solution temperature decreases, adding kombucha fermentation starter liquid; adding the starter culture and covering with breathable cloth to promote the growth of acetic acid bacteria; on days 2- 4 of fermentation, gradually adding the remaining sugar while monitoring the pH to maintain it between 3.0 and 3.3; maintaining a micro-oxygen environment of 3%-5% by controlling the oxygen supply; introducing lactic acid bacteria during the secondary fermentation stage; and extending the fermentation time at low temperature: on day 8, setting the fermentation temperature to 12°C and extending the fermentation time to 15 days. This invention, using a blend of black tea and sugar, significantly increases the organic acids and exhibits high antibacterial properties compared to kombucha products made with green tea. The method is simple, with high processing efficiency. Abstract This invention belongs to the field of beverage technology and provides a method for preparing high-antibacterial kombucha liquid using blended black tea, mixed sugar, and micro-oxygen technology. The process involves heating water, adding part of the mixed sugar, and infusing with highland blended black tea; natural cooling and continued steeping; removing the tea leaves; after the solution temperature decreases, adding kombucha fermentation starter liquid; adding the starter culture and covering with breathable cloth to promote the growth of acetic acid bacteria; on days 2- 4 of fermentation, gradually adding the remaining sugar while monitoring the pH to maintain it between 3.0 and 3.3; maintaining a micro-oxygen environment of 3%-5% by controlling the oxygen supply; introducing lactic acid bacteria during the secondary fermentation stage; and extending the fermentation time at low temperature: on day 8, setting the fermentation temperature to 12°C and extending the fermentation time to 15 days. This invention, using a blend of black tea and sugar, significantly increases the organic acids and exhibits high antibacterial properties compared to kombucha products made with green tea. The method is simple, with high processing efficiency. 1 20 24 27 82 65 10 D ec 2 02 4 1 0 D e c 2 0 2 4 A b s t r a c t 2 0 2 4 2 7 8 2 6 5 1 0 D e c 2 0 2 4 A b s t r a c t 2 0 2 4 2 7 8 2 6 5

Description

TECHNICAL FIELD The present invention belongs to the field of beverage technology, and particularly relates to a method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology. BACKGROUND OF THE INVENTION Kombucha is a fermented beverage made from tea extract and sugar as substrates, using a symbiotic culture of bacteria and yeast. Kombucha not only retains some of the active components of tea, such as tea polyphenols, caffeine, and alkaloids, but also contains a variety of bioactive substances produced by microbial metabolism during fermentation, including organic acids, vitamins, amino acids, and glucuronic acid. It exhibits multiple health benefits, such as antioxidants, anti-inflammatory, immune-regulatory, liver-protective effects, as well as prevention and treatment of atherosclerosis, hypertension, and cancer. Additionally, the antimicrobial proteins, organic acids, and tea polyphenols produced during kombucha fermentation exhibit a broad-spectrum antibacterial effect, making it a promising natural food preservative. Kombucha can be used in food storage, as supportive therapy for gastrointestinal infections, and to combat antibiotic resistance, serving as an important source of novel antibacterial agents. CN116584563A discloses a method for preparing kombucha with high antioxidant activity and antibacterial properties, belonging to the field of food fermentation processing technology. The method includes the following steps: (1) preparation of tea-sugar solution: sucrose and crushed tea powder are mixed with water in a certain proportion, heated, cooled, and filtered to obtain tea extract; (2) mixed inoculation: kombucha strains are inoculated into the tea-sugar solution prepared in step (1); (3) 2024278265   10 Dec 2024 fermentation: the mixture is statically fermented in the dark at 25°C for 7 days, resulting in kombucha with high antibacterial and antioxidant activity. This invention shortens fermentation time, and the kombucha produced has a clear color, balanced taste, and rich aroma while demonstrating excellent antioxidant capacity, high polyphenol content, and high organic acid content. It also effectively reduces the sucrose content in the beverage. Additionally, the product exhibits strong antibacterial activity, with good inhibitory effects on Escherichia coli and Staphylococcus aureus, meeting contemporary consumer demand for healthy, nutritious, and palatable functional beverages. However, this method primarily uses a single type of black tea as the tea raw material, and research on the organic acid content and antibacterial properties of kombucha products is limited. This lack of research leads to inconsistent product quality, posing significant challenges to the large-scale production of kombucha. Therefore, there is an urgent need to develop a method to enhance the organic acid content and antibacterial properties