High-tea-substrate-concentration-resistant lactic acid bacteria and preparation method of high-substrate-concentration fermented tea of high-tea-substrate-concentration-resistant lactic acid bacteria

Abstract

The invention discloses a strain of Lactobacillus pentosus B5, which is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No. 66054. The invention also discloses a method for preparing the Lactobacillus pentosus B5. The Lactobacillus pentosus B5 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the Lactobacillus pentosus B5 is GDMCC No. 66054. The strain is separated from the surfaces of fresh tea leaves, has good tolerance to high tea substrate concentration, and is suitable for fermentation of various tea leaves such as green tea, black tea and oolong tea. The strain can stably grow and efficiently produce acid at a high tea substrate concentration of 8-10%. By combining with an optimized high-substrate-concentration fermentation process, the fermentation efficiency can be effectively improved, release and conversion of active ingredients of the tea leaves are promoted, the polyphenol content is increased, the proportion of ester catechin is reduced, and the flavor and antioxidant activity of the product are improved. Meanwhile, the high-concentration tea substrate fermentation can reduce the moisture proportion, improve the concentration of effective components in unit volume, optimize the production cost, and enhance the functionality and market competitiveness of the final product. The strain is derived from tea leaves, is used for fermenting the tea leaves, and has relatively high safety and wide industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of lactic acid bacteria tolerant to high tea concentrations and its application in fermenting tea with high substrate concentrations. Background Technology

[0002] Lactic acid bacteria fermented tea utilizes lactic acid bacteria to ferment tea leaves or tea infusion, producing a unique flavor and a series of beneficial metabolites. It combines the probiotic properties of the fermentation strains with the health benefits of tea itself, thus possessing both the health benefits of tea and the probiotic effects of lactic acid bacteria. As a raw material for producing related fermented tea beverages, lactic acid bacteria fermented tea has received increasing attention and application in recent years. With the increasing demand for healthy drinks and skincare products, the market for functional beverages is expanding. Due to the beneficial components produced by lactic acid bacteria during fermentation, lactic acid bacteria fermented tea has become an ideal raw material for fermented beverage products. As a raw material for tea beverages, lactic acid bacteria fermented tea aligns with modern consumers' demand for functional and healthy drinks. Against the backdrop of the growing popularity of healthy eating and "probiotic drinks," the market demand for lactic acid bacteria fermented tea has risen significantly.

[0003] The core of lactic acid bacteria fermentation technology for tea lies in selecting lactic acid bacteria fermentation strains with superior characteristics. Because tea contains abundant polyphenols, tannins, caffeine, and other substances, which can inhibit the growth of lactic acid bacteria and affect the smooth progress of the fermentation process, the selected fermentation strains must be able to tolerate tea components, grow stably at high tea concentrations, and ferment efficiently, producing acid and maintaining the activity of the lactic acid bacteria to ensure the quality of the final product. Higher tea substrate concentrations can improve fermentation efficiency, promote better release of active ingredients from the tea, accelerate the conversion of tea polyphenols and enhance their biological activity, thereby improving their health benefits.

[0004] Using high substrate concentrations as the culture medium for fermentation offers significant advantages. Firstly, high substrate concentrations increase the concentration of active ingredients per unit volume, leading to a more concentrated fermentation product and thus enhancing its functionality and bioactivity. Secondly, high-concentration culture media promote the release and transformation of active ingredients in tea. Furthermore, the reduced moisture content during fermentation decreases energy consumption in subsequent concentration and drying processes, optimizing production costs and improving industrial production efficiency.

[0005] Currently, lactic acid bacteria strains capable of adapting to high tea substrate concentrations during fermentation are relatively scarce, and related research reports are also limited. This invention successfully obtained a lactic acid bacteria strain capable of stable growth at high tea substrate concentrations through screening from the surface of tea leaves. This strain can ferment efficiently in high-concentration tea liquid, producing acid while maintaining high activity. After optimizing the fermentation process, this strain can stably produce lactic acid bacteria fermented tea base material, which not only possesses excellent flavor and functional properties but can also be used as a raw material for the production of lactic acid bacteria fermented tea beverages. Summary of the Invention

[0006] The purpose of this invention is to provide a lactic acid bacterium screened from fresh tea leaves and its application in fermented tea. The lactic acid bacterium has the ability to tolerate high tea concentration fermentation, rapid proliferation, acid production and good fermentation stability. After fermentation, it can increase the polyphenol content in the fermented sample, significantly reduce the ester catechin content, improve bitterness and astringency, increase the content of volatile components, improve and increase aroma, and enhance the antioxidant capacity of fermentation products.

