Paracaseicillus casei and application thereof in fermented fruit kombucha

Abstract

The present application relates to a strain of paracasei lactis and its application in fermented fruit kombucha, belonging to the field of bioengineering technology. The present application is a kind of lactic acid bacteria isolated and screened from natural kombucha fermentation broth, which has been preserved, and the preservation number is CGMCC No.32759. The present application forms kombucha starter by compounding paracasei lactis XT-5 with a certain proportion of saccharomyces cerevisiae and tissieria equisimilis CGMCC 22548, and uses tea sugar water and fruit juice as raw materials to ferment and produce fruit kombucha beverage. The obtained kombucha beverage has mellow flavor, moderate sweet and sour taste, and has high total flavonoid content, total polyphenol content and strong antioxidant activity, and has good quality.

Description

Technical Field

[0001] This invention belongs to the field of microbial application and food brewing, and relates to a strain of lactic acid bacteria and its application, specifically a strain of Lacticaseibacillus paracasei XT-5 with good fermentation characteristics, producing a lot of flavor substances, and suitable for brewing kombucha beverages. Background Technology

[0002] *Lactobacillus paracasei* is a Gram-positive homofermenting lactic acid bacterium commonly found in the human gut and oral cavity. It is frequently used in fermentation and possesses abundant probiotic properties. *Lactobacillus paracasei* can regulate the immune system, improve lipid metabolism, and has antioxidant and antibacterial effects. Due to these probiotic properties, *Lactobacillus paracasei* can effectively maintain host health, thus playing a role in the food and medical fields. Furthermore, studies have shown that *Lactobacillus paracasei* can promote the formation of aromatic substances, including acids, alcohols, and esters. When added in small proportions, it can be used in the production of traditional fermented foods to significantly enhance their flavor.

[0003] Kombucha, an ancient beverage originating in China, also known as black tea or kombucha, is a uniquely flavored tea drink made by fermenting tea and sugar water with a symbiotic microbial community mainly composed of yeast, acetic acid bacteria, and lactic acid bacteria. Kombucha is loved not only for its sweet and sour taste, rich bubbles, and low alcohol content, but also for its various disease prevention and health benefits. However, naturally fermented kombucha strains are complex, containing many miscellaneous bacteria, resulting in low fermentation efficiency, long fermentation times, and the potential production of harmful substances. Traditionally cultivated kombucha strains exhibit significant differences in fermentation and probiotic properties. Naturally fermented kombucha drinks cannot fully utilize the nutrients in the tea water; moreover, fermentation quality cannot be well controlled—short fermentation times result in incomplete fermentation, while long fermentation times lead to an overly sour taste. Artificially constructing synthetic microbial communities for kombucha fermentation is a good way to improve fermentation efficiency and achieve controllable fermentation. Lactic acid bacteria, as one of the main microorganisms in kombucha fermentation, are of great importance in selecting lactic acid bacteria with good fermentation characteristics for use in kombucha fermentation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a strain of *Lactobacillus paracasei* and its application in fermented fruit kombucha. This invention isolates, screens, and identifies a strain of *Lactobacillus paracasei* XT-5 with good acid and ethanol tolerance and excellent fermentation ability from traditional kombucha fermentation broth. This strain, along with *Saccharomyces cerevisiae* and *Bacillus thuringiensis*, forms a synthetic microbial community for kombucha fermentation. The resulting kombucha beverage has a rich flavor, a balanced sweet and sour taste, and high levels of total flavonoids and polyphenols, as well as strong antioxidant activity, demonstrating excellent quality.

[0005] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a strain of Lactobacillus paracasei XT-5, which is classified and named Lactobacillus paracasei. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 32759 and deposit date of November 25, 2024.

[0007] In a second aspect, the present invention provides a microbial inoculant, comprising the aforementioned *Lactobacillus paracasei* XT-5.

