Composite strain, microbial inoculant and application thereof in preparation of fermented perilla frutescens tea

Abstract

The application belongs to the field of microorganisms, and provides a composite strain, a bacterial agent and application of the composite strain in preparation of fermented east perilla tea. The composite strain comprises Lactiplantibacillus plantarum 2HDS3-La5 with a preservation number of CGMCC NO.34339, Pediococcus acidilactici 2HDS3-SM6 with a preservation number of CGMCC NO.34337, and Weizmannia coagulans N2-La1 with a preservation number of CGMCC NO.34338. The application also provides application of the composite strain or the bacterial agent containing the composite strain in preparation of fermented east perilla tea. The composite strain is applied in preparation of fermented east perilla tea, significantly improves the content of substances with anti-inflammatory, antioxidant and immune-regulating effects in east perilla tea, and the improvement multiple is significant. The prepared fermented east perilla tea can significantly improve cell viability.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a compound bacterial strain, a bacterial agent, and its application in the preparation of fermented perilla tea. Background Technology

[0002] Matrine is an alkaloid extracted from legumes such as Sophora flavescens and Sophora alopecuroides, possessing anti-inflammatory, antiviral, antitumor, and immunomodulatory effects. Sesamol is a phenolic compound and a potent antioxidant with antioxidant and anti-inflammatory capabilities. It can scavenge free radicals, fight tumors, and has neuroprotective and other anti-inflammatory and soothing effects. Witch hazel tannins are a group of structurally complex hydrolyzed tannins with antioxidant and anti-inflammatory effects. Garcinia cambogia, a polycyclic flavonoid, has anti-inflammatory, antioxidant, and antibacterial effects. Rosmarinic acid has antioxidant, anti-inflammatory, and anti-aging effects, as well as strong weight-loss and lipid-lowering effects, and is used to treat cardiovascular diseases and has anti-cancer properties.

[0003] Dongzisu tea has the effects of relieving exterior syndromes, raising clear yang, clearing liver heat, dispersing exterior syndromes, regulating qi and stomach. It is mainly used for symptoms such as colds, sore throats, headaches, tonsillitis, pediatric stomatitis, toothaches, conjunctivitis, hepatitis, and indigestion.

[0004] Currently, most Dongzi perilla tea is fermented using a single strain of bacteria, which is a relatively simple fermentation method. However, the conversion of nutrients is insufficient, and the functional properties are lacking. Furthermore, market research indicates that the flavor and taste of fermented liquid made from a single strain are monotonous and unappealing to consumers. Its efficacy needs further development. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a compound bacterial strain, a microbial agent, and their application in the preparation of fermented Dongzi perilla tea. The compound bacterial strain and microbial agent of this invention, when applied to the preparation of fermented Dongzi perilla tea, significantly increase the content of substances with anti-inflammatory, antioxidant, and immunomodulatory effects in Dongzi perilla tea, with a significantly higher increase factor. The prepared fermented Dongzi perilla tea can significantly improve cell viability.

[0006] The purpose of this invention is to provide a composite bacterial strain comprising *Lactobacillus plantarum* 2HDS3-La5 (CGMCC NO.34339), *Pediococcus lactis* 2HDS3-SM6 (CGMCC NO.34337), and *Weizmannii coagulans* N2-La1 (CGMCC NO.34338). *Lactobacillus plantarum* 2HDS3-La5, *Pediococcus lactis* 2HDS3-SM6, and *Weizmannii coagulans* N2-La1 were deposited with the China General Microbiological Culture Collection Center on April 25, 2025.

[0007] Another objective of this invention is to provide a bacterial agent containing the above-mentioned compound strain.

[0008] Preferably, the strain number ratio of *Lactobacillus plantarum* 2HDS3-La5, *Pediococcus lactis* 2HDS3-SM6, and *Weizmannii coagulans* N2-La1 is 0.5-3:0.5-3:0.5-3.

[0009] The preparation method of the above-mentioned microbial agent includes the steps of statically fermenting and growing the above-mentioned compound strain at 30℃-37℃ for 1-5 days. Preferably, the above-mentioned strain has an OD of 600 The value was 0.6-1, and the bacterial count was 10. 7 -10 9 CFU / ml.

[0010] The present invention also aims to provide an application of the above-mentioned compound strain or the above-mentioned microbial agent in the preparation of fermented perilla tea.