[0007] The following technical solution is adopted: A type of Lactobacillus pentosus B5 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66054 and deposit date of March 26, 2025.

[0008] This invention discloses a strain of Lactobacillus pentosus ( Lactiplantibacillus pentosus Lactic acid bacteria strain B5, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 66054), is isolated from the surface of fresh tea leaves. It exhibits good tolerance to high tea substrate concentrations and is suitable for fermenting various teas, including green tea, black tea, and oolong tea. This strain can stably grow and efficiently produce acid at a high tea substrate concentration of 8-10%. Combined with an optimized high substrate concentration fermentation process, it can effectively improve fermentation efficiency, promote the release and transformation of active ingredients in tea, increase polyphenol content, reduce the proportion of ester-type catechins, and improve the flavor and antioxidant activity of the product. Simultaneously, high-concentration tea substrate fermentation can reduce the moisture content, increase the concentration of effective ingredients per unit volume, optimize production costs, and enhance the functionality and market competitiveness of the final product. The resulting lactic acid bacteria fermented tea base can serve as a core ingredient in functional tea beverages, meeting consumers' demand for high-nutritional-value healthy drinks. This invention's strain, derived from tea leaves and used for tea fermentation, possesses high safety and broad industrial application prospects.

[0009] An application of the above-mentioned Lactobacillus pentosus B5 is characterized in that it is used in fermented tea.

[0010] Preferably, the fermented tea is obtained by fermentation with the Lactobacillus pentosus B5 fermenting agent; the fermentation substrate of the fermented tea includes tea leaves.

[0011] Preferably, the tea leaves in the fermentation substrate have a mass concentration of 8-10%; the tea leaves include one or more of green tea, black tea, and oolong tea.

[0012] Preferably, the preparation of the fermented tea includes the following steps: A. Activate the Lactobacillus pentosus B5 to obtain the activated bacterial solution; B. Prepare a fermentation medium, which includes one or more of tea leaves, glucose, sucrose, sodium chloride, yeast extract, sodium dihydrogen phosphate, and distilled water, and then adjust the pH and sterilize it. C. Inoculate the activated bacterial solution into the fermentation medium and carry out fermentation to obtain fermented material; D. The fermented material is subjected to solid-liquid separation, and the remaining liquid is homogenized to obtain the fermented tea.

[0013] Preferably, in step B, the mass-to-volume ratio of the tea leaves, glucose, sucrose, sodium chloride, yeast extract, sodium dihydrogen phosphate, and distilled water in the fermentation culture medium is 2-10 g: 0-2 g: 0-2 g: 0.1-0.5 g: 0.02-0.1 g: 0.001-0.005 g: 100 mL. 8-10 g of tea leaves are added per 100 mL of water, resulting in a tea leaf concentration (mass fraction) of 8-10%.

[0014] Preferably, in step A, the activation conditions include: static incubation in MRS liquid medium at 28-40 °C for 24-48 h; in step B, the sterilization conditions include: sterilization at 100-115 °C for 10-15 min; in step C, the fermentation conditions include: static fermentation at 28-40 °C for 3-7 days; and in step D, the solid-liquid separation method includes filtration.

[0015] Preferably, in step A, the MRS liquid culture medium contains 1-5% tea leaves; in step D, the filter cloth used for filtration comprises a 60-120 mesh filter cloth.

[0016] Preferably, in step A, the concentration of the activated bacterial solution includes 10. 8 ~10 9 CFU / mL; in step B, the pH of the fermentation medium is 6.0-7.0; in step C, the volume ratio of the activated bacterial solution to the fermentation medium is 1-5%:1; in step D, the homogenization conditions are: homogenization at 20-100 MPa for 5-45 min, repeated 1-3 times.

[0017] A fermented tea obtained by the above application.

[0018] Compared with the prior art, implementing the present invention has the following beneficial effects: (1) The present invention obtains a strain of Lactobacillus pentosus with strong tolerance from the surface of fresh tea leaves. It can tolerate the antibacterial components in tea leaves, grow stably under high tea substrate concentration, and produce acid efficiently, ensuring smooth fermentation.

