A strain of Lactiplantibacillus argentoratensis SSR and its application in fermented Chinese medicinal residue.

Abstract

The present invention discloses a Lactiplantibacillus argentoratensis SSR strain and its application. This strain has been deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC M 20252330. The strain of the present invention is isolated from the medicinal residues of Lizhong Powder, and it is Gram-positive, non-hemolytic, and has good growth and acid production capabilities. The core of the present invention is that by fermenting the medicinal residues of Lizhong Powder, Sijunzi Powder and Huanglian Jiedu Powder with the Lactiplantibacillus argentoratensis SSR strain, the contents of active substances such as polyphenols, flavonoids and proteins in the fermentation products of the medicinal residues can be significantly increased, the antioxidant activity of the fermented medicinal residues is significantly improved, and it has good prebiotic activity. It provides an effective microbial transformation path for the resource utilization of traditional Chinese medicine medicinal residues with high value, and has broad application prospects in the field of functional feed additives.

Description

Technical Field

[0001] The present invention relates to the fields of microbial technology and utilization of traditional Chinese medicine resources. Specifically, it relates to a new Lactiplantibacillus argentoratensis SSR strain screened from the medicinal residues of Lizhong Powder, and its application in fermenting the medicinal residues of Lizhong Powder, Sijunzi Powder, and Huanglian Jiedu Powder to increase the content of their active substances. Background Art

[0002] Lizhong Powder, Sijunzi Powder, Huanglian Jiedu Powder, etc. are classic traditional Chinese medicine compound prescriptions, which are widely used in the clinical and health product fields. During the production process of traditional Chinese medicine preparations, a large amount of medicinal residues are generated. These medicinal residues are usually treated as waste, which not only causes resource waste but also may bring environmental pressure. However, the medicinal residues still contain abundant utilizable active ingredients such as polysaccharides, proteins, polyphenols, and flavonoids.

[0003] Microbial fermentation technology is one of the effective means to improve and enhance the value of traditional Chinese medicine. Probiotic fermentation, due to its safe and mild characteristics, is widely used in the food and health product industries. Probiotic fermentation can decompose plant cell walls, release the encapsulated active substances, and at the same time produce new beneficial components such as organic acids and bacteriocins through its metabolic activities, thereby enhancing the biological activity and utilization rate of raw materials.

[0004] At present, although there are studies on using probiotics to ferment traditional Chinese medicine, there is still a blank in screening exclusive probiotic strains with high-efficiency and broad-spectrum conversion ability for specific types of traditional Chinese medicine compound prescriptions (such as invigorating the spleen and benefiting qi type and clearing heat and removing dampness type) of medicinal residues, and systematically studying the changes in active substances before and after fermenting different medicinal residues. The purpose of the present invention is to fill this blank and provide a special probiotic strain and method that have a significant improvement effect on various traditional Chinese medicine medicinal residues. Summary of the Invention

[0005] The purpose of the present invention is to provide a new Lactiplantibacillus argentoratensis SSR strain independently screened from the medicinal residues of Lizhong Powder. This strain has good acid-producing ability and safety, can effectively ferment various traditional Chinese medicine medicinal residues, especially the medicinal residues of prescriptions for invigorating the spleen and benefiting qi (such as Lizhong Powder, Sijunzi Powder) and clearing heat and removing dampness (such as Huanglian Jiedu Powder), significantly increase the content of active substances such as polyphenols, flavonoids, and proteins in the medicinal residues, enhance the antioxidant activity of the fermentation products and have prebiotic activity, providing a highly applicable solution for the high-value utilization of traditional Chinese medicine medicinal residues.

[0006] The present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a strain of Lactiplantibacillus argentoratensis SSR, which was deposited at the China Center for Type Culture Collection (CCTCC) on October 24, 2025, with accession number CCTCC M20252330.

[0008] Secondly, the present invention provides the application of the Lactiplantibacillus argentoratensis SSR strain in fermented Chinese medicine residue, wherein the Chinese medicine residue is preferably the residue of Chinese medicine compound formulas that invigorate the spleen and replenish qi and clear heat and remove dampness.

[0009] Thirdly, the present invention provides a method for increasing the content of active substances in traditional Chinese medicine residue, comprising the following steps:

[0010] 1. Dry, pulverize, and sieve the Chinese medicine residue (such as the residue of Lizhong San, Sijunzi San, or Huanglian Jiedu San), add water to prepare a certain concentration of residue culture medium, and then sterilize it.

