Compound microbial agent and plant fermentation liquor

Abstract

The application provides a composite microbial inoculant and a plant fermentation liquor, the composite microbial inoculant comprising at least two of Lactiplantibacillus plantarum, Wickerhamomyces anomalus and Weizmannia coagulans; the Lactiplantibacillus plantarum is preserved on April 25, 2025, and has a preservation number of CGMCC No.34339; the Wickerhamomyces anomalus is preserved on May 9, 2025, and has a preservation number of CGMCC No.34474; the Weizmannia coagulans is preserved on April 25, 2025, and has a preservation number of CGMCC No.34338; the strains are all preserved in the China General Microbiological Culture Collection Center; the plant fermentation liquor is obtained by inoculating the composite microbial inoculant into a plant stock solution and performing fermentation; and the composite microbial inoculant promotes the plant stock solution to greatly improve effective components in the plant stock solution through fermentation.

Description

Technical Field

[0001] This invention relates to a compound microbial agent and plant fermentation broth, belonging to the field of microbial application technology. Background Technology

[0002] Based on the tradition and widespread use of traditional Chinese medicine, using plants for daily health maintenance has become a simple and convenient way for people to maintain their health. By rationally selecting plant-based powders and beverages, minor physical discomforts can be alleviated to some extent, such as reducing swelling and detoxifying, dispersing wind-heat, stopping diarrhea, antibacterial and anti-inflammatory effects, lowering blood lipids, detoxifying and beautifying the skin, and enhancing immunity. As a result, preparations or beverages made from a variety of natural plants or traditional Chinese herbs have gained widespread market popularity. Therefore, how to improve the effectiveness of these active ingredients and enhance their effects has become a topic of even greater interest. 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 compound microbial agent that promotes the fermentation of plant extracts and greatly enhances the effective components therein.

[0004] The second objective of this invention is to provide a plant fermentation liquid with a high content of aloe-emodin and syringin, which has anti-inflammatory, antioxidant and immunomodulatory effects.

[0005] The first objective of this invention can be achieved by adopting the following technical solution: a compound microbial agent, comprising at least two of *Lactobacillus plantarum*, *Wickham's yeast*, and *Weizmann's coagulans*; *Lactobacillus plantarum*, classified and named as *Lactobacillus plantarum*, with the Latin scientific name: Lactiplantibacillus plantarum The deposit date is April 25, 2025, and the accession number is CGMCC No. 34339; *Saccharomyces cerevisiae*, classified and named as *Saccharomyces cerevisiae*, with the Latin scientific name: Wickerhamomyces anomalus The deposit date is May 9, 2025, and the accession number is CGMCC No. 34474; *Weizmannii coagulans*, classification and name: *Weizmannii coagulans*, Latin scientific name: Weizmannia coagulans The preservation date is April 25, 2025, and the preservation number is CGMCC No. 34338. All strains are preserved at the China General Microbiological Culture Collection Center, located at No. 3, No. 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 *Wickham's aberrant yeast* is shown in SEQ ID NO.2; and the 16S rDNA of *Weizmann's coagulation* is shown in SEQ ID NO.3.

[0007] Furthermore, the compound microbial agent includes *Lactobacillus plantarum*, *Wickham's abnormal yeast*, and *Weizmann's coagulans*.

[0008] Furthermore, the ratio of viable counts of *Lactobacillus plantarum*, *Wickham's anomalous*, and *Weizmann's coagulans* was (1-3):(2-4):(1-2).

[0009] The second objective of this invention can be achieved by adopting the following technical solution: a plant fermentation liquid, wherein the plant fermentation liquid is obtained by inoculating a compound microbial agent into a plant stock solution and then fermenting it;

[0010] The compound microbial agent includes at least two of the following: *Lactobacillus plantarum*, *Wickham's yeast aberrante*, and *Weizmann's coagulans*. The *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; the *Wickham's yeast aberrante* was deposited on May 9, 2025, with accession number CGMCC No. 34474; and the *Weizmann's coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338. All strains are deposited at the China General Microbiological Culture Collection Center.

[0011] Furthermore, the raw materials for preparing the plant extract include at least one of the following: mulberry leaves, lemongrass, ginger, lemon leaves, peppermint leaves, basil leaves, pandan leaves, and Longjing tea leaves.

[0012] Furthermore, the plant extract is prepared by the following method: 0.4-2 parts by weight of mulberry leaves, 0.4-2 parts by weight of lemongrass, 0.4-2 parts by weight of ginger, 0.5-3 parts by weight of lemon leaves, 0.5-3 parts by weight of peppermint leaves, 0.5-3 parts by weight of basil leaves, 0.4-3 parts by weight of pandan leaves and 0.4-3 parts by weight of Longjing tea leaves are added to a solvent and heated, and then the liquid is filtered out to obtain the plant extract.

[0013] Furthermore, the compound bacterial agent is cultured at a temperature of 35-38℃ for 2-4 days, and then inoculated into the plant stock solution.

[0014] Furthermore, the compound microbial agent is inoculated into the plant stock solution at 0.1-0.3 wt% for fermentation.