[0019] (2) This invention uses *Lactobacillus pentosus* as the fermentation strain, which can be used for fermentation in tea concentrations ranging from 0% to 10%. Currently, there are no reports of lactic acid bacteria being able to tolerate fermentation in tea substrate concentrations of 8% to 10%. The optimized fermentation process can promote the release of active ingredients in tea, increase the polyphenol content in the fermented sample, and promote the conversion and bioactivity of tea polyphenols. Simultaneously, this process can significantly reduce the content of ester-type catechins, reducing bitterness; increase the content of volatile components, enhancing aroma; and improve the antioxidant capacity of the fermentation products, thereby increasing the health value and flavor of the final product.

[0020] (3) This strain is derived from fresh tea leaves and is used in the fermentation process of tea, which meets the requirements of natural food processing, avoids the food safety risks that may be caused by the introduction of exogenous strains, and has high safety.

[0021] (4) This strain can be used for the fermentation of a variety of common teas and has a wide range of adaptability to fermented tea substrate raw materials.

[0022] (5) The method provided by this invention can use a higher concentration of tea substrate for fermentation, which can increase the amount of material fed at one time and accelerate the fermentation process. High-concentration tea substrate fermentation can reduce the water content, increase the concentration of effective ingredients per unit volume, optimize production costs, improve fermentation efficiency, and enhance the functionality and market competitiveness of the final product. The fermentation process of this invention optimizes the growth environment of lactic acid bacteria, improves the survival rate and fermentation efficiency of the strains under high tea substrate concentration, and enables them to stably produce high-quality lactic acid bacteria fermented tea base material, which is suitable for large-scale industrial production. Attached Figure Description

[0023] Figure 1 Colony morphology of strain B5 Figure 2 Cell morphology of strain B5 after Gram staining Figure 3 Alignment and phylogenetic tree of 16S rDNA sequence of strain B5 Figure 4 Multiplex PCR identification results of strain B5 Figure 5 Fermentation concentrations of strain B5 under different tea concentration conditions. Note: Lowercase letters in the figure indicate significant differences between different concentrations. p <0.05) Figure 6 Acid production and pH changes during the fermentation process of strain B5 Figure 7 Changes in viable cell count during fermentation of strain B5 Figure 8 The catechin content in tea infusions after fermentation with strain B5 and control strains E9 and L16, and in unfermented tea. Note: Lowercase letters in the figure indicate significant differences between different concentrations. p <0.05) Figure 9 Percentages of volatile components in tea infusions after fermentation with strain B5 and control strains E9 and L16, compared to unfermented tea. Note: Lowercase letters in the figure indicate significant differences between different concentrations. p <0.05). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] Unless otherwise specified, the raw materials and reagents used in the following embodiments are commercially available, and the methods and equipment used are conventional in the art.

[0026] Example 1 This example illustrates the process of isolating, purifying, screening, and identifying strain B5.

[0027] 1.1 Enrichment, Isolation and Purification of Lactic Acid Bacteria Take 50g of fresh tea leaves, aseptically cut them into 1cm pieces, mix them with 100mL of 1% sterile glucose solution, adjust the pH to 5 with 0.1mol / L HCl, transfer to an anaerobic culture bag, and incubate at 30℃ for 3 days. Take 1mL of the cultured bacterial solution and add it to a new mixture containing 50g of fresh tea leaves and 100mL of 1% sterile glucose, put it into a new anaerobic culture bag again, and incubate at 30℃ for 3 days. The resulting culture solution is used for the isolation of lactic acid bacteria.

[0028] Take the above culture medium and perform 10-fold serial dilutions with sterile physiological saline. Inject 1 mL of each appropriate dilution into sterile petri dishes, and aseptically add MRS agar medium containing 1% CaCO3 melted. Pour the medium into the dishes and mix well. After the medium solidifies, invert the dishes and incubate at 35 ℃ for 48 h. Pick colonies with a CaCO3 lysis zone around them and streak them onto MRS plates for purification. Take the purified colonies, Gram-stain them, examine them under a microscope, and retain the Gram-positive strains for preservation on MRS slant agar.

[0029] 1.2 Initial screening of lactic acid bacteria Prepare 100 mL of MRS liquid medium, add 5 g of tea leaves and 1.5 g of agar, sterilize at 115 ℃ for 20 min, pour into sterile Petri dishes to make plates containing 5% tea concentration, streak the strains to be screened on the above plates, incubate at 35 ℃ for 24-48 h, observe the growth of the strains, and select strains that can grow rapidly and form full colonies of 1-2 mm for the next screening step.

