Cane sugar-consuming klebsiella zhanfangensis zfh h1 and application thereof and preparation method of black tea fermented beverage

Abstract

This invention discloses a *Sucrose-eating bacterium* ZFHH1 strain and its application in the preparation of fermented black tea beverages. The invention provides a *Sucrose-eating bacterium* ZFHH1 strain, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:2026292. It also discloses a method for preparing a fermented black tea beverage, comprising the following steps: Step S1: preparing black tea infusion; Step S2: reviving and culturing *Sucrose-eating bacterium* ZFHH1 to obtain a seed culture; Step S3: inoculating the seed culture into the black tea infusion for single-strain fermentation to obtain a fermented black tea broth; Step S4: filtering and bottling the fermented black tea broth to obtain the fermented black tea beverage. The fermented black tea beverage prepared using the ZFHH1 strain of this invention has a harmonious sweet and sour flavor, a mellow tea taste, a rich aroma, and low bitterness; the single-strain fermentation process is highly controllable.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a *Sucrose-eating bacterium* ZFHH1 and its application in the preparation of fermented black tea beverages. Background Technology

[0002] Black tea, as one of the world's most consumed tea beverages, is beloved by consumers for its rich flavor and abundant bioactive components. Traditional black tea drinks are mostly brewed, while fermented black tea beverages, through microbial metabolism and transformation of nutrients in the tea leaves, can further enhance the flavor and functionality of the drinks, becoming an innovative direction in the tea beverage industry.

[0003] Currently, the production of fermented black tea beverages mostly adopts a mixed-culture fermentation system. However, this model has the following drawbacks: On the one hand, the metabolic synergy among mixed cultures is complex, and key parameters such as pH value and metabolic product concentration are difficult to control precisely during the fermentation process, resulting in significant flavor differences between batches and poor quality stability. On the other hand, the utilization efficiency of nutrients in the black tea matrix by mixed cultures is uneven, and some raw materials are not fully converted, resulting in resource waste and potentially producing undesirable flavor substances such as bitterness and off-flavors.

[0004] Single-strain fermentation technology has gradually become the preferred choice for the industrial production of functional beverages due to its advantages such as well-defined metabolic pathways, easy control of the fermentation process, and stable product quality. Among them, *Sucrose-eating bacillus*, a type of microorganism that can efficiently utilize carbohydrates such as sucrose, produces flavor substances such as organic acids and esters during its metabolism and exhibits strong adaptability to tea substrates. However, the application of existing *Sucrose-eating bacillus* strains in black tea substrate fermentation has not been reported. There is a lack of dedicated strains optimized for black tea fermentation, with outstanding fermentation performance and the ability to improve beverage flavor, which fails to meet the needs of industrial production. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art by providing a method for preparing fermented black tea beverages using *Sucrose-eating Bacillus ZFHH1* and its application, which has the advantages of strong controllability of single-strain fermentation, harmonious sweet and sour flavor of the product, reduced bitterness, and suitability for industrial production.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a Komagataeibacter saccharivorans ZFHH1, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:2026292, January 30, 2026.

[0007] Application of Sucrose-eating Bacteroides ZFHH1 in the preparation of fermented black tea beverages.

[0008] A method for preparing a fermented black tea beverage includes the following steps: Step S1: Prepare the black tea infusion; Step S2: The *Sucrose-eating Bacillus ZFHH1* as described in claim 1 is revived and cultured to obtain a seed culture; Step S3: Inoculate the seed liquid into black tea infusion for single-strain fermentation to obtain black tea fermentation liquid; Step S4: The fermented black tea liquid is aseptically filtered and aseptically filled to obtain the fermented black tea beverage.

[0009] Optionally, in step S1, the method for preparing black tea infusion is as follows: boil purified water, add tea leaves, extract the tea leaves, filter out the tea leaves, add sucrose, stir to dissolve, and then cool.

[0010] Optionally, the ratio of purified water, tea leaves and sucrose used is 1L : 5g : 70g.

[0011] Optionally, the specific steps for resuscitation and cultivation in step 2 are as follows: Take out *Sucrose-eating bacillus* from the cryopreservation tube and inoculate it into HS liquid medium. Streak the cultured bacterial solution on HS solid medium and incubate at 30°C for 24 hours. Then pick a single colony and inoculate the selected single colony into fresh HS liquid medium. Continue to incubate at 30°C for 24 hours to obtain activated bacterial solution. Take the above activated bacterial solution, count the bacteria using the dilution plating method, and store it in a refrigerator; Take 8 mL of bacterial culture, centrifuge at 5000 rpm for 3 min, remove the supernatant culture medium, add black tea infusion, mix well, and centrifuge again. Repeat the operation several times. After the last centrifugation, discard the supernatant, add 8 mL of black tea infusion to resuspend, and obtain the seed culture.

