A rod-shaped Lactobacillus putrefaciens CY-06 and its use in fermenting and converting ginsenoside Re to prepare rare ginsenosides

Abstract

A rod-shaped lactobacillus coryniformis CY-06 and its use in fermenting and converting ginsenoside Re to prepare rare ginsenosides belong to the field of microbial fermentation. The rod-shaped lactobacillus coryniformis CY-06 has been deposited with the China Center for Type Culture Collection on February 25, 2025, with a deposit number of CCTCC NO: M2025299. The rod-shaped lactobacillus coryniformis CY-06 can ferment and convert ginsenoside Re to prepare rare ginsenosides. The present invention uses ginsenoside Re as a raw material and utilizes the rod-shaped lactobacillus coryniformis CY-06 to ferment and convert rare ginsenosides Rg2, Rh1, Rg6, and F4. After fermentation, ginsenoside Re is completely converted into Rg2, Rh1, Rg6, and F4. This method, which eliminates the need for alkaline hydrolysis, high-temperature, high-pressure, or steam treatment, offers the advantages of a mild reaction, environmental friendliness, low cost, and high product content. This invention provides a new pathway for the targeted synthesis of the rare ginsenosides Rg2, Rh1, Rg6, and F4, significantly enhancing their value in pharmaceutical research and development and industrialization.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to the use of a rod-shaped lactobacillus (Loigolactobacillus coryniformis) CY-06 strain in fermenting and converting ginsenoside Re in the preparation of rare ginsenosides. Background Art

[0002] Ginseng (Panax ginseng C. mey.), a medicinal plant of the Araliaceae family, is known for its key active ingredients, ginsenosides, which exhibit biological activities such as alleviating physical fatigue and regulating glucose and lipid metabolism and immune function. Compared to primary ginsenosides, rare ginsenosides, due to their lipid solubility, can more easily penetrate biological membranes and exert anti-tumor and immunomodulatory effects. However, their extremely low natural abundance makes them difficult to obtain on a large scale. Current industrial production relies primarily on acid-base hydrolysis and thermal cracking techniques, but high temperatures and pressures can easily lead to fragmentation of the saponin backbone and inactivation of functional groups. Traditional enzymatic hydrolysis, while capable of targeted cleavage of sugar chains, faces application bottlenecks such as difficulty purifying enzymes and high operating costs. While intestinal microbiota can catalyze the conversion of ginsenosides, the heterogeneity of individual microbiomes results in instability in the biotransformation pathway. Using in vitro microbial fermentation technology, by mimicking intestinal metabolic mechanisms, precise regulation of glycosylation can be achieved at room temperature and pressure. Probiotic fermentation technology demonstrates unique advantages: its substrate-specific recognition allows for the selective preservation of ginsenoside active nuclei, avoiding the formation of byproducts common in chemical methods. Its environmentally friendly process eliminates the need for toxic reagents, significantly reducing the burden of waste treatment. Compared to traditional enzymatic hydrolysis, this technology leverages the synergistic action of microbial intracellular enzymes to maintain the structural integrity of saponins while enhancing the bioavailability of fermentation products. This economically viable biocatalytic model offers a new path for the development of high-purity rare saponins.

[0003] Lactobacillus coryniformis is a highly adaptable microorganism with a wide distribution, having been isolated from fermented foods, animal intestines, and the natural environment. Numerous studies have demonstrated its potential probiotic properties, including acid and bile salt tolerance, adhesion, antioxidant activity, antibacterial activity, anti-inflammatory activity, and lipid-lowering properties, promising widespread applications in food and pharmaceuticals. However, it is noteworthy that despite its numerous probiotic properties, its application in the bioconversion of ginsenosides has not been reported. To this end, this study employed the CY-06 strain of Lactobacillus coryniformis for the first time, using ginsenoside Re as a substrate for targeted fermentation. After 14 days of bioconversion, the product successfully transformed into a series of rare saponins, including Rg2, Rg6, F4, and Rh1. This process is suitable for large-scale industrial production. The rare ginsenosides Rg2, Rg6, F4 and Rh1 prepared are widely used and have broad market prospects in the fields of cardiovascular disease treatment and functional food development. Summary of the Invention

[0004] The present invention aims to provide a use of a strain of Lactobacillus putrefaciens CY-06 in fermenting and converting ginsenoside Re to prepare rare ginsenosides, thereby providing a new approach for the preparation of rare ginsenosides Rg2, Rg6, F4 and Rh1.