[0030] 1.3 Secondary screening of lactic acid bacteria 50 mL of MRS liquid culture medium was prepared and diluted with 50 mL of distilled water. Then, 5 g of tea leaves were added to the diluted medium (100 mL). The medium was sterilized at 115 °C for 20 min, aseptically filtered, and the resulting liquid culture medium was used to prepare a secondary screening medium for lactic acid bacteria. The strains to be screened were inoculated into the MRS liquid culture medium and activated at 35 °C for 24 h to obtain seed culture. Each activated bacterial culture was then inoculated into the secondary screening medium at a 2% inoculation rate and incubated at 35 °C for 24 h. The OD value of each strain's culture was measured at 600 nm. The results showed that among the 15 strains to be screened, strain B5 had the highest OD value (0.983 ± 0.087), indicating that B5 has the ability to tolerate high concentrations of tea for growth.

[0031] Example 2 This example illustrates the identification and classification of B5.

[0032] 2.1 Colony and microscopic morphological characteristics of strain B5 The selected strain B5 was streaked on MRS agar plates and cultured statically at 35°C for 48 hours for purity testing. At the same time, the colony morphology was photographed and recorded. Gram staining was then performed, and the morphology of the stained cells was observed under a microscope.

[0033] Strain B5 colonies are small, smooth, round, grayish-white, with regular edges, and a diameter of approximately 1-2 mm. Figure 1 Gram staining revealed that strain B5 was Gram-positive, non-spore-forming, and rod-shaped with blunt ends. The cell diameter was approximately 1 μm, with a length-to-width ratio of 3:1-4:1. The cells were arranged in clusters rather than chains. Figure 2 ).

[0034] 2.2 Physiological and biochemical characteristics of strain B5 The culture and basic physiological and biochemical characteristics experiments were performed in accordance with the reference: *Handbook of Systematic Identification of Common Bacteria*, Science Press, 2001. The results are shown in Table 1.

[0035] Table 1. Culture and physiological and biochemical characteristics of strain B5

[0036] The experimental results of the physiological and biochemical characteristics of strain B5 are shown in Table 1. Its non-spore-forming, non-motile, catalase-negative, and Gram-positive characteristics conform to the general characteristics of lactic acid bacteria. B5 can ferment various sugars such as lactose, mannitol, cellobiose, mesotriose, and xylose to produce acid, indicating that it can utilize multiple carbon sources for metabolism. Furthermore, the hemolytic test results show that B5 is non-hemolytic, indicating good safety.

[0037] 2.3 Molecular identification of strain B5 Table 2 Primers used in this embodiment

[0038] 2.3.1 16S rDNA sequence alignment and phylogenetic analysis Genomic DNA was extracted from B5 cells and amplified using universal primers 27F and 1541R for bacterial 16S rDNA. The amplification system consisted of 3 μL 10 mmol / mL MgCl2, 5 μL 10×PCR buffer, 4 μL dNTPs (2.5 μmol / L), 0.25 μL Taq polymerase (5 U / μL), 1 μL each of upstream and downstream primers (2.5 μmol / L), 1 μL template DNA, and finally, double-distilled water to a final volume of 50 μL. The mixture was then placed in a PCR instrument for denaturation at 95 °C for 5 min, followed by denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min, for a total of 30 cycles. The final extension at 72 °C for 10 min completed the reaction.

[0039] PCR products were subjected to 1% agarose gel electrophoresis, and the results were observed and photographed using a gel imaging system. The PCR products were then purified by gel extraction using a gel extraction kit. Sequencing was performed by Shanghai Sangon Biotech Co., Ltd. Homology analysis was conducted between the 16S DNA sequence and nucleotide sequences in the NCBI database. A phylogenetic tree was constructed using the Neighbour-joining method with MEGA X software, and bootstrap analysis was performed, with 1000 replicates.

[0040] A phylogenetic tree was constructed using strain B5 and closely related typical strains of *Lactobacillus* after comparison. The results are as follows: Figure 3 As shown, strain B5 and Lactobacillus pentosus Lactobacillus pentosus 124-2 (NR 029133.1) form a group, and the closest other group is... Lactobacillus plantarum NBRC 15891 (NR 113338.1), and is relatively far from other Lactobacillus strains. Based on this result, strain B5 can be preliminarily identified as belonging to Lactobacillus pentosus (NBRC 15891 (NR 113338.1)). Lactobacillus pentosus ).

