Compound fermented Kangpu tea as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fermented tea beverages, and particularly discloses compound fermented Kangpu tea as well as a preparation method and application thereof. According to the compound fermented Kangpu tea, black tea water is inoculated with compound bacteria for fermentation, the compound bacteria are composed of foal bacillus medialis, saccharomyces cerevisiae and sake lactobacillus, the mass ratio of the foal bacillus medialis to the saccharomyces cerevisiae to the sake lactobacillus is 2: 2: (1-2), the preservation number of the foal bacillus medialis is WDCM No. JCM 16936, the preservation number of the saccharomyces cerevisiae is CGMCC No. 2.3973, and the preservation number of the sake lactobacillus is CGMCC No. 2.3973. The sake lactobacillus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34016. Compared with single-bacterium or two-bacterium composite inoculated fermentation, the growth activity, the acid production capacity and the reducing sugar consumption capacity of the Kangpu tea are better, the content of flavor substances such as aldehydes and esters is improved through the GC-IMS technology, and the flavor quality of the Kangpu tea is improved.

Description

Technical Field

[0001] This invention relates to the field of fermented tea beverage technology, and in particular to a compound fermented kombucha, its preparation method, and its application. Background Technology

[0002] Kombucha, also known as kombu tea or Haibao, is a traditional fermented tea beverage with a long history. It is mainly made by mixing tea leaves, water, and sucrose in a certain proportion and fermenting them with various microorganisms such as yeast, acetic acid bacteria, and lactic acid bacteria. It has a pleasant taste, is rich in tea polyphenols, organic acids, and other active functional ingredients, and is highly nutritious, making it very popular among consumers.

[0003] Traditional kombucha beverages are typically produced using a home-culture method with a mixed natural microbial strain. This method suffers from long fermentation cycles, difficulty in controlling fermentation conditions, and susceptibility to contamination by other microorganisms and strain degeneration, leading to unstable product quality and failing to fundamentally guarantee the quality and safety of kombucha, thus limiting its application and development. In the field of traditional fermented foods, inventors often employ modern bio-fermentation technology, selecting superior strains for inoculation and fermentation. This approach effectively addresses the shortcomings of traditional natural fermentation methods, inhibits the proliferation of harmful microorganisms during fermentation, improves the quality and safety of fermented products, and promotes flavor development. However, existing inventions primarily focus on the isolation, screening, and inoculation fermentation of single-species dominant strains, and on optimizing flavor-oriented inoculation fermentation processes. Inventions utilizing combined fermentation of dominant microorganisms to improve the flavor and quality of kombucha are rarely reported. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a compound fermented kombucha, its preparation method, and its application. This invention optimizes the traditional kombucha processing technology using modern bio-fermentation techniques. It utilizes a symbiotic system composed of dominant bacteria such as acetic acid bacteria, yeast, and lactic acid bacteria isolated and purified from commercially available high-quality kombucha products. A rapid fermentation technology based on multi-dominant bacteria inoculation is developed to achieve high-quality, high-efficiency, and high-stability large-scale processing of kombucha, laying a theoretical foundation for the standardized, large-scale, and industrialized production of kombucha beverages.

[0005] The first objective of this invention is to provide a method for preparing compound fermented kombucha, which involves inoculating black tea water with a compound bacteria for fermentation, wherein the compound bacteria are *Bacillus intermedius* (…). K. intermedius ), brewer's yeast ( S. cerevisiae ) and sake lactobacillus ( L. sakeiThe composition of the samples was as follows: The mass ratio of the samples was 2:2:(1~2). The *Intermediate-sized *Bacillus intermedius* was purchased from Beijing Bio-Tech Biotechnology Co., Ltd., with accession number WDCM No. JCM 16936. The *Saccharomyces cerevisiae* was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 2.3973. The *Lactobacillus sakei* was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34016.

[0006] Furthermore, the mass ratio of the intermediate *Bacillus intermedius*, *Saccharomyces cerevisiae*, and *Lactobacillus sakei* is 2:2:1.

