A bacterial composition and a wild rice fermentation liquor

Abstract

The present application provides a bacterial composition and wild chrysanthemum rice fermentation liquor, the bacterial composition comprises at least two of lactobacillus plantarum, weizmannia conjuncta and lactobacillus pentosus; the preservation date of lactobacillus plantarum is April 25, 2025, and the preservation number is CGMCC No.34339; the preservation date of weizmannia conjuncta is April 25, 2025, and the preservation number is CGMCC No.34338; the preservation date of lactobacillus pentosus is May 9, 2025, and the preservation number is CGMCC No.34471; the wild chrysanthemum rice fermentation liquor is obtained by inoculating the bacterial composition into wild chrysanthemum rice stock solution for fermentation; the bacterial composition promotes the wild chrysanthemum rice stock solution to greatly improve the effective components therein through fermentation.

Description

Technical Field

[0001] This invention relates to a bacterial composition and wild chrysanthemum rice fermentation broth, belonging to the field of microbial application technology. Background Technology

[0002] Wild chrysanthemum buds, the flower buds of wild chrysanthemum, have demonstrated rich efficacy and effects in both traditional medicine and modern nutrition. In traditional Chinese medicine, they are slightly cold in nature and bitter in taste, entering the lung and liver meridians, possessing multiple effects such as dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and detoxifying. They are generally used for making tea, cooking porridge, or in medicinal cuisine, with relatively limited daily applications. Furthermore, wild chrysanthemum buds are rich in protein, chrysanthemum extract, flavonoids, chrysanthemum lactones, amino acids, and various trace elements. These components work together to supplement the body with essential nutrients. Despite being a valuable ingredient, the utilization of wild chrysanthemum buds remains limited. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide a bacterial composition that promotes the fermentation of wild chrysanthemum rice extract and greatly enhances its effective components.

[0004] The second objective of this invention is to provide a wild chrysanthemum rice fermentation liquid containing high levels of bergenin, matrine, and phloretin, which can alleviate chronic inflammation and regulate immunity.

[0005] The first objective of this invention can be achieved by adopting the following technical solution: a bacterial composition comprising at least two of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosus*; *Lactobacillus plantarum* classification and nomenclature: *Lactobacillus plantarum*, Latin scientific name: Lactiplantibacillus plantarum The deposit date is April 25, 2025, and the accession number is CGMCC No. 34339; *Weizmannii coagulans*, classification and name: *Weizmannii coagulans*, Latin scientific name: Weizmannia coagulans The deposit date is April 25, 2025, and the accession number is CGMCC No. 34338; *Lactobacillus pentosaccharis*, taxonomic name: *Lactobacillus pentosaccharis*, Latin scientific name: Lactiplantibacillus pentosus The preservation date is May 9, 2025, and the preservation number is CGMCC No. 34471. All strains are preserved at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Furthermore, the 16S rDNA of *Lactobacillus plantarum* is shown in SEQ ID NO.1; the 16S rDNA of *Weizmannii coagulans* is shown in SEQ ID NO.2; and the 16S rDNA of *Lactobacillus pentosus* is shown in SEQ ID NO.3.

[0007] Furthermore, the bacterial composition includes *Lactobacillus plantarum*, *Weizmannia coagulans*, and *Lactobacillus pentosus*.

[0008] Furthermore, the ratio of viable counts of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosus* was (1-2):(3-5):(1-3).

[0009] The second objective of this invention can be achieved by adopting the following technical solution: a wild chrysanthemum rice fermentation liquid, wherein the wild chrysanthemum rice fermentation liquid is obtained by inoculating a bacterial composition into wild chrysanthemum rice stock solution for fermentation;

[0010] The bacterial composition includes at least two of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosaccharis*; the deposit date of *Lactobacillus plantarum* is April 25, 2025, with accession number CGMCC No. 34339; the deposit date of *Weizmannii coagulans* is April 25, 2025, with accession number CGMCC No. 34338; the deposit date of *Lactobacillus pentosaccharis* is May 9, 2025, with accession number CGMCC No. 34471; all strains are deposited at the China General Microbiological Culture Collection Center.