[0008] Thirdly, the present invention provides the application of the aforementioned *Lactobacillus paracasei* XT-5 or microbial inoculant in the food industry. Further, the application refers to the fermentation preparation of fruit kombucha beverages.

[0009] Fourthly, the present invention provides a method for producing fruit kombucha beverage by fermentation of the above-mentioned *Lactobacillus paracasei* XT-5, comprising the following steps: Step 1: Slant culture: Inoculate the bacterial strain onto a solid slant culture medium and incubate at 32-37℃ for 2-3 days to obtain the activated bacterial strain in the slant culture, which is then ready for use. Step 2: Shake-flask culture expansion: Take the activated strain obtained in Step 1 and add sterile water to prepare a bacterial suspension; inoculate the bacterial suspension into the expansion medium, the inoculation amount is 5-10% of the volume of the liquid seed expansion medium, the culture temperature is 32-37℃, the shaking speed is 150-250 r / min, and the culture time is 1-2 days to obtain the seed liquid; Step 3: Use the seed liquid prepared in Step 2 as seeds for the fermentation production of fruit kombucha beverage.

[0010] As a further optimization of the above method, in step two, the formula of the liquid seed expansion culture medium is as follows: per 1000 mL of culture medium, there are 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of triamine citrate, 5 g of sodium acetate, 1 mL of Tween 80, 0.58 g of MgSO4, 0.25 g of MnSO4, and the balance is water;

[0011] As a further optimization of the above method, in step three, the fermentation production method includes the following steps: Step (1), after boiling water, add tea leaves at a ratio of 10-20 g / L water and add 80-100 g / L sucrose to prepare tea sugar water; then add fruit juice to the tea sugar water at a volume ratio of 10%-20% to prepare fermentation culture medium; Step (2): Inoculate Lactobacillus paracasei XT-5 and Saccharomyces cerevisiae into the fermentation medium prepared in step (1) and ferment at 25-30℃ for 1 day; Step (3): Inoculate the fermentation broth obtained in step (2) with Bacillus subtilis CGMCC 22548 and ferment at 25-30℃ for 1-3 days; Step (4): Separate the fermentation broth and let it stand for 5-10 days to mature.

[0012] As a further optimization of the above method, in step (1), the tea leaves are at least one of green tea, black tea, and Pu-erh tea. The fruit juice is any one or a combination of apple juice, grape juice, and pear juice.

[0013] As a further optimization of the above method, in step (2), the inoculation ratio of Lactobacillus paracasei XT-5 is 2%-4% and the inoculation ratio of Saccharomyces cerevisiae is 1%-3%; in step (3), the inoculation ratio of Bacillus humifusa CGMCC22548 is 3%-5%.

[0014] Beneficial Effects: This invention isolated, screened, and identified a strain of *Lactobacillus paracasei* XT-5 with good acid and ethanol tolerance and excellent fermentation ability from traditional kombucha fermentation broth. Further, by adding *Lactobacillus paracasei* XT-5, along with the auxiliary addition of *Saccharomyces cerevisiae* and *Bacillus simonii* CGMCC 22548, the fruit kombucha beverage exhibits superior flavor compared to traditional fermented kombucha, possessing a pleasant sour and sweet aroma, fruity and honey notes, and a subtle tea fragrance. Through fermentation, the antioxidant activity, total flavonoid content, and total polyphenol content of the fruit kombucha beverage are significantly enhanced compared to the unfermented form, resulting in greater health benefits. Unlike traditional fermentation methods, this invention features a shorter fermentation cycle, higher product quality, and easier control of the fermentation process, improving the utilization rate of the microbial strain and possessing certain industrial application value.