[0041] 2.3.2 Multiplex PCR Identification To further verify the results of the identification of strain B5 using the 16S rDNA sequence alignment method, and to distinguish it as belonging to *Lactobacillus plantarum* (…), Lactobacillus plantarum ) or belongs to Lactobacillus pentosus ( Lactobacillus pentosus The method described in the reference (Appl. Environ. Microbiol. 2001, 67(8):3450) uses a method based on... rec Primers paraF, pentF, planF, and pREV were used to amplify the B5 genome using multiplex PCR. The recA gene sequence from *Lactobacillus plantarum* was amplified to 318 bp, and the recA gene sequence from *Lactobacillus pentosus* was amplified to 218 bp. The amplification conditions were: 94℃ pre-denaturation for 3 min, followed by 30 cycles of 94℃ denaturation for 30 s, 56℃ annealing for 10 s, and 72℃ extension for 30 s, with a final extension at 72℃ for 5 min to terminate the PCR amplification. The products were observed and photographed using a gel imaging system after 2% agarose gel electrophoresis.

[0042] Multiplex PCR amplification results as follows Figure 4 As shown in the figure, clear and bright bands can be observed at 200bp and 300bp in the amplification product. Sequencing revealed that the amplification product of B5 was 218bp. Based on this result, strain B5 can be identified as *Lactobacillus pentosus* (…). Lactobacillus pentosus ).

[0043] Based on the comprehensive morphological characteristics, culture and physiological and biochemical characteristics, as well as the results of molecular biological identification, strain B5 was identified as *Lactobacillus pentosus*. Lactobacillus pentosus ).

[0044] Example 3 This example is used to compare the ability of B5 and other different lactic acid bacteria strains to grow under high tea concentration conditions.

[0045] The tested strains were derived from lactic acid bacteria isolated from fermented plant materials, as well as standard strains from public collections. These included: *Lactobacillus fermentum* (…). Lactobacillus fermentum T9, Lactobacillus fermentum ( Lactobacillus fermentum D11, Lactobacillus brevis ( Lactobacillus brevis CICC6239, Lactobacillus brevis ( Lactobacillus brevis J25, Lactobacillus plantarum ( Lactobacillus plantarum ATCC8014, Lactobacillus plantarum ( Lactobacillus plantarum ACCC11095, Lactobacillus plantarum ( Lactobacillus plantarum L5, Lactobacillus plantarum ( Lactobacillus plantarum L16, Lactobacillus plantarum (Lactobacillus plantarum W30, Lactobacillus plantarum ( Lactobacillus plantarum K6, Lactobacillus pentosus ( Lactobacillus paracasei B5, Lactobacillus pentosus ( Lactobacillus paracasei E9, Lactobacillus paracasei ( Lactobacillus paracasei E8, Lactobacillus paracasei ( Lactobacillus paracasei B27, Leuconostoc mesenteroides ( Leuconostoc mesenteroides A6. Leuconostoc mesentery ( Leuconostoc mesenteroides G16, Pediococcus pentosaceus ( Pediococcus pentosaceus R12, Pediococcus pentosaceus ( Pediococcus pentosaceus )W5.

[0046] Tea culture medium: Weigh 6g of green tea sample and add it to a flask containing 100mL of distilled water. Mix well and incubate in a boiling water bath for 15min. Filter to remove tea residue. Add 1g glucose, 0.5g sucrose, 0.3g sodium chloride, 0.05g yeast extract, and 0.002g sodium dihydrogen phosphate to the filtrate. Add distilled water to bring the volume to 100mL. Sterilize at 108℃ for 25min after preparation.

[0047] Each strain was inoculated into MRS liquid medium and cultured at 30℃ for 36 h. Then, 2% of the strain was inoculated into the above tea medium and cultured at 30℃. Samples were taken at 24 h and 48 h to measure the absorbance at 600 nm to evaluate the growth ability of the strains.

[0048] The experimental results are shown in Table 3. Significant differences were observed in the growth of the 18 tested lactic acid bacteria strains at a 6% tea concentration. Among them, *Lactobacillus pentosus* B5 exhibited the best growth performance, with OD values ​​of 0.926 and 1.108 at 24 h and 48 h, respectively, both significantly higher than other strains. p <0.05). The second most abundant strain was *Lactobacillus pentosaccharide* E9, with corresponding OD values ​​of 0.473 and 0.733. *Lactobacillus plantarum* L16 ranked third, with OD values ​​of 0.324 and 0.3590. *Lactobacillus plantarum* W30 ranked fourth, with OD values ​​of 0.207 and 0.389. The growth of the remaining strains was significantly inhibited, with OD values ​​below 0.2 and 0.35 at 24 h and 48 h, respectively. These results indicate that *Lactobacillus pentosaccharide* B5 exhibits strong tolerance to high substrate concentrations.