of kombucha products. STATEMENT OF INVENTION To address the issues identified in the background technology, the present invention provides a method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology, resulting in kombucha with high organic acid content and exceptional antibacterial properties. The specific technical solution is as follows: A method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology, comprising the following processes: Si, Preparation of the sugar solution: The water is heated, and a portion of the mixed sugar is added and stirred until fully dissolved; At this temperature, highland blended black tea is added for steeping; The heat source is then turned off, allowing the tea to cool naturally while continuing to steep. The tea leaves are then removed; Specifically, in step Si, the water is heated to 90°C, and 45% of the total sugar mass 2024278265   10 Dec 2024 of the mixed sugars is added, resulting in an initial sugar solution concentration of 35.2 g / L; Highland blended black tea is added at a concentration of 14.5 g / L and steeped for 6 minutes; the heat source is then turned off, and the tea is steeped for an additional 5 minutes. The mixed sugars comprise the following components by mass percentage: Refined white sugar: 48%; Glucose: 21%; Fructose: 16%; Brown Sugar: 10%; Honey: 5%. The highland blended black tea comprises highland black tea from the Waikato region and highland black tea from the Canterbury region of New Zealand, mixed at a mass ratio of 3:1.7. S2. Cooling and inoculation: The sugared tea solution is cooled to room temperature and then transferred to a temperature-controlled cabinet for further cooling; Once the solution temperature has decreased, kombucha starter culture is added; The starter microbial culture is added, and the container is covered with breathable fabric to promote the growth of acetic acid bacteria; Furthermore, in step S2, the temperature-controlled cabinet lowers the solution temperature to 16°C, the kombucha starter culture is added at a concentration of 83 ml / L, and the solution pH is maintained within the range of 3.0-3.3; The starter microbial culture is added at a concentration of 36 g / L and gently placed on the surface of the solution. S3, Gradual addition of sugar and pH control: During days 2 to 4 of fermentation, the remaining sugar is gradually added, and the pH is monitored to ensure it remains within the range of 3.0-3.3. In step S3, sugar is added on days 2 to 4 in proportions of 25%, 20%, and 10% of the total mass, respectively. When the pH reaches 3.1, the addition of sugar is reduced or 2024278265   10 Dec 2024 paused. S4> Microaerobic fermentation assistance; Microaerobic fermentation environment control: the oxygen supply is regulated to maintain a microaerobic environment with an oxygen concentration of 3%—5%; In the secondary fermentation stage, Lactobacillus plantarum is introduced on the 6th day of fermentation to produce more lactic acid and acetic acid while stabilizing the pH level; Low-temperature extended fermentation: on the 8th day, the fermentation temperature is set to 12°C, extending the fermentation duration to 15 days. The starter microbial culture comprises the following components by mass percentage: Komagataeibacterxylinus ATCC 53524 (Primarily produces acetic acid and glucuronic acid): 50%; Acetobacter pasteurianus JCM 7686 (High-yield acetic acid bacteria): 25%; Leuconostocmesenteroides ATCC 8293(Supports fermentation and stabilizes acidity): 12%; Zygosaccharomyces bailii CBS 680(Acid-tolerant yeast, aids in acetic acid production): 8%; Lactobacillus plantarum (Added during the secondary fermentation stage): 5%. The method for preparing kombucha described in the present invention utilizes blended black tea and sugars, significantly enhancing the organic acid content and antibacterial properties of kombucha products made from black tea. The method is simple to operate, highly efficient, and suitable for large-scale industrial production. 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, 2024278265   10 Dec 2024 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 as described below: The raw materials required for this embodiment include: (1) Types of Black Tea: Highland black tea from the Waikato region of New Zealand and highland black tea from the Canterbury region are blended in a quality ratio of 3:1.7. (2) Fermentation microorganism (mass percentage) pomposition: Komagataeibacterxylinus ATCC 53524 (primarily produces acetic acid and glucuronic acid): 50%; Acetobacter pasteurianus JCM 7686 (high-acid-producing acetic acid bacteria): 25%; Leuconostocmesenteroides ATCC 8293 (auxiliary fermentation, stabilizing acidity): 12%; Zygosaccharomyces bailii CBS 680 (acid-tolerant yeast, aiding in acetic acid production): 8%; Lactobacillus plantarum (added during the secondary fermentation stage): 5%0 (3)Mixed sugar (mass percentage): Refined white sugar: 48%; Glucose: 21%; Fructose: 16%; Brown sugar: 10%; Honey: 5%. The continuous treatment method for the kombucha fermentation starter liquid is as follows: Add to the starter liquid: New Zealand highland black tea blend