Engineering bacterium Lactobacillus plantarum JY002 for fermenting traditional Chinese medicinal materials

Abstract

The invention discloses an engineering bacterium Lactobacillus plantarum JY002 for fermenting traditional Chinese medicinal materials, the preservation number of the engineering bacterium is CCTCC (China Center For Type Culture Collection) NO: M 2025585, and the preservation date is March 25, 2025. Compared with the existing commercial strain lactobacillus plantarum, the preserved strain has ligninase activity, pectinase activity and alcohol resistance. The saccharomyces cerevisiae NJ01 has better ligninase activity and wine resistance than commercial strain saccharomyces cerevisiae NJ01, can be used as a traditional Chinese medicinal material fermentation engineering bacterium, especially can maintain high ligninase and pectinase activity under the yellow wine processing condition, and can be used for traditional Chinese medicinal material fermentation or wine-processed traditional Chinese medicinal material fermentation.

Description

Technical Field

[0001] This invention belongs to the field of preservation microorganisms and fermentation engineering technology, and more specifically, relates to a fermentation engineered bacterium for traditional Chinese medicine, Lactobacillus plantarum JY002. Background Technology

[0002] Processing of traditional Chinese medicine is a key technique for enhancing efficacy, reducing toxicity, and altering medicinal properties. Among these processes... Fermentation is one of the traditional methods of processing Chinese medicine. Originally, microbial fermentation was used to brew wine, and later, the resulting wine and microbial fermentation were applied to the processing of Chinese medicine. In recent years, the targeted fermentation of Chinese medicinal materials by inoculating them with exogenous probiotics has become a research hotspot in modern Chinese medicine processing. However, in this process, the structure of the cell walls of Chinese medicinal materials, especially the dense network composed of cellulose, hemicellulose, and lignin, is the main physical barrier restricting the release of active ingredients from within the cells. Lignin, as a complex aromatic polymer, is one of the most difficult components to degrade in plant cell walls. Therefore, microorganisms capable of efficiently degrading lignin have a unique advantage in disrupting the cell structure of medicinal materials and promoting the dissolution of active ingredients.

[0003] In existing technologies, some studies have attempted to use known lignin-degrading bacteria, such as white-rot fungi (e.g., Phanerochaetechrysosporium), for the pretreatment of traditional Chinese medicine fermentation, achieving some success. However, most known lignin-degrading bacteria (such as white-rot fungi) are wild-type filamentous fungi, with long fermentation cycles, difficult-to-control conditions, and potential safety hazards related to toxin production, failing to meet the requirements of "safety, controllability, and efficiency" for production strains in traditional Chinese medicine processing. While existing probiotics (such as lactic acid bacteria and yeast) are highly safe, they generally lack the ability to efficiently consume and degrade lignin, resulting in poor fermentation effects of existing probiotics on traditional Chinese medicines with high lignification levels, such as roots, stems, and bark.

[0004] To facilitate the dissolution of active ingredients in traditional Chinese medicinal materials, processing techniques involving alcohol (such as alcohol-roasting, alcohol-stewing, and alcohol-steaming) can be employed. These processing methods typically require the use of rice wine (huangjiu), but the ethanol (usually 10%-15% vol) in huangjiu strongly inhibits the growth and metabolic activity of most microorganisms. This causes a sharp decline or even inactivation of the enzyme activity of the vast majority of lignin-degrading bacteria, including some fungi, under huangjiu processing conditions, making them ineffective. Currently, there are few reported microbial strains that can simultaneously tolerate huangjiu and maintain high levels of lignin-degrading activity. This challenge makes it difficult to combine highly efficient lignin-degrading bacteria with traditional Chinese medicine processing techniques, limiting the development of high-quality fermented huangjiu products. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a fermentation engineered strain of *Lactobacillus plantarum* JY002. The purpose is to isolate and screen a novel *Lactobacillus plantarum* strain possessing ligninase, pectinase, and alcohol resistance from the intestines of healthy humans. This strain was deposited on March 25, 2025, with the accession number CCTCCNO:M2025585. This solves the technical problem that existing commercial strains of *Lactobacillus plantarum* lack ligninase activity, making them unsuitable as fermentation engineered strains for highly lignified medicinal materials such as roots, stems, and bark.