[0049] Table 3. Growth of different lactic acid bacteria strains in high tea substrate concentration culture medium

[0050] Note: Different lowercase letters indicate significant differences in the growth results of different strains. p <0.05) Example 4 This example illustrates the tolerance of strain B5 to different tea concentrations during fermentation using different types of tea as raw materials.

[0051] Weigh 2-12g of different tea samples and add them to flasks containing 100mL of distilled water. Mix well and incubate in a boiling water bath for 10min. Filter to remove tea residue. Add 1g glucose, 0.5g sucrose, 0.3g sodium chloride, 0.05g yeast extract, and 0.002g sodium dihydrogen phosphate to the filtrate, and add distilled water to bring the volume to 100mL. Sterilize at 108℃ for 25min after preparation.

[0052] Strain B5 was inoculated into MRS liquid medium containing 2% tea leaves and cultured statically at 35°C for 48 h to obtain activated seed liquid. The seed liquid was then inoculated into tea mediums of different concentrations at an inoculation rate of 1% and cultured at 35°C for 24 h. Using the uninoculated medium as a blank, the absorbance value at 600 nm of the tea fermentation liquid at different concentrations was measured.

[0053] The results of strain B5's tolerance to different tea concentrations are as follows: Figure 5 As shown in the figure. This example selected three types of tea: green tea, black tea, and oolong tea, and investigated the changes in cell concentration during fermentation within a concentration range of 2%-12%. At concentrations of 2% and 6%, the cell concentration of strain B5 was less affected by the tea concentration, and strain B5 in all three types of tea showed good growth. When the concentration reached 8%, the cell concentration in black tea and oolong tea decreased compared to the previous three concentrations, but their OD values ​​remained above 0.9, while the cell concentration in green tea was above 0.8. When the tea concentration increased to 10%, the green tea fermentation sample had the lowest cell concentration among the three types of tea, with an OD value of 0.685, while the OD values ​​of black tea and oolong tea remained above 0.8, indicating that the strain could still grow and ferment at a tea concentration of 10%. However, when the tea concentration was further increased to 12%, the OD values ​​of the cell concentration in all three types of tea dropped below 0.4, indicating that high concentrations of tea substrate significantly inhibited cell fermentation growth. In summary, strain B5 was able to tolerate fermentation at three different tea concentrations of up to 10% under the experimental conditions.

[0054] Example 5 This example illustrates the acid-producing ability of strain B5 during fermentation in tea culture medium.

[0055] Following the method described in Example 3, a culture medium with a tea concentration of 6% was prepared. The activated strain B5 was inoculated at a rate of 2% and cultured at 35°C. Samples were taken at different time points to determine the titratable acidity (TTA) and pH value, and the viable cell count was determined using the plate count method.

[0056] Figure 6This chart shows the changes in pH and titratable acidity (TTA) over time during the fermentation of strain B5. Initially, the pH was approximately 6.6, gradually decreasing as fermentation progressed. This indicates that the bacteria were producing lactic acid, leading to the pH decrease. The pH reached its lowest point around 24 hours of fermentation and then stabilized at approximately 4.0. Titratable acidity was low initially and gradually increased over time. The increase in TTA indicates the accumulation of organic acids during fermentation, reaching its peak around 48 hours. The change in viable cell count over time during fermentation is shown below. Figure 7 As fermentation time increased, the viable cell count rapidly increased after 12 hours, reaching a peak at approximately 24 hours. Afterward, the viable cell count remained stable until fermentation reached 60 hours. These results indicate that strain B5 possesses highly efficient acid-producing capacity. This strain can rapidly and significantly lower the pH value, making it suitable for fermentation processes requiring rapid acidification, and it exhibits good stability during long-term fermentation. The strain demonstrates rapid proliferation in the early stages of fermentation, quickly reaching the required viable cell count. Once a certain viable cell count is reached, the strain can maintain stability, contributing to the stability of the fermentation process. In summary, this strain exhibits rapid proliferation, acid production, and good fermentation stability, making it suitable for fermentation processes requiring rapid start-up and maintenance of high microbial activity.

[0057] Example 6 This embodiment illustrates the changes in the content of polyphenols, flavonoids, catechins, volatile components, and antioxidant activity in fermented tea infusion after fermentation using B5 and other comparative lactic acid bacteria strains.