[0015] Furthermore, the fermentation temperature is 28-38℃.

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

[0017] 1. The compound bacterial agent obtained by the present invention through the selection and compounding of strains has good stability and can promote the fermentation of various plant extracts, thereby greatly enhancing the effective components and having significant market value.

[0018] 2. The plant fermentation liquid of the present invention has a high content of aloe-emodin and syringin, which have anti-inflammatory, antioxidant and immunomodulatory effects, and can promote cell growth and enhance cell vitality.

[0019] 3. The plant fermentation liquid of the present invention, through static fermentation with compound microbial agents, not only increases the content of active substances with anti-inflammatory and immune-enhancing properties in the plant original liquid, but also promotes the growth of NRK-52E cells, providing a practical solution and method for improving the effective components and enhancing their effects. Attached Figure Description

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

[0021] The *Wickerhamomyces anomalus* involved in this protocol was deposited on May 9, 2025, with accession number CGMCC No. 34474.

[0022] The *Weizmannia coagulans* strain involved in this protocol was deposited on April 25, 2025, with accession number CGMCC No. 34338.

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

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

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

[0026] Figure 3 The colony morphology of the abnormal Wickham yeast strain;

[0027] Figure 4 Microscopic morphology of abnormal Wickham yeast strain;

[0028] Figure 5 The colony morphology of *Weizmannii* strains;

[0029] Figure 6 The microscopic morphology of the *Weizmannii* strain. 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 -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. 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, *Wickham's yeast* HDS3-24, and *Weizmann's coagulans* N2-La1 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 abnormal Wickham yeast are as follows. Figures 3-4 As shown; the colony morphology and microscopic morphology of *Weizmannii* coagulation are as follows: Figures 5-6 As shown.

[0037] Example 2:

[0038] 1) Preparation of compound microbial agents:

[0039] Lactobacillus plantarum, Wickham's yeast, and Weizmann's coagulans (OD600nm adjusted to 1.0, bacterial count approximately 10^8) were mixed at a live bacterial count ratio of 1.8:2.2:1.8 and cultured at 37℃ for 3 days to obtain a composite bacterial solution.

[0040] Among them, *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; *Wickham's anomalous yeast* was deposited on May 9, 2025, with accession number CGMCC No. 34474; and *Weizmann's coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338. 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 *Wickhamia lanceolata* is shown in SEQ ID NO.2; and the 16S rDNA of *Weizmannia coagulans* is shown in SEQ ID NO.3.

[0042] 2) Preparation of plant extract: Add 1 part by weight of mulberry leaves, 1 part of lemongrass, 1 part of ginger, 1.5 parts of lemon leaves, 1.5 parts of mint leaves, 1.5 parts of basil leaves, 2 parts of pandan leaves and 2 parts of Longjing tea leaves to purified water, with a solid-liquid ratio of 5-20g:1L, heat to 100℃ for 30 minutes, then filter out the liquid to obtain the plant extract;

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

[0044] Comparative Example 1:

[0045] The compound microbial agent was prepared using commonly used commercial strains such as *Lactobacillus plantarum*, *Wickham's yeast*, and *Weizmann's coagulation*. Other steps were the same as in Example 2 to prepare the plant fermentation broth.

[0046] Detection:

[0047] 1. Content detection:

[0048] The plant fermentation broths 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 plant fermentation broths of Example 2 and Comparative Example 1 revealed that the syringin content in the plant fermentation broth of Example 2 was 31.47 times higher than that in the original plant extract, while in Comparative Example 1, which used a commercially available compound strain, the syringin content was reduced to 0.78 times compared to the original extract. Furthermore, the aloesin content in the plant fermentation broth of Example 2 was 172.69 times higher than that in Comparative Example 1, which was 2.09 times higher, indicating a significant increase in both syringin and aloesin levels in Example 2.

[0053] Syringin is an important precursor in the synthesis of lignin in plants and has been extensively studied due to its various biological activities in the human body. For example:

[0054] Antioxidant activity;

[0055] Anti-inflammatory activity: It reduces the production of pro-inflammatory factors (such as TNF-α and IL-6) by inhibiting inflammatory pathways such as NF-κB;

[0056] Immune regulation: It has a bidirectional regulatory effect, which can both enhance the phagocytic function of macrophages and inhibit excessive immune responses;

[0057] Neuroprotective effects: the potential to protect neurons and improve memory and cognitive function;

[0058] Adaptogenic effects: Similar to ginsenosides, it can help the body resist fatigue and stress, and has a certain anti-fatigue effect.

[0059] Aloe-emodin is broken down by bacterial enzymes in the intestines and converted into aloe-emodin. Aloe-emodin can stimulate intestinal neurons in the large intestine wall, promote intestinal peristalsis, and has a certain inhibitory effect on various bacteria (such as Staphylococcus aureus and Streptococcus) and fungi (such as Candida albicans).

[0060] Aloe vera extract can inhibit the production of some key inflammatory factors (such as TNF-α, IL-1β, IL-6) and affect inflammatory pathways such as COX-2, thereby exhibiting anti-inflammatory activity.