[0007] Furthermore, the *Intermediate Oligobacterium intermedius* is inoculated into acetic acid bacteria liquid culture medium for activation and expansion culture to prepare *Intermediate Oligobacterium intermedius* seed culture; the *Saccharomyces cerevisiae* is inoculated into YPD liquid culture medium for activation and expansion culture to prepare *Saccharomyces cerevisiae* seed culture; and the *Lactobacillus sakei* is inoculated into MRS liquid culture medium for activation and expansion culture to prepare *Saccharomyces cerevisiae* seed culture.

[0008] Furthermore, the black tea water is prepared by boiling water and black tea leaves with sugar.

[0009] Furthermore, the mass ratio of the black tea leaves, water, and sugar is (1~2):100:10.

[0010] Furthermore, the inoculation amount of the compound bacteria into the black tea water for fermentation is 5% to 6% by volume.

[0011] Furthermore, the process of inoculating the compound bacteria into the black tea water for fermentation is as follows: constant temperature and light protection, static fermentation at 25℃~26℃ for 8~9 days.

[0012] Furthermore, the pH value of the fermentation broth obtained after inoculating the compound bacteria into black tea water for fermentation is 3.0~3.5.

[0013] A second objective of this invention is to provide a compound fermented kombucha prepared using the method described above.

[0014] A third objective of this invention is to provide a beverage prepared using the aforementioned compound fermented kombucha.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the strain *Intermediate Corymbospora* (… K. intermedius ), brewer's yeast ( S. cerevisiae ) and sake lactobacillus ( L. sakeiAfter cultivation, it was co-inoculated and fermented with kombucha. Compared with single or two strains, it was found to have better growth activity, acid production capacity and reducing sugar consumption capacity. GC-IMS technology showed that the content of flavor substances such as aldehydes and esters was increased, and the flavor quality of kombucha was improved. Attached Figure Description

[0016] Figure 1 Graph showing the change in biomass of fermented kombucha after inoculation with different dominant bacteria; Figure 2 A graph showing the pH changes in kombucha fermented with different dominant bacteria. Figure 3 Graph showing the change in total acid content of kombucha fermented with different dominant bacteria; Figure 4 Graph showing the change in reducing sugar content in kombucha fermented with different dominant bacteria; Figure 5 Electronic nose LDA for inoculating fermented kombucha with different dominant bacteria; Figure 6 The results of electronic tongue analysis of kombucha inoculated with different dominant bacteria; Figure 7 The kombucha flavor component analysis diagram provided by this invention; Figure 8 A phylogenetic tree diagram of Lactobacillus sago. Detailed Implementation

[0017] To more clearly explain the technical solution and beneficial effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the described accompanying drawings are only some embodiments of the present invention and are used only to explain the present invention, and should not be construed as limiting the present invention. Unless otherwise specified, the equipment and reagents used in the present invention are commercially available products conventional in this technical field.

[0018] The black tea used in this invention is Jin Jun Mei black tea, purchased from Fujian Lixiang Tea Industry Co., Ltd.

[0019] The detailed information of the strains used in this invention is as follows: The intermediate *Bacillus intermedius* was purchased by the applicant from Beijing Bio-Biotech Co., Ltd., with accession number WDCMNo. JCM 16936; the *Saccharomyces cerevisiae* was purchased by the applicant from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 2.3973; and the *Lactobacillus sakei* was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34016.

