Kangpu tea fermentation liquor and preparation method thereof

Abstract

The invention belongs to the technical field of fermented drinks, and particularly relates to a Kangpu tea fermentation broth and a preparation method thereof, the preparation method comprises the following steps: S1, soaking tea leaves in water, and filtering to obtain tea soup; s2, dissolving a carbon source and a nitrogen source into the tea soup, and sterilizing to obtain sterile tea sugar water; the carbon source comprises white granulated sugar and concentrated apple juice, and the addition amount of the carbon source is 5-12wt% based on the mass of the tea sugar water; s3, cooling the sterile tea sugar water to room temperature, inoculating compound bacterium powder, standing in a fermentation container, and fermenting in a dark place to obtain fermentation liquor; the mass ratio of the lactobacillus to the saccharomycetes in the compound bacterium powder is (1.0-1.5): (0.8-1.6); s4, centrifuging, filling, sterilizing and cooling the Kangpu tea fermentation liquor to obtain the Kangpu tea fermentation liquor. The preparation method disclosed by the invention is rapid, short in fermentation time and easy in process control, and the Kangpu tea fermentation liquor is clear and transparent in color and luster, free of precipitation impurities, fresh and compound in fragrance, sour, sweet and balanced in taste, fine and smooth in bubble feeling and high in overall acceptability.

Description

Technical Field

[0001] This invention belongs to the field of fermented beverage technology, specifically relating to a kombucha fermentation liquid and its preparation method. Background Technology

[0002] Kombucha, a traditional fermented beverage originating in my country, is a distinctive fermented tea drink typically made from tea leaves and white sugar as the main fermentation substrate. It is produced through static fermentation using a symbiotic culture of bacteria and yeast (SCOBY) as the starter culture. Studies have shown that kombucha is rich in fermentation metabolites, containing various nutrients and active ingredients such as tea polyphenols, organic acids, amino acids, minerals, and vitamins. It possesses numerous health benefits, including antibacterial, anti-inflammatory, antioxidant, blood pressure-lowering, liver-protective, and immunity-boosting effects, thus holding an important position in the beverage industry and receiving widespread attention.

[0003] However, traditional kombucha production mainly relies on natural fermentation. The core fermenting agent, SCOBY, has a complex and unstable microbial composition. The microbial community structure of SCOBY from different sources and batches varies significantly, and the succession of the microbial community fluctuates with the change of the cultivation cycle. This directly leads to poor consistency and low reproducibility of the final product in terms of flavor, taste, content of active ingredients, and even safety, which seriously restricts its standardization and large-scale industrial production.

[0004] In recent years, the design of synthetic microbial communities (SMCs) through the interaction of core microorganisms has demonstrated excellent performance in improving the quality of traditional fermented foods and regulating product quality. Therefore, designing suitable synthetic microbial communities for kombucha fermentation by screening specific combinations of commercially available strains is an effective strategy for maintaining product flavor stability and achieving industrialized production. Furthermore, precise control of fermentation process parameters, fundamentally addressing the shortcomings of traditional processes, is also key to improving product stability, safety, flavor quality, functional properties, and production efficiency.

[0005] While the aforementioned directions hold great promise, the key technological challenge remaining to be overcome in this field is how to construct a simplified, efficient, and synergistic combination of specific microbial strains (SMCs) and match it with suitable fermentation process parameters to systematically address core industrialization issues such as flavor controllability, production efficiency, and safety. Therefore, developing a method for preparing kombucha fermentation broth that combines simplified commercial microbial composition, stable flavor output, and high-efficiency production is of great significance for promoting its industrialization. Summary of the Invention

[0006] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for preparing kombucha fermented liquid. By employing a specific combination of commercial microbial strains, carbon sources, and nitrogen supplements, it effectively improves the problems of long fermentation cycles, complex microbial communities, high uncontrollability of fermentation, and unstable products in traditional kombucha production. This achieves controllability and standardization of the fermentation process, ensuring uniform and stable product quality. The technical solution provided by this invention makes the production process of kombucha fermented liquid simple and efficient, resulting in excellent product quality, and is suitable for large-scale preparation of kombucha fermented liquid.