[0011] The aforementioned compound bacterial strains or agents are used to increase the content of at least one of the anti-inflammatory, antioxidant, and antibacterial substances in Dongzisu tea. Preferably, the aforementioned compound bacterial strains or agents are used to increase the content of at least one of the following substances in Dongzisu tea: matrine, sesamol, witch hazel tannin, rosmarinic acid, and gambogeylic acid.

[0012] The above-mentioned fermented perilla tea is used in the preparation of medicines or health products for the treatment or prevention of gastrointestinal diseases or neurodegenerative diseases.

[0013] The application method described above involves adding the aforementioned compound bacterial strain or inoculant to a mixture containing perilla tea for cultivation.

[0014] Preferably, the above-mentioned microbial agent is added to the Dongzisu tea stock solution at an addition rate of 0.0001-0.2 g / ml, with the liquid content accounting for 70-75% of the container. Subsequently, fructooligosaccharides are added at an amount of 0.1-5% of the volume of the Dongzisu tea stock solution. The container is sealed, and the liquid in the bottle is shaken and the gas is released every 8-15 hours. The mixture is then incubated at 25-33℃ for 3-10 days.

[0015] The preparation method of Dongzisu tea concentrate involves mixing Dongzisu tea with water and heating it to boiling. The ratio of Dongzisu tea to water is 1g-10g: 1ml-2000mL.

[0016] Preferably, the pH of the original extract of Dongzi perilla tea is maintained between 5.6 and 6.5.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The compound strain of the present invention, when used to prepare fermented Dongzi perilla tea, significantly increases the content of anti-inflammatory, active, antioxidant, and immunomodulatory substances in Dongzi perilla tea. Tests show that GES-1 (human gastric mucosal cells) cultured in the fermentation supernatant obtained by using the compound strain of the present invention for Dongzi perilla tea can achieve a cell viability of over 188.78%, and BV2 cells cultured in the fermentation supernatant obtained by using the compound strain of the present invention for Dongzi perilla tea can achieve a cell viability of over 119.88%. The prepared fermented Dongzi perilla tea has a significant effect on promoting cell growth and cell viability.

[0019] (2) This invention ferments Dongzisu tea using a compound strain. By selecting and compounding strains, a stable and complementary microbial community is formed, reducing interference from miscellaneous bacteria, making the fermentation process more controllable, and the product quality more stable. At the same time, under the synergistic effect of multiple microorganisms, proteins, monosaccharides and other substances can be more thoroughly converted into more beneficial metabolites (such as flavonoids, triterpenoids, etc.), enhancing the health benefits of the fermentation liquid. Finally, the compound strain produces richer and more coordinated aroma components (alcohols, esters, etc.), reducing bitterness and bringing a more mellow and refreshing flavor. It avoids the off-flavors that may be caused by a single strain, making it easier to gain market recognition and having significant market value. Attached Figure Description

[0020] Figure 1 The experimental procedure for screening and isolating compound bacterial strains;

[0021] Figure 2 This is a colony morphology diagram of Lactobacillus plantarum 2HDS3-La5.

[0022] Figure 3 This is a colony morphology diagram of Pediococcus lactis 2HDS3-SM6.

[0023] Figure 4 This is a colony morphology diagram of Weizmann's N2-La1 coagulation bacteria. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Fermentation, screening, and identification of strains

[0026] The experimental procedure for screening and isolating compound bacterial strains is as follows: 1. Dilution, 2. Spreading, 3. Purification, 4. Isolation, 5. Preservation of strains, 6. Cell culture, 7. Conducting cell experiments, as follows: Figure 1 As shown in AG.

[0027] The specific steps for screening and isolating the compound bacterial strains are as follows: 100 μL of the fermentation broth is extracted and added to 900 μL of sterile water, and then the sample is diluted to 10 μL. -4 10 -5 For concentration, 100 μL was spread onto an MRS plate and incubated in an anaerobic incubator at 37°C for 48 hours. Bacterial growth was observed. After colonies appeared, a typical single colony was streaked onto MRS solid medium for bacterial isolation and purification. The single colony was inoculated into 5 mL of sterilized MRS medium and incubated statically at 37°C for approximately 48 hours. The culture was then stored at -80°C with 50% glycerol for later use. MRS medium was purchased from Qingdao Haibo Biotechnology.

[0028] The isolated and purified single colonies were sent to Zhejiang Shangya Biotechnology Co., Ltd., and the identification primers were:

[0029] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (sequence shown in SEQ ID NO.4);

[0030] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (Sequence as shown in SEQ ID NO.5).