[0020] Isolation and identification of Lactobacillus in sake One sample of traditional fermented kombucha from each of six different provinces (Liaoning, Hebei, Shandong, Shaanxi, Henan, and Guangdong) was purchased and transported to the laboratory within 2 days via a 4℃ cold chain. 100 μL of each sample was aspirated and incubated on acetic acid bacteria plates at 30℃ for 2–3 days. Plates with a certain number of non-overlapping colonies were selected, and colony morphology, color, and size were recorded. Typical colonies were streaked to obtain single colonies (M). DNA was extracted from the activated strains, and PCR amplification of the bacterial 16S rDNA was performed using universal primers 27F and 1492R. The PCR products were detected by electrophoresis on a 1.5% agarose gel, and PCR products with clear bands and no significant tailing were sequenced, followed by sequence alignment analysis. Based on this, a phylogenetic tree (e.g., ) was constructed using the neighbor-joining method. Figure 8 As shown), the strain was analyzed and compared with Lactobacillus sakei ( Lactobacillus sakei The homology reached 99%, and it was identified as *Lactobacillus sakei* ( Lactobacillus sakei The 16S rDNA sequence of this *Lactobacillus sakei* is shown in SEQ ID NO. 1. This *Lactobacillus sakei* was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named *Lactobacillus sakei*. Latilactobacillussakei The accession number is CGMCC No.34016.

[0021] The specific preparation method of the compound fermented kombucha provided by this invention is as follows: (1) Strain K. intermedius , S. cerevisiae , L. sakei The revival and cultivation of the strain K. intermedius After inoculating into acetic acid bacteria liquid culture medium, the strain was cultured; S. cerevisiae After inoculation into YPD liquid medium, the strain was cultured; L. sakei After inoculation into MRS liquid medium, culture was carried out.

[0022] (2) Boil water and black tea leaves together for 15 minutes, then filter out the tea leaves; (3) Add white sugar to step (2) and stir until dissolved, then let stand and cool to room temperature; (4) Pour the room temperature tea sugar water from step (3) into the prepared sterilized fermentation tank, and then pour the bacterial liquid into the fermentation bottle and mix. It is essential to let it stand during the fermentation process. (5) Seal with eight layers of sterilized gauze, leaving room for ventilation (the lid should not be sewn closed to prevent insects). (6) Ferment in a constant temperature incubator at 25℃ in the dark for about 8 days. When the pH of the fermentation liquid reaches 3.0 and becomes acidic, the fermentation is complete.

[0023] In the above technical solutions, strain (1) K. intermedius Inoculation amount: 1% (v / v), incubation at 30℃ for 24 h; strain S. cerevisiae Inoculation amount: 1% (v / v), incubation at 30℃ for 24 h; strain L. sakei The inoculum was 1% (v / v), and the culture was carried out at 37°C for 48 h.

[0024] Example 1 This embodiment provides a compound fermented kombucha, the preparation method of which is as follows: (1) The strain K. intermedius Inoculate the acetic acid bacteria liquid medium at a 1% (v / v) inoculum and incubate at 30°C for 24 h; then... S. cerevisiae Inoculate the strain into YPD liquid medium at a 1% (v / v) inoculum and incubate at 30°C for 24 h; L. sakei Inoculate 1% (v / v) into MRS liquid medium and incubate at 37°C for 48 h.

[0025] (2) Boil 250mL of water and 2.5g of black tea together, then filter out the tea leaves.

[0026] (3) Add 25g of white sugar to step (2) and stir until dissolved. Let stand and cool to room temperature.

[0027] (4) Pour the room temperature tea sugar water from step (3) into the prepared sterilized fermentation tank.

[0028] (5) Inoculate the tea infusion from step (4) with 5% (v / v) of K. intermedius + S. cerevisiae + L. Sakei The compound bacteria (2:2:1, v / v) were fermented statically in the dark at room temperature (25℃) for 8 days and named MS2.

[0029] Example 2 This embodiment provides a compound fermented kombucha, the preparation method of which is as follows: It is basically the same as Example 1, except that: K. intermedius + S. cerevisiae + L. Sakei Compound bacteria (2:2:2, v / v).

[0030] Example 3 This embodiment provides a compound fermented kombucha, the preparation method of which is as follows: It is basically the same as Example 1, except that: K. intermedius + S. cerevisiae + L. Sakei Compound bacteria (2:2:1.5, v / v) Comparative Example 1 The difference from Example 1 is that in step (5), 2% (v / v) of the solution is inoculated. K. intermedius A single bacterium, named KI.