[0058] Weigh 6g of tea sample and add it to a beaker containing 80mL of distilled water. Mix well and incubate in a boiling water bath for 10min. Add 1g glucose, 0.5g sucrose, 0.3g sodium chloride, 0.05g yeast extract, and 0.002g sodium dihydrogen phosphate. Add distilled water to bring the volume to 100mL. Sterilize at 108℃ for 25min after preparation.

[0059] Lactobacillus pentosus B5, Lactobacillus pentosus E9, and Lactobacillus plantarum L16 were inoculated at a rate of 3% and fermented at 30℃. Samples were taken for analysis after 60 hours of fermentation. The determination of tea polyphenols and catechins was performed in accordance with GB / T 8313—2018 "Determination Method of Tea Polyphenols and Catechins in Tea"; the determination of flavonoids was performed in accordance with the literature: Food and Fermentation Industries, 2023, 49(14):280-287; the determination of volatile components was performed in accordance with the literature: Food Industry Technology, 2023, 44(01):96-108; the determination of antioxidant activity was performed in accordance with the literature: Modern Food Science and Technology, 2022, 38(12):290-299.

[0060] 1. Polyphenol content The polyphenol and flavonoid content of the samples fermented with strain B5 was tested, and the results are shown in Table 4.

[0061] Table 4 shows the polyphenol and flavonoid content of samples after fermentation with strain B5.

[0062] Note: Different letters indicate that the results of different treatments are significantly different. p <0.05) As shown in Table 4, compared with the unfermented samples and the samples fermented with strains E9 and L16, the polyphenol content in the tea infusion significantly increased after fermentation with strain B5, with an increase of approximately 30%. In contrast, the polyphenol content of the tea infusion fermented with strains E9 and L16 only increased slightly, showing little difference from the unfermented blank sample. Regarding flavonoid content, the flavonoid content after fermentation with strain B5 was significantly higher than that of the unfermented samples and the samples fermented with strains E9 and L16. However, there was no significant difference in flavonoid content between the unfermented samples and the samples fermented with strains E9 and L16. These results indicate that fermentation with strain B5 is beneficial for the dissolution of polyphenols and flavonoids in tea, and that fermentation with strain B5 significantly improves the polyphenol extraction rate.

[0063] 2. Catechin content The results are as follows Figure 7 As shown, compared with unfermented tea infusion, among the eight catechin components measured after fermentation with strain B5, gallatechin (GC), epigallocatechin (EGC), and epicatechin (EC) showed a significant increase, while the ester-type catechins epigallocatechin gallate (EGCG), gallatechin gallate (GCG), epicatechin gallate (ECG), and catechin gallate (CG) showed a significant decrease; no significant difference was found in catechin (C). However, after fermentation with control strains E9 and L16, compared with the unfermented control, only a slight increase in non-ester-type catechins EGC and EC was observed; the other catechin components showed no significant changes.

[0064] Ester-type catechins typically possess a strong bitterness and astringency, contributing significantly to the bitterness of tea infusions; while non-ester-type catechins exhibit weaker bitterness and a more refreshing taste. Simple catechins play a crucial role in balancing the bitterness of tea, resulting in a more harmonious and balanced flavor. B5 fermentation alters the structure and composition of catechins, effectively reducing bitterness and enhancing the refreshing taste of the beverage. Therefore, using B5 fermentation has a significant flavor-enhancing effect in preparing fermented tea beverages.

[0065] 3. Comparison of volatile content like Figure 9 As shown in the figure, compared with the unfermented group, the content of volatile substances such as alcohols, aldehydes, terpenes, heterocyclic compounds, and esters increased significantly after fermentation with B5 inoculation. Terpenes and esters showed the most significant increases, and these substances are mostly contributors to the aroma of tea. The content of volatile substances such as amines, aromatics, sulfur-containing compounds, ethers, acids, hydrocarbons, and ketones did not change significantly. When fermented with control strains E9 and L16, compared with the unfermented control group, only a slight increase in heterocyclic compounds was observed after fermentation with strain E9; no other significant differences were observed in the other components.

[0066] The above results indicate that, overall, compared to unfermented tea, the tea liquor produced by B5 fermentation is richer in aroma components, and fermentation is beneficial for improving and enhancing the aroma of the tea liquor. Sensory evaluation shows that the fermented tea liquor has a better floral and fruity aroma than Phoenix Dancong tea, and incorporates the aroma of lactic acid bacteria fermentation.

