Fungus composition and chrysanthemum indicum seed fermentation liquor

Abstract

The invention provides a bacterial composition and chrysanthemum indicum seed fermentation liquor. The bacterial composition comprises at least two of plant lactobacillus, Witzia ciliaris and plant lactobacillus pentosus. The preservation date of the plant lactobacillus is April 25, 2025, and the preservation number of the plant lactobacillus is CGMCC (China General Microbiological Culture Collection Center) No.34339; the preservation date of the Witzia ciliaris is April 25, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.34338; the preservation date of the lactobacillus pentosus is May 9, 2025, and the preservation number of the lactobacillus pentosus is CGMCC (China General Microbiological Culture Collection Center) No.34471; the chrysanthemum indicum bud fermentation liquor is obtained by inoculating a bacterial composition into a chrysanthemum indicum bud stock solution for fermentation; the bacterium composition promotes the chrysanthemum indicum bud stock solution to be fermented, so that the effective components in the chrysanthemum indicum bud stock solution are greatly increased.

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] Further, the bacterial composition comprises Lactobacillus plantarum, Weizmannia coagulans and Lactobacillus pentosus.

[0008] Further, the ratio of the viable bacterial count of Lactobacillus plantarum, Weizmannia coagulans and Lactobacillus pentosus is (1-2):(3-5):(1-3).

[0009] The second object of the present application can be achieved by adopting the following technical solution: a wild chrysanthemum rice fermentation liquor, wherein the wild chrysanthemum rice fermentation liquor is obtained by inoculating a bacterial composition into wild chrysanthemum rice stock solution and then performing fermentation; The bacterial composition comprises at least two of Lactobacillus plantarum, Weizmannia coagulans 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 coagulans 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; and the strains are all preserved in the General Microbiological Center of China Microbial Culture Collection Committee.

[0010] Further, the wild chrysanthemum rice stock solution is prepared by the following method: adding wild chrysanthemum rice into a solvent and heating, the mass percentage of the wild chrysanthemum rice is 1-3 wt%, and then filtering out the liquid to obtain the wild chrysanthemum rice stock solution.

[0011] Further, the bacterial composition is cultured at a temperature of 35-38℃ for 2-4 days, and then inoculated into the wild chrysanthemum rice stock solution.

[0012] Further, the bacterial composition is inoculated into the wild chrysanthemum rice stock solution for fermentation at a proportion of 0.1-0.3 wt%.

[0013] Further, the fermentation temperature is 30-38℃.

[0014] Further, the application mode of the wild chrysanthemum rice fermentation liquor is external application and / or oral administration.

[0015] Compared with the prior art, the present application has the following beneficial effects: 1. The bacterial composition obtained by optimizing and compounding the strains has good stability, promotes the fermentation of the wild chrysanthemum rice stock solution, greatly improves the effective components therein, and has important market value; 2. The wild chrysanthemum rice fermentation liquor has high bergenin, sophocarpine and phloretin, has the effects of relieving chronic inflammation, immune regulation, promoting cell growth and improving cell vitality; 3. The wild chrysanthemum rice fermentation liquor of the present application is used to unlock more use scenarios of wild chrysanthemum rice, and the use of the bacterial composition for fermentation of the wild chrysanthemum rice original liquid can improve the active substances originally contained in the wild chrysanthemum rice, thereby providing a new scheme and idea for further development of wild chrysanthemum rice. BRIEF DESCRIPTION OF DRAWINGS

[0016] The plant lactobacillus involved in the present scheme was preserved on April 25, 2025, and the preservation number is CGMCC No. 34339; The preservation date of the weizmannia conjuncta involved in the present scheme is April 25, 2025, and the preservation number is CGMCC No. 34338; The preservation date of the lactobacillus pentosus involved in the present scheme is May 9, 2025, and the preservation number is CGMCC No. 34471; The strains are preserved in the China General Microbiological Culture Collection Center.

[0017] Figure 1 The colony morphology of the plant lactobacillus; Figure 2 The microscopic morphology of the plant lactobacillus; Figure 3 The colony morphology of the weizmannia conjuncta; Figure 4 The microscopic morphology of the weizmannia conjuncta; Figure 5 The colony morphology of the lactobacillus pentosus; Figure 6 The microscopic morphology of the lactobacillus pentosus. DETAILED DESCRIPTION

[0018] The present application will be further described below in combination with the drawings and specific embodiments: Example 1: The screening and separation of the strains are as follows: 100 uL of the sample liquid is taken in 900 uL of sterile water, and then the sample is diluted to 10 -4 , 10 -5 concentration, 100 uL is taken and coated on an MRS plate, and placed in a 37℃ anaerobic incubator for 48h, the bacterial growth on the plate is observed, and after the colonies grow, a typical single colony is picked and streaked on an MRS solid culture medium for strain separation and purification. The single colony is inoculated into 5mL of sterilized MRS medium, and cultured at 37℃ for about 48h, and stored in 50% v / v glycerol at -80℃ for standby. The MRS medium is purchased from Qingdao Haibo Biology.