[0031] Comparative Example 2 The difference from Example 1 is that in step (5), 2% (v / v) of the solution is inoculated. S. cerevisiae A single bacterium, named SC.

[0032] Comparative Example 3 The difference from Example 1 is that in step (5), 4% (v / v) of the solution is inoculated. K. intermedius + S. cerevisiae The compound bacteria (1:1, v / v) was named MS1.

[0033] Comparative Example 4 The difference from Example 1 is that in step (5), the tea sugar water was not inoculated with bacteria.

[0034] To better illustrate the compound fermented kombucha provided by this invention, the following research was also conducted: (1) Determination of growth curve On days 0, 2, 4, 6, and 8 of fermentation, 10 mL of each kombucha fermentation liquid from Examples 1-5 was taken, and the absorbance at a wavelength of 600 nm was measured using a visible spectrophotometer.

[0035] like Figure 1 As shown, the OD of each inoculated group during the entire fermentation process 600 nm The values ​​showed a trend of first increasing and then decreasing; among them, the KI group reached its maximum value (0.53 g / L) on day 2 of fermentation, while the SC, MS1, and MS2 groups reached their maximum values ​​on day 4 of fermentation (0.54 g / L, 0.61 g / L, and 0.73 g / L, respectively). Subsequently, the OD values ​​of each inoculated group... 600 nm The values ​​gradually decreased, decreasing by 43.1%, 35.3%, 20.6%, and 22.5% respectively by the end of fermentation. The OD values ​​of each inoculated group in the mid-to-late stages of fermentation (>4 days) were... 600 nm The decrease in OD values ​​may be due to the gradual consumption of readily available carbon sources in the fermentation broth as fermentation progresses, thus inhibiting cell growth. However, the OD values ​​of the MS2 group... 600 nm The value was higher than that of other inoculated groups throughout the fermentation process, indicating better growth activity.

[0036] (2) pH value determination On days 0, 2, 4, 6, and 8 of fermentation, 10 mL of kombucha fermentation liquid from Examples 1-5 was taken and the pH value was measured using a pH meter.

[0037] likeFigure 2 The figure shows the pH changes of kombucha fermented with different dominant bacteria. Throughout the fermentation process, the pH of each inoculated group gradually decreased. At the end of fermentation, the pH of each group decreased by 48.5%, 45.3%, 49.4%, and 40.5% respectively compared with the initial value (0 d). Among them, the pH of MS2 group was significantly lower than that of other groups throughout the fermentation process (p < 0.05), showing strong acid production performance. This may be because the inoculated lactic acid bacteria can use amino acids such as glutamic acid, arginine, and histidine, as well as metabolites such as vitamins produced by yeast and acetic acid bacteria to grow and reproduce, and convert the sugar in the fermentation broth into lactic acid.

[0038] (3) Determination of total acidity On days 0, 2, 4, 6, and 8 of fermentation, 30 mL of kombucha fermentation liquid from Examples 1-5 was taken and the total acid content was determined.

[0039] like Figure 3 The graph shows the changes in total acid content of kombucha fermented with different dominant bacteria. As fermentation progressed, the total acid content in each inoculation group gradually increased, reaching 1.49 g / L, 1.16 g / L, 2.71 g / L, and 3.28 g / L at the end of fermentation, respectively. The total acid content in groups MS1 and MS2 was significantly higher than other inoculation groups throughout the entire fermentation process (p < 0.05). At the end of fermentation, the total acid content in group MS1 was 54.6% higher than that in group KI. This may be due to the mutualistic symbiotic relationship between yeast and acetic acid bacteria during fermentation; that is, the ethanol produced by yeast during its growth stage enables acetic acid bacteria to produce more acetic acid, and acetic acid, in turn, promotes the production of ethanol by yeast. Meanwhile, the total acid content in group MS2 was 17.5% higher than that in group MS1, possibly because some lactic acid bacteria can utilize glucose to produce lactic acid through glycolysis (embden-meyerhof-parnas, EMP) or the pentose phosphate pathway, further increasing the total acid content.