[0015] Strain Preservation Notes: Lactobacillus paracasei XT-5, classified as Lactobacillus paracasei, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32759 and deposit date of November 25, 2024. Attached Figure Description

[0016] Figure 1 This is a colony diagram of Lactobacillus paracasei XT-5; Figure 2 This is a Gram-stained microscopic image of Lactobacillus paracasei XT-5 cells; Figure 3 It is a phylogenetic tree of Lactobacillus paracasei XT-5; Figure 4 This is a growth curve of Lactobacillus paracasei XT-5; Figure 5 This is the acid production curve of Lactobacillus paracasei XT-5; Figure 6 This is a schematic diagram of kombucha prepared through fermentation. Detailed Implementation

[0017] I. Screening and identification of a lactic acid-producing microbial strain, the specific steps of which are as follows:

[0018] In this invention, 10 mL of fermentation broth from a natural kombucha fermentation tank is added to 90 mL of sterile water and shaken well. After shaking, 1 mL of the sample is added to 9 mL of sterile water and mixed using a vortex mixer. Then, another 1 mL of the sample is added to 9 mL of sterile water, and so on. Three suitable concentration gradients are sequentially spread onto solid culture plates (each 1000 mL of culture medium contains 10 g peptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g K₂HPO₄, 2 g triamine citrate, 5 g sodium acetate, 1 mL Tween 80, 0.58 g MgSO₄, 0.25 g MnSO₄, 20 g agar, and 15 g CaCO₃, with the remainder being water). The plates are incubated at 37°C for 36-48 h. Strains that produce a large transparent zone on the plates are selected for later use. The strains selected in the initial screening were inoculated onto solid culture medium plates for secondary screening (each 1000 mL of medium contained 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 2 g K2HPO4, 2 g triamine citrate, 5 g sodium acetate, 1 mL Tween 80, 0.58 g MgSO4, 0.25 g MnSO4, 20 g agar, and 15 g CaCO3, with the remainder being water), and incubated at 37°C for 36-48 h. The strains with better growth were then selected. The selected strains were inoculated into a secondary screening liquid medium (each 1000 mL of medium contained 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 2 g K₂HPO₄, 2 g triamine citrate, 5 g sodium acetate, 1 mL Tween 80, 0.58 g MgSO₄, 0.25 g MnSO₄, with the remainder being water) and cultured in shake flasks at 37 °C and 60 rpm for 15 h. The total acid content (calculated as lactic acid) was determined by titration with sodium hydroxide. After two rounds of secondary screening, a lactic acid-producing strain XT-5 was finally obtained (see...). Figure 1 The bacterium was Gram-positive (see [link to article]). Figure 2 It has a lactic acid production of 2.13 g / 100 mL and can tolerate 10% ethanol.

[0019] This invention screened and obtained the lactic acid-producing microorganism XT-5. The strain was sent to BGI Genomics for sequencing, and the sequencing results were submitted to the NCBI database for comparison. The results showed that the 16S rDNA sequence of strain XT-5 had a homology of up to 99% with Lacticaseibacillus paracasei JCM8130. A phylogenetic tree of strain XT-5 was constructed using MEGA 7.0 software. Combined with physiological and biochemical identification according to Bergey's Manual of Bacteriological Identification (8th edition), this bacterium is identified as Lacticaseibacillus paracasei, belonging to the genus Lacticaseibacillus of the family Lactobacteriaceae in the order Lactobacillales. This bacterium is rod-shaped, Gram-positive, with small colonies and a smooth surface at isolation. It is heterotrophic, utilizes glucose for growth, and is an aerobic bacterium. The optimal temperature for growth is 32–37℃, and the optimal pH for growth is 5.5–8.0.

[0020] II. A fermentation process for a fermented fruit kombucha beverage, the specific steps of which are as follows:

[0021] (1) Slant solid culture

[0022] Lactobacillus paracasei XT-5 was inoculated onto a solid slant culture medium and cultured at 32–37°C for 2–3 days to obtain an activated strain of bacteria in solid slant culture for later use.