[0011] Furthermore, the wild chrysanthemum rice stock solution is prepared by the following method: wild chrysanthemum rice is added to a solvent and heated, the mass percentage of wild chrysanthemum rice is 1-3 wt%, and then the liquid is filtered out to obtain the wild chrysanthemum rice stock solution.

[0012] Furthermore, the bacterial composition was cultured at a temperature of 35-38℃ for 2-4 days, and then inoculated into the wild chrysanthemum rice stock solution.

[0013] Furthermore, the bacterial composition was inoculated into the wild chrysanthemum rice stock solution at 0.1-0.3 wt% for fermentation.

[0014] Furthermore, the fermentation temperature is 30-38℃.

[0015] Furthermore, the wild chrysanthemum rice fermentation liquid can be applied externally and / or taken internally.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The bacterial composition obtained by the present invention through the selection and compounding of strains has good stability and promotes the fermentation of wild chrysanthemum rice extract, thereby greatly enhancing the effective components therein and having significant market value.

[0018] 2. The wild chrysanthemum rice fermentation liquid of the present invention has high levels of bergenin, matrine and phloretin, which can relieve chronic inflammation and regulate immunity, and can also promote cell growth and enhance cell vitality.

[0019] 3. The wild chrysanthemum rice fermentation liquid of the present invention unlocks more application scenarios for wild chrysanthemum rice. It uses a bacterial composition to ferment the original wild chrysanthemum rice liquid, thereby enhancing the original active substances in wild chrysanthemum rice and providing new solutions and ideas for the further development of wild chrysanthemum rice. Attached Figure Description

[0020] The Lactobacillus plantarum involved in this protocol was deposited on April 25, 2025, with accession number CGMCC No. 34339;

[0021] The *Weizmannii coagulans* strain involved in this protocol was deposited on April 25, 2025, with accession number CGMCC No. 34338.

[0022] The *Lactobacillus pentosaccharide* involved in this protocol was deposited on May 9, 2025, with accession number CGMCC No. 34471.

[0023] All strains are deposited at the China General Microbiological Culture Collection Center of the China Association for the Preservation and Management of Microbial Culture Collections.

[0024] Figure 1 The colony morphology of *Lactobacillus plantarum*;

[0025] Figure 2 Microscopic morphology of *Lactobacillus plantarum*;

[0026] Figure 3 The colony morphology of *Weizmannii* coagulation;

[0027] Figure 4 Microscopic morphology of *Weizmannii* coagulation bacteria;

[0028] Figure 5 The colony morphology of *Lactobacillus pentosus*;

[0029] Figure 6 The microscopic morphology of Lactobacillus pentosus. Detailed Implementation

[0030] The present invention will now be further described with reference to the accompanying drawings and specific embodiments:

[0031] Example 1:

[0032] For the screening and isolation of strains, 100 μL of the fermentation broth was taken and placed in 900 μL of sterile water, and then the sample was diluted to 10 μL. -4 10 -5For 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. It was then stored at -80°C with 50% v / v glycerol for later use. MRS medium was purchased from Qingdao Haibo Biotechnology.

[0033] The isolated and purified single colonies were sent to Zhejiang Shangya Biotechnology Co., Ltd., and the identification primers were:

[0034] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO.4)

[0035] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 5).

[0036] Identification results: The 16S rDNA sequences of *Lactobacillus plantarum* 2HDS3-La5, *Weizmannii coagulans* N2-La1, and *Lactobacillus pentosus* HDS3-8 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the colony morphology and microscopic morphology of *Lactobacillus plantarum* are shown in... Figures 1-2 As shown; the colony morphology and microscopic morphology of *Weizmannii* coagulation are as follows: Figures 3-4 As shown; the colony morphology and microscopic morphology of *Lactobacillus pentosus* are as follows. Figures 5-6 As shown.