[0040] (4) Determination of reducing sugar content On days 0, 2, 4, 6, and 8 of fermentation, 30 mL of kombucha fermentation liquid from Examples 1-5 was taken, and the reducing sugar content was determined by the 3,5-dinitrosalicylic acid colorimetric method.

[0041] Changes in reducing sugar content in kombucha fermented with different dominant bacteria, such as Figure 4As shown, the reducing sugar content in each inoculation group showed a trend of first increasing and then decreasing, reaching its extreme value at 4 days of fermentation, which were 1.06 g / L, 7.08 g / L, 6.41 g / L and 5.89 g / L, respectively. Among them, the reducing sugar content in the KI group remained at a low level (≤1.56 g / L) throughout the fermentation process, with no significant difference from the control group (p>0.05). This may be because acetic acid bacteria cannot directly utilize sucrose, and yeast needs to play a "promoter" role in the early stage of kombucha fermentation to hydrolyze the sucrose before utilization. The reducing sugar content in the SC, MS1 and MS2 groups increased significantly in the early stage of fermentation (≤4 days) (p<0.05), increasing by 90.2%, 88.4% and 87.6%, respectively. This may be because the yeast present in each group multiplied rapidly in the early stage of fermentation, and under the action of invertase, hydrolyzed sucrose into reducing sugars such as glucose and fructose. In the middle and late stages of fermentation (>4 d), the levels of reducing sugar in the SC, MS1 and MS2 groups decreased significantly (p<0.05), decreasing by 41.3%, 42.6% and 48.5% respectively at the end of fermentation compared with the middle stage (4 d). This may be because the large presence of reducing sugar in the fermentation broth caused acetic acid bacteria to start growing and multiplying rapidly, and the reducing sugar was consumed as a carbon source to generate metabolites such as acetic acid and gluconic acid.

[0042] (5) Measurement of electronic nose Take 3 mL of the samples from day 8 of Examples 1-5 and day 0 of Example 5 and seal them in headspace vials for later use. Measure the samples using the direct headspace method at room temperature (20°C).

[0043] like Figure 5 The results show the electronic nose LDA of fermentation groups inoculated with different dominant bacteria. The variance contribution rates of LD_1 and LD_2 were 61.6% and 22.8%, respectively, with a total variance contribution rate of 84.4%, indicating that LDA can effectively distinguish the odor differences between fermentation groups inoculated with different dominant bacteria. The odor response values ​​of each group of samples showed a certain distance along the LD_1 direction, without overlap or intersection, clearly distinguishing the six groups of samples. This indicates that different inoculation and fermentation treatments have a significant impact on the volatile flavor components of kombucha. Furthermore, although the samples MS1-8 and MS2-8 in the combined inoculation group were close together along the LD_1 direction, they were far apart along the LD_2 direction, indicating that the addition of lactic acid bacteria in the MS2 group significantly altered the volatile flavor characteristics of kombucha.

[0044] (6) Measurement of electronic tongue The samples from Example 1, Comparative Examples 1-4 on day 8, and Example 5 on day 0 were centrifuged to remove the bacterial cells and raw materials. 120 mL of the supernatant was taken and the five tastes (sour, bitter, astringent, umami, and salty) of different single or compound fermentation broths were measured using different sensors of an electronic tongue instrument.