[0023] The formulation of the slant solid culture medium is as follows: per 1000 mL of culture medium, there are 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of triamine citrate, 5 g of sodium acetate, 1 mL of Tween 80, 0.58 g of MgSO4, 0.25 g of MnSO4, 20 g of agar, and 15 g of CaCO3, with the remainder being water;

[0024] (2) Shake flask culture of microbial strains

[0025] Take the Lactobacillus paracasei XT-5 obtained in (1), add sterile water to make a bacterial suspension, add 3-5 mL of sterile water to one test tube, inoculate the bacterial suspension into the expansion medium, the inoculation amount is 5-10% of the liquid medium volume, the culture temperature is 32-37℃, the shaking speed is 150-250 r / min, the culture time is 1-2 days, and the seed liquid is obtained as the seed for fermenting kombucha;

[0026] The formula for the liquid seed culture medium is as follows: per 1000 mL of culture medium, there are 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of triamine citrate, 5 g of sodium acetate, 1 mL of Tween 80, 0.58 g of MgSO4, 0.25 g of MnSO4, and the balance is water;

[0027] (3) Fermentation of fruit kombucha beverages

[0028] After boiling the water, add tea leaves at a ratio of 10-20 g / L water and 80-100 g / L sucrose, then filter out the tea leaves; add fruit juice to the tea-sugar water at a ratio of 10%-20%; inoculate with *Lactobacillus paracasei* XT-5 and *Saccharomyces cerevisiae* in a specific ratio; ferment at 25-30℃ for 1 day; inoculate with *Bacillus hummica* CGMCC 22548 in a specific ratio; ferment at 25-30℃ for 3-5 days; separate the fermentation liquid and allow it to stand for 5-10 days to mature.

[0029] III. ABTS Free Radical Scavenging Capacity Test

[0030] Sample preparation: Take 2.5 mL of sample and add it to a 50 mL volumetric flask, then dilute to volume with water. Then take 1 mL of the sample and add 9 mL of distilled water to dilute it to obtain a sample solution diluted 200 times. Take 1 mL of the diluted sample solution and 4 mL of ABTS solution as the experimental group, and use distilled water as a blank control. Measure the absorbance at 734 nm and calculate the ABTS free radical scavenging rate.

[0031] IV. Determination of Volatile Flavor Compounds

[0032] The headspace-solid phase microextraction-gas chromatography-mass spectrometry method was used.

[0033] (1) Extraction conditions: First, age the extraction head at 250℃ in the gas chromatograph injection port until no impurity peaks are observed. Add 6 mL of sample (with 10 μL of 100 mg / L 2-octanol as an internal standard) to a 15 mL solid-phase microextraction sample bottle, add 1 g of sodium chloride, add a magnetic stir bar, and close the lid. Place the bottle in a 40℃ constant temperature water bath and equilibrate for 10 min. Insert the SPME extraction head into the headspace portion of the sample through the bottle cap, push out the fiber head, allow headspace adsorption for 40 min, then pull back the fiber head, remove the extraction head from the sample bottle, insert the extraction head into the gas chromatograph injection port of the GC-MS instrument, push out the fiber head, desorb at 250℃ for 5 min, pull back the fiber head, and simultaneously start the instrument to acquire data.

[0034] (2) Instrument conditions: Chromatographic conditions: TG-WAXMS capillary column, column length 30μm, inner diameter 0.25mm, liquid film thickness 0.25μm, carrier gas: He gas, flow rate 1mL / min, split flow rate: 50mL / min, purge flow rate: 3mL / min, injection port temperature: 250℃.

[0035] Temperature program: Initial temperature 35℃, hold for 5 min; increase to 180℃ at 4℃ / min, hold for 2 min; then increase to 220℃ at 5℃ / min, hold for 5 min, for a total of 56.25 min.

[0036] Mass spectrometry conditions: transfer line temperature: 250℃, ion source temperature: 280℃, ionization mode: EI ionization source, electron energy: 70eV, mass scan range: 33-450amu.