[0005] The technical solutions adopted by the present invention to solve the technical problems are as follows:

[0006] The rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) CY-06 provided by the present invention has been deposited in the China Center for Type Culture Collection on February 25, 2025, with a deposit number of CCTCC NO: M 2025299, and the deposit address is Wuhan, China.

[0007] The present invention provides a use of a β-glucosidase-producing rod-shaped lactobacillus putrefaciens CY-06 strain for fermenting and converting ginsenoside Re in preparing rare ginsenosides.

[0008] As a preferred embodiment, the rare ginsenosides include Rg2, Rg6, F4 and Rh1.

[0009] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides Rg2, Rg6, F4 and Rh1 is as follows: ginsenoside Re is hydrolyzed at the C-20 position of glucose under the action of Loigolactobacillus coryniformis CY-06 to produce rare ginsenoside Rg2, which is further dehydrated to produce rare ginsenoside F4 and Rg6. At the same time, rare ginsenoside Rg2 can remove a rhamnose at the C-6 position to produce rare ginsenoside Rh1.

[0010] As a preferred embodiment, the present invention provides a method for fermenting and converting ginsenoside Re into rare ginsenosides by a strain of Lactobacillus coryniformis CY-06, which specifically comprises the following steps:

[0011] Ginsenoside Re was dissolved in 2 mL of dimethyl sulfoxide, and then heated in a 50°C water bath for 10 min. The mixture was added to the sterilized liquid fermentation medium at a dosage of 0.5 g / L and sterilized by microporous filtration. Loigolactobacillus coryniformis CY-06 was prepared into a bacterial suspension, and the bacterial suspension was diluted to 2 × 10 7 The inoculum size was 500 CFU / ml and inoculated into a liquid fermentation medium supplemented with ginsenoside Re, and anaerobically fermented at 37±0.5°C for 7 to 14 days. After the fermentation, the fermentation product was extracted with water-saturated n-butanol, and then concentrated under reduced pressure at low temperature (50°C) and freeze-dried (-80°C, 5 Pa). The residue was dissolved in methanol and centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant was decompressed and the solvent was recovered, followed by vacuum freeze-drying (-80°C, 5 Pa) to obtain a dry product, which contained rare ginsenosides Rg2, Rh1, Rg6 and F4.

[0012] As a preferred embodiment, the liquid fermentation medium comprises: 1 g / L glucose, 1 g / L tryptone, 0.5 g / L yeast extract powder and 1 g / L sodium chloride; pH 6.0-7.0.

[0013] As a preferred embodiment, the specific process of preparing the bacterial suspension of Loigolactobacillus coryniformis CY-06 is as follows:

[0014] Loigolactobacillus coryniformis CY-06 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24–48 h. The bacterial precipitate was collected by centrifugation at 8000–12000 rpm for 5–10 min at 4°C and then suspended in sterile PBS to adjust the viable bacterial count to 1.0 × 10 7 ~1.0×10 8 CFU / ml, and obtain the suspension of Loigolactobacillus coryniformis CY-06 bacteria.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention screened and obtained a rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) CY-06 from a sample of traditionally fermented sauerkraut made by farmers in Jilin City, Jilin Province. The strain can produce β-glucosidase and bioconvert ginsenoside Re into rare ginsenosides Rg2, Rh1, Rg6 and F4. The strain CY-06 was also identified as Loigolactobacillus coryniformis. The strain was deposited in the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025299. The present invention determines a new process for preparing rare ginsenosides Rg2, Rh1, Rg6 and F4 by fermenting and converting ginsenoside Re with Loigolactobacillus coryniformis CY-06, and simultaneously identifies the converted rare ginsenosides Rg2, Rh1, Rg6 and F4 by HPLC chromatographic analysis. This process utilizes the β-glucosidase-producing ability of Lactobacillus coryniformis CY-06 to ferment and convert ginsenoside Re into the rare ginsenosides Rg2, Rh1, Rg6, and F4. HPLC chromatographic analysis of the converted ginsenosides Rg2, Rh1, Rg6, and F4 revealed that the retention time of the converted ginsenoside Re product was consistent with that of the standards of the rare ginsenosides Rg2, Rh1, Rg6, and F4. This process offers the advantages of mild reaction conditions, environmental friendliness, and high conversion efficiency, significantly different from existing bioconversion routes. This process is suitable for large-scale industrial production, and the resulting rare ginsenosides Rg2, Rh1, Rg6, and F4 have broad applications and promising prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a color diagram of the rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) CY-06 with high β-glucosidase production on the screening culture medium.