[0019] The single colony after separation and purification is sent to Zhejiang Shangya Biotechnology Co., Ltd., and the identification primer is: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 4) 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 5).

[0020] Identification results: the 16s rDNA sequences of Lactiplantibacillus plantarum 2HDS3-La5, Weizmannia coagulans N2-La1 and Lactiplantibacillus pentosus HDS3-8 are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 respectively; the colony morphology and microscopic morphology of Lactiplantibacillus plantarum are shown in Figures 1-2 ; the colony morphology and microscopic morphology of Weizmannia coagulans are shown in Figures 3-4 ; the colony morphology and microscopic morphology of Lactiplantibacillus pentosus are shown in Figures 5-6 .

[0021] Example 2: 1) Preparation of bacterial composition: Lactiplantibacillus plantarum, Weizmannia coagulans and Lactiplantibacillus pentosus (OD600nm adjusted to 1.0, bacterial number about 10^8) were mixed in the proportion of 1.5:4:1.5 according to the number of viable bacteria, and cultured at a temperature of 36℃ for 2d to obtain a composite bacterial liquid.

[0022] Among them, the preservation date of Lactiplantibacillus plantarum is April 25, 2025, and the preservation number is CGMCC No. 34339; the preservation date of Weizmannia coagulans is April 25, 2025, and the preservation number is CGMCC No. 34338; the preservation date of Lactiplantibacillus pentosus is May 9, 2025, and the preservation number is CGMCC No. 34471; all strains are preserved in the General Microbiological Center of China Microbial Culture Collection Management Committee.

[0023] Among them, the 16s rDNA of Lactiplantibacillus plantarum is shown in SEQ ID NO. 1; the 16s rDNA of Weizmannia coagulans is shown in SEQ ID NO. 2; the 16s rDNA of Lactiplantibacillus pentosus is shown in SEQ ID NO. 3; 2) Preparation of wild chrysanthemum rice stock solution: wild chrysanthemum rice was added to the solvent and boiled for 30min, the mass percentage of wild chrysanthemum rice was 2wt%, then the liquid was filtered out to obtain the wild chrysanthemum rice stock solution; 3) Preparation of wild chrysanthemum rice fermentation liquid: the composite bacterial liquid was inoculated in the wild chrysanthemum rice stock solution at 0.2wt%, 2wt% of fructooligosaccharide was added, and the fermentation was carried out at a temperature of 30℃ for 7d, then inactivated at 100℃ to obtain the wild chrysanthemum rice fermentation liquid.

[0024] Comparative Example 1: The preparation of the complex bacterial agent uses commercial strains of Lactobacillus plantarum, Weizmannia coagulans and Lactobacillus pentosus, and other steps are the same as in Example 2 to prepare the wild chrysanthemum rice fermentation liquor.

[0025] Detection: 1. Content detection: The wild chrysanthemum rice fermentation liquor of Example 2 and the comparative example was stored at -80°C, and dry ice was used to send the sample to Shanghai ZK New Life Biotechnology Co., Ltd. (APTBIO) for LC-MS liquid chromatography mass spectrometry non-target metabolite detection. The results were analyzed for differential metabolites, and the results are shown in Table 1: Table 1 Relative content of metabolites

[0026] Note: The data is the relative content of each metabolite in the corresponding sample, and has no specific unit.

[0027] Differential metabolite analysis of the supernatant of the wild chrysanthemum rice fermentation liquor of Example 2 and Comparative Example 1 found that the content of bergenin and matrine in the wild chrysanthemum rice fermentation liquor of Example 2 was increased by 541.95 and 180.82 times compared with the wild chrysanthemum rice original liquid, while the commercial complex strain used in Comparative Example 1 only increased bergenin and matrine by 2.81 and 2.12 times compared with the original liquid. In addition, the content of phloretin in the wild chrysanthemum rice fermentation liquor of Example 2 was increased by 39.57 times, while that of Comparative Example 1 was increased by 7 times. The content of bergenin and matrine in Example 2 was significantly higher than that in the original liquid and Comparative Example 1.

[0028] Bergenin is a natural organic compound, an active ingredient extracted from plants, which has various biological activities, including anti-inflammatory, antioxidant, anti-tumor and immunomodulatory effects.