[0007] According to a first aspect of the present invention, a method for preparing kombucha fermentation liquid is provided, comprising the following steps:

[0008] S1. Brewing tea: Soak tea leaves in water, then filter to obtain tea soup;

[0009] S2. Preparation of tea sugar water: Dissolve carbon source and nitrogen source into tea soup and sterilize to obtain sterile tea sugar water; the carbon source is white sugar and concentrated apple juice, and the amount of carbon source added is 5-12 wt% based on the mass of tea sugar water; the nitrogen source is a mixture of various inactivated yeasts.

[0010] S3. Inoculation and Fermentation: Cool the sterile tea sugar water obtained in step S2 to room temperature, inoculate it with compound bacterial powder, and place it in a fermentation container to carry out anaerobic fermentation in the dark to obtain fermentation liquid; in the compound bacterial powder, the mass ratio of Lactobacillus powder and yeast powder is (1.0-1.5):(0.8-1.6).

[0011] S4. Post-processing: After centrifuging, filling, sterilizing and cooling the kombucha fermentation liquid obtained in step S3, kombucha fermentation liquid is prepared.

[0012] In existing kombucha-related technologies, research and development largely focus on controlling performance indicators such as acidity generation rate, sensory flavor (e.g., sweet-sour balance, floral and fruity aromas), or shortening the fermentation cycle during fermentation. However, these are often limited to the immediate quality at the end of fermentation, generally neglecting the evolution of the product's physicochemical properties during subsequent storage. In fact, even with good initial sensory performance, kombucha fermentation liquid prepared by traditional methods is still prone to problems such as darkening of color, significant precipitation, decreased clarity, and even the development of unpleasant off-odors during storage, severely restricting its commercial application and shelf-life assurance. This stability deficiency stems from the inherent uncontrollability of the fermentation system, especially the combined effects of complex microbial metabolic byproducts, high content of oxidatively sensitive substances, and the instability of the colloidal system.

[0013] This invention proposes a stability-oriented, controllable fermentation overall solution. This solution is not a localized optimization of a single factor, but rather, based on the stability degradation mechanism throughout the fermentation and storage process of kombucha, it synergistically designs a specific composition of functional microbial communities, a specific type of complex nitrogen source, and a synergistically proportioned complex carbon source, and matches them with suitable process conditions. By precisely controlling the metabolic flow of microorganisms, it promotes the formation of pure flavor and structurally stable main components, while effectively inhibiting adverse reaction pathways that lead to browning, flocculation, and off-flavors, thereby improving the intrinsic stability of the fermentation broth at its source. This not only achieves good fermentation efficiency and sensory quality, but more importantly, it endows the product with excellent storage stability, maintaining a clear color, high clarity, and long-lasting flavor without the addition of additional stabilizers or post-processing.

[0014] In some specific embodiments, the tea leaves in step S1 are selected from at least one of green tea, black tea, oolong tea, jasmine tea, or pu-erh tea.

[0015] In some specific implementations, the mass ratio of tea leaves to water in step S1 is (1.0-3.5):100.

[0016] In some specific implementations, the tea brewing conditions in step S1 are 75-90℃ for 5-20 minutes.

[0017] In some specific implementations, the filtration in step S1 uses a 200-mesh filter cloth.

[0018] In some specific embodiments, the mass ratio of white sugar to concentrated apple juice in step S2 is (1.0-3.0):(1.0-5.0).

[0019] In some specific embodiments, the nitrogen source in step S2 is a yeast extract powder composed of a mixture of multiple inactivated yeasts. In this invention, this nitrogen source can promote the synergistic growth of the complex microorganisms and improve fermentation efficiency without having any negative impact on the aroma or flavor of the fermentation broth. Its addition amount is 0.01-0.2% of the final weight of the aseptic tea syrup.

[0020] In some specific implementations, the sterilization conditions in step S2 are: 85-95℃ for 300 s or 118-121℃ for 5-15 s.

[0021] In some specific embodiments, the lactobacillus powder includes at least one of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus fermentum*, *Lactobacillus helveticus*, and *Lactobacillus paracasei*, and may optionally include Bifidobacterium powder (such as *Bifidobacterium lactis*, *Bifidobacterium animalis*, etc.). The yeast powder includes at least one of *Kluyveromyces martensii* and *Pichia pastoris*.

[0022] In some specific embodiments, in the preferred compound bacterial powder in step S3, the weight ratio of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus fermentum is (0.8-1.2):(0.1-0.3):(0.1-0.2).