[0012] Optionally, in step 3, the inoculation amount of seed liquid is 1%~5% (v / v), the fermentation is static fermentation, the fermentation temperature is 28~32℃, and the fermentation time is 5~9 days.

[0013] Optionally, in step 4, a 0.22μm sterile filter membrane is used for filtration.

[0014] A fermented black tea beverage, prepared by the method described above.

[0015] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The *Sucrose-eating Bacillus* ZFHH1 strain of this invention exhibits good regulatory ability in the fermentation of black tea substrate, achieving a coordinated metabolism of sugars in a high-acid environment. This results in a product with a balanced sweet-sour ratio, producing fermented black tea beverages with a harmonious sweet-sour taste, rich tea flavor, strong aroma, and low bitterness. The fermentation products show superior sensory quality and consumer acceptance compared to other strains of the same genus. Furthermore, this single-strain fermentation process offers strong controllability, high flavor stability, and excellent product quality, demonstrating broad market application prospects. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a column chart comparing the pH values ​​of fermented black tea beverages in Example 1 of this invention with those in Comparative Examples 1-4. Figure 2 This is a bar chart comparing the total acid content in fermented black tea beverages of Example 1 and Comparative Examples 1-4 of the present invention; Figure 3 This is a bar chart comparing the total sugar content in fermented black tea beverages of Example 1 and Comparative Examples 1-4 of the present invention; Figure 4 This is a bar chart comparing the total phenol content in fermented black tea beverages of Example 1 and Comparative Examples 1-4 of the present invention; Figure 5 Radar image of electronic nose and electronic tongue analysis of the fermented black tea beverage prepared in Example 1 of the invention; Figure 6 This invention presents a phylogenetic tree constructed based on the 16S rRNA gene sequence of *Sucrose-eating Corydalis* ZFHH1 and related strains. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0018] The *Sucrose-eating bacterium* used in this invention ( Komagataeibacter saccharivoransZFHH1 was isolated from naturally fermented tea samples. Species identification was performed using 16S rRNA gene sequence analysis, confirming the strain as *Sucrose-eating Corydalis*. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:2026292.

[0019] See Figure 6 ZFHH1 and the K1~K4 strains used in the comparative example both belong to acetic acid bacteria and have a certain phylogenetic relationship; however, ZFHH1 and K1~K4 are in different evolutionary branches and are genetically distant, indicating that they are not completely the same, and ZFHH1 has a unique genetic background.

[0020] For ease of understanding, several embodiments of this application are described in detail below. Example 1

[0021] A method for preparing a fermented black tea beverage includes the following steps: Step 1. Preparation of black tea infusion Take 1L of purified water, boil it, and then add 5g of tea leaves (purchased from Xiamen Wuyishan Yiming Ecological Tea Co., Ltd.). Steep for 15 minutes and then filter out the tea leaves. Add 70g of sucrose (from Shanghai Sugar, Tobacco & Alcohol Co., Ltd.), stir to dissolve, and let stand to cool to obtain the black tea infusion.

[0022] Step 2. Resuscitation and culture of *Sucrose-eating Bacillus* ZFHH1 strain was extracted from a cryovial with accession number CCTCC NO:2026292 and inoculated into HS liquid medium at a 2% inoculum. The cultured K1 was then streaked onto HS solid medium and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated into HS liquid medium, and incubated again at 30°C for 24 hours to obtain activated bacterial culture.

[0023] The above-mentioned active bacterial suspension was collected, and the ZFHH1 strain was counted using the dilution plating method. The suspension was then stored at 4°C. 8 mL of the active bacterial suspension was centrifuged at 5000 rpm for 3 minutes, the supernatant was removed, and the culture medium was mixed with black tea infusion. The mixture was centrifuged again, and this process was repeated three times to wash away the culture medium. After the final centrifugation, 8 mL of black tea infusion was added to resuspend the bacterial cells, thus obtaining the seed culture.

[0024] Step 3. Single-strain fermentation Take 800mL of black tea infusion into a fermentation tank and inoculate it with 8mL of *Sucrose-eating bacillus* K1 (10⁸ CFU / mL). Cover the opening of the tank with three layers of gauze and place the fermentation tank in a 30℃ constant temperature incubator for 7 days to obtain the black tea fermentation liquid.