[0045] like Figure 6 The table shows the characteristic signal response values ​​of fermentation groups inoculated with different dominant bacteria. Sourness is the most important component of kombucha's flavor profile. The sourness response values ​​of the inoculated groups were 26.09, 25.42, 27.99, and 28.64, respectively. Among them, the sourness response values ​​of MS1-8 and MS2-8 were significantly higher than those of KI-8 and SC-8 (p < 0.05), indicating that the combined inoculation group had a stronger acid-producing capacity. Furthermore, the sourness response value of MS2-8 was higher than that of MS1-8, suggesting that the addition of lactic acid bacteria may have promoted a further decrease in the pH value of the fermentation system. Richness is also an important indicator reflecting the flavor quality of kombucha. The richness response values ​​of the inoculated groups were 0.22, 0.25, 0.44, and 2.61, respectively. Among them, the richness response value of MS2-8 was significantly higher than that of the other groups (p < 0.01), indicating that the inoculation of *L. sakei* in kombucha significantly improved the richness of flavor compounds. Meanwhile, the bitterness and astringency in KI-8, SC-8, MS1-8, and MS2-8 were significantly lower than those in the uninoculated group (CK-8) (p < 0.05); among them, the bitterness and astringency response values ​​in MS2-8 were reduced by 16.7% and 33.3% respectively compared with MS1-8. This may be because the addition of L. sakei in MS2-8 promoted the generation of metabolites such as lipids, phenols, and alcohols that can improve flavor, and reduced the production of bitterness and astringency substances.

[0046] (7) GC-IMS determination Take 3.0 mL of the samples from Example 1, Comparative Examples 1-4 (day 8), and Example 5 (day 0) and place them into 20 mL headspace vials. Set the incubation temperature to 60°C, the incubation speed to 500 r / min, and the incubation time to 20 min. Use automatic headspace sampling mode with an injection volume of 500 μL. Set up three parallel replicates for each sample and perform analysis and determination under the following conditions: GC conditions: MXT-WAX column (15 m × 0.53 mm × 1 μm), column temperature: 40℃. Run time: 30 min, carrier gas initial flow rate 2 mL / min, hold for 2 min, increase to 10 mL / min within 10 min, increase to 100 mL / min within 20 min, and increase to 150 mL / min within 30 min.

[0047] IMS conditions: drift tube length 9.8 cm; linear voltage inside the tube 400 V / cm; temperature 45℃; drift gas flow rate 150 mL / min (N2, purity >99.99%). Qualitative analysis: Material spectrum analysis was performed using the FlavorSpec® flavor analyzer, and qualitative analysis was performed using the GC×IMSLibrary Search software with built-in NIST and IMS databases.

[0048] During the fermentation of kombucha, a large number of flavor compounds such as alcohols, aldehydes, acids, and esters are formed under the combined influence of various microorganisms, thus endowing the product with unique sensory flavor qualities. The volatile composition of the samples from each inoculation group is shown in Table 3. A total of 32 volatile compounds were detected, including 21 esters, 3 alcohols, 2 alkanes, 2 aldehydes, and 4 others. Fingerprint spectral results ( Figure 7 It visually demonstrates the distribution of volatile compounds.

[0049] Esters are mainly formed by the esterification of alcohols and acids. They constitute a significant portion of the flavor components in kombucha and play a crucial role in the product's flavor profile. Esters containing short-chain acids often have fruity aromas, while those containing long-chain acids tend to have a milder oily flavor. Figure 7 It was found that KI-8 and SC-8 contained 3 and 2 types of esters, respectively. The composite inoculation group samples (MS1-8 and MS2-8) contained more esters than the single inoculation group samples (KI-8 and SC-8), with 13 and 16 types, respectively. The newly added esters were isoamyl isovalerate, methyl hexanoate, 3-methylbutyrate, methyl octanoate, hexyl isovalerate, amyl butyrate, isopropyl acetate, methyl 3-methylthiopropionate, butyl isovalerate, isobutyl isovalerate, diethyl adipate, ethyl 2-methylbutyrate, isobutyl crotonate, butyl valerate, methyl valerate, and isoamyl hexanoate, resulting in a significant increase in odor richness. Meanwhile, MS2-8 showed the addition of three ester compounds compared to MS1-8: 3-methylbutyrate-2-methylbutanoate (fruity aroma), methyl hexanoate (pineapple aroma), and isoamyl isovalerate (apple aroma), further enhancing the complex fruity aroma. Furthermore, the signal intensities of butyl valerate (ether aroma), isobutyl crotonate (fruity aroma), diethyl adipate (acetic acid aroma), butyl isovalerate (banana aroma), and methyl 3-methylthiopropionate (pineapple aroma) in MS2-8 were significantly higher than those in MS1-8. This may be because organic acids such as acetic acid, lactic acid, and malic acid produced by the metabolism of *L. sakei* inoculated in MS2-8 participate in subsequent esterification reactions, promoting the synthesis of esters such as butyl valerate and butyl isovalerate.