[0023] In some specific embodiments, in the preferred compound bacterial powder in step S3, the yeast powder is Kluyveromyces martensii and Pichia pastoris in a weight ratio of (0.1-0.5):(1.0-1.5).

[0024] In some specific embodiments, in step S3, the total amount of the compound bacterial powder inoculated is 0.02-0.1% of the weight of the sterile tea sugar water, and the initial total number of viable compound bacteria in the sterile tea sugar water is maintained at 10 after inoculation. 5 -10 8 CFU / mL.

[0025] In some specific embodiments, the conditions for static fermentation in the fermentation container in step S3, which is carried out in the dark, are 25-32°C for 2-5 days.

[0026] In some specific implementations, the condition for terminating fermentation in step S3 is that the pH of the fermentation system drops to 3.2-3.6.

[0027] In some specific embodiments, the centrifugation in step S4 is performed using a disc centrifuge with a feed flow rate of 1.0-3.0 t / h.

[0028] In some specific embodiments, the sterilization conditions for step S4 are 105-115℃ for 10-30 s or 85-90℃ for 15-30 min.

[0029] According to a second aspect of the present invention, a kombucha fermentation liquid is provided, which is prepared by the kombucha fermentation liquid preparation method as described in any one of the first aspects.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The method for preparing kombucha fermentation liquid provided by the present invention uses a compound microbial agent composed of lactobacillus and yeast, and uses specific carbon and nitrogen sources and fermentation process conditions. Compared with the traditional kombucha process, this preparation method is fast, convenient, has a short fermentation time, and the processing technology is simple and easy to control, and can be applied to large-scale industrial production.

[0032] (2) The kombucha fermentation liquid provided by this invention has a clear and bright color, without any sediment or impurities, a fresh and complex aroma, a balanced sweet and sour taste, a delicate effervescence, and excellent floral and fruity aroma intensity, with high overall acceptability. It meets other hygienic physicochemical indicators and microbiological indicators, and complies with the GB / T31121-2014 Hygienic Standard for Fruit and Vegetable Juices and Beverages. Attached Figure Description

[0033] Figure 1 The graphs show the changes in acidity of the fermentation broth over time during the fermentation process for the preparation methods in Examples 1-4.

[0034] Figure 2 The graphs show the changes in acidity of the fermentation broth over time during the fermentation process of the preparation methods in Example 1 and Comparative Examples 1-10. Detailed Implementation

[0035] To illustrate the technical content, objectives, and effects of this invention in detail, the following description is provided in conjunction with the embodiments and accompanying drawings. These embodiments are merely illustrative of this application and should not be construed as limiting the scope of this application.

[0036] Other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of this invention.

[0037] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The *Lactobacillus plantarum* (Harvest LB-1), *Lactobacillus acidophilus* (LA-5), *Pichia kluyveromyces* (FrootZen), and *Kluyveromyces martensii* (LAF-4) used in the following examples were all purchased from Chr. Hansen Beijing Trading Co., Ltd. *Lactobacillus fermentum* (JYLF-315) was purchased from Minsheng Zhongke Jiayi (Shandong) Biotechnology Co., Ltd.

[0038] The nitrogen source used in this invention is yeast extract purchased from Chr. Hansen Beijing Trading Co., Ltd., under the product name Bactiv-Aid 2.0. Peptone was purchased from Beijing Luqiao Technology Co., Ltd., and is a conventional commercial nitrogen source for microbial culture.

[0039] Example 1

[0040] A method for preparing kombucha fermented liquid, the specific steps of which are as follows:

[0041] S1. Brewing tea: Weigh green tea leaves according to a mass ratio of 2.5:100, steep at 75℃ for 10 minutes, and filter through a 200-mesh filter cloth to obtain tea soup.

[0042] S2. Preparation of tea sugar water: Add a carbon source to the tea infusion. Use white sugar and concentrated apple juice with a soluble solids content of 70% (the weight ratio of white sugar to concentrated apple juice is 1.0:3.0) as a composite carbon source. The amount added is 8 wt% (based on the final weight of the tea sugar water). Weigh 0.12% of the final weight of the sterile tea sugar water as a nitrogen source, stir to dissolve, and heat the tea sugar water to 90℃ and maintain it for 300 s to obtain sterile tea sugar water.