[0025] Step 4. Finished Product Preparation The fermented black tea liquid was filtered through a 0.22μm sterile filter membrane to remove bacterial cells and impurities. The clarified fermented liquid was then aseptically bottled to obtain the fermented black tea beverage of this embodiment, which is named ZFHH1.

[0026] Comparative Example 1 The strains K1, K2, K3, and K4 used in Comparative Examples 1-4 are all real acetic acid bacteria, isolated from kombucha from four different regions of my country. Currently, K1-K4 are all preserved in the brewing laboratory of the College of Food Science and Technology, Yangzhou University. Each comparative example used the exact same fermentation conditions and operating steps as Example 1, except that the ZFHH1 strain was replaced with K1, K2, K3, and K4, respectively. The resulting fermented black tea beverages were designated K1, K2, K3, and K4, respectively.

[0027] The difference between this embodiment and Embodiment 1 is that the ZFHH1 strain is replaced with the K1 strain, and the prepared black tea fermented beverage is denoted as K1.

[0028] Comparative Example 2 The difference between this embodiment and Embodiment 1 is that the ZFHH1 strain is replaced with the K2 strain, and the prepared black tea fermented beverage is denoted as K2.

[0029] Comparative Example 3 The difference between this embodiment and Embodiment 1 is that the ZFHH1 strain is replaced with the K3 strain, and the prepared black tea fermented beverage is denoted as K3.

[0030] Comparative Example 4 The difference between this embodiment and Embodiment 1 is that the ZFHH1 strain is replaced with the K4 strain, and the prepared black tea fermented beverage is denoted as K4.

[0031] See Figures 1 to 4 After testing the relevant indicators of the black tea fermented beverages in the five examples, it was found that compared with the K1~K4 strains of the same genus, the ZFHH1 group had the lowest pH value (2.74) and the highest total acid content (3.4 g / L), and formed a coordinated sweet-sour ratio with a moderate total sugar content (13.4 g / L). The total sugar content refers to the residual sugar content after fermentation, which is the sum of soluble sugars such as sucrose, glucose, and fructose in the fermentation broth, and can fully reflect the utilization of sugar by the ZFHH1 strain. As can be seen from the above, ZFHH1 can efficiently produce acid while moderately retaining sugar, thus forming a metabolic characteristic of high acid and medium sugar. This gives the beverage a refreshing sour taste to inhibit miscellaneous bacteria, while retaining a moderate amount of sweetness to neutralize the sour taste, thereby achieving a palatable sweet-sour ratio, which reflects the precision of the strain's metabolic regulation.

[0032] Its total phenolic content (511.7 mg / L) falls within an optimal range that balances the preservation of tea flavor with the reduction of bitterness, effectively balancing the unique mellowness of black tea with the bitterness brought by phenolic substances. This is because ZFHH1 can secrete enzymes such as polyphenol oxidase and glycosyltransferase during metabolism, converting the more bitter ester-type catechins into less bitter oxidation products or glycoside conjugates; at the same time, the organic acids produced by fermentation can promote the binding of phenolic substances with proteins and polysaccharides, reducing the perceived bitterness of free phenols.

[0033] Therefore, as can be seen from the table above, the fermented black tea beverage prepared by *Sucrose-eating Bacterium sucrase* ZFHH1 in Example 1 has superior overall performance. To further verify these results, an electronic nose and electronic tongue were used to test the fermented black tea beverage.

[0034] See Figure 5 As can be seen from the radar chart of the electronic nose, after 7 days of fermentation, the response values ​​of the W1W sensor (sensitive to alcohols and aldehydes), the W2W sensor (sensitive to organosulfur compounds and terpenes), and the W5S sensor (sensitive to ketones and esters) increased significantly. This indicates that the ZFHH1 strain produced a wealth of flavor compounds such as alcohols and esters during fermentation, giving the product a rich floral and fruity aroma and ester aroma.

[0035] The radar chart from the electronic tongue shows that, in terms of taste, bitterness, umami, and saltiness decreased, while sourness gradually turned positive and significantly increased with increasing fermentation days, reaching a peak of 12.0 at 7 days. In summary, the significant decrease in bitterness effectively alleviated the inherent bitterness of the black tea base, making the product smoother and more mellow on the palate; the significant increase in sourness gave the product a refreshing sweet and sour taste, which is both appetizing and helps to inhibit the growth of unwanted bacteria. The synergistic effect of decreasing bitterness and increasing sourness, combined with a moderate total sugar content (13.4 g / L), i.e., the residual sugar content after fermentation, together achieved a harmonious sweet and sour flavor profile with low bitterness, enhancing the consumer's drinking experience and acceptance.

