Engineering bacterium Lactobacillus plantarum JY003 for fermenting wine-processed traditional Chinese medicine decoction pieces

Abstract

The invention discloses a fermentation engineering bacterium Lactobacillus plantarum JY003 for wine-processed traditional Chinese medicine decoction pieces, the preservation number of the fermentation engineering bacterium is CCTCC (China Center For Type Culture Collection) NO: M 20252157, the preservation date is September 29, 2025, and the classification name of the fermentation engineering bacterium is Lactobacillus plantarum JY003. The preserved strain can tolerate alcoholic strength of 10-15%, has both pectinase activity and cellulase activity, can keep high cellulase activity and pectinase activity under the yellow wine processing condition, further promotes plant cell wall degradation, enables active ingredients in cells to be released and dissolved out, can be used as a wine-processed traditional Chinese medicine decoction piece fermentation engineering strain, and has a wide application prospect. The method is used for preparing wine-processed traditional Chinese medicine decoction pieces.

Description

Technical Field

[0001] This invention belongs to the field of preservation microbial fermentation technology, and more specifically, relates to an engineered microorganism Lactobacillus plantarum JY003 for fermenting medicinal herbs used in winemaking. Background Technology

[0002] Probiotics mainly include Lactobacillus, Bifidobacterium, Bacillus, and yeast, and their core functions include biotransformation, metabolic transformation, and immune regulation. With their multiple functions such as biotransformation, metabolic transformation, and immune regulation, probiotics have become a key biological tool for improving the efficiency of traditional Chinese medicine processing and promoting the release of active ingredients. For example, in the article reviewing the research progress of probiotic fermentation of traditional Chinese medicine by Academician Yin Yulong et al. (DOI:https: / / doi.org / 10.1002 / imt2.93), it was mentioned that Lactococcus can effectively degrade cellulose. Fermentation of Astragalus membranaceus with Lactococcus increased the content of crude polysaccharides, total flavonoids, and total saponins in the roots, stems, and leaves of Astragalus membranaceus. Fermentation of traditional Chinese medicines such as Vaccaria segetalis and Leonurus japonicus with probiotics such as Lactobacillus casei, Enterococcus faecalis, and Candida utilis increased the content of soluble total flavonoids, total alkaloids, crude polysaccharides, and total saponins. After fermentation by Lactobacillus pentosus, the content of quercetin and kaempferol in the extract of Broomcorn millet was increased.

[0003] However, the processing of medicinal herbs in alcohol-based preparations often involves alcohol-containing steps such as steaming, roasting, and soaking. For example, the processing of Cistanche deserticola in alcohol involves slicing Cistanche deserticola, mixing it with rice wine, steaming, drying (low-temperature drying or sun-drying), and then quality testing and packaging to obtain the Cistanche deserticola slices. However, the optimal growth environment for most classic probiotic strains with application value (such as lactic acid bacteria and bifidobacteria) is alcohol-free, and their tolerance to ethanol concentrations is usually low (generally below 5% vol). The rice wine commonly used in the processing of traditional Chinese medicine typically has an alcohol content between 10% and 20% vol. In high-concentration steaming, roasting, or soaking, the survival rate of probiotics decreases, enzyme activity is significantly inhibited, leading to incomplete cell wall degradation and difficulty in releasing active ingredients. Therefore, screening for probiotics with both plant-degrading enzyme activity and alcohol tolerance holds promise for developing fermentation engineered bacteria for alcohol-based medicinal herbs, which could improve the bioavailability of these herbs. Summary of the Invention

[0004] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a fermentation engineered bacterium, Lactobacillus plantarum JY003, for fermenting medicinal herbs in alcoholic beverages. The purpose is to address the fact that existing commercial strains with plant-degrading enzymes generally have poor alcohol tolerance, while this strain (accession number CCTCCNO: M20252157) shows improved activity after alcohol treatment, demonstrating good alcohol tolerance. Furthermore, it possesses both pectinase and cellulase activities, making it suitable as a fermentation engineered bacterium for fermenting medicinal herbs in alcoholic beverages. This solves the technical problem that existing commercial strains with plant-degrading enzymes have poor alcohol tolerance and cannot be used as fermentation engineered bacteriums for fermenting medicinal herbs in alcoholic beverages.

[0005] To achieve the above objectives, according to one aspect of the present invention, a fermentation engineered microorganism for processing Chinese medicinal herbs into alcoholic beverages is provided, with accession number CCTCCNO:M20252157, accession date of September 29, 2025, and classification name LactobacillusplantarumJY003.

[0006] Preferably, the 16S rDNA sequence of the fermentation engineered bacteria for the wine-processed Chinese herbal medicine slices is shown in SEQ ID NO:1.