[0043] S3. Inoculation and Fermentation: Cool the sterile tea sugar water to 25-30℃. Weigh out 0.08% of the weight of the sterile tea sugar water using compound bacterial powder, in which the mass ratio of Lactobacillus powder to yeast powder is 1.3:1.6. The mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 1.0:0.2:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.5:0.1. Inoculate the compound bacterial powder into the sterile tea sugar water and stir to mix well. Place the fermentation container at 30℃ in the dark for 3 days of anaerobic fermentation. Terminate fermentation when the pH of the fermentation system drops to 3.2-3.6.

[0044] S4. Post-processing: The fermentation broth was centrifuged and clarified using a disc centrifuge at a feed rate of 3.0 t / h. The supernatant was then collected and bottled. The fermentation broth was sterilized at 90℃ for 20 min, and then cooled to room temperature to obtain kombucha fermentation broth a1.

[0045] Example 2

[0046] S1. Brewing tea: Weigh green tea leaves according to a mass ratio of 2.5:100, steep at 75℃ for 10 minutes, and filter through a 200-mesh filter cloth to obtain tea soup.

[0047] S2. Preparation of tea sugar water: Add a carbon source to the tea soup. Use white sugar and concentrated apple juice with 70% soluble solids (the weight ratio of white sugar to concentrated apple juice is 1.0:1.0) as a composite carbon source. The amount added is 8 wt% (based on the final weight of the tea sugar water). Weigh 0.1% of the final weight of the sterile tea sugar water as a nitrogen source, stir to dissolve, and heat the tea sugar water to 90℃ and maintain it for 300 s to obtain sterile tea sugar water.

[0048] S3. Inoculation and Fermentation: Cool the sterile tea sugar water to 25-30℃. Weigh 0.06% of the weight of the sterile tea sugar water into a compound bacterial powder, wherein the mass ratio of Lactobacillus powder to yeast powder is 1.3:1.2. The mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 1.0:0.2:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.0:0.2. Inoculate the compound bacterial powder into the sterile tea sugar water and stir to mix well. Incubate the fermentation container at 32℃ in the dark for 2 days. Terminate the fermentation when the pH of the fermentation system drops to 3.2-3.6.

[0049] S4. Post-processing: The fermentation broth was centrifuged and clarified using a disc centrifuge at a feed rate of 3.0 t / h. The supernatant was then collected and bottled. The fermentation broth was sterilized at 90℃ for 15 min, and then cooled to room temperature to obtain kombucha fermentation broth a2.

[0050] Example 3

[0051] A method for preparing kombucha fermented liquid, the specific steps of which are as follows:

[0052] S1. Brewing tea: Weigh out black tea leaves at a mass ratio of 2.0:100, steep at 90℃ for 10 minutes, and filter through a 200-mesh filter cloth to obtain tea soup.

[0053] S2. Preparation of tea sugar water: Add a carbon source to the tea infusion. Use white sugar and concentrated apple juice with a soluble solids content of 70% (the weight ratio of white sugar to concentrated apple juice is 3.0:2.0) as a composite carbon source. The amount added is 5 wt% (based on the final weight of the tea sugar water). Weigh 0.15% of the final weight of the sterile tea sugar water as a nitrogen source, stir to dissolve, and heat the tea sugar water to 95℃ and maintain it for 300 s to obtain sterile tea sugar water.

[0054] S3. Inoculation and Fermentation: Cool the sterile tea sugar water to 25-30℃. Weigh 0.04% of the weight of the sterile tea sugar water into a compound bacterial powder, wherein the mass ratio of Lactobacillus powder to yeast powder is 1.15:1.2. The mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 0.8:0.25:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.0:0.2. Inoculate the compound bacterial powder into the sterile tea sugar water and stir to mix well. Incubate the fermentation container at 30℃ in the dark for 2 days. Terminate the fermentation when the pH of the fermentation system drops to 3.2-3.6.

[0055] S4. Post-processing: The fermentation broth was centrifuged and clarified using a disc centrifuge at a feed rate of 2.0 t / h. The supernatant was then collected for bottling. The fermentation broth was sterilized at 85℃ for 25 min, and then cooled to room temperature to obtain kombucha fermentation broth a3.

[0056] Example 4

[0057] A method for preparing kombucha fermented liquid, the specific steps of which are as follows:

[0058] S1. Brewing tea: Weigh out oolong tea leaves at a mass ratio of 3.0:100, steep at 85℃ for 8 minutes, and filter through a 200-mesh filter cloth to obtain tea soup.