[0011] 2. The Lactiplantibacillus argentoratensis SSR strain described in claim 1 is inoculated into MRS liquid medium for activation to prepare a seed culture.

[0012] 3. Inoculate the seed culture from step 2 into the sterilized drug residue culture medium from step 1 at a certain inoculation amount, and carry out fermentation at a suitable temperature.

[0013] 4. After fermentation, the fermentation products are post-processed to obtain fermented Chinese medicine residue rich in active substances.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. Novel strain and unique origin: The Lactiplantibacillus argentoratensis SSR strain provided by this invention was directly screened from the special environment of medicinal residue in Lizhongsan, and has good adaptability to the medicinal residue environment.

[0016] 2. Broad application potential: This strain not only has a good effect on the residue of Lizhongsan, but also significantly increases the content of active substances in the residue of other types of traditional Chinese medicine such as Sijunzisan and Huanglian Jiedusan, showing broad application potential.

[0017] 3. High-efficiency conversion: The fermentation of this strain can significantly increase the content of polyphenols, flavonoids and proteins in the residues of different Chinese medicinal herbs, realizing the value-added utilization of the residues.

[0018] 4. High safety: This strain was confirmed as Gram-⁺ bacteria by Gram staining, and the Columbia blood plate test showed that it has no β-hemolytic activity, which meets the general requirements for food safety strains.

[0019] 5. Broad application prospects: The content of active ingredients in fermented Chinese herbal medicine residue is greatly increased, the antioxidant activity is significantly improved, and it also has prebiotic activity. It can be used as a functional feed additive, organic fertilizer, or further extracted for use in health products and other fields, turning waste into treasure, which is economical and environmentally friendly. Attached Figure Description

[0020] Figure 1 Phylogenetic tree (constructed based on 16S rRNA gene sequence) and Gram-stained microscopic images of the Lactiplantibacillus argentoratensis SSR strain of this invention.

[0021] Figure 2 This invention presents the growth curve and acid production curve (pH change curve) of the Lactiplantibacillus argentoratensis SSR strain.

[0022] Figure 3 The results of the hemolytic activity test of the Lactiplantibacillus argentoratensis SSR strain of this invention on Columbia blood agar plates (showing no hemolytic zone).

[0023] Figure 4 Bar chart comparing the content of active substances (including polysaccharides, polyphenols, flavonoids, and proteins) in different Chinese herbal medicine residues (Lizhong San, Sijunzi San, and Huanglian Jiedu San) before and after fermentation.

[0024] Figure 5 The effects of different Chinese herbal medicine residues (Sijunzi San, Lizhong San, and Huanglian Jiedu San) on DPPH and ABTS before and after fermentation + Comparison chart of free radical scavenging effects.

[0025] Figure 6 The effects of different Chinese herbal medicine residues (Sijunzi San, Lizhong San, and Huanglian Jiedu San) on the growth-promoting effect on *Lactobacillus plantarum* and the inhibitory effect on *Escherichia coli* before and after fermentation: A. Growth-promoting effect of Sijunzi San residue fermentation products on *Lactobacillus plantarum*; B. Growth-promoting effect of Lizhong San residue fermentation products on *Lactobacillus plantarum*; C. Growth-promoting effect of Huanglian Jiedu San residue fermentation products on *Lactobacillus plantarum*; D. Growth-inhibiting effect of Sijunzi San residue fermentation products on *Escherichia coli*; E. Growth-inhibiting effect of Lizhong San residue fermentation products on *Escherichia coli*; F. Growth-inhibiting effect of Huanglian Jiedu San residue fermentation products on *Escherichia coli*. Detailed Implementation

[0026] The present invention will be described in detail below with reference to embodiments to facilitate a further understanding of the invention, but the scope of protection of the present invention is not limited thereto.

[0027] Example 1:

[0028] Strain screening and identification:

[0029] Samples were taken from the naturally fermented residue of Lizhongsan (a traditional Chinese medicine formula), and spread onto MRS agar plates using the dilution plating method. The plates were then incubated at 37°C for 48 hours. Single colonies were picked and purified. The 16S rRNA gene sequence of a rapidly growing strain (SSR) was analyzed. The 16S rRNA gene was amplified by PCR and sequenced. The sequence was compared with the EzBioCloud database, showing the highest homology (>99.5%) with *Lactiplantibacillus argentoratensis* DSM 16365. Therefore, this strain was identified as *Lactiplantibacillus argentoratensis* SSR. Figure 1 A).