[0018] Figure 2 HPLC chromatograms of ginsenoside Re fermentation on day 0 and day 14; wherein, a is the HPLC chromatogram of ginsenoside Re fermentation on day 0, and b is the HPLC chromatogram of ginsenoside Re fermentation on day 14.

[0019] Figure 3 This is a biotransformation synthesis route for the fermentation and conversion of ginsenoside Re into rare ginsenosides Rg2, Rh1, Rg6 and F4 by Loigolactobacillus coryniformis CY-06. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] 1. Isolation of strains

[0022] In November 2024, samples of sauerkraut made by traditional fermentation from farmers in Jilin City, Jilin Province were selected. The sauerkraut stock solution was dipped and inoculated on an MRS medium plate containing bromocresol purple using the plate streak method. It was cultured at 37°C for 24 hours. According to the morphology of the single colony, the colonies that can make bromocresol purple turn yellow were picked for microscopic examination. The strains that were Gram-positive were selected and cultured at 37°C for 24 hours. The streak inoculation was continued until the pure isolate was isolated and purified. It was further inoculated on aesculin screening medium (used to isolate strains that produce β-glucosidase, add esculin 1 g / L and ferric citrate 0.5 g / L to MRS agar, and sterilize under high pressure at 121°C for 20 minutes). The strains were surrounded by a reddish-brown to dark brown circle, which was a positive strain (such as Figure 1 ), indicating that the strain has the ability to produce β-glucosidase. The pure culture was inoculated into liquid MRS medium and cultured. After adding 80% glycerol, the strain was stored in a -80°C freezer. One of the strains, Loigolactobacillus coryniformis, was designated CY-06.

[0023] 2. Identification of strains

[0024] 1. The physiological and biochemical identification results of strain CY-06 are as follows:

[0025] Gram-positive, it stains positive for esculin, maltose, mannitol, sorbitol, sucrose, raffinose, and inulin among 11 carbohydrates. It is negative for cellobiose, salicin, lactose, and 1% sodium hippurate. Its optimal growth temperature is 37°C; its optimal pH is 6.0-7.0; it tolerates 6.5% NaCl. It grows uniformly turbid in liquid MRS medium, with a white precipitate forming upon prolonged growth.

[0026] 2. Molecular biological identification and results are as follows:

[0027] The target strain was inoculated into fresh MRS liquid medium and cultured for 24 hours. DNA was extracted using a kit from Tiangen Biochemical Technology Co., Ltd., and 16S rDNA sequence amplification was performed. The genus-universal primers, 1492R and 27F, were used for amplification. Electrophoresis of the 16S rDNA PCR product of strain CY-06 revealed a single, highly specific band at approximately 1500 bp, consistent with the expected molecular weight. Sequencing was performed, and the sequence is shown as SEQ ID NO. 1 in the sequence listing. Comparison of the sequence with the 16S rDNA gene sequences of selected strains accessed at http: / / www.ncbi.nlm.nih.gov revealed that strain CY-06 shared 99.59% homology with Loigolactobacillus coryniformis subsp. torquens (NR_029018.1). Based on these results, strain CY-06 was identified as a member of the rod-shaped Lactobacillus species (Loigolactobacillus coryniformis).

[0028] The primer pair sequence information is as follows:

[0029] 27sF: 5′-agagttgatcctggctcag-3′; 1492R: 5′-ggttaccttgttacgactt-3′; PCR amplification conditions were as follows: initial denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; final extension at 72°C for 2 min, and storage at 4°C.

[0030] 3. Preservation of strains

[0031] The rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) CY-06 of the present invention has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on February 25, 2025. The address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), and the preservation number is: CCTCC NO: M 2025299.

