Composite strain, microbial agent and application of composite strain and microbial agent in preparation of fermented East purple perilla tea

Abstract

The invention belongs to the field of microorganisms. The invention provides a compound strain, a microbial inoculum and application of the compound strain in preparation of fermented East purple perilla tea. The compound strain comprises phytobacterium plantarum 2HDS3-La5 with the preservation number of CGMCC NO.34339, pediococcus acidilactici 2HDS3-SM6 with the preservation number of CGMCC NO.34337, Witzmania clostridium N2-La1 with the preservation number of CGMCC NO.34338, and the microbial inoculum is applied to preparation of fermented East purple perilla tea. The invention also provides application of the compound strain or a microbial inoculum containing the compound strain in preparation of fermented East purple perilla tea. The compound strain disclosed by the invention is applied to preparation of the fermented Dongbao tea, the content of substances with anti-inflammatory, anti-oxidation and immunoregulation effects in the Dongbao tea is remarkably increased by multiple times, and the prepared fermented Dongbao tea can remarkably improve the cell activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a composite strain, a bacterial agent and application thereof in preparation of fermented east perilla tea. BACKGROUND

[0002] ‌Matrine is extracted from leguminous plants such as Sophora flavescens, and has the effects of anti-inflammatory, anti-virus, anti-tumor and immune regulation. Sesamol is a phenolic compound and a strong antioxidant, and has the effects of anti-oxidation, anti-inflammation, free radical scavenging, anti-tumor and neuroprotection. Witch hazel tannin is a group of complex hydrolyzed tannins, and has the effects of anti-oxidation and anti-inflammation. Gambogic acid is a polycyclic flavone, and has the effects of anti-inflammation, anti-oxidation and anti-bacteria. Rosemary acid has the effects of anti-oxidation, anti-inflammation, anti-aging, weight loss and lipid reduction, treatment of cardiovascular disease and anti-cancer.

[0003] East perilla tea has the effects of relieving superficies, ascending clear yang, clearing liver heat, dispersing superficies, regulating qi and harmonizing stomach, and is mainly used for cold, sore throat, headache, tonsillitis, pediatric stomatitis, toothache, conjunctivitis, hepatitis and indigestion.

[0004] At present, east perilla tea is mainly fermented by a single strain, and the fermentation form is relatively simple, the conversion of nutritional components is insufficient, and the functionality is relatively insufficient. According to market research, the flavor and taste of the fermentation liquid of the single strain are single, and consumers do not like it, and the effect thereof needs to be further developed. SUMMARY

[0005] In view of the above problems, the present application provides a composite strain, a bacterial agent and application thereof in preparation of fermented east perilla tea. The composite strain and the bacterial agent of the present application are applied to preparation of fermented east perilla tea, significantly improve the content of substances with the effects of anti-inflammation, anti-oxidation and immune regulation in east perilla tea, and the improvement multiple is significantly high, and the prepared fermented east perilla tea can significantly improve cell viability.

[0006] The present application aims to provide a composite strain, which comprises plant lactobacillus 2HDS3-La5 with a preservation number of CGMCC NO.34339, lactococcus 2HDS3-SM6 with a preservation number of CGMCC NO.34337 and weizmannia conjuncta N2-La1 with a preservation number of CGMCC NO.34338. The plant lactobacillus 2HDS3-La5 was sent to the China General Microbiological Culture Collection Center on April 25, 2025, the lactococcus 2HDS3-SM6 was sent to the China General Microbiological Culture Collection Center on April 25, 2025, and the weizmannia conjuncta N2-La1 was sent to the China General Microbiological Culture Collection Center on April 25, 2025.

[0007] The application also aims to provide a microbial agent containing the complex strain.

[0008] Preferably, the strain number ratio of Lactobacillus plantarum 2HDS3-La5, Pediococcus acidilactici 2HDS3-SM6 and Weizmannia conjuncta N2-La1 is 0.5-3:0.5-3:0.5-3.