[0050] Aldehydes mainly originate from lipid oxidation and amino acid degradation, exhibiting citrus and waxy aromas. Two aldehydes were detected in the samples: decanal and citronellol. Decanal appeared only in the single-inoculation group samples, with a weak signal intensity; while citronellol was abundant in the multi-inoculation group samples, and its signal intensity in MS2-8 was significantly higher than that in MS1-8, indicating that the addition of *L. sakei* may have further promoted citronellol synthesis. Three alcohols were detected: nerol, 1-octen-3-ol, and 2-methyl-1-butanol, with generally weak signal intensities. 1-Octen-3-ol is considered to be commonly found in fermented beverages such as wine and beer, possessing aromas of mushroom, lavender, rose, and hay, and is one of the important flavor compounds in fermented beverages; in this invention, it was only present in small amounts in samples KI-8 and SC-8, indicating a significant flavor difference between kombucha and traditional fermented beverages.

[0051] (8) Sensory evaluation Sensory evaluation scores were assigned based on Table 1.

[0052]

[0053] The effects of different dominant bacteria inoculation on the sensory scores of fermented kombucha are shown in Table 2.

[0054] Table 2.

[0055] For any points not covered above, existing technologies shall apply.

[0056] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a compound fermented kombucha, characterized in that, The compound bacteria were inoculated into black tea water for fermentation, and the compound bacteria consisted of *Bacillus intermedius* (…). K. intermedius ), brewer's yeast ( S. cerevisiae ) and sake lactobacillus ( L. sakei The composition is as follows: the mass ratio of the components is 2:2:(1~2). The *Intermediate-classical* was purchased from Beijing Bio-Tech Biotechnology Co., Ltd., with accession number WDCM No. JCM 16936. The *Saccharomyces cerevisiae* was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 2.3973. The *Lactobacillus sakei* was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34016.

2. The preparation method according to claim 1, characterized in that, The mass ratio of *Intermediate Oligobacterium*, *Saccharomyces cerevisiae*, and *Lactobacillus sakei* is 2:2:

1.

3. The preparation method according to claim 1 or 2, characterized in that, The *Intermediate Oligobacterium* was inoculated into acetic acid bacteria liquid culture medium for activation and expansion culture to prepare *Intermediate Oligobacterium* seed culture; the *Saccharomyces cerevisiae* was inoculated into YPD liquid culture medium for activation and expansion culture to prepare *Saccharomyces cerevisiae* seed culture; The *Lactobacillus sacchariformis* was inoculated into MRS liquid medium for activation and expansion culture to prepare *Saccharomyces cerevisiae* seed culture.

4. The preparation method according to claim 3, characterized in that, The black tea water is prepared by boiling water and black tea leaves with sugar.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the black tea leaves, water, and sugar is (1~2):100:

10.

6. The preparation method according to claim 5, characterized in that, The inoculation amount of the compound bacteria into black tea water for fermentation is 5%~6% by volume.

7. The preparation method according to claim 6, characterized in that, The process of inoculating the compound bacteria into black tea water for fermentation is as follows: constant temperature and avoidance of light, static fermentation at 25℃~26℃ for 8~9 days.

8. The preparation method according to claim 7, characterized in that, The pH value of the fermentation broth obtained after inoculating the compound bacteria into black tea water for fermentation is 3.0~3.

5.

9. A compound fermented kombucha, characterized in that, It is prepared by any one of the preparation methods according to claims 1-8.

10. A beverage, characterized in that, It is prepared using the compound fermented kombucha described in claim 9.

Images