[0037] (3) Data Processing and Analysis: The spectral libraries were searched using replib and mainlab. Compounds with a volatile component matching degree greater than 800 were detected, with the highest matching degree being 1000. The relative peak area ratio of all components was calculated using the peak area normalization method. The concentration of flavor substances in each component of the sample was calculated using the formula: Ci=Ai / As×Cs; Where: Ci: concentration of each volatile flavor compound in the sample, μg / L; Cs: 2-Octanol concentration, μg / L; Ai: The chromatographic peak area corresponding to the analyte in the sample; As: Peak area of ​​the internal standard.

[0038] V. Determination of Total Flavonoid Content

[0039] Take 1 mL of sample into a 10 mL colorimetric tube, add 60% ethanol to make the total volume of all colorimetric tubes 5 mL, add 0.3 mL of 5% sodium nitrite solution, shake well and let stand for 5 minutes, then add 0.3 mL of 10% aluminum nitrate solution, shake well with a shaker and let stand for 6 minutes, then add 4 mL of 1 mol / L sodium hydroxide solution, and finally make up the volume with 60% ethanol. Let stand for 15 minutes, measure the absorbance at a wavelength of 510 nm, and calculate the total flavonoid content according to the standard curve.

[0040] VI. Determination of Total Polyphenol Content

[0041] Take 1.00 mL of sample into a 25 mL volumetric flask, add 0.65 mL of 0.80 mol / L Folin reagent, react in the dark, add 4 mL of 20% sodium carbonate solution, dilute to volume with distilled water, incubate at 60℃ for 60 min, take 2 mL of the reacted sample, dilute with 2 mL of distilled water twice, measure the absorbance at 720 nm, and calculate the total polyphenol content.

[0042] This invention utilizes *Lactobacillus paracasei* XT-5, which can be combined with a certain proportion of *Saccharomyces cerevisiae* and *Bacillus simonii* CGMCC 22548 to form a kombucha starter. Using green tea syrup and apple juice as raw materials, fermentation for 5-7 days produces an apple kombucha beverage. The fermented apple kombucha beverage showed an increase of 80 flavor compounds, with the ABTS free radical scavenging rate increasing from 52.43% to 83.76%, the total flavonoid content increasing from 0.46 mg / mL to 0.69 mg / mL, and the total phenolic content increasing from 0.43 mg / mL to 0.81 mg / mL. This invention primarily uses strains with proprietary intellectual property rights to ferment fruit kombucha beverages, significantly improving the flavor components and nutritional value of the kombucha beverage and increasing the added value of the tea.

[0043] The applicant will now provide a detailed description of the preparation and application processes of the product of this invention, using specific embodiments, to facilitate a clear understanding of the invention by those skilled in the art. However, it should be understood that the following embodiments should not be construed as limiting the scope of protection claimed in this application.

[0044] Example 1: Explanation of how to screen for and obtain Lactobacillus paracasei XT-5

[0045] (1) Initial screening on plates: 10 mL of the fermentation tank of naturally fermented kombucha was added to 90 mL of sterile water and shaken well. After shaking, 1 mL of the sample was added to 9 mL of sterile water and mixed on a vortex mixer. Then, 1 mL of the sample was added to 9 mL of sterile water, and so on. Three suitable concentration gradients were selected and sequentially spread onto solid culture plates (each 1000 mL of culture medium contained 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of triamine citrate, 5 g of sodium acetate, 1 mL of Tween 80, 0.58 g of MgSO4, 0.25 g of MnSO4, 20 g of agar, and 15 g of CaCO3, with the remainder being water). The plates were incubated at 37℃ for 36-48 h, and strains that produced larger transparent zones on the plates were selected. The strains selected in the initial screening were inoculated onto solid culture medium plates for secondary screening (each 1000 mL of medium contained 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 2 g K2HPO4, 2 g triamine citrate, 5 g sodium acetate, 1 mL Tween 80, 0.58 g MgSO4, 0.25 g MnSO4, 20 g agar, and 15 g CaCO3, with the remainder being water), and incubated at 37°C for 36-48 h. The strains with better growth were then selected.