[0032] 4. Preparation of Loigolactobacillus coryniformis CY-06 bacterial suspension

[0033] The obtained rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) CY-06 was inoculated into liquid MRS medium and cultured at 37°C for 24 h. The culture was centrifuged at 4°C and 8000 rpm for 10 min, and the bacterial precipitate was collected. The culture was suspended in sterile 0.85% saline solution and the viable bacterial count was adjusted to 1.0×10 9 CFU / mL, and obtain the suspension of Loigolactobacillus coryniformis CY-06.

[0034] 5. Preparation of rare ginsenosides by fermentation of ginsenoside Re by Loigolactobacillus coryniformis CY-06

[0035] 1. Prepare liquid fermentation medium: 1 g / L glucose, 1 g / L tryptone, 0.5 g / L yeast extract powder, and 1 g / L sodium chloride; pH 6.0-7.0, sterilize at 121°C for 15 min.

[0036] 2. Fermentation conversion: Ginsenoside Re (purchased from Chengdu Aifa Biotechnology Co., Ltd., HPLC ≥ 98%) was added to the liquid fermentation medium and dissolved in 2 mL of dimethyl sulfoxide, wherein the amount of ginsenoside Re added was 0.5 g / L, filtered through a 0.22 μM microporous membrane for sterilization, and then heated to 2 × 10 7A suspension of Loigolactobacillus coryniformis CY-06 was inoculated with an inoculum of 100 CFU / mL and anaerobically fermented at 37°C for 14 days. After fermentation, the resulting fermentation product was extracted three times with water-saturated n-butanol. The resulting fermentation product was concentrated under reduced pressure at 50°C and vacuum freeze-dried (-80°C, 5 Pa). The product was then extracted with methanol and centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant was decompressed and the solvent was recovered, followed by vacuum freeze-drying (-80°C, 5 Pa) to obtain a dried product containing rare ginsenosides Rg2, Rh1, Rg6, and F4.

[0037] 6. Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0038] High-performance liquid chromatography (HPLC) was used to detect the rare ginsenosides Rg2, Rh1, Rg6, and F4: Ginsenoside Re and the dried product obtained above were dissolved in methanol, filtered through a 0.22 μm microporous membrane, and then analyzed by HPLC. The HPLC analysis was performed using an Agilent pursuit5 SB C18 column, with an injection volume of 20 μL, an elution rate of 1.0 mL / min, a column temperature of 30°C, and a detection wavelength of 203 nm. The mobile phase was water (A) and acetonitrile (C) with gradient elution as follows: 0 min, 81.50%A, 18.50%C; 20 min, 79.50%A, 20.50%C; 30 min, 70%A, 30%C; 45 min, 65%A, 35%C; 60 min, 55%A, 45%C; 70 min, 40%A, 60%C; 80 min, 30%A, 70%C; 90 min, 20%A, 80%C; 91 min, 81.50%A, 18.50%C; 95 min, 81.50%A, 18.50%C.

[0039] In the process of preparing rare ginsenosides Rg2, Rh1, Rg6 and F4 by fermenting ginsenoside Re with Loigolactobacillus coryniformis CY-06 suspension, the biotransformation synthesis route is as follows: Figure 2As shown in the figure: under the action of Loigolactobacillus coryniformis CY-06, ginsenoside Re hydrolyzes the glucose at the C-20 position to produce rare ginsenoside Rg2, which is further dehydrated to produce rare ginsenoside F4 and Rg6. At the same time, rare ginsenoside Rg2 can also remove a rhamnose at the C-6 position to produce rare ginsenoside Rh1.

[0040] HPLC chromatographic identification results are as follows Figure 3 As shown in the figure, qualitative and quantitative analysis of ginsenoside components in the products before and after fermentation showed that the retention time of the conversion product of ginsenoside Re was consistent with that of the standards of rare ginsenosides Rg2, Rg6, F4, and Rh1. This proves that ginsenoside Re can be converted into rare ginsenosides Rg2, Rg6, F4, and Rh1 by fermentation with β-glucosidase-producing Lactobacillus coryniformis CY-06. This proves that ginsenoside Re can be converted into rare ginsenosides Rg2, Rg6, F4, and Rh1 by fermentation with Lactobacillus coryniformis CY-06.

[0041] The present invention discloses the use of a strain of Lactobacillus coryniformis (Loigolactobacillus coryniformis) CY