[0031] Identification results: The 16S rDNA sequences of *Lactobacillus plantarum* 2HDS3-La5, *Pediococcus lactis* 2HDS3-SM6, and *Weizmannii coagulans* N2-La1 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The colony morphologies are shown in... Figure 2 , Figure 3 , Figure 4 As shown.

[0032] *Lactobacillus plantarum* 2HDS3-La5 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC NO.34339. *Pediococcus lactis* 2HDS3-SM6 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC NO.34337. *Weizmannii coagulans* N2-La1 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC NO.34338.

[0033] Among them, Lactiplantibacillus plantarum 2HDS3-La5 was classified as Lactiplantibacillus plantarum;

[0034] The classification name of *Pediococcus acidilactici* 2HDS3-SM6 is *Pediococcus acidilactici*.

[0035] The classification name of Weizmannia coagulans is Weizmannia coagulans.

[0036] Example 2 Preparation of compound strain A

[0037] The strains *Lactobacillus plantarum* 2HDS3-La5, *Pediococcus lactis* 2HDS3-SM6, and *Weizmannii coagulans* 2HDS3-SM6 were respectively adjusted (OD600nm of the above strains was adjusted to 1.0, and the bacterial concentration was approximately 10). 8 The mixture (CFU / ml) was prepared at a ratio of 1:1:1 and allowed to ferment at 37 ℃. After 3 days of growth, the mixture was removed and named compound strain A.

[0038] Comparative Example 1: Preparation of Compound Strains B

[0039] Commonly used commercial fermentation strains, including *Lactobacillus plantarum* 1.38, *Pediococcus lactis* 1.263, and *Weizmannii coagulans* 1.42, were compounded in a ratio of 1:1:1 using the method described above, and named compound strain B.

[0040] Application Example 1

[0041] 1. Preparation of Dongzisu Tea Extract (Fermentation Base)

[0042] Take 5g of Dongzisu tea (origin: Yunnan), add 500mL of purified water, then place it in a heating device and boil it to obtain Dongzisu tea stock solution. Then test the initial pH of the Dongzisu tea and adjust the pH of the stock solution with food-grade citric acid or sodium hydroxide to maintain the pH between 5.6 and 6.5.

[0043] 2. Compound bacterial strains were used in the preparation of fermented perilla tea.

[0044] Compound strains A and B were added to the Dongzisu tea stock solution at a dosage of 0.002 g / ml, with the total liquid content reaching 70-75% of the container. Subsequently, fructooligosaccharides were added at a dosage of 2% of the Dongzisu tea stock solution volume. The container was sealed (the liquid in the bottle was shaken every 12 hours, and the cap of the blue-capped bottle was gently tightened to release the gas), and incubated in an incubator at 28-33℃ for 7 days.

[0045] Substance content test

[0046] The fermentation supernatant of compound strain A and commercially available compound strain B prepared in step 2 of Application Example 1 were sterilized at 100℃ for 30 min. Subsequently, the original solution (referred to as YSC), the fermentation supernatant of compound strain A (referred to as YSCJ), and the fermentation supernatant of commercially available compound strain B (referred to as YSCFJ) were taken out separately, stored at -80℃, and sent to Shanghai APTBIO Biotechnology Co., Ltd. for LC-MS detection of non-target metabolites using dry ice. Differential metabolic analysis was performed simultaneously on the results.

[0047] The test results are shown in the table below:

[0048]

[0049] Note: The data represents the relative content of each metabolite in the corresponding sample, without specific units.

[0050] Differential metabolic analysis of the original extract of Dongzi perilla tea, the fermentation supernatant of compound strain A, and the fermentation supernatant of commercially used compound strain B revealed that after fermentation by compound strain A, the content of matrine in the fermentation supernatant of compound strain A increased by 252.46 times compared with the original extract, while the content of matrine in the fermentation supernatant of commercially used compound strain B increased by only 2.52 times compared with the original extract. The content of matrine in the fermentation supernatant of compound strain A increased by 100.37 times compared with the fermentation supernatant of commercially used compound strain B, which is a significant increase.

[0051] Furthermore, compared to the original solution, the sesamol content in the fermentation supernatant of compound strain A increased by 51.76 times, while the sesamol content in the fermentation supernatant of commercially commonly used compound strain B increased by 41.01 times. The witch hazel tannin content in the fermentation supernatant of compound strain A increased by 24.31 times, while the witch hazel tannin content in the fermentation supernatant of commercially commonly used compound strain B increased by only 3.94 times compared to the original solution. Compared to the fermentation supernatant of commercially commonly used compound strain B, the witch hazel tannin content in the fermentation supernatant of compound strain A increased by 6.16 times.