powder, with a concentration of 8.4 g / L; mixed sugar, with a concentration of 62 g / L; storage temperature: 2°C, for later use. The preparation process for fermenting highland blended black tea and mixed sugar to produce antibacterial kombucha liquid is as follows: S1 > Preparation of the sugar solution: 2024278265   10 Dec 2024 Heat the water to 90°C, add 45% of the total sugar amount, and stir until completely dissolved. The initial concentration of the sugar solution is 35.2 g / L; At this temperature, add the black tea leaves with a concentration of 14.5 g / L and steep for 6 minutes; After turning off the heat source, allow the black tea to cool naturally and steep for an additional 5 minutes, then remove the tea leaves. S2> Cooling and inoculation: Cool the sugar tea solution to room temperature (approximately 22°C), then transfer the solution to a temperature-controlled cabinet set at 16°C. When the solution temperature drops to 16°C, add the kombucha fermentation starter liquid at a concentration of 83 ml / L. The pH should then be between 3 and 3.3. Add the starter culture (SCOBY) at a concentration of 36 g / L, gently placing it on the surface of the solution, cover with breathable cloth to promote the growth of acetic acid bacteria. S3, Gradually add sugar (to control pH): Day 1: 45% of the total sugar has been added, with an initial sugar solution concentration of 35.2 g / L. Days 2-4: Gradually add the remaining sugar in the following proportions: 25%, 20%, and 10%. Monitor the pH daily and ensure the sugar is completely dissolved. When the pH approaches 3.1, reduce or pause the sugar addition. Gradually adding sugar helps prevent the rapid metabolism of sugars, regulates the acid production rate, and ensures that the pH remains between 3.0 and 3.3. S4 > Innovative microaerobic fermentation assistance: To further enhance the production and stability of organic acids, the following auxiliary techniques are employed: Microaerobic fermentation environment control: By regulating the oxygen supply, a micro-oxygen environment of 3%-5% is maintained to promote acetic acid bacteria in producing more acetic acid while preventing the inhibition of yeast fermentation. This environment can be achieved through a valve device at the top of the fermentation vessel. 2024278265   10 Dec 2024 Introduce lactic acid bacteria during the secondary fermentation stage: on day 6 of fermentation, add Lactobacillus plantarum. This helps generate more lactic acid and acetic acid while stabilizing the pH and preventing excessive decline. Lactobacillus plantarum is highly acid-resistant and can coexist with other fermentation microorganisms, increasing the yield of organic acids. Low-temperature extended fermentation time: On day 8, set the fermentation temperature to 12°C, extending the fermentation period to 15 days. This temperature helps slow down the fermentation process while producing more stable and higher concentrations of organic acids, improving the flavor and antibacterial properties of the fermentation liquid. Functional test results: The antibacterial activity analysis results of the kombucha liquid obtained in this embodiment are as follows (unit: mm): Staphylococcus aureus (S. aureus MCTIC 4163) : 16.00±0.00a Escherichia coli (E. coli NCTIC 8196) : 18.00±0.00cd Pseudomonas aeruginosa(P. aeruginosa MUA26): 8.00±0.00 Bacillus cereus(B. cereus MU-A44): 14.00±0.00 Table 1 shows the results of 17 experimental groups conducted, each using different fermentation parameters. The fermentation liquids obtained from these groups were tested and compared. The different parameters include: varying fermentation time, temperature, starter liquid, and fermentation processes. Additionally, a full antibacterial (positive) control is included. In the table, K11 refers to the antibacterial test results of the kombucha liquid obtained using the technology of this invention. Table1 Antibacterial test results The antibacterial activity analysis results of the kombucha ____________________liquid (mm)____________________ Sample S. aureus MCTIC 4163 E. coli NCTIC 8196 P. aeruginosa MUA26 B. cereus MU-A44 K1 6.00±0.00c 6.00±0.00g 0.00 6.00±0.00 K2 6.00±0.00c 6.00±0.00g 0.00 6.00±0.00 K3 6.00±0.00c 9.00±0.00f 0.00 6.00±0.00 2024278265   10 Dec 2024 K4 6.00±0.00c 13.33±1.15bc 0.00 8.00±0.00 K5 6.00±0.00c 12.67±0.58bcd 0.00 8.67±0.58 K6 6.00±0.00c 11.00±0.00de 0.00 6.00±0.00 K7 6.00±0.00c 12.00±0.00cd 0.00 8.67±0.58 K8 6.00±0.00c 6.00±0.00g 0.00 6.00±0.00 K9 6.00±0.00c 6.00±0.00g 0.00 6.00±0.00 K10 6.00±0.00c 6.00±0.00g 0.00 6.00±0.00 K11 16.00±0.00a 18.00±0.00cd 0.00 14.00±0.00 K12 6.00±0.00c 11.67±0.58cde 0.00 8.67±0.58 K13 6.00±0.00c 11.67±0.58cde 0.00 8.67±0.58 K14 6.00±0.00c 10.00±0.00ef 0.00 6.00±0.00 K15 8.33±0.57b 14.00±0.00b 0.00 9.33±1.16 K16 6.00±0.00c 12.00±0.00cd 0.00 8.33±0.58 K17 6.00±0.00c 12.67±2.08bcd 0.00 9.00±0.00 Positive 52.00 25.00 20.00 control 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 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. Such modifications or substitutions do not depart from the scope of the technical solutions of the present invention as described in the embodiments.