[0067] 4. Antioxidant activity Table 5. DPPH scavenging rate, ABTS scavenging rate, and Fe scavenging rate of samples after fermentation with strain B5. 2+ Chelation force determination

[0068] Table 5 shows a comparison of antioxidant capacity indices after fermentation with and without strain B5. Compared with the control without fermentation, the antioxidant capacity indices after fermentation with strain B5 increased significantly, including DPPH scavenging rate, ABTS scavenging rate, and Fe2+ scavenging rate. 2+ Significant increases in chelating power were observed in all samples, with the DPPH and ABTS scavenging rates showing the largest increases. While fermentation with control strains E9 and L16 resulted in increases in all three antioxidant capacity indicators compared to the unfermented control group, the increases were smaller than those observed with B5 fermentation, indicating a limited effect on enhancing the antioxidant capacity of the fermented tea infusion samples.

[0069] The DPPH scavenging rate test is mainly used to measure the ability of antioxidants to directly donate hydrogen atoms or electrons to neutralize free radicals. The ABTS scavenging rate method can be used to determine the antioxidant capacity of both hydrophilic and lipophilic substances and can reflect the overall antioxidant performance of the sample. The results in this section show that after fermentation with B5 inoculation, both DPPH and ABTS scavenging rates showed significant increases, indicating that the fermented product has broad-spectrum antioxidant activity, can combat multiple types of free radicals, and can effectively capture and neutralize free radicals, thereby preventing oxidation reactions and playing a crucial role in preventing cell damage caused by free radicals. 2+ Chelating ability refers to the ability of antioxidants to bind with transition metal ions (such as iron ions) and reduce their catalytic ability to generate free radicals (e.g., through the Fenton reaction). After fermentation, the Fe content of the sample... 2+ A significant increase in chelation capacity was also observed, suggesting that the post-fermentation sample helps reduce cell damage caused by metal-catalyzed oxidation processes, which may be of significance in preventing diseases caused by excessive oxidative stress.

[0070] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A type of Lactobacillus pentosus B5, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 66054.

2. An application of Lactobacillus pentosus B5 as described in claim 1, characterized in that, Used in fermented tea.

3. The application as described in claim 2, characterized in that, The fermented tea is obtained by fermentation with the Lactobacillus pentosus B5 fermentation agent; the fermentation substrate of the fermented tea includes tea leaves.

4. The application as described in claim 3, characterized in that, The tea leaves in the fermentation substrate have a mass concentration of 8-10%; the tea leaves include one or more of green tea, black tea, and oolong tea.

5. The application as described in claim 2, characterized in that, The preparation of the fermented tea includes the following steps: A. Activate the Lactobacillus pentosus B5 to obtain the activated bacterial solution; B. Prepare a fermentation medium, which includes one or more of tea leaves, glucose, sucrose, sodium chloride, yeast extract, sodium dihydrogen phosphate, and distilled water, and then adjust the pH and sterilize it. C. Inoculate the activated bacterial solution into the fermentation medium and carry out fermentation to obtain fermented material; D. The fermented material is subjected to solid-liquid separation, and the remaining liquid is homogenized to obtain the fermented tea.

6. The application as described in claim 5, characterized in that, In step B, the mass-to-volume ratio of the tea leaves, glucose, sucrose, sodium chloride, yeast extract, sodium dihydrogen phosphate, and distilled water in the fermentation culture medium is 2~10g: 0~2g: 0~2g: 0.1~0.5g: 0.02~0.1g: 0.001~0.005g: 100mL.

7. The application as described in claim 5, characterized in that, In step A, the activation conditions include: static incubation in MRS liquid medium at 28-40 ℃ for 24-48 h; in step B, the sterilization conditions include: sterilization at 100-115 ℃ for 10-15 min; in step C, the fermentation conditions include: static fermentation at 28-40 ℃ for 3-7 days; in step D, the solid-liquid separation method includes filtration.

8. The application as described in claim 7, characterized in that, In step A, the MRS liquid culture medium contains 1-5% tea leaves; in step D, the filter cloth used for filtration comprises 60-120 mesh filter cloth.

9. The application as described in claim 5, characterized in that, In step A, the concentration of the activated bacterial solution includes 10. 8 ~10 9 CFU / mL; In step B, the pH of the fermentation medium includes 6.0~7.0; In step C, the volume ratio of the activated bacterial solution to the fermentation medium is 1-5%:1; in step D, the homogenization conditions include: homogenizing at 20-100 MPa for 5-45 min, repeated 1-3 times.

10. A fermented tea obtained by the application as described in claim 2.

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