[0006] To achieve the above objectives, according to one aspect of the present invention, a fermentation engineered microorganism for traditional Chinese medicine is provided, with accession number CCTCCNO:M2025585, accession date March 25, 2025, and classification name LactobacillusplantarumJY002.

[0007] Preferably, the 16S rDNA sequence of the fermentation engineered bacteria for the Chinese medicinal materials is shown in SEQ ID NO: 1.

[0008] According to another aspect of the present invention, the application of the fermentation engineered bacteria of Chinese medicinal materials as described in the present invention in the fermentation of Chinese medicinal materials or the fermentation of Chinese medicinal materials in wine is also provided. Lactobacillus plantarum JY002 is used as the fermentation engineered bacteria of Chinese medicinal materials to degrade lignin and promote the dissolution of active ingredients of Chinese medicinal materials.

[0009] Preferably, in the application, the medicinal materials include one or more of the following: Cistanche deserticola, radish seed, peach kernel, astragalus, codonopsis, and tangerine peel.

[0010] Preferably, in the application, the active ingredients include polysaccharides, polyphenols, and flavonoids.

[0011] In summary, compared with the prior art, the technical solutions conceived in this invention, by screening and obtaining a new strain of *Lactobacillus plantarum* with ligninase activity, can achieve the following beneficial effects: The fermentation strain of traditional Chinese medicine provided by this invention is a preserved strain, with accession number CCTCCNO:M2025585, preservation date March 25, 2025, and classification name Lactobacillus plantarum JY002. Compared with the existing commercial strain Lactobacillus plantarum (which does not have ligninase activity), this preserved strain has ligninase activity, as well as pectinase activity and alcohol resistance, exhibiting excellent lignin decomposition ability and thus can be used as a fermentation strain for traditional Chinese medicine. When traditional Chinese medicine is fermented using this preserved strain, the amount of active ingredients dissolved increases significantly, especially the amount of polyphenols and flavonoids dissolved, making it suitable for fermentation of traditional Chinese medicine or fermentation of traditional Chinese medicine for alcoholic beverages. Attached Figure Description

[0012] Figure 1 The Congo red staining method is used to determine pectinase activity.

[0013] Figure 2 This study investigates the effect of fermentation of this preserved strain on the dissolution of active ingredients in traditional Chinese medicine. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0015] Most existing probiotics, such as commercial strains of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, and Bacillus coagulans, do not have ligninase activity. However, Chinese medicinal materials are mostly roots, stems, and bark, which have a high degree of lignification. Lignin is the most difficult component to degrade in plant cell walls. Since current probiotic fermentation and processing methods cannot effectively degrade lignin, the dissolution rate of active ingredients in Chinese medicinal materials remains low.

[0016] To address the high degree of lignification in traditional Chinese medicinal materials, this invention isolates and screens strains with ligninase activity from fecal samples of healthy individuals. Unexpectedly, a new strain of *Lactobacillus plantarum* with both ligninase and pectinase activities was discovered. This strain also exhibits good alcohol tolerance and can be used as an engineered microbial culture for fermenting traditional Chinese medicine or for fermenting medicinal materials in alcoholic beverages. The isolated and purified *Lactobacillus plantarum* strain was deposited on March 25, 2025, at the China Center for Type Culture Collection (Wuhan University Collection Center), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Its accession number is CCTCCNO:M2025585, the deposit date is March 25, 2025, and its classification name is *Lactobacillus plantarum* JY002.

[0017] Based on this, the present invention provides a fermentation engineered microorganism for Chinese medicinal materials, with the preservation number CCTCCNO:M2025585, the preservation date being March 25, 2025, and the classification name being LactobacillusplantarumJY002.

[0018] The 16S rDNA sequence of Lactobacillusplantarum JY002 is shown in SEQ ID NO: 1.