[0052] Compared with the original solution, the rosmarinic acid content in the fermentation supernatant of compound strain A increased by 11.618 times.

[0053] Compared with the fermentation supernatant of commercially used compound strain B, the content of gambogeylic acid in the fermentation supernatant of compound strain A was increased by 12.78 times.

[0054] Therefore, it can be seen that the strains isolated and screened by this invention, when combined and used in the fermentation of Dongzisu tea, significantly increase the content of substances with anti-inflammatory, antioxidant, and immunomodulatory effects in Dongzisu tea.

[0055] Cellular experimental testing

[0056] BV2 cell experiment (mouse microglia)

[0057] BV2 is a microglia cell line derived from mice that is widely used in neuroscience research. This immortalized cell line can serve as an in vitro model to study neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis) and related cellular conditions and processes, such as neuroinflammation. Furthermore, BV2 cells are considered an alternative model system for primary microglia.

[0058] The specific testing steps are as follows:

[0059] 1. Take out the original liquid of Dongzisu tea, the fermentation supernatant of compound strain A, and the fermentation supernatant of commercially used compound strain B for later use.

[0060] 2. Preparation of BV2 cell culture medium: DMEM high glucose medium + 10-15% FBS (fetal bovine serum) + 1% P / S

[0061] 3. BV2 cell resuscitation: Thaw cryovials containing 1 mL of cell suspension rapidly by shaking in a 37°C water bath. Add the thawed cells to centrifuge tubes containing 4-6 mL of culture medium and mix thoroughly. Centrifuge at 1000 RPM for 3-5 min, discard the supernatant, and resuspend the cells in the culture medium. Then, add the cell suspension to a culture flask containing 5 mL of culture medium and incubate at 37°C with 5% CO2 for 48 h. Observe the cell concentration.

[0062] 4. Passaging of BV2 cells: When the BV2 cell concentration reaches 8-10 × 10⁵ cells / mL, passage is performed. Discard the culture medium from the original cell culture flask, add 1.5 mL of trypsin, and incubate at 37 ℃ in a 5% CO₂ incubator for 2 min to digest. Remove and discard the culture medium, add 1.5 mL of DMEM high glucose + 10% FBS to terminate digestion, and pipette to form a cell suspension. Add 4 mL of culture medium to a cell culture dish, and seed 0.5 mL of the 1.5 mL cell suspension into a 6 cm cell culture dish. After completion, incubate the cell culture flask at 37 ℃ in a 5% CO₂ incubator until the cell count reaches 8-10 × 10⁵ cells / mL, then proceed with experiments.

[0063] 5. Plating BV2 cells: After digesting the cells, add 10 μL of cell suspension to a 96-well cell culture plate, followed by 190 μL of culture medium, and incubate overnight at 37 °C with 5% CO2.

[0064] 6. Sample addition: Discard the cell supernatant from overnight culture, then add the stock solution, fermentation supernatant of compound strain A, and fermentation supernatant of model compound strain B to the wells at a 20% addition rate. Then, use culture medium to make up the remaining volume and incubate overnight at 37°C with 5% CO2.

[0065] 7. Following the instructions, use the CCK-8 kit to determine the cell viability in 96-well cell cultures and to ascertain the effects of the stock solution, fermentation supernatant of compound strain A, and fermentation supernatant of commercially available compound strain B on the cells.

[0066] GES-1 cell experiments (human gastric mucosal cells)

[0067] GES-1 (Gastric Epithelial Cell Line-1) is an immortalized human gastric mucosal epithelial cell line, established through genetic modification of normal gastric mucosal tissue. Its most significant characteristic is the preservation of the morphological and functional features of normal gastric epithelial cells. Compared to tumor-derived gastric cells (such as AGS and MKN45), GES-1 has not undergone malignant transformation and can more accurately reflect the physiological state of normal gastric mucosa, making it an ideal model for studying the early mechanisms of gastritis, gastric ulcers, and gastric cancer.

[0068] The testing method is the same as that for GES-1 cells and BV2 cells, using the CCK-8 kit.

[0069] Test Results

[0070] The test results of the two cell experiments are shown in the table below:

[0071]

[0072] Note: OD 450

[0073] Tests revealed that the cell viability of GES-1 (human gastric mucosal cells) cultured in the fermentation supernatant of compound strain A was 188.78%, while the cell viability of GES-1 treated with commercially available compound strain B was 94.77%, and the cell viability of GES-1 treated with the original solution was 98.23%.