[0046] (2) Shake flask re-screening: The selected strains were inoculated into re-screening liquid culture medium (each 1000 mL of culture medium contains 10 g peptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g K2HPO4, 2 g triamine citrate, 5 g sodium acetate, 1 mL Tween 80, 0.58 g MgSO4, 0.25 g MnSO4, with the remainder being water) and cultured in shake flasks at 37 °C and 60 r / min for 15 h. The total acid content (calculated as lactic acid) was determined by titration with sodium hydroxide.

[0047] (3) After two rounds of screening, a lactic acid-producing strain XT-5 was finally obtained (see Figure 1 The bacterium was Gram-positive (see [link to article]). Figure 2 It has good resistance to ethanol and strong acid production ability.

[0048] Implementation Case 2: How to identify Lactobacillus paracasei XT-5

[0049] (1) Single-colony slant cultures were isolated and purified, and sent to BGI Genomics in Wuhan for sequencing. The sequencing results were submitted to the NCBI database for comparison. The results showed that the 16S rDNA sequence of strain XT-5 had a homology of up to 99% with that of Lacticaseibacillus paracasei JCM8130. A phylogenetic tree of XT-5 was constructed using MEGA 7.0 software, the NJ method, and an expansion value of 3000 (see...). Figure 3 ).

[0050] (2) Based on the morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence of strain XT-1, XT-5 was identified as Lacticaseibacillus paracasei.

[0051] (3) The strain XT-5, identified as Lactobacillus paracasei, was deposited at the China General Microbiological Culture Collection Center (CGMCC) at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number was CGMCC No. 32759 and the deposit date was November 25, 2024.

[0052] (4) The 16S rDNA sequence of the Lactobacillus paracasei XT-5 is shown in SEQ ID NO: 01.

[0053] Example 3 illustrates how to ferment apple kombucha beverages with Lactobacillus paracasei XT-5.

[0054] (1) Preparation of seed culture: XT-1 slant culture stored at 4℃ was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL (1L liquid culture medium formula: 10g peptone, 10g beef extract, 5g yeast powder, 20g glucose, 2g K2HPO4, 2g triamine citrate, 5g sodium acetate, 1mL Tween 80, 0.58g MgSO4, 0.25g MnSO4, with the remainder being water), and cultured at 37℃ and 80r / min for 36h; Saccharomyces cerevisiae was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL YPD medium and cultured at 30℃ and 180r / min for 24h; Bacillus thuringiensis CGMCC 22548 slant culture was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL GYE medium and cultured at 28℃ and 80r / min for 36h.

[0055] (2) Preparation of tea sugar water: Boil 1000mL of water to sterilize it. Weigh out 15g of tea leaves (green tea) and add them to the boiling water. Then add 80g of white sugar and dissolve it completely. Prepare a sterilized container, filter the tea sugar water into the container, and let it cool to room temperature.

[0056] (3) Add apple juice: Add 100mL of sterilized apple juice to the tea sugar water and mix well.

[0057] (4) Fermentation: Lactobacillus paracasei XT-5: Saccharomyces cerevisiae were inoculated into the fermentation medium at a ratio of 3%:1% and fermented at 30°C for 1 day; Bacillus thymosa CGMCC 22548 was inoculated into the fermentation liquid at a ratio of 3% and fermented at 30°C for 3 days.

[0058] (5) Post-filtration maturation: Take the fermented kombucha liquid, filter it, and then pasteurize it. After pasteurization, the kombucha fermentation liquid can be post-matured at room temperature in the dark for 5 days.

[0059] (6) The antioxidant properties, total flavonoid content and total polyphenol content of the fermented kombucha liquid were determined, with unfermented tea sugar water and apple juice mixture as controls. The results are shown in Table 1.

[0060] Table 1 Nutritional Indicators of Example 3

[0061] Example 4 illustrates how to ferment apple kombucha beverages with Lactobacillus paracasei XT-5.