[0037] Example 2:

[0038] 1) Preparation of the bacterial composition:

[0039] Lactobacillus plantarum, Weizmannii coagulans, and Lactobacillus pentosus (OD600nm adjusted to 1.0, bacterial count approximately 10^8) were mixed at a viable ratio of 1.5:4:1.5 and cultured at 36℃ for 2 days to obtain a composite bacterial solution.

[0040] Among them, *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; *Weizmannii coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338; and *Lactobacillus pentosaccharide* was deposited on May 9, 2025, with accession number CGMCC No. 34471. All strains are deposited at the China General Microbiological Culture Collection Center.

[0041] Among them, the 16S rDNA of *Lactobacillus plantarum* is shown in SEQ ID NO.1; the 16S rDNA of *Weizmannii coagulans* is shown in SEQ ID NO.2; and the 16S rDNA of *Lactobacillus pentosus* is shown in SEQ ID NO.3.

[0042] 2) Preparation of wild chrysanthemum rice stock solution: Add wild chrysanthemum rice to a solvent and heat to boiling for 30 minutes. The mass percentage of wild chrysanthemum rice is 2wt%. Then filter out the liquid to obtain wild chrysanthemum rice stock solution.

[0043] 3) Preparation of wild chrysanthemum rice fermentation broth: Inoculate the compound bacterial solution into the wild chrysanthemum rice stock solution at 0.2wt%, add 2wt% of oligofructose, ferment at 30℃ for 7 days, and then inactivate at 100℃ to obtain wild chrysanthemum rice fermentation broth.

[0044] Comparative Example 1:

[0045] The preparation of the compound microbial agent uses commonly used commercial strains such as *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosus*. Other steps are the same as in Example 2 to prepare the wild chrysanthemum rice fermentation broth.

[0046] Detection:

[0047] 1. Content detection:

[0048] The fermentation broths of Chrysanthemum indicum from Example 2 and the comparative example were stored at -80℃ and transported to Shanghai APTBIO Biotechnology Co., Ltd. (APTBIO) using dry ice for LC-MS liquid chromatography-mass spectrometry detection of non-targeted metabolites. Differential metabolite analysis was also performed, and the results are shown in Table 1.

[0049] Table 1 Relative content of metabolites

[0050]

[0051] Note: The data represents the relative content of each metabolite in the corresponding sample, without specific units.

[0052] Differential metabolite analysis of the supernatant from the fermentation broths of *Chrysanthemum indicum* from Example 2 and Comparative Example 1 revealed that the fermentation broth from Example 2 showed a 541.95-fold and 180.82-fold increase in the content of bergenin and matrine, respectively, compared to the original fermentation broth. In contrast, Comparative Example 1, using a commercially available compound strain, only showed a 2.81-fold and 2.12-fold increase in bergenin and matrine, respectively, compared to the original broth. Furthermore, the fermentation broth from Example 2 showed a 39.57-fold increase in phloretin, while Comparative Example 1 showed a 7-fold increase. The increases in bergenin and matrine in Example 2 were significantly greater than those in the original broth and Comparative Example 1.

[0053] Bergenin is a natural organic compound, an active ingredient extracted from plants, with a variety of biological activities, including anti-inflammatory, antioxidant, anti-tumor and immunomodulatory effects.

[0054] Matrine is a natural alkaloid with broad-spectrum biological activity. Its mechanisms of action include antiviral, antitumor, and anti-inflammatory effects, alleviating symptoms of rheumatoid arthritis, dermatitis, and improving allergic reactions.

[0055] Phloretin is a natural flavonoid compound extracted from plants, which has antioxidant, whitening, anti-inflammatory, and blood sugar and lipid-regulating effects.

[0056] Therefore, Example 2, through the fermentation treatment of wild chrysanthemum rice extract, significantly increased the concentration of active ingredients, resulting in high levels of anti-inflammatory, antioxidant, and immunomodulatory effects. It can be applied topically to the skin to improve its condition, or taken internally to improve internal bodily functions.