[0074] BV2 cells cultured in the fermentation supernatant of compound strain A had a cell viability of 119.88%, while GES-1 cells treated with commercially available compound strain B had a cell viability of 97.63% and a cell viability of 83.14% after treatment with the original solution.

[0075] Therefore, it can be seen that the fermentation supernatant of compound strain A can promote the growth of GES-1 (human gastric mucosal cells), and has a protective effect on the health of the stomach and gastrointestinal tract, and can prevent the occurrence of gastrointestinal diseases such as gastritis and gastric ulcers. According to the results in the table, the fermentation supernatant of compound strain A can improve the cell viability of BV2 and promote the growth of BV2 cells, suggesting that the fermentation broth of the compound strain of this invention has a preventive effect on neurodegenerative diseases.

[0076] This invention utilizes a compound bacterial strain to ferment Dongzi perilla tea. By selecting and combining specific functional strains, a stable and complementary microbial community is formed, reducing interference from other microorganisms, making the fermentation process more controllable, and resulting in more stable product quality. Simultaneously, the synergistic effect of multiple microorganisms more thoroughly transforms proteins and monosaccharides into more beneficial metabolites (such as flavonoids and triterpenoids), enhancing the health benefits of the fermentation liquid. Finally, the compound bacterial system produces richer and more harmonious aroma components (alcohols, esters, etc.), reducing bitterness and resulting in a more mellow and refreshing flavor, avoiding the off-flavors that may result from a single bacterial strain, thus gaining market acceptance.

[0077] The compound microbial agent of this invention is used to prepare fermented perilla tea, which significantly increases the content of anti-inflammatory, bioactive, antioxidant, and immunomodulatory substances in perilla tea. The fermented perilla tea prepared has a significant promoting effect on cell growth and cell vitality.

[0078] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. A composite bacterial strain, characterized in that, It comprises Lactiplantibacillus plantarum 2HDS3-La5 with the preservation number of CGMCC NO.34339, Pediococcus acidilactici 2HDS3-SM6 with the preservation number of CGMCC NO.34337 and Weizmannia coagulans N2-La1 with the preservation number of CGMCC NO.34338.

2. A microbial agent containing the composite strain of claim 1.

3. The complex bacterial strain according to claim 1, characterized by the fact that, The ratio of the number of bacteria of the Lactiplantibacillus plantarum 2HDS3-La5, the Pediococcus acidilactici 2HDS3-SM6 and the Weizmannia coagulans N2-La1 is 0.5-3:0.5-3:0.5-3.

4. The bacterial agent of claim 2, wherein The ratio of the number of bacteria of the Lactiplantibacillus plantarum 2HDS3-La5, the Pediococcus acidilactici 2HDS3-SM6 and the Weizmannia coagulans N2-La1 is 0.5-3:0.5-3:0.5-3.

5. The bacterial agent of claim 4, characterized in that, The preparation method of the microbial agent comprises the step of: allowing the composite strain to stand and ferment at 30-37°C, and obtaining after 1-5 days of growth.

6. The bacterial agent of claim 5, characterized in that, The strain OD600 in the bacterial agent is 0.6-1, and the bacterial number is 10 7 -10 9 CFU / ml.

7. Use of the composite strain of claim 1 in the preparation of fermented Perilla frutescens tea.

8. Use according to claim 7, characterized in that, The composite strain is used to increase the content of at least one of matrine, sesamol, witch hazel tannin, rosemary acid and gambogic acid in the Perilla frutescens tea.

9. Use of the microbial agent of claim 2 in the preparation of fermented Perilla frutescens tea.

10. Use according to claim 9, characterized in that, The microbial agent is used to increase the content of at least one of matrine, sesamol, witch hazel tannin, rosemary acid and gambogic acid in the Perilla frutescens tea.

11. Use according to claim 9, characterized in that, The application method is that the microbial agent is added to the Perilla frutescens tea stock solution at an addition amount of 0.0001-0.2 g / ml of the Perilla frutescens tea stock solution, and fructooligosaccharide is added, the amount of fructooligosaccharide being 0.1-5% of the volume of the Perilla frutescens tea stock solution, and the mixture is cultured at 25-33°C for 3-10 days.

12. Use according to claim 11, characterized in that, The preparation method of the Perilla frutescens tea stock solution is that Perilla frutescens tea is mixed with water, and heated to boiling, the amount ratio of Perilla frutescens tea to water being 1-10 g:1-2000 mL, and the pH of the Perilla frutescens tea stock solution is maintained at 5.6-6.5.

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