[0059] S2. Preparation of tea sugar water: Add a carbon source to the tea soup. Use white sugar and concentrated apple juice with 70% soluble solids (the weight ratio of white sugar to concentrated apple juice is 2.0:1.0) as a composite carbon source. The amount added is 6 wt% (based on the final weight of the tea sugar water). Weigh 0.1% of the final weight of the sterile tea sugar water as a nitrogen source, stir to dissolve, and heat the tea sugar water to 90℃ and maintain it for 300 s to obtain sterile tea sugar water.

[0060] S3. Inoculation and Fermentation: Cool the sterile tea sugar water to 25-30℃. Weigh 0.05% of the final weight of the sterile tea sugar water using compound bacterial powder, wherein the mass ratio of Lactobacillus powder to yeast powder is 1.0:1.0. The mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 1.2:0.2:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.0:0.5. Inoculate the compound bacterial powder into the sterile tea sugar water and stir to mix well. Incubate the fermentation vessel at 30℃ in the dark for 3 days. Terminate the fermentation when the pH of the fermentation system drops to 3.2-3.6.

[0061] S4. Post-processing: The fermentation broth was centrifuged and clarified using a disc centrifuge at a feed rate of 1.5 t / h. The supernatant was then collected and bottled. The fermentation broth was sterilized at 85℃ for 15 min, and then cooled to room temperature to obtain kombucha fermentation broth a4.

[0062] Comparative Example 1

[0063] The only difference between this comparative example and the kombucha fermentation liquid preparation method in Example 1 is that no nitrogen source is added during the fermentation process. Kombucha fermentation liquid b1 was specifically prepared using this comparative example.

[0064] Comparative Example 2

[0065] The only difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is that only Lactobacillus powder was used during the fermentation process. Kombucha fermentation broth b2 was specifically prepared according to this comparative example.

[0066] Comparative Example 3

[0067] The only difference between this comparative example and the kombucha fermentation liquid preparation method in Example 1 is that only yeast powder was used during the fermentation process. Kombucha fermentation liquid b3 was specifically prepared by this comparative example.

[0068] Comparative Example 4

[0069] The only difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is that only white sugar is used as the carbon source. Kombucha fermentation broth b4 was specifically prepared according to this comparative example.

[0070] Comparative Example 5

[0071] The only difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is that the carbon source used is only concentrated apple juice. Kombucha fermentation broth b5 was specifically prepared according to this comparative example.

[0072] Comparative Example 6

[0073] The only difference between this comparative example and the kombucha fermentation liquid preparation method in Example 1 is that the brewing conditions are 60°C for 10 min. The kombucha fermentation liquid b6 was obtained through this comparative example.

[0074] Comparative Example 7

[0075] This comparative sample was prepared using traditional fermentation methods, and the specific steps are as follows.

[0076] S1. Brewing tea: Weigh green tea leaves according to a mass ratio of 2.5:100, steep at 75℃ for 10 minutes, and filter through a 200-mesh filter cloth to obtain tea soup.

[0077] S2. Preparation of tea sugar water: Add white sugar to the tea soup at an amount of 8 wt% (based on the final weight of the tea sugar water), stir to dissolve, heat the tea sugar water to 90℃ and maintain for 300 s to obtain sterile tea sugar water.

[0078] S3. Inoculation and Fermentation: Cool the sterile tea sugar water to 25-30℃. Inoculate the sterile tea sugar water with traditional mother liquor at an inoculation rate of 10-20%, and stir well. Incubate the fermentation vessel at 25-30℃ in the dark for 7-14 days. Terminate fermentation when the pH of the fermentation system drops to 3.2-3.6.

[0079] S4. Post-processing: The fermentation broth was centrifuged and clarified using a disc centrifuge at a feed rate of 2.5 t / h. The supernatant was then collected and bottled. The fermentation broth was sterilized at 90℃ for 15 min, and then cooled to room temperature to obtain kombucha fermentation broth b7.

[0080] Comparative Example 8

[0081] The only difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is that peptone is used as the nitrogen source. Kombucha fermentation broth b8 was specifically prepared according to this comparative example.