[0061] As an anthraquinone compound, aloe vera extract can scavenge free radicals, such as superoxide anions and hydroxyl radicals, and has a certain antioxidant capacity, which helps to reduce the damage of oxidative stress to cells.

[0062] Therefore, Example 2, through the fermentation treatment of plant extracts, significantly increased the concentration of active ingredients, resulting in higher anti-inflammatory, antioxidant, and immunomodulatory effects. The fermented plant extract can be further formulated and taken orally to improve internal bodily functions.

[0063] 2. Cellular experimental tests:

[0064] 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.

[0065] Preparation of NRK-52E cell culture medium: DMEM high glucose medium + 10-15% FBS (fetal bovine serum) + 1% P / S.

[0066] 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 plant stock solution, the plant fermentation broth of Example 2, and the plant 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.

[0067] Cell viability in 96-well cell cultures was measured using a CCK-8 assay kit to determine the effects of the plant stock solution, the plant fermentation broth from Example 2, and the plant fermentation broth from Comparative Example 1 on the cells. The results are shown in Table 2.

[0068] Table 2 Cell Viability Values

[0069]

[0070] The average cell viability of NRK-52E (rat kidney cells) cultured in the plant fermentation broth of Example 2 was 124.34%, while the cell viability of NRK-52E cells cultured in the plant fermentation broth of Comparative Example 1 was 81.51%, and the cell viability of cells cultured in the plant stock solution was 102.50%. Only the plant fermentation broth of Example 2 could promote the growth of rat kidney cells, indicating that the plant 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. Example 2 combines various plant raw materials in a certain proportion and ferments them statically with a compound microbial agent. This not only increases the content of active substances with anti-inflammatory and immune-enhancing properties in the plant stock solution, but also promotes the growth of NRK-52E cells, providing a practical solution and method for improving the effective components and enhancing their effects.

[0071] 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 compound microbial agent, characterized in that, The compound microbial agent is composed of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ), abnormal Wickham yeast ( Wickerhamomyces anomalus ) and Weizmann's coagulans ( Weizmannia coagulans The composition includes: *Lactobacillus plantarum*, deposited on April 25, 2025, with accession number CGMCC No. 34339; *Wickham's anomalous yeast*, deposited on May 9, 2025, with accession number CGMCC No. 34474; and *Weizmann's coagulans*, deposited on April 25, 2025, with accession number CGMCC No. 34338. All strains are deposited at the China General Microbiological Culture Collection Center. The ratio of viable Lactobacillus plantarum, Wickham's yeast anomala, and Weizmann's coagulans was 1.8:2.2:1.

8.

2. The compound microbial agent as described in claim 1, characterized in that, The 16S rDNA of *Lactobacillus plantarum* is shown in SEQ ID NO. 1; the 16S rDNA of *Wickham's abnormal yeast* is shown in SEQ ID NO. 2; and the 16S rDNA of *Weizmann's coagulation* is shown in SEQ ID NO.

3.

3. A plant fermentation liquid, characterized in that, The plant fermentation broth is obtained by inoculating a compound microbial agent into a plant stock solution and then fermenting it. The compound microbial agent consists of *Lactobacillus plantarum*, *Wickham's yeast*, and *Weizmann's coagulans*. The *Lactobacillus plantarum* was deposited on April 25, 2025, with accession number CGMCC No. 34339; the *Wickham's yeast* was deposited on May 9, 2025, with accession number CGMCC No. 34474; and the *Weizmann's coagulans* was deposited on April 25, 2025, with accession number CGMCC No. 34338. All strains are deposited at the China General Microbiological Culture Collection Center. The viable cell ratio of *Lactobacillus plantarum*, *Wickham's yeast*, and *Weizmann's coagulans* was 1.8:2.2:1.

8. The raw materials for preparing the plant extract consist of: mulberry leaves, lemongrass, ginger, lemon leaves, peppermint leaves, basil leaves, pandan leaves, and Longjing tea leaves.

4. The plant fermentation liquid as described in claim 3, characterized in that, The plant extract was prepared by the following method: 0.4-2 parts by weight of mulberry leaves, 0.4-2 parts by weight of lemongrass, 0.4-2 parts by weight of ginger, 0.5-3 parts by weight of lemon leaves, 0.5-3 parts by weight of peppermint leaves, 0.5-3 parts by weight of basil leaves, 0.4-3 parts by weight of pandan leaves and 0.4-3 parts by weight of Longjing tea leaves were added to a solvent and heated. The liquid was then filtered out to obtain the plant extract.

5. The plant fermentation liquid as described in claim 3, characterized in that, The compound bacterial agent is cultured at a temperature of 35-38℃ for 2-4 days, and then inoculated into the plant stock solution.

6. The plant fermentation liquid as described in claim 3, characterized in that, The compound microbial agent is inoculated into the plant stock solution at 0.1-0.3 wt% for fermentation.

7. The plant fermentation liquid as described in claim 3, characterized in that, The fermentation temperature is 28-38℃.

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