[0057] 2. Cellular experimental tests:

[0058] Rat renal proximal tubular epithelial cells (NRK-52E) are an epithelial cell line isolated from the proximal convoluted tubules of the rat kidney and are spontaneously immortalized. NRK-52E is a commonly used kidney cell model, helpful in elucidating the development mechanisms of kidney diseases (such as renal fibrosis, sodium metabolism disorders, and diabetes), and in assessing the nephrotoxicity of drugs. It is of great significance for developing new treatment strategies and evaluating drug safety.

[0059] Preparation of NRK-52E cell culture medium: DMEM high glucose medium + 10-15% FBS (fetal bovine serum) + 1% P / S.

[0060] NRK-52E cells were cultured, revived, and passaged until the cell number reached 8-10 × 10⁻⁶. 5 cell / mL, after plating: Discard the supernatant of cells cultured overnight, add the wild chrysanthemum rice stock solution, wild chrysanthemum rice fermentation broth of Example 2, and wild chrysanthemum rice fermentation broth of Comparative Example 1 to the wells (add according to 20% of the total volume of the wells), and then use culture medium to make up the remaining volume. Incubate overnight at 37°C with 5% CO2.

[0061] Cell viability in 96-well cell cultures was measured using a CCK-8 assay kit to determine the effects of the original Wild Chrysanthemum rice solution, the Wild Chrysanthemum rice fermentation broth from Example 2, and the Wild Chrysanthemum rice fermentation broth from Comparative Example 1 on the cells. The results are shown in Table 2.

[0062] Table 2 Cell Viability Values

[0063]

[0064] The average cell viability of NRK-52E (rat kidney cells) cultured in the wild chrysanthemum rice fermentation broth of Example 2 was 122.47%, while the cell viability of NRK-52E cells cultured in the wild chrysanthemum rice fermentation broth of Comparative Example 1 was 99.42%, and the cell viability of cells cultured in the original wild chrysanthemum rice solution was 103.45%. Only the wild chrysanthemum rice fermentation broth of Example 2 could promote the growth of rat kidney cells, indicating that the wild chrysanthemum rice fermentation broth of Example 2 is of great significance for the prevention of kidney diseases, the development of new treatment strategies, and the evaluation of drug safety.

[0065] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A bacterial composition, characterized in that, The bacterial composition includes *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ), Weizmann's coagulation bacteria ( Weizmannia coagulans ) and Pentosacchari lactobacillus ( Lactiplantibacillus pentosus The *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; the *Weizmannii coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338; and the *Lactobacillus pentosaccharide* was deposited on May 9, 2025, with accession number CGMCC No. 34471. All strains were deposited at the China General Microbiological Culture Collection Center. The viable count ratio of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosaccharide* was 1.5:4:1.

5.

2. A wild chrysanthemum rice fermentation liquid, characterized in that, The fermented wild chrysanthemum rice broth is obtained by culturing the bacterial composition at a temperature of 35-38℃ for 2-4 days, and then inoculating it into the original wild chrysanthemum rice broth at a rate of 0.1-0.3wt% for fermentation; the fermentation temperature is 30-38℃. The bacterial composition comprises *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosaccharis*; the *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; the *Weizmannii coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338; and the *Lactobacillus pentosaccharis* was deposited on May 9, 2025, with accession number CGMCC No. 34471; all strains are deposited at the China General Microbiological Culture Collection Center; the viable count ratio of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosaccharis* is 1.5:4:1.

5.

3. The wild chrysanthemum rice fermentation liquid as described in claim 2, characterized in that; The wild chrysanthemum rice stock solution is prepared by the following method: wild chrysanthemum rice is added to a solvent and heated, the mass percentage of wild chrysanthemum rice is 1-3 wt%, and then the liquid is filtered out to obtain the wild chrysanthemum rice stock solution.

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