[0019] The screening was conducted using a lignin-based screening medium with lignin as the sole carbon source. The results showed that commercial strains of Lactobacillus plantarum could not grow on the lignin-based screening medium, while Lactobacillus plantarum JY002, screened by this invention, could grow well on the lignin-based screening medium.

[0020] Furthermore, the activities of ligninase and pectinase in different strains were compared using the plate hydrolysis circle method. The results showed that compared with the commercial strain Saccharomyces cerevisiae NJ01, the preserved strain had higher ligninase activity and also had pectinase activity, which is more conducive to degrading plant cell walls and thus promoting the dissolution of active ingredients in Chinese medicinal materials. It can be used as an engineered strain for fermentation of Chinese medicinal materials.

[0021] Furthermore, this invention also discovered that the preserved strain possesses alcohol tolerance. Compared to the commercial strain *Saccharomyces cerevisiae* NJ01, this preserved strain exhibits superior alcohol tolerance and can be used as an engineered fermentation microorganism for alcohol-based traditional Chinese medicine. The alcohol-based traditional Chinese medicine is obtained by adding rice wine during the processing for steaming, soaking, or roasting.

[0022] The present invention also provides an application of the fermentation engineered bacteria for Chinese medicinal materials as described in the present invention in the fermentation of Chinese medicinal materials, using Lactobacillus plantarum JY002 as the fermentation engineered bacteria for degrading lignin and promoting the dissolution of active ingredients in Chinese medicinal materials.

[0023] The medicinal materials include one or more of the following: Cistanche deserticola, radish seed, peach kernel, astragalus, codonopsis, and tangerine peel. In some embodiments, Lactobacillus plantarum JY002 is used as the fermentation engineered bacteria for the medicinal materials, which include Cistanche deserticola, radish seed, peach kernel, astragalus, codonopsis, and tangerine peel, to promote the dissolution of their active ingredients such as polysaccharides, flavonoids, and polyphenols.

[0024] The following are examples. The strains involved in this invention are derived from commercial strains or obtained through routine isolation and screening. The isolated strains are deposited with the China Center for Type Culture Collection (CCTCC). In use, all microorganisms undergo three consecutive cultures, followed by incubation in sterile MRS broth at an inoculum size of 5%. Fresh fermentation broth is obtained after 24 hours of incubation at 37°C for testing.

[0025] Example 1: Isolation, purification, and identification of strains (1) Isolation of strains Fecal samples were prepared by mixing fecal matter with physiological saline. The fecal concentration in the fecal sample solution was 5 g / ml. The fecal sample solution was then serially diluted with physiological saline to a concentration of 10 g / ml. -1 10 -2 10 -3 10 -410 -5 Diluted samples were spread onto MRS basal broth medium and placed directly in an aerobic environment (ordinary incubator) at 37°C for 3-5 days.

[0026] Single colonies were selected based on colony morphology, color, and size and streaked on three zones of MRS basic broth agar plates. Single colonies were repeatedly selected for purification until pure strains were isolated and numbered 24 and 191.

[0027] (2) Molecular biological identification The obtained single colonies were scraped and subjected to 16s identification for preservation. The morphological characteristics and molecular biological identification of the strains were combined as follows: Genomic DNA was extracted from strains 24 and 191 strictly following the steps of the bacterial DNA extraction kit, and then used as a template for polymerase chain reaction (PCR) amplification. The forward primer for the amplification reaction was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0028] PCR reaction system: 25 μL of 2×EcoTaqPCRSuper-Mix, 1 μL each of forward and reverse primers, 1 μL of genomic DNA, and ddH2O to make up to 50 μL. PCR reaction program: 95℃ for 10 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for a total of 30 cycles; final extension at 72℃ for 10 min.

[0029] The PCR products were directly sequenced by Qingke Biotechnology Co., Ltd., and the gene sequences were compared with BLAST in NCBI. The gene sequences of the strains with the highest homology were then identified in GenBank to further confirm the isolated strains.