[0082] Comparative Example 9

[0083] The difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is only that the proportion of yeast powder in the compound microbial powder is higher. Specifically, the mass ratio of Lactobacillus powder to yeast powder is 1.0:2.0, where the mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 1.0:0.2:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.5:0.1. Kombucha fermentation broth b9 was prepared from this comparative example.

[0084] Comparative Example 10

[0085] The difference between this comparative example and the kombucha fermentation broth preparation method in Example 1 is only that the proportion of Lactobacillus powder in the compound microbial powder is higher. Specifically, the mass ratio of Lactobacillus powder to yeast powder is 1.0:0.5, where the mass composition of the Lactobacillus powder is *Lactobacillus plantarum*: *Lactobacillus acidophilus*: *Lactobacillus fermentum* = 1.0:0.2:0.1, and the mass composition of the yeast powder is *Pichia pastoris*: *Kluyveromyces martensii* = 1.5:0.1. Kombucha fermentation broth b10 was prepared from this comparative example.

[0086] Test Example 1

[0087] Sensory evaluation of kombucha fermentation liquid

[0088] Sensory evaluation tests were conducted on the kombucha fermented liquids prepared in Examples 1-4 and Comparative Examples 1-7. Thirty volunteers who passed the sensory sensitivity test were recruited as judges, including 15 aged 30 and above and 15 under 30 years old. The fermented liquids were evaluated on four indicators: color (color, clarity), aroma (fermentation aroma, floral and fruity aroma, tea aroma), taste (sweetness and acidity, body, balance), and overall acceptability, using a scale of 1-9. The higher the overall acceptability score, the more popular the product / service. For the other six indicators, 5 points was considered just right, 9 points was considered very strong, and 1 point was considered very weak. The results are shown in Table 1.

[0089] Table 1. Sensory Survey Results of Kombucha Fermentation Broth

[0090] As shown in Table 1, the sensory evaluation results indicate that the kombucha fermented liquids a1-a4 prepared in the embodiments of the present invention are significantly superior to all comparative examples in terms of color, aroma, taste and overall acceptability.

[0091] Specifically, Comparative Example 1 (without added nitrogen source) exhibited a slow fermentation process, resulting in insufficient aroma compound formation and a significantly lower aroma intensity score. This demonstrates that the addition of an appropriate amount of nitrogen source is a crucial and necessary condition for ensuring the full formation of the characteristic aroma of kombucha. Comparative Examples 2 and 3 show that using only Lactobacillus or yeast for fermentation has a significant impact on the aroma and taste of the fermented liquid. In particular, product b2 has a slightly stronger sour and astringent taste, and the floral and fruity aromas produced during fermentation are very weak, resulting in a lower overall score. This confirms the key role of the compound microbial strains used in this invention in enhancing the flavor of kombucha fermented liquid. Comparative Examples 4 and 5 highlight that a compound carbon source is the core for ensuring flavor balance. In particular, product b5, which was fermented using only concentrated apple juice, has a stronger sour and astringent taste and insufficient sweetness, resulting in a lower score in taste and overall acceptability. The score of Comparative Example 6 indicates that the brewing process conditions have a certain impact on the color, aroma, and taste of the kombucha fermented liquid product. Comparative Example 7, due to the use of traditional mother liquor fermentation for kombucha, exhibited a noticeable sour and rancid odor, resulting in low overall acceptance. This low acceptance underscores the significant progress made by this invention in overcoming the defects of traditional processes and improving flavor purity. Comparative Example 8, using peptone as a nitrogen source, showed a more pronounced odor, darker color, and higher acidity compared to Example 1, leading to even lower overall acceptance. Comparative Examples 9 and 10 adjusted the ratio of lactic acid bacteria and yeast in the compound probiotic powder. While the color was similar to Example 1, the aroma was significantly reduced, and the taste was worse, resulting in even lower overall acceptance than Example 1. This demonstrates that the composition of the compound probiotic powder plays a crucial role in the quality of the kombucha fermentation liquid in this invention. Overall, the reviewers showed high acceptance of the kombucha fermentation liquids from each example.

[0092] Test Example 2

[0093] Determination of acidification curve of kombucha fermentation broth

[0094] The kombucha fermentation broths prepared in Examples 1-4 and Comparative Examples 1-10 were used as test samples. Acidity was determined according to the national standard GB 5009.239-2016. Samples were taken and tested daily during fermentation, and a curve showing the change in acidity of the kombucha fermentation broth over time was finally plotted. Figure 1 and Figure 2 As shown.