[0062] (1) Preparation of seed culture: XT-5 slant culture stored at 4℃ was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL (1L liquid culture medium formula: 10g peptone, 10g beef extract, 5g yeast powder, 20g glucose, 2g K2HPO4, 2g triamine citrate, 5g sodium acetate, 1mL Tween 80, 0.58g MgSO4, 0.25g MnSO4, with the remainder being water), and cultured at 37℃ and 80r / min for 36h; Saccharomyces cerevisiae was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL YPD medium and cultured at 30℃ and 180r / min for 24h; Bacillus thuringiensis CGMCC 22548 slant culture was inoculated into 250mL Erlenmeyer flasks with a volume of 100mL GYE medium and cultured at 28℃ and 80r / min for 36h.

[0063] (2) Preparation of tea sugar water: Boil 1000mL of water to sterilize it. Weigh out 15g of tea leaves (green tea) and add them to the boiling water. Then add 80g of white sugar and dissolve it completely. Prepare a sterilized container, filter the tea sugar water into the container, and let it cool to room temperature.

[0064] (3) Add apple juice: Add 200mL of sterilized apple juice to the tea sugar water and mix well.

[0065] (4) Fermentation: Lactobacillus paracasei XT-5: Saccharomyces cerevisiae were inoculated into the fermentation medium at a ratio of 3%:1% and fermented at 30℃ for 1 day; Bacillus thymosa CGMCC 22548 was inoculated into the fermentation liquid at a ratio of 3% and fermented at 30℃ for 5 days.

[0066] (5) Post-filtration maturation: Take the fermented kombucha liquid, filter it, and then pasteurize it. After pasteurization, the kombucha fermentation liquid can be post-matured at room temperature in the dark for 8 days.

[0067] (6) The antioxidant properties, total flavonoid content and total polyphenol content of the fermented kombucha liquid were determined, with unfermented tea sugar water and apple juice mixture as controls. The results are shown in Table 2.

[0068] Table 2 Nutritional Indicators of Example 4

[0069] Analysis of the volatile flavor compounds in this embodiment revealed a total of 28 alcohol compounds, 13 aldehyde / ketone compounds, 17 organic acids, and 22 ester compounds, totaling 80 volatile flavor compounds. The specific compounds are shown in Table 4.

[0070] Example 5 illustrates how to ferment apple kombucha beverage using Lactobacillus plantarum CGMCC 24035.

[0071] (1) Preparation of seed culture: Lactobacillus plantarum CGMCC 24035 slant culture stored at 4℃ was inoculated into a 250mL Erlenmeyer flask with a volume of 100mL (1L liquid culture medium formula: 10g peptone, 10g beef extract, 5g yeast powder, 20g glucose, 2g K2HPO4, 2g triamine citrate, 5g sodium acetate, 1mL Tween 80, 0.58g MgSO4, 0.25g MnSO4, the remainder being water), and cultured at 37℃ and 80r / min for 36h; Saccharomyces cerevisiae was inoculated into a 250mL Erlenmeyer flask with a volume of 100mL YPD medium and cultured at 30℃ and 180r / min for 24h; Bacillus thuringiensis CGMCC 22548 slant culture was inoculated into a 250mL Erlenmeyer flask with a volume of 100mL GYE medium and cultured at 28℃ and 80r / min for 36h.

[0072] (2) Preparation of tea sugar water: Boil 1000mL of water to sterilize it. Weigh out 15g of tea leaves (green tea) and add them to the boiling water. Then add 80g of white sugar and dissolve it completely. Prepare a sterilized container, filter the tea sugar water into the container, and let it cool to room temperature.

[0073] (3) Add apple juice: Add 200mL of sterilized apple juice to the tea sugar water and mix well.

[0074] (4) Fermentation: Lactobacillus plantarum CGMCC 24035: Saccharomyces cerevisiae were inoculated into the fermentation medium at a ratio of 3%:1% and fermented at 30℃ for 1 day; Bacillus thuringiensis CGMCC 22548 was inoculated into the fermentation liquid at a ratio of 3% and fermented at 30℃ for 5 days.