[0029] Matrine is a natural alkaloid with broad-spectrum biological activity, and its mechanism of action includes anti-viral, anti-tumor, anti-inflammatory, and reducing rheumatoid arthritis, dermatitis and other symptoms, and improving allergic reactions.

[0030] ‌Phloretin is a natural flavonoid compound extracted from plants, which has antioxidant, whitening, anti-inflammatory, blood glucose and lipid regulation effects.

[0031] Therefore, by fermenting the wild chrysanthemum rice original liquid in Example 2, the content of the effective components is greatly increased, and it has high anti-inflammatory, antioxidant and immunomodulatory effects. It can be used externally to improve the skin condition, or taken internally to improve the internal function of the body.

[0032] 2. Cell experiment test: Rat renal proximal tubular epithelial cells NRK-52E is a cell line with epithelial morphology isolated from rat renal proximal tubule, which is a spontaneously immortalized cell. NRK-52E is a commonly used kidney cell model, which helps to reveal the development mechanism of kidney diseases (such as renal fibrosis, sodium metabolism disorder, diabetes, etc.), and evaluate the renal toxicity of drugs, which is of great significance for the development of new treatment strategies and drug safety evaluation.

[0033] Preparation of NRK-52E cell culture medium: DMEM high-sugar medium + 10-15% FBS (fetal bovine serum) + 1% P / S.

[0034] NRK-52E cells were cultured, recovered, and subcultured to a cell number of 8-10 x 10 5 After the cell number reached 8-10 x 10

[0035] The CCK-8 kit was used to determine the cell viability in the 96-well cell culture, and the effects of wild chrysanthemum original liquid, wild chrysanthemum fermentation liquid of Example 2, and wild chrysanthemum fermentation liquid of Comparative Example 1 on cells were determined. The results are shown in Table 2: Table 2 Cell viability values

[0036] The average cell viability of NRK-52E (rat kidney cells) cultured with the wild chrysanthemum fermentation liquid of Example 2 was 122.47%, while the cell viability of NRK-52E cultured with the wild chrysanthemum fermentation liquid of Comparative Example 1 was 99.42%, and the cell viability after culture with wild chrysanthemum original liquid was 103.45%. Only the wild chrysanthemum fermentation liquid of Example 2 can promote the growth of rat kidney cells, indicating that the wild chrysanthemum fermentation liquid of Example 2 is of great significance for the prevention of kidney diseases, the development of new treatment strategies, and drug safety evaluation.

[0037] For those skilled in the art, other various corresponding changes and modifications can be made according to the above described technical solutions and concepts, and all of these changes and modifications should belong to the protection scope of the claims of the present application.

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 At least two of the following strains are included: *Lactobacillus plantarum*, whose preservation date is April 25, 2025, and preservation number is CGMCC No. 34339; *Weizmannii coagulans*, whose preservation date is April 25, 2025, and preservation number is CGMCC No. 34338; and *Lactobacillus pentosaccharide*, whose preservation date is May 9, 2025, and preservation number is CGMCC No. 34471; all strains are deposited at the China General Microbiological Culture Collection Center.

2. The bacterial composition according to claim 1, characterized in that, 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.

3. The bacterial composition according to claim 1, characterized in that, The bacterial composition includes *Lactobacillus plantarum*, *Weizmannia coagulans*, and *Lactobacillus pentosus*.

4. The bacterial composition according to claim 3, characterized in that, The ratio of viable counts of *Lactobacillus plantarum*, *Weizmannii coagulans*, and *Lactobacillus pentosus* is (1-2):(3-5):(1-3).

5. A wild chrysanthemum rice fermentation liquid, characterized in that, The wild chrysanthemum rice fermentation liquid is obtained by inoculating the bacterial composition into the wild chrysanthemum rice stock solution and then fermenting it. The bacterial composition comprises at least two of *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.

6. The wild chrysanthemum rice fermentation liquid as described in claim 5, 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.

7. The wild chrysanthemum rice fermentation liquid as described in claim 5, characterized in that, The bacterial composition was cultured at 35-38℃ for 2-4 days, and then inoculated into the wild chrysanthemum rice stock solution.

8. The wild chrysanthemum rice fermentation liquid as described in claim 5, characterized in that, The bacterial composition was inoculated into the wild chrysanthemum rice stock solution at 0.1-0.3 wt% for fermentation.

9. The wild chrysanthemum rice fermentation liquid as described in claim 5, characterized in that, The fermentation temperature is 30-38℃.

10. The wild chrysanthemum rice fermentation liquid as described in claim 5, characterized in that, The wild chrysanthemum rice fermentation liquid is applied externally and / or taken internally.

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