[0095] Depend on Figure 1 and Figure 2 As shown, compared with the comparative example, the fermentation process of Example 1 of the present invention has a shorter cycle and higher efficiency, and shows a significant advantage in acid production rate. This reflects the unexpected technical effect brought about by the synergistic effect of specific strains and nutrient system.

[0096] Specifically, compared to Comparative Example 1 (without added nitrogen source), Example 1 significantly improved the acid production rate by supplementing a key nitrogen source, providing the necessary foundation for microbial growth and metabolism. Compared to Comparative Examples 2 and 3 (fermentation using a single type of microbial agent), the compound microbial agent used in Example 1 further increased acidity through synergistic effects of the microbial community. Comparative Example 4 (using only white sugar) resulted in significantly insufficient acidity, while Comparative Example 5 (using only concentrated apple juice), although having a higher final acidity, had a lower sensory score for the final product. Comparative Example 6 illustrates that the tea brewing process has a certain impact on the fermentation effect. Key data shows that the acidity of Example 1 reached approximately 65°T on the 3rd day of fermentation, while Comparative Example 7 (using traditional mother liquor fermentation), representing the traditional process, even after 7 days of fermentation, had an acidity (approximately 34°T) that was about half that of Example 1. Comparative Example 8 used peptone as a nitrogen source. Although its final acid production rate was even higher than that of Example 1 (reaching 77.3°T on day 3), its sensory evaluation was extremely poor. This indicates that excessively high acidity is not an indicator of excellent product quality. The specific nitrogen source selected in this invention can achieve the best flavor balance while ensuring efficient fermentation. Comparative Examples 9 and 10, due to the imbalance of the compound microbial powder ratio, had acceptable acid production efficiencies (58.7°T and 65.41°T on day 3, respectively), but still lower than that of Example 1. Moreover, their sensory quality decreased significantly. This result confirms that this invention, through the synergy of specific microbial strains and nutrient systems, can achieve higher acid production efficiency, which is crucial for forming a final product with a full flavor and stable quality.

[0097] Test Example 3

[0098] Stability determination of kombucha fermentation broth

[0099] The kombucha fermentation broths prepared in Examples 1-4 and Comparative Examples 1-10 were used as test samples and stored under accelerated degradation conditions at 37°C. Samples were taken on days 0, 7, 14, 21, and 28 to determine their color, sedimentation, and flavor, in order to evaluate the overall stability of the products. The results are recorded in Table 2.

[0100] Table 2. Stability Record of Kombucha Fermentation Broth

[0101] Table 2. Stability Record of Kombucha Fermentation Broth (Continued)

[0102] Table 2. Stability Record of Kombucha Fermentation Broth (Continued)

[0103] Table 2. Stability Record of Kombucha Fermentation Broth (Continued)

[0104] According to the statistical results in the table above, the following can be observed: Examples a1 and a2, using green tea flavored raw materials, only changed from "light yellow" to "yellow" throughout the observation period, showing slight color change and good color stability. Examples a3 and a4, using black tea or oolong tea as raw materials, only changed from "brown" to "dark reddish-brown" throughout the observation period, also showing slight color change and good color stability. Comparative examples b1-b7 generally turned "yellow" or "yellowish-brown" within 14 days, with a significant color deepening, and most turned "yellowish-brown" at the experimental endpoint, indicating that their color stability was significantly inferior to the examples. Examples a1, a2, a3, and a4 only showed "a small amount of precipitation" until day 28, indicating slow precipitation formation. Comparative examples b1-b7 generally showed precipitation on day 14 or earlier, and most developed into "more precipitation" or "a large amount of precipitation" in the later stages of the experiment, indicating a more prominent precipitation problem. Examples a1, a2, a3, and a4 maintained normal flavor throughout the entire observation period. Comparative products b1, b2, b3, b5, and b7 all exhibited a "slight off-odor" on day 28, demonstrating inferior flavor retention compared to the example. Comparative product b8, using peptone as a nitrogen source, exhibited an unstable yellow color from the outset and rapidly produced significant precipitation and a noticeable off-odor during storage, exhibiting extremely poor overall stability. This illustrates the crucial role of the specific nitrogen source selected in this invention in ensuring long-term product stability. Comparative products b9 and b10, while initially in good condition, also developed substantial precipitation and a slight off-odor in the later stages of storage (days 21-28), resulting in an overall stability assessment of "unstable." This indicates that deviating from the compound microbial powder formulation range defined in this invention, even if fermentation is completed, will compromise the product's shelf-life stability.