[0075] (5) Post-filtration maturation: Take the fermented kombucha liquid, filter it, and then pasteurize it. After pasteurization, the kombucha fermentation liquid can be post-matured at room temperature in the dark for 8 days.

[0076] (6) The antioxidant properties, total flavonoid content and total polyphenol content of the fermented kombucha liquid were determined, and the results are shown in Table 3.

[0077] Table 3 Nutritional Indicators of Example 5

[0078] Analysis of the volatile flavor compounds in this embodiment revealed a total of 73 volatile flavor compounds, including 23 alcohols, 13 aldehydes / ketones, 16 organic acids, and 21 esters, after data processing. Specific substances are shown in Table 4.

[0079] By measuring three nutritional indicators—ABTS free radical scavenging rate, total flavonoid content, and total polyphenol content—in Examples 3 and 4, it was found that their contents were significantly higher than those of unfermented tea. This indicates that by purifying kombucha fermentation, the active ingredients in tea and fruit can be better converted and utilized, thereby increasing the added value of tea and fruit.

[0080] By measuring four nutritional indicators—ABTS free radical scavenging rate, total flavonoid content, total polyphenol content, and volatile flavor compounds—in Examples 4 and 5, it was found that their contents were all higher than those of apple kombucha beverage fermented with Lactobacillus plantarum CGMCC 24035. This indicates that Lactobacillus paracasei XT-5 can better transform and utilize the active ingredients in tea and fruit, thereby increasing the added value of tea and fruit.

[0081] Table 4. Volatile flavor compounds in Examples 4 and 5

[0082] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A method for fermenting and producing fruit kombucha beverage, characterized in that, The Lactobacillus paracasei used ( Lacticaseibacillus paracasei XT-5 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32759 and deposit date of November 25, 2024. The method includes the following steps: Step 1: Slant culture: Inoculate the bacterial strain onto a solid slant culture medium and incubate at 32-37℃ for 2-3 days to obtain the activated bacterial strain in the slant culture, which is then ready for use. Step 2: Shake-flask culture of microbial strain: Take the activated microbial strain obtained in Step 1 and add sterile water to prepare a bacterial suspension; inoculate the bacterial suspension into the expansion medium, the inoculation amount is 5-10% of the volume of the liquid seed expansion medium, the culture temperature is 32-37℃, the shaking speed is 150-250 r / min, and the culture time is 1-2 days to obtain the seed liquid; Step 3: Use the seed liquid prepared in Step 2 as seeds for the fermentation production of fruit kombucha beverage; Fermentation production includes the following steps: Step (1): After boiling the water, add green tea leaves at a ratio of 10-20g / L water and 80-100g / L sucrose to prepare tea sugar water; then add apple juice to the tea sugar water at a volume ratio of 10%-20% to prepare fermentation culture medium. Step (2): Inoculate *Lactobacillus paracasei* XT-5 and *Saccharomyces cerevisiae* into the fermentation medium prepared in step (1), and ferment at 25-30℃ for 1 day; the inoculation ratio of *Lactobacillus paracasei* XT-5 is 2%-4%, and the inoculation ratio of *Saccharomyces cerevisiae* is 1%-3%. Step (3): Inoculate the fermentation broth obtained in step (2) with *Bacillus simonii* (…). Komagataeibacter oboediens CGMCC 22548 was fermented at 25-30℃ for 5 days; the inoculation ratio of the docile *C. truncatula* CGMCC 22548 was 3%-5%. Step (4) Separate the fermentation liquid and let it stand for 5-10 days to ripen.

2. The method according to claim 1, characterized in that: In step two, the formula for the liquid seed culture medium is as follows: per 1000 mL of culture medium, there are 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of triamine citrate, 5 g of sodium acetate, 1 mL of Tween 80, 0.58 g of MgSO4, 0.25 g of MnSO4, and the balance is water.

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