[0030] Example 2:

[0031] Study on the basic biological characteristics of the strain:

[0032] 1. Growth and acid production characteristics: After activation and subculturing of strain SSR, it was inoculated into MRS liquid medium and cultured at 37℃. Its OD was measured every 2 hours. 600 nm Values ​​and pH values. Results are as follows: Figure 2 As shown, this strain has a short lag phase, grows rapidly, and has a strong acid-producing capacity; the pH can drop below 3.5 after 24 hours of fermentation. Figure 2 ).

[0033] 2. Gram staining: Gram staining was performed according to standard methods. Microscopic examination revealed short, blue-purple rod-shaped bacteria, confirming them as Gram-positive (G⁺) bacteria. Figure 1 B).

[0034] 3. Hemolytic activity test: After activation and subculturing of strain SSR, the bacteria were streaked onto Columbia blood agar plates and anaerobically incubated at 37°C for 48 h. Observation showed no clear hemolytic zone (γ-hemolysis) around the colonies, indicating that the strain is non-hemolytic and has good safety. Figure 3 ).

[0035] Example 3:

[0036] Determination of fermented Lizhongsan / Sijunzisan / Huanglian Jiedusan dregs and active substances:

[0037] 1. Preparation of medicinal residue culture medium: Take dried medicinal residue, crush it through an 80-mesh sieve, add distilled water at a material-to-liquid ratio of 1:10 (g / mL), and autoclave at 121℃ for 15 min.

[0038] 2. Fermentation: The activated SSR seed culture was inoculated into sterilized medicinal residue culture medium at a 10% inoculation rate and fermented at 37℃ for 48 h. Uninoculated sterilized medicinal residue culture medium was used as a blank control.

[0039] 3. Determination: After fermentation, the fermentation residue and a blank control were subjected to water extraction, alcohol extraction, and alkali extraction. The supernatant was collected by centrifugation, and the polysaccharide content was determined using the phenol-sulfuric acid method, the total polyphenol content using the Folin-Ciocalteu method, the total flavonoid content using the aluminum nitrate colorimetric method, and the soluble protein content using the BCA method, respectively. Moisture content determination: Clean, dry weighing bottles were numbered and weighed. An appropriate amount of sample was then placed in the weighing bottle and weighed again. The weighing bottle was then placed in an oven at 105℃ for 4 hours with the cap open, and weighed again. The two weighing data and the weight of the weighing bottle were recorded. The ratio of the difference between the two weighings to the difference between the first weighing and the weighing bottle was the moisture content by weight. Crude ash content was determined using the high-temperature ignition method; crude fat content was determined using the Soxhlet extraction method; and crude fiber content was determined using the polyester fiber filter bag method.

[0040] 4. Results: such as Figure 4 As shown in A-4C, compared with the blank control, the content of various active substances in the supernatant of the three kinds of Chinese herbal medicine residues after SSR fermentation was significantly increased (p < 0.05). As shown in Table 1, the moisture and ash content increased significantly after fermentation, the crude fiber content decreased significantly, and the crude fat content decreased slightly with no significant difference from that before fermentation.

[0041] Table 1. Comparison of basic substances in the residue of three kinds of traditional Chinese medicine before and after fermentation.

[0042] Note: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0043] Example 4:

[0044] Antioxidant activity determination of fermented residues of Lizhongsan, Sijunzisan, and Huanglian Jiedusan:

[0045] 1. Sample Preparation: Take the fermented residue from Example 3 and the corresponding unfermented blank residue, add 20 times the volume of deionized water, heat under reflux for 2 hours to obtain the extract, filter, centrifuge, collect the supernatant, and concentrate to obtain the concentrate. Freeze-dry the concentrate to obtain the lyophilized powder, dissolve it in distilled water and dilute it appropriately before use.

[0046] 2. DPPH free radical scavenging rate determination: Take 2.0 mL of the diluted sample solution, add 2.0 mL of 0.1 mmol / L DPPH-ethanol solution, mix well, and react at room temperature in the dark for 30 min. Using anhydrous ethanol as a reference, measure the absorbance value (A) at a wavelength of 517 nm. sample Simultaneously, the absorbance (A) of the sample solution mixed with anhydrous ethanol was measured. bg ), and the absorbance (A) of the DPPH solution mixed with anhydrous ethanol. control The formula for calculating the clearance rate is: Clearance rate (%) = [1 − (A)] sample - A bg ) / A control ]× 100.