[0030] The 16S rDNA sequence of strain 191, obtained through laboratory isolation, purification, and identification, is shown in SEQ ID NO: 1. The 16S rDNA sequence was compared using BLAST on NCBI, and the results showed that the 16S rDNA sequence of strain 191 had a 99.89% similarity to the sequence of *Lactobacillus plantarum* (OR502092.1), confirming strain 191 as a novel strain of *Lactobacillus plantarum*.

[0031] The 16S rDNA sequence of strain number 24 was compared with that of Lactobacillus plantarum (OR481916.1) using BLAST on NCBI. The results showed that the 16S rDNA sequence of strain number 24 was 100% similar to that of Lactobacillus plantarum (OR481916.1).

[0032] Example 2: Screening of probiotics with ligninase activity The strains numbered 24 and 191, isolated and purified in Example 1, were compared with commercial strains to screen for probiotics that possess both ligninase activity and alcohol tolerance, as detailed below: (1) Screening strains with lignin degradation capabilities Prepare a lignin screening medium with lignin as the sole carbon source: 4g lignin, 0.266g ammonium nitrate, 0.1g magnesium sulfate, 2g potassium dihydrogen phosphate, 0.04g disodium hydrogen phosphate, 4g agar, 200mL distilled water, pH 7.5-8.0.

[0033] Strain activation: The strains to be screened were removed from the -80°C freezer. Before use, all microorganisms were activated by three consecutive cultures, and then cultured in sterile MRS broth at an inoculum of 5% for 24 hours at 37°C.

[0034] ① Initial screening of ligninase: Use a disposable inoculation loop to dip the activated bacterial solution of the strain, streak it on the lignin screening medium plate, place it in a constant temperature incubator, set the temperature to 37℃ and incubate for 1-7 days. Observe whether colonies grow on each plate every 24 hours. The results are shown in Table 1.

[0035] Table 1. Results of initial screening for ligninase

[0036] In the table, "+" indicates "colony growth" and "-" indicates "no colony growth".

[0037] As shown in Table 1, none of the existing commercial strains of *Lactobacillus plantarum* can grow on a medium with lignin as the sole carbon source. However, a new strain of *Lactobacillus plantarum*, numbered 191, was unexpectedly found to grow on a medium with lignin as the sole carbon source. It is speculated that this new strain of *Lactobacillus plantarum* has ligninase activity and can degrade recalcitrant lignin into a usable carbon source.

[0038] ② Ligninase rescreening: Prepare aniline blue agar medium (aniline blue 0.2g, potassium dihydrogen phosphate 6g, magnesium sulfate 3g, agar 40g, distilled water 2000mL, pH 7.5-8.0), and use the aniline blue plate decolorization test.

[0039] Commercial strain Saccharomyces cerevisiae NJ1 and screened and purified strain 191 were respectively inoculated into aniline blue agar medium plates and incubated in a constant temperature incubator at 30℃ for 3 days. The diameter of the decolorization zone was measured and the results are shown in Table 2.

[0040] Manganese chloride agar medium (MnCl2·4H2O 20.3g, potassium dihydrogen phosphate 6g, magnesium sulfate 3g, agar 40g, distilled water 2000mL, pH 7.5-8.0) was prepared, and the colorimetric assay was performed using manganese chloride plates. Commercial strain *Saccharomyces cerevisiae* NJ1 and the screened and purified strain 191 were respectively inoculated into manganese chloride agar plates and incubated at 30℃ for 3 days. The diameter of the colorimetric zone was then measured, and the results are shown in Table 2.

[0041] Table 2 Results of the secondary screening of probiotics that degrade lignin

[0042] As shown in Table 2, compared with the commercial strain Saccharomyces cerevisiae NJ1, the selected Lactobacillus plantarum strain numbered 191 has higher ligninase activity and better ability to degrade lignin.

[0043] (2) Comparison of pectinase activity of different strains A pectin selection medium (K₂HPO₄: 1g, MgSO₄: 0.5g, NaNO₃: 3g, FeSO₄·7H₂O: 0.01g, pectin 2g, agar 15g, distilled water 1000mL, pH 5.5) was prepared with pectin as the sole carbon source. Using the Congo red staining method, commercial strain *Saccharomyces cerevisiae* NJ1 and the screened and purified strain 191 were separately inoculated into petri dishes of the pectin selection medium and incubated at 37℃ for 3 days. The colony diameters were then measured. Next, 1mg / mL Congo red staining solution was added to the petri dishes, and staining was performed for approximately 40 minutes. The Congo red staining solution was discarded, and a thin layer of Congo red on the surface was rinsed off with a small amount of distilled water. Distilled water was added again for destaining for 10 minutes, and the presence of a clear hydrolysis zone was observed under light. The results are as follows: Figure 1 As shown.

[0044] Depend on Figure 1 The results showed that the commercial strain *Saccharomyces cerevisiae* NJ1 did not exhibit a hydrolysis zone, while *Lactobacillus plantarum* strain number 191 showed a distinct hydrolysis zone (3.79 cm in diameter). This indicates that *Lactobacillus plantarum* strain number 191 possesses the activities of both ligninase and pectinase, two plant degrading enzymes, and has a better ability to decompose plant cell walls. This is more conducive to promoting the release and dissolution of active ingredients in the cells of Chinese medicinal materials. It can be used as an engineered fermentation strain for Chinese medicinal materials or fermented Chinese medicinal materials to promote the release and dissolution of active ingredients in the cells of Chinese medicinal materials.

[0045] (3) Compare the alcohol tolerance of different strains Strain activation: Commercial strains and screened purified strains were removed from the -80°C freezer. Before use, all microorganisms underwent three consecutive incubations for activation, followed by incubation in sterile MRS broth at 5% inoculum for 24 hours at 37°C.

[0046] To compare the alcohol tolerance of different strains: the activated strains were centrifuged to remove the supernatant, and the cells were washed with buffer and then an equal amount of rice wine (alcohol content: 10-15%) was added. The mixture was mixed and allowed to stand for 3 hours. The activity of the strains was detected by plate counting method. The results are shown in Table 3.

[0047] Table 3 Comparison of alcohol tolerance among different strains

[0048] As shown in Table 3, neither the commercial Lactobacillus plantarum P01 nor the screened Lactobacillus plantarum 24 can tolerate alcohol. After treatment with rice wine, the viable cell counts of commercial Lactobacillus plantarum LP90 and Saccharomyces cerevisiae NJ01 decreased significantly, indicating that the activity of the strains was inhibited. However, after treatment with rice wine, the viable cell count of the newly screened Lactobacillus plantarum strain 191 did not change, indicating that the activity of the strain was not inhibited. This suggests that Lactobacillus plantarum strain 191 has good alcohol tolerance and can tolerate 10-15% alcohol.

[0049] Based on the above experimental results, compared with the existing commercial strain Saccharomyces cerevisiae NJ01, the new strain Lactobacillus plantarum, numbered 191, has both ligninase and pectinase activities, and its ligninase activity and alcohol tolerance are better. It can be used as an engineered fermentation strain for Chinese medicinal materials or Chinese medicinal materials for wine production.

[0050] The newly isolated strain of *Lactobacillus plantarum*, numbered 191, was deposited on March 25, 2025, at the China Center for Type Culture Collection (Wuhan University Collection Center), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Its accession number is CCTCCNO:M2025585, the deposit date is March 25, 2025, and its classification name is *Lactobacillus plantarum* JY002.

[0051] Example 3 verifies that Lactobacillus plantarum JY002 can be used as an engineered microorganism for fermentation of traditional Chinese medicine. Culture medium for traditional Chinese medicine formula: 30g of Cistanche deserticola, 30g of radish seed, 15g of peach kernel, 20g of Astragalus membranaceus, 20g of Codonopsis pilosula, and 10g of dried tangerine peel. Mix and grind into powder, weigh out 5g, add 55ml of pure water, sterilize at 121℃ for 20min, and then cool for later use.

[0052] (1) Activation of strain: The preserved strain Lactobacillusplantarum JY002 was taken out at -80℃. When using it, it was inoculated into MRS broth medium at a 5% inoculation rate and placed at 37℃ for 24 hours. The inoculation was repeated three times. Then it was cultured in sterile MRS broth at a 5% inoculation rate for 24 hours at 37℃.

[0053] (2) Fermentation of traditional Chinese medicine: The activated strain LactobacillusplantarumJY002 was inoculated into the culture medium of traditional Chinese medicine formula at an inoculation amount of 6% and cultured at 37℃ for 24 hours to obtain the fermented product of traditional Chinese medicine.

[0054] (3) Detect the number of viable Lactobacillus plantarum JY002 cells before and after fermentation. Take out the fermented Chinese medicine obtained in step (2), weigh out 0.15g, add 2 steel balls and 1.5ml of PBS buffer solution, place in a grinder, grind evenly at 70HZ for 60s, and perform serial dilution (e.g., 10). -1 10 -2 10 -3 …), take 0.1 ml of each dilution sample and spread it evenly on the surface of the MRS solid culture medium. Invert the culture dish and incubate it in a 37℃ constant temperature incubator for 48 h. Remove the culture dish, observe and record the colony count at each dilution. Repeat three times. The results are shown in Table 4.

[0055] Table 4. Results of viable cell counts of *Lactobacillus plantarum* JY002 before and after fermentation.

[0056] As shown in Table 4, the number of viable LactobacillusplantarumJY002 cells increased after fermentation compared to the initial inoculum amount, confirming that the preserved strain LactobacillusplantarumJY002 can grow well in the culture medium of traditional Chinese medicine formulas and can be used as an engineered microorganism for fermentation of traditional Chinese medicine materials.

[0057] (4) Detection of dissolution of active ingredients The fermented herbal product (liquid mixture containing herbal powder) obtained in step (2) was taken out, centrifuged at 5000 rpm for 6 min, and the supernatant was collected. The contents of polysaccharides, flavonoids, and polyphenols in the supernatant were detected. This was repeated three times, with a herbal formula without any probiotic fermentation as a control. The effect of the preserved strain Lactobacillus plantarum JY002 as the fermentation engineered bacteria on the dissolution of active ingredients in herbal materials was studied. The results are as follows: Figure 2 .

[0058] Depend on Figure 2The results showed that, compared with traditional Chinese medicine formulas without any probiotic fermentation, the dissolution of active ingredients such as flavonoids, polysaccharides, and polyphenols was significantly increased after fermentation with the preserved strain LactobacillusplantarumJY002. In particular, the dissolution of flavonoids and polyphenols increased significantly. This confirms that the preserved strain LactobacillusplantarumJY002 can be used as a fermentation engineer for traditional Chinese medicine to promote the dissolution of active ingredients such as flavonoids, polysaccharides, and polyphenols in traditional Chinese medicine.

[0059] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation engineered microbial culture for traditional Chinese medicinal materials, characterized in that, Its accession number is CCTCCNO:M2025585, the accession date is March 25, 2025, and the classification name is LactobacillusplantarumJY002.

2. The engineered microbial culture for fermenting Chinese medicinal materials as described in claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The application of the engineered microorganism for fermenting Chinese medicinal materials as described in claim 1 in the fermentation of Chinese medicinal materials or the fermentation of Chinese medicinal materials for winemaking, characterized in that, Lactobacillus plantarum JY002 was used as a fermentation engineer for traditional Chinese medicine to degrade lignin and promote the dissolution of active ingredients in traditional Chinese medicine.

4. The application as described in claim 3, characterized in that, The medicinal materials mentioned include one or more of the following: Cistanche deserticola, radish seed, peach kernel, astragalus, codonopsis, and tangerine peel.

5. The application as described in claim 4, characterized in that, The active ingredients include polysaccharides, polyphenols, and flavonoids.

Images