[0105] Therefore, under accelerated conditions, the products (a1-a4) of this invention exhibit significantly better color, sedimentation control, flavor retention, and overall stability than the comparative products (b1-b10), demonstrating the superior effect of this patented technology in improving product stability.

[0106] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing kombucha fermented liquid, characterized in that, Includes the following steps: S1. Brewing tea: Soak tea leaves in water, then filter to obtain tea soup; S2. Preparation of tea sugar water: Dissolve carbon source and nitrogen source into the tea soup, sterilize and obtain sterile tea sugar water; the carbon source is white sugar and concentrated apple juice, and the weight ratio of the two is (1.0-3.0):(1.0-5.0). S3. Inoculation and Fermentation: Cool the sterile tea sugar water to room temperature, inoculate with compound bacterial powder, and carry out anaerobic fermentation under constant temperature, static and light-proof conditions to obtain fermentation liquid; the compound bacterial powder includes lactobacillus powder and yeast powder in a mass ratio of (1.0-1.5):(0.8-1.6); S4. Post-processing: The fermentation liquid is centrifuged, filled, sterilized and cooled to obtain kombucha fermentation liquid.

2. The preparation method according to claim 1, characterized in that, In step S1, the tea leaves are selected from at least one of green tea, black tea, oolong tea, jasmine tea, or pu-erh tea.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of tea leaves to water is (1.0-3.5):

100.

4. The preparation method according to claim 1, characterized in that, In step S1, the tea is brewed at 75-90℃ for 5-20 minutes.

5. The preparation method according to claim 1, characterized in that, In step S1, the filtration uses a 200-mesh filter cloth.

6. The preparation method according to claim 5, characterized in that, In step S2, the amount of carbon source added is 5-12 wt% based on the mass of the tea sugar water.

7. The preparation method according to claim 1, characterized in that, In step S2, the nitrogen source is yeast extract powder, which is a mixture of various inactivated yeasts, and its addition amount is 0.01-0.2% of the final weight of the sterile tea sugar water.

8. The preparation method according to claim 1, characterized in that, In step S2, the sterilization conditions are: 85-95℃ for 300 s, or 118-121℃ for 5-15 s.

9. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the compound bacterial powder, lactobacillus powder and yeast powder is (1.0-1.5):(0.8-1.6).

10. The preparation method according to claim 9, characterized in that, In step S3, the lactobacillus powder includes at least one of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus helveticus, and Lactobacillus paracasei.

11. The preparation method according to claim 9, characterized in that, In step S3, the yeast powder includes at least one of Kluyveromyces maculae and Pichia pastoris.

12. The preparation method according to claim 10, characterized in that, In step S3, the lactobacillus powder is composed of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus fermentum, and the weight ratio of the three is (0.8-1.2):(0.1-0.3):(0.1-0.2).

13. The preparation method according to claim 11, characterized in that, In step S3, the yeast powder is composed of Kluyveromyces martensii and Pichia pastoris, and the weight ratio of the two is (0.1-0.5):(1.0-1.5).

14. The preparation method according to claim 1 or 9 to 13, characterized in that, In step S3, the total amount of compound bacterial powder inoculated is 0.02-0.1% of the final weight of the aseptic tea sugar water, and the initial total number of viable compound bacteria in the aseptic tea sugar water after inoculation is 10. 5 -10 8 CFU / mL.

15. The preparation method according to claim 1, characterized in that, In step S3, the fermentation conditions are 25-32℃ for 2-5 days.

16. The preparation method according to claim 1, characterized in that, In step S3, the fermentation is terminated when the pH of the fermentation system drops to 3.2-3.

6.

17. The preparation method according to claim 1, characterized in that, In step S4, the centrifugation is carried out using a disc centrifuge with a feed flow rate of 1.0-3.0 t / h.

18. The preparation method according to claim 1, characterized in that, In step S4, the sterilization conditions are 105-115℃ for 10-30 s or 85-90℃ for 15-30 min.

19. A kombucha fermentation liquid, characterized in that, It is prepared by the preparation method according to any one of claims 1-18.

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