[0036] To further verify the sensory quality of the fermented black tea beverage of the present invention, a sensory evaluation experiment was conducted, and the results are shown in the table below:

[0037] As shown in the table above, the product's clarity and color scored an average of 9.8 points (close to full marks), indicating excellent visual quality. It is free of sediment and has a bright color, giving consumers a good first impression. The overall flavor scored an average of 7.7 points, reaching the "very much liked" level, with a harmonious blend of sweetness, slight acidity, and tea aroma, demonstrating outstanding flavor coordination. The overall taste scored an average of 7.6 points, with a balanced sweet and sour flavor, smooth texture, and no off-flavors or roughness, providing a good drinking experience. The overall preference score was an average of 6.3 points, falling between "like / acceptable" and "very much liked," indicating that most consumers recognize and are willing to try the product, demonstrating good market acceptance.

[0038] In summary, the *Sucrose-eating Bacillus ZFHH1* strain of the present invention exhibits superior fermentation control performance in the preparation of fermented black tea beverages. It can precisely achieve a harmonious balance of sweet and sour flavors, as well as a balance between tea flavor and bitterness. Furthermore, it can selectively enrich characteristic flavor substances such as floral and fruity aromas and ester aromas, thereby enhancing the sensory quality and consumer acceptance of fermented black tea beverages.

[0039] In addition, the single-strain fermentation process of the sucrose-eating bacterium ZFHH1 is highly controllable and the product quality is stable, providing core strain support for the industrialized and standardized production of fermented tea beverages. It solves the problems of poor fermentation flavor control and weak product market adaptability of similar strains, and has outstanding patent application value and industrialization advantages.

[0040] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A Komagataeibacter saccharivorans ZFHH1, characterized in that: The *Sucrose-eating bacterium* ZFHH1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:2026292.

2. The application of *Sucrose-eating Bacteroides ZFHH1* as described in claim 1 in the preparation of fermented black tea beverages.

3. A method for preparing a black tea fermented beverage, characterized by, Includes the following steps: Step S1: Prepare the black tea infusion; Step S2: The *Sucrose-eating Bacillus ZFHH1* as described in claim 1 is revived and cultured to obtain a seed culture; Step S3: Inoculate the seed liquid into black tea infusion for single-strain fermentation to obtain black tea fermentation liquid; Step S4: The fermented black tea liquid is aseptically filtered and aseptically filled to obtain the fermented black tea beverage.

4. The preparation method of black tea fermentation beverage according to claim 3, characterized in that, In step S1, the method for preparing black tea infusion is as follows: boil purified water, add tea leaves, extract the tea leaves, filter out the tea leaves, add sucrose, stir to dissolve, and then cool.

5. The preparation method of black tea fermentation beverage according to claim 4, characterized in that: The ratio of purified water, tea leaves, and sucrose used is 1L : 5g : 70g.

6. The preparation method of black tea fermentation beverage according to claim 3, characterized in that, The specific steps for resuscitation and cultivation in step 2 are as follows: Take out *Sucrose-eating bacillus* from the cryopreservation tube and inoculate it into HS liquid medium. Streak the cultured bacterial solution on HS solid medium and incubate at 30°C for 24 hours. Then pick a single colony and inoculate the selected single colony into fresh HS liquid medium. Continue to incubate at 30°C for 24 hours to obtain activated bacterial solution. Take the above activated bacterial solution, count the bacteria using the dilution plating method, and store it in a refrigerator; Take 8 mL of bacterial culture, centrifuge at 5000 rpm for 3 min, remove the supernatant culture medium, add black tea infusion, mix well, and centrifuge again. Repeat the operation several times. After the last centrifugation, discard the supernatant, add 8 mL of black tea infusion to resuspend, and obtain the seed culture.

7. The preparation method of black tea fermentation beverage according to claim 3, characterized in that: In step 3, the inoculation amount of seed liquid is 1%~5% (v / v), the fermentation is static fermentation, the fermentation temperature is 28~32℃, and the fermentation time is 5~9 days.

8. The preparation method of black tea fermentation beverage according to claim 3, characterized in that: In step 4, a 0.22μm sterile filter membrane is used for filtration.

9. A black tea fermented beverage, characterized by, The black tea fermented beverage is prepared by the method described in any one of claims 3 to 8.

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