[0007] Preferably, the fermentation engineered bacteria used in the wine-processed Chinese herbal medicine slices are used to promote the dissolution of polysaccharides and flavonoids in the Chinese herbal medicines during fermentation.

[0008] Preferably, the fermentation engineered bacteria used in the wine-making process are capable of tolerating an alcohol content of 10-15%.

[0009] Preferably, the fermentation engineered bacteria for traditional Chinese medicine in winemaking can tolerate rice wine with an alcohol content of 10-15%.

[0010] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The preserved strain Lactobacillus plantarum JY003 provided by this invention is a novel strain of Lactobacillus plantarum, possessing both pectinase and cellulase activities. Compared to existing commercial strains with plant-degrading enzyme activities, the activity of this preserved strain is not only not inhibited after alcohol treatment, but is actually enhanced, exhibiting good alcohol tolerance. It can be used as an engineered fermentation strain for alcohol-processed traditional Chinese medicine (TCM) decoction pieces to promote the dissolution of polysaccharides and flavonoids in TCM decoction pieces, thereby improving the bioavailability of TCM decoction pieces. Attached Figure Description

[0011] Figure 1 These are the results of pectinase screening.

[0012] Figure 2 These are the results of cellulase screening.

[0013] Figure 3 This study compares the dissolution effects of active ingredients (polysaccharides and flavonoids) in Chinese medicinal materials before and after fermentation using Lactobacillus plantarum JY003. 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] While existing commercial bacterial strains possess certain plant-degrading enzyme activity, they generally cannot tolerate alcohol. Considering the characteristics of alcohol-processed Chinese medicinal herbs, this invention aims to screen for probiotics with plant-degrading enzyme activity that can tolerate alcohol for use in the fermentation of alcohol-processed Chinese medicinal herbs.

[0016] This invention isolates and screens bacterial strains from fecal samples of healthy individuals. By comparing the plant-degrading enzyme activities and alcohol tolerance of different strains, strain number 187 was identified as a novel *Lactobacillus plantarum* strain. This strain exhibits pectinase and cellulase activities, and its activities are not only not inhibited but actually enhanced after alcohol treatment, demonstrating good alcohol tolerance. Therefore, it can be used as an engineered fermentation strain in alcohol-processed traditional Chinese medicine decoction pieces. The newly isolated strain number 187 was deposited on September 29, 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:M20252157, the deposit date is September 29, 2025, and its classification name is *Lactobacillus plantarum* JY003.

[0017] Based on this, the present invention provides an engineered microbial culture for fermenting Chinese medicinal herbs in alcoholic beverages, with accession number CCTCCNO:M20252157, accession date of September 29, 2025, and classification name LactobacillusplantarumJY003.

[0018] The 16S rDNA sequence of Lactobacillusplantarum JY003 is shown in SEQ ID NO:1.

[0019] The pectinase activity of *Lactobacillus plantarum* JY003 was qualitatively and semi-quantitatively assessed using the plate hydrolysis zone method to evaluate the ability of the screened strain to secrete pectinase. After being cultured on agar plates with pectin as the sole carbon source, the target strain formed clear and broad hydrolysis zones. The diameter of the hydrolysis zone was measured to be 3.82 cm. This result provides direct and strong evidence that this strain possesses the ability to efficiently secrete extracellular pectinase.

[0020] The cellulase activity of *Lactobacillus plantarum* JY003 was qualitatively and semi-quantitatively assessed using the plate hydrolysis zone method to evaluate the cellulase secretion ability of the selected strain. After being cultured on agar plates with cellulose as the sole carbon source, the target strain formed clear and broad hydrolysis zones. The diameter of these hydrolysis zones was measured to be 2.2 cm. This result provides direct and strong evidence that this strain possesses the ability to efficiently secrete extracellular cellulase.

[0021] The alcohol tolerance of *Lactobacillus plantarum* JY003 was assessed by centrifuging the activated strain, removing the supernatant, washing the cells with buffer, adding an equal volume of rice wine (10-15% alcohol), mixing thoroughly, and allowing to stand for 3 hours. The bacterial activity was then detected using the plate count method. If the number of colonies detected by the plate count method increased after rice wine treatment compared to before treatment, it indicates that the strain has a certain degree of alcohol tolerance and can withstand 10-15% alcohol, especially rice wine with an alcohol content of 10-15%.

[0022] The preferred method is to use LactobacillusplantarumJY003 as an engineered fermentation microorganism for fermenting Chinese medicinal herbs, which promotes the dissolution of polysaccharides and flavonoids in the herbs.

[0023] The following are examples. Based on a literature review, probiotics containing plant-degrading enzymes include *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, *Streptococcus thermophilus*, *Angelica dahurica*, *Lactobacillus acidophilus*, *Lactobacillus pentosus*, and *Lactobacillus brevis*. In this embodiment, strains were isolated and purified from fecal samples from healthy individuals, and all isolated strains were stored at -80°C using 25% glycerol.