[0047] 3. Determination of ABTS⁺ free radical scavenging rate: Mix an equal volume of 7.4 mmol / L ABTS solution with 2.6 mmol / L potassium persulfate solution and incubate at room temperature in the dark for 12-16 h to prepare ABTS⁺ stock solution. Before use, dilute with phosphate buffer (0.01 M, pH 7.4) to a absorbance of 0.70 ± 0.02 at 734 nm to obtain the working solution. Take 0.1 mL of sample solution, add 3.9 mL of ABTS⁺ working solution, mix well, and react at room temperature in the dark for 6 min. Immediately measure the absorbance at 734 nm (A). sample Phosphate buffer was used as a control instead of the sample (A). control The formula for calculating the clearance rate is: Clearance rate (%) = (A) control -A sample / A control × 100.

[0048] 4. Results: such as Figure 5 As shown, compared with the unfermented blank control, the DPPH and ABTS⁺ free radical scavenging rates of the extracts of Lizhongsan, Sijunzisan and Huanglian Jiedusan after SSR fermentation were significantly increased (p < 0.05), indicating that fermentation treatment effectively enhanced the antioxidant activity of the medicinal residues.

[0049] Example 5:

[0050] Prebiotic activity determination of fermented residues from Lizhongsan, Sijunzisan, and Huanglian Jiedusan:

[0051] 1. A basal culture medium was prepared by adding 5% of the freeze-dried extracts of Lizhongsan, Sijunzisan, and Huanglian Jiedusan as the sole carbon source, and a culture medium with glucose as the sole carbon source was used as a control. The effects of the fermented extracts on promoting the growth of Lactobacillus plantarum and inhibiting Escherichia coli were evaluated.

[0052] 2. Results: such as Figure 6 As shown, compared with the culture medium using glucose as the sole carbon source, the culture medium containing the fermentation extract effectively promoted the growth of *Lactobacillus plantarum* and inhibited the growth of *Escherichia coli*. This indicates that the fermentation product has good prebiotic activity.

Claims

1. A *Lactiplantibacillus argentoratensis* SSR strain, characterized in that, This strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 20252330.

2. The Lactiplantibacillus argentoratensis SSR strain as described in claim 1, characterized in that, This strain was isolated from the residue of traditional Chinese medicine.

3. A microbial preparation, characterized in that, It comprises at least one of the Lactiplantibacillus sargentoratensis SSR strains as described in claim 1 or 2, its culture or fermentation product, and a pharmaceutically, foodologically, or agriculturally acceptable carrier.

4. The application of the Lactiplantibacillus argentoratensis SSR strain as described in claim 1 or 2 in fermentation of plant raw materials.

5. The application as described in claim 4, characterized in that, The plant materials are Chinese medicine residues, including Lizhong San, Sijunzi San, and Huanglian Jiedu San, but not limited to the three compound formulas mentioned above.

6. A method for increasing the content of active substances in the residues of Lizhong San, Sijunzi San, and Huanglian Jiedu San, characterized in that, Includes the following steps: a) Provide a culture medium formed by mixing the residues of Lizhongsan, Sijunzisan, and Huanglian Jiedusan with water; b) Inoculate the culture medium with the Lactiplantibacillus argentoratensis SSR strain as described in claim 1 or 2, and carry out fermentation; c) Obtain a fermentation product; wherein, compared with the unfermented Lizhongsan residue, the fermentation product has an increased content of at least one active substance among polysaccharides, polyphenols, flavonoids, and proteins; d) Obtain a fermentation product; wherein, after fermentation, DPPH and ABTS are present in the fermentation product. + The free radical scavenging ability was significantly improved; e) Fermentation products were obtained: all of which had a growth-promoting effect on Lactobacillus plantarum and a growth-inhibiting effect on Escherichia coli.

7. The method as described in claim 6, characterized in that, In step b), the fermentation is static fermentation, the fermentation temperature is 30-37℃, and the fermentation time is 24-72 h.

8. The method as described in claim 6, characterized in that, In step c), the fermentation product is dried.

9. A composition, characterized in that, Prepared by the method described in any one of claims 6 to 8.

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