Claims

2024278265   10 Dec 2024WHAT WE CLAIM IS1 , A method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology, characterized by the following processes:S1 , Preparation of the sugar solution:The water is heated, and a portion of the mixed sugar is added and stirred until fully dissolved;At this temperature, highland blended black tea is added for steeping;The heat source is turned off, allowing the tea to cool naturally while continuing to steep, the tea leaves are then removed;S2, Cooling and inoculation:The sugared tea solution is cooled to room temperature and then transferred to a temperature-controlled cabinet for further cooling;Once the solution temperature has decreased, kombucha starter culture is added; The starter microbial culture is added, and the container is covered with breathable fabric to promote the growth of acetic acid bacteria;S3 , Sugar is gradually added, and the pH is carefully controlled;During days 2 to 4 of fermentation, the remaining sugar is gradually added, and the pH is monitored to ensure it remains within the range of 3.0-3.3;S4, Microaerobic fermentation assistance;Microaerobic fermentation environment control: the oxygen supply is regulated to maintain a microaerobic environment with an oxygen concentration of 3%—5%;In the secondary fermentation stage, Lactobacillus plantarum is introduced on the 6th day of fermentation, promoting the production of additional lactic acid and acetic acid while stabilizing the pH level;Low-temperature extended fermentation: on the 8th day, the fermentation temperature is set to 12°C, extending the fermentation duration to 15 days.2, According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that, in step S1, water is heated to 90°C, and 45% of the total sugar mass of the mixed sugars is added, resulting in an initial sugar solution concentration of 35.2 g / L;The highland blended black tea is added at a concentration of 14.5 g / L and steeped for 6 minutes; the heat source is then turned off, and the tea is steeped for an additional 5 minutes.3,According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that the mixed sugars comprise the following components in percentage by mass:Refined white sugar: 48%;Glucose: 21%;Fructose: 16%;Brown sugar: 10%;2024278265   10 Dec 2024Honey: 5%4, According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that the highland blended black tea comprises highland black tea from the Waikato region and highland black tea from the Canterbury region of New Zealand, mixed at a mass ratio of 3:1.7.

5. According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that, in step S2, the temperature-controlled cabinet lowers the solution temperature to 16°C, the kombucha starter culture is added at a concentration of 83 ml / L, and the solution pH is maintained within the range of 3.0-3.3;The starter microbial culture is added at a concentration of 36 g / L and gently placed on the surface of the solution.6> According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that the starter microbial culture comprises the following components by mass parts:Komagataeibacterxylinus ATCC 53524: 50 parts, used for producing acetic acid andglucuronic acid;Acetobacter pasteurianus JCM 7686:25 parts, serves as a high-yield acetic acid bacterium;Leuconostocmesenteroides ATCC 8293:12 parts, supports fermentation and stabilizes acidity;Zygosaccharomyces bailii CBS 680:8%, serves as an acid-tolerant yeast to assist in acetic acid production.7 ^ According to Claim 1, the method for preparing high-antibacterial kombucha beverage using blended black tea, mixed sugars, and microaerobic technology is characterized in that, in step S3, sugar is added on days 2 to 4 in proportions of 25%, 20%, and 10% of the total mass, respectively.