[0024] 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 -4 10-5 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.

[0025] Single colonies were selected based on their morphology, color, and size and streaked on three zones of MRS basal broth agar plates. Single colonies were repeatedly selected for purification until pure strains were isolated and numbered.

[0026] (2) Molecular biological identification The obtained single colonies were scraped and subjected to 16s identification for preservation. Based on the morphological characteristics and molecular biological identification of the strains, multiple strains of Lactobacillus were obtained, as follows: Genomic DNA of the isolated bacterial strain was extracted strictly according to the steps of the bacterial DNA extraction kit, and polymerase chain reaction (PCR) amplification was performed using it as a template. The forward primer for the amplification reaction was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0027] PCR reaction system: 2×EcoTaqPCRSuper-Mi×25μL, 1μL each of forward and reverse primers, 1μL of genomic DNA, and ddH2O to make up to 50μL. 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.

[0028] 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 found in GenBank to further confirm the isolated strains. The results are shown in Table 1.

[0029] Table 1. Identification results of isolated strains

[0030] The 16S rDNA sequence of strain number 187 is shown in SEQ ID NO:1. The obtained 16S rDNA sequence was compared with BLAST on NCBI. The results showed that the 16S rDNA sequence of strain number 187 was 99.68% similar to the sequence of Lactobacillus plantarum (PQ451450.1). Strain number 187 was identified as a new strain of Lactobacillus plantarum.

[0031] (3) Performance testing of strains The strains isolated and purified in the examples were cultured three times consecutively, and then cultured in sterile MRS broth with an inoculum of 5%. After culturing at 37°C for 24 hours, the inoculum was adjusted to 10.9 CFU / ml, poured into a sterile spray bottle for later use.

[0032] (1) Pectinase activity assay A selection medium with added substrate was prepared (K₂HPO₄: 1g, MgSO₄: 0.5g, NaNO₃: 3g, FeSO₄·7H₂O: 0.01g, pectin 2g, agar 15g, distilled water 1000mL, pH 5.5). Using the Congo red staining method, the purified and preserved strain *Lactobacillus plantarum* JY003 was inoculated into pectin agar plates and incubated at 37℃ for 3 days. The colony diameter was then measured. Next, 1mg / mL Congo red staining solution was added to the plates, and staining was performed for approximately 40 minutes. The Congo red staining solution was discarded, and a thin layer of surface Congo red was rinsed off with a small amount of distilled water. Distilled water was added again for destaining for 10 minutes. A clear hydrolysis zone was observed under light. Figure 1 As shown.

[0033] (2) Cellulase activity assay Prepare a selection medium with added substrate (5g yeast extract, 5g carboxymethyl cellulose, 15g agar powder, 1000mL distilled water). Using the Congo red staining method, inoculate the purified strain *Lactobacillus plantarum* JY003 into pectin agar plates and incubate at 37℃ for 3 days. Measure the colony diameter. Then, add 1mg / mL Congo red staining solution to the plates and stain for approximately 40 minutes. Discard the Congo red staining solution, rinse off the thin layer of Congo red on the surface with a small amount of distilled water, and add distilled water to decolorize for 10 minutes. A clear hydrolysis zone will be visible under light. Figure 2 As shown.

[0034] Depend on Figure 1 and Figure 2 The results showed that strain 187 exhibited a distinct hydrolysis zone, demonstrating both pectinase and cellulase activity, and exhibiting good hydrolytic activity against plant cell walls. All strains were stored at -80°C using 25% glycerol. Before use, all microorganisms underwent three consecutive cultures, followed by incubation in sterile MRS broth at a 5% inoculum size. Fresh fermentation broth was obtained after 24 hours of incubation at 37°C for testing.

[0035] Example 2: Screening of alcohol-tolerant probiotics The strains isolated and purified in Example 1 were compared with commercial strains to screen for strains that could tolerate alcohol. All strains used in the experiment are shown in Table 2.

[0036] Table 2. Strain Information

[0037] 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.

[0038] Screening of alcohol-tolerant probiotics: The activated strains were centrifuged to remove the supernatant, and the bacterial cells were washed with buffer and then an equal amount of rice wine (10-15% alcohol) was added. The mixture was mixed and allowed to stand for 3 hours. The bacterial activity was detected by plate counting method. The results are shown in Table 3.

[0039] Table 3 Screening of alcohol-resistant strains

[0040] As shown in the table above, compared with other strains, the strain numbered 187, which was unexpectedly isolated, not only did not have its activity inhibited after alcohol treatment, but its activity was actually enhanced. This indicates that the strain has good alcohol tolerance and can withstand 10-15% alcohol.