[0009] The preparation method of the microbial agent comprises the step of allowing the complex strain to ferment and grow at 30-37°C, and obtaining after 1-5 days of growth. Preferably, the OD of the strain is 0.6-1, and the bacterial number is 10 600 -10 7 -10 9 CFU / ml.

[0010] The application also aims to provide the use of the complex strain or the microbial agent in the preparation of fermented perilla tea.

[0011] The complex strain or the microbial agent is used to increase the content of at least one of anti-inflammatory, antioxidant and antibacterial substances in the perilla tea. Preferably, the complex strain or the microbial agent is used to increase the content of at least one of matrine, sesamol, witch hazel tannin, rosmarinic acid and gambogic acid in the perilla tea.

[0012] The fermented perilla tea is used in the preparation of a medicine or health product for treating or preventing gastrointestinal diseases or neurodegenerative diseases.

[0013] The application method of the use is to add the complex strain or the microbial agent to a mixture containing perilla tea for culture.

[0014] Preferably, the microbial agent is added to the perilla tea stock solution at an addition amount of 0.0001-0.2 g / ml of the perilla tea stock solution, the liquid content is 70-75% of the container, then fructooligosaccharide is added, the amount of fructooligosaccharide is 0.1-5% of the volume of the perilla tea stock solution, the container is sealed, the liquid in the bottle is shaken and gas is released every 8-15 hours, and the culture is carried out at 25-33°C for 3-10 days.

[0015] The preparation method of the perilla tea stock solution is to mix perilla tea and water, and heat to boiling, and the amount ratio of perilla tea to water is 1g-10g:1ml-2000ml.

[0016] Preferably, the pH of the perilla tea stock solution is maintained at 5.6-6.5.

[0017] The application has the following beneficial effects: (1) The compound strain is used for preparing fermented east perilla tea, and the content of anti-inflammatory, active, antioxidant and immune-regulating substances in the east perilla tea is significantly improved. Tests show that the cell viability of GES-1 (human gastric mucosa cells) cultured by the fermented supernatant of the east perilla tea fermented by the compound strain can reach 188.78% or more, and the cell viability of BV2 cells cultured by the fermented supernatant of the east perilla tea fermented by the compound strain can reach 119.88% or more, and the prepared fermented east perilla tea has a significant effect on promoting cell growth and cell viability. (2) The east perilla tea is fermented by the compound strain, the strain is optimized and compounded, a stable and complementary microbial community is formed, the interference of miscellaneous bacteria is reduced, the fermentation process is more controllable, the product quality is more stable, and under the synergistic action of a plurality of microorganisms, protein, monosaccharide and other substances can be more completely converted into more beneficial metabolites (such as flavonoids and triterpenoids), the health care value of the fermented liquid is improved, finally, the compound strain produces more rich and coordinated aroma components (alcohols and esters), reduces the bitter taste, brings more mellow and refreshing flavor, avoids the peculiar smell caused by single strain, is more easily accepted by the market, and has important market value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the experimental process for screening and separating the compound strain; Figure 2 It is a colony morphology diagram of plant lactobacillus 2HDS3-La5; Figure 3 It is a colony morphology diagram of lactococcus lactis 2HDS3-SM6; Figure 4 It is a colony morphology diagram of weizmannia conjuncta N2-La1. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0020] Example 1 Fermentation, screening and identification of strain The experimental process for screening and separating the compound strain is as follows: 1, dilution, 2, coating, 3, purification, 4, separation, 5, preservation of the strain, 6, culture of cells, 7, performance of cell experiments, which are shown in A-G of Figure 1 .

[0021] The specific steps of screening and separating the compound strain are as follows: 100 uL sample liquid is taken from the fermentation sample liquid in 900 uL sterile water, and then the sample is diluted to 10 -4 ,10 -5 concentration, 100 uL is coated on the MRS plate, and placed in a 37℃ anaerobic incubator for 48h, and the bacterial growth on the plate is observed. After the colonies grow, a typical single colony is picked and streaked on the MRS solid medium for strain separation and purification. The single colony is inoculated into 5 mL sterilized MRS medium, and incubated at 37℃ for about 48h, and stored in a-80℃ refrigerator with 50% glycerol for standby. The MRS medium is purchased from Qingdao Haibo Biology.

[0022] The single colony after separation and purification is sent to Zhejiang Shangya Biotechnology Co., Ltd., and the identification primer is: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (the sequence is shown as SEQ ID NO. 4); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (the sequence is shown as SEQ ID NO. 5).

[0023] The identification results are as follows: the 16s rDNA sequences of Lactiplantibacillus plantarum 2HDS3-La5, Pediococcus acidilactici 2HDS3-SM6 and Weissella coagulans N2-La1 are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and the colony morphologies are shown as Figure 2 、 Figure 3 、 Figure 4 respectively.

[0024] Lactiplantibacillus plantarum 2HDS3-La5 was deposited with the China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC NO. 34339. Pediococcus acidilactici 2HDS3-SM6 was deposited with the China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC NO. 34337. Weissella coagulans N2-La1 was deposited with the China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC NO. 34338.

[0025] Among them, the taxonomic name of Lactiplantibacillus plantarum 2HDS3-La5 is Lactiplantibacillus plantarum; the taxonomic name of Pediococcus acidilactici 2HDS3-SM6 is Pediococcus acidilactici; The taxonomic designation of Weizmannia coagulans N2-La1 is Weizmannia coagulans.

[0026] Example 2 Preparation of compound strain A Strains of Lactiplantibacillus 2HDS3-La5, Pediococcus acidilactici 2HDS3-SM6, Weizmannia coagulans 2HDS3-SM6 (OD600nm of the above strains is adjusted to 1.0, and the bacterial concentration is about 10 8 CFU / ml), are mixed at a ratio of 1:1:1 at 37°C and allowed to ferment and grow statically. After 3 days of growth, the mixture is taken out and named as compound strain A.

[0027] Comparative Example 1 Preparation of compound strain B Commercially common fermentation strains of Lactiplantibacillus 1.38, Pediococcus acidilactici 1.263, and Weizmannia coagulans 1.42 are compounded according to the above method at a ratio of 1:1:1, and named as compound strain B.

[0028] Application Example 1 1. Preparation of East Perilla Tea Stock Solution (Fermentation Substrate) 5 g of East Perilla Tea (origin: Yunnan) is taken, 500 mL of purified water is added, and then the mixture is placed in a heating device and boiled to obtain an East Perilla Tea stock solution. The initial pH of the East Perilla Tea is then detected, and food-grade citric acid or sodium hydroxide is used to adjust the pH of the stock solution to maintain the pH between 5.6 and 6.5.

[0029] 2. Application of Compound Strains to Prepare Fermented East Perilla Tea Compound strains A and B are respectively added to the East Perilla Tea stock solution at an addition amount of 0.002 g / ml, and the total liquid content is 70-75% of the vessel. Then, 2% of the volume of the East Perilla Tea stock solution of fructooligosaccharides is added, the vessel is sealed (the liquid in the bottle is shaken every 12 h, and the blue cap bottle cap is twisted lightly to release the gas), and the culture is incubated in a 28-33°C incubator. After 7 days, the culture is taken out.

[0030] Material Content Test The fermentation supernatant of compound strain A prepared in step 2 of Application Example 1 and the fermentation supernatant of the commonly used commercial compound strain B are sterilized at 100°C for 30 min. Then, the stock solution (referred to as YSC), the fermentation supernatant of compound strain A (referred to as YSCJ), and the fermentation supernatant of the commonly used commercial compound strain B (referred to as YSCFJ) are respectively taken out, placed in -80°C storage, and sent to Shanghai ZK New Life Biological Technology Co., Ltd. (APTBIO) for LC-MS liquid chromatography mass spectrometry non-target metabolite detection. The results are subjected to differential metabolite analysis.

[0031] The test results are shown in the table below:

[0032] Note: The data represents the relative content of each metabolite in the corresponding sample, without specific units.

[0033] 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.

[0034] 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.

[0035] Compared with the original solution, the rosmarinic acid content in the fermentation supernatant of compound strain A increased by 11.618 times.

[0036] 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.

[0037] 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.

[0038] Cellular experimental testing BV2 cell experiment (mouse microglia) 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.

[0039] The specific testing steps are as follows: 1. Take out the original solution of Dongzisu tea, the fermentation supernatant of compound strain A, and the fermentation supernatant of commercial commonly used compound strain B for standby.

[0040] 2. Configuration of BV2 cell culture medium: DMEM high-sugar medium + 10-15% FBS (fetal bovine serum) + 1% P / S 3. Resuscitation of BV2 cells: quickly shake the cryopreservation tube containing 1 mL of cell suspension in a 37°C water bath to thaw, and add it to a centrifuge tube containing 4-6 mL of culture medium and mix evenly. Centrifuge at 1000 RPM for 3-5 min, discard the supernatant, and complete the resuspension of the cells in the culture medium. Then add the cell suspension to a culture flask containing 5 mL of culture medium at 37°C, 5% CO2, for 48 h, and observe the cell concentration.

[0041] 4. Passage of BV2 cells: when the culture concentration of BV2 cells grows to 8-10 × 105 cell / mL, passaging is performed. Pour out the culture medium in the original cell flask, add 1.5 mL of trypsin, and place it in a 37°C, 5% CO2 incubator for 2 min of digestion, then remove and discard, add 1.5 mL of DMEM high-sugar + 10% FBS to terminate digestion, and blow to form a cell suspension. Add 4 mL of culture medium to a cell culture dish, and inoculate 0.5 mL of the 1.5 mL cell suspension into a 6 cm cell culture dish, and then place the cell culture flask in a 37°C, 5% CO2 incubator for culture until the cell number reaches 8-10 × 105 cell / mL before performing the experiment.

[0042] 5. Plating of BV2 cells: after cell digestion, add 10 uL of cell suspension to a 96-well cell culture plate, then add 190 uL of culture medium, and incubate overnight at 37°C, 5% CO2.

[0043] 6. Addition of samples: discard the supernatant of the overnight cultured cells, then add the original solution, the fermentation supernatant of compound strain A, and the fermentation supernatant of model compound strain B to the wells according to a 20% addition amount, then use culture medium to make up the remaining volume, and incubate overnight at 37°C, 5% CO2.

[0044] 7. According to the instructions, use the CCK-8 kit to determine the cell viability in the 96-well cell culture, and determine the effect of the original solution, the fermentation supernatant of compound strain A, and the fermentation supernatant of commercial commonly used compound strain B on cells.

[0045] GES-1 cell experiment (human gastric mucosa cells) GES-1 (Gastric Epithelial Cell Line-1) is a kind of immortalized human gastric mucosa epithelial cell line, which is established by gene modification technology from normal gastric mucosa tissue. Its biggest feature is to retain the morphological and functional characteristics of normal gastric epithelial cells. Compared with tumor-derived gastric cells (such as AGS, MKN45), GES-1 has not been malignant transformed, and can more truly reflect the physiological state of normal gastric mucosa, which is an ideal model for studying the early mechanism of gastritis, gastric ulcer and gastric cancer.

[0046] The test method is the same as the GES-1 cell viability test method and BV2 cell, which is detected by CCK-8 kit.

[0047] Test results The test results of the two cell experiments are shown in the following table:

[0048] Note: OD 450 It is found through detection that the cell viability of GES-1 (human gastric mucosa cells) cultured by the fermentation supernatant of the compound strain A is 188.78%, while the cell viability of GES-1 treated by the commercial commonly used compound strain B is 94.77%, and the cell viability after treatment by the original solution is 98.23%.

[0049] The cell viability of BV2 cells cultured by the fermentation supernatant of the compound strain A is 119.88%, while the cell viability of GES-1 treated by the commercial commonly used compound strain B is 97.63%, and the cell viability after treatment by the original solution is 83.14%.

[0050] Therefore, the fermentation supernatant of the compound strain A can promote the growth of GES-1 (human gastric mucosa cells), has a protective effect on the stomach and gastrointestinal health, and can prevent the occurrence of gastritis, gastric ulcer and other gastrointestinal diseases; according to the table results, the fermentation supernatant of the compound strain A can improve the cell viability of BV2 and promote the growth of BV2 cells, and it is speculated that the fermentation broth of the compound strain of the application has a preventive effect on neurodegenerative diseases.

[0051] The present application ferments the east perilla tea by the compound strain, optimizes and compounding specific functional strains to form a stable and complementary microbial community, reduces the interference of miscellaneous bacteria, makes the fermentation process more controllable, and the product quality more stable. Meanwhile, under the synergistic action of multiple microorganisms, protein, monosaccharide and other substances can be more completely converted into more beneficial metabolites (such as flavonoids and triterpenes), the health care value of the fermentation broth is improved, and finally more rich and harmonious aroma components (alcohols, esters, etc.) are produced by the compound strain, the bitter taste is reduced, the flavor is more mellow and refreshing, the peculiar smell caused by single strain is avoided, and market recognition is obtained.

[0052] The compound microbial agent is used for preparing fermented Perilla frutescens tea, and the content of anti-inflammatory, active, antioxidant and immune-regulating substances in the Perilla frutescens tea is significantly improved, and the prepared fermented Perilla frutescens tea has obvious promoting effect on promoting cell growth and cell activity.

[0053] The above examples are only used for illustrating the present application, and are not limited to the present application. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the present application. Therefore, all equivalent technical solutions should belong to the scope of the present application, which is limited by the claims.

Claims

1. A compound bacterial strain, characterized in that, These include *Lactobacillus plantarum* 2HDS3-La5 with accession number CGMCC NO.34339, *Pediococcus lactis* 2HDS3-SM6 with accession number CGMCC NO.34337, and *Weizmannii coagulans* N2-La1 with accession number CGMCC NO.34338.

2. A bacterial agent containing the compound strain as described in claim 1.

3. The compound strain according to claim 1 or the microbial agent according to claim 2, characterized in that, The bacterial count 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.

4. The microbial agent according to claim 3, characterized in that, The preparation method of the bacterial agent includes the steps of statically fermenting and growing the compound bacterial strain at 30℃-37℃ for 1-5 days.

5. The microbial agent according to claim 4, characterized in that, The bacterial strain in the bacterial agent has an OD600 of 0.6-1 and a bacterial count of 10-1. 7 -10 9 CFU / ml.

6. The application of the compound strain as described in claim 1 or the microbial agent as described in claim 2 in the preparation of fermented perilla tea.

7. The application according to claim 6, characterized in that, The compound strain or inoculant is used to increase the content of at least one of the anti-inflammatory, antioxidant, and immunomodulatory substances in Dongzisu tea.

8. The application according to claim 6, characterized in that, The fermented perilla tea is used in the preparation of medicines or health products for the treatment or prevention of gastrointestinal diseases or neurodegenerative diseases.

9. The application according to claim 6, characterized in that, The application method is as follows: add the bacterial agent to the Dongzisu tea stock solution at an addition amount of 0.0001-0.2 g / ml, and add fructooligosaccharides at an amount of 0.1-5% of the volume of the Dongzisu tea stock solution, and incubate at 25-33℃ for 3-10 days.

10. The application according to claim 9, characterized in that, The preparation method of the Dongzisu tea stock solution is as follows: Dongzisu tea is mixed with water and heated to boiling. The ratio of Dongzisu tea to water is 1g-10g:1ml-2000mL. The pH of the Dongzisu tea stock solution is maintained between 5.6 and 6.5.

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