[0041] The newly isolated strain, numbered 187, was deposited on September 29, 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:M20252157, the deposit date is September 29, 2025, and its classification name is LactobacillusplantarumJY003.

[0042] Example 3: Detection of viable bacteria and extractables in wine-processed Cistanche deserticola slices In this embodiment, the preserved strain Lactobacillus plantarum JY003 was used as the fermentation engineered bacteria to study the viable cell count and extracts in the wine-processed Cistanche deserticola slices before and after fermentation, as detailed below: (1) Preparation of bacterial suspension: The preserved strain Lactobacillus plantarum JY003 was taken out at -80℃. Before use, this strain was cultured three times consecutively, and then cultured in sterile MRS broth with an inoculum of 5%. It was cultured at 37℃ for 24 hours, placed in a centrifuge tube, centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the suspension was resuspended with an equal proportion of physiological saline for use.

[0043] (2) Inoculation and cultivation: The dried Cistanche deserticola slices were divided into 10 sterilized stainless steel steaming trays at a standard of 30 grams each. Each tray was evenly sprayed with 6 ml of rice wine (10-15% alcohol) and 1.8 ml of bacterial suspension. After spraying, the Cistanche deserticola slices were covered with a damp gauze and placed in a constant temperature incubator at 37℃ for 4 hours.

[0044] (3) Detection of viable bacteria of Lactobacillus plantarum JY003 Take out the Cistanche deserticola slices fermented for 4 hours in step (2), weigh a certain amount of Cistanche deserticola slices, cut them into small pieces with sterile scissors, add steel balls and PBS buffer solution, place them in a grinder, grind them evenly at 70 Hz for 60 seconds, and perform serial dilution with PBS buffer (e.g., 10-fold). -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 the process multiple times. The results are shown in Table 4.

[0045] Table 4. Results of viable cell counts of *Lactobacillus plantarum* JY003 before and after fermentation.

[0046] (4) Detection of the dissolution of active ingredients Take out the Cistanche deserticola slices fermented for 4 hours in step (2), transfer them all to a steamer, steam for 4 hours, then transfer them to an oven, set the temperature to 65℃, and dry for 24~48 hours before taking them out to complete the secondary drying and obtain the fermented Cistanche deserticola slices.

[0047] Weigh 5 grams of fermented Cistanche deserticola slices, place them in a container, add 100 ml of water, and simmer over low heat for 30 minutes. Concentrate the decoction to 50 ml and transfer it to a centrifuge tube for later use. Detect the polysaccharide and flavonoid content in the concentrate, repeating the process three times. The results are as follows: Figure 3 As shown.

[0048] Depend on Figure 3 The results showed that there was no statistically significant difference in the content of extracts (such as polysaccharides and flavonoids) between the experimental group (yellow wine + PBS) which added PBS, a solvent without live bacteria, and the control group (yellow wine group) which prepared Cistanche deserticola using the traditional processing method. This indicates that adding PBS, a solvent without live bacteria, did not affect the dissolution of active ingredients (such as polysaccharides and flavonoids) in Cistanche deserticola prepared by the traditional processing method.

[0049] Compared to the control group (yellow wine group) of Cistanche deserticola prepared by traditional processing, the Cistanche deserticola prepared by fermenting Cistanche deserticola slices with preserved strain LactobacillusplantarumJY003 (yellow wine + 187) using traditional processing showed a significant increase in the active ingredient polysaccharide, and a significant increase in echinacoside and flavonoids. This indicates that the addition of preserved strain LactobacillusplantarumJY003 during fermentation can promote the release and dissolution of active ingredients such as polysaccharides and flavonoids in Cistanche deserticola prepared by fermentation. It can be used as an engineered fermentation strain for preparing traditional Chinese medicine in wine, which is beneficial to improving the dissolution rate of its active ingredients (such as polysaccharides and flavonoids).

[0050] 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 processing traditional Chinese medicine slices into alcohol, characterized in that, Its accession number is CCTCCNO:M20252157, the accession date is September 29, 2025, and the classification name is LactobacillusplantarumJY003.

2. The engineered fermentation bacteria for processing Chinese medicinal herbs into wine, as described in claim 1, is characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The engineered fermentation bacteria for processing Chinese medicinal herbs into wine, as described in claim 2, is characterized in that... It is used to promote the dissolution of polysaccharides and flavonoids in Chinese medicinal materials during fermentation.

4. The engineered fermentation bacteria for alcoholic beverages as described in claim 3, characterized in that, It can tolerate an alcohol content of 10-15%.

5. The engineered microbial culture for fermenting traditional Chinese medicine in alcohol production as described in claim 4, characterized in that, It can tolerate rice wine with an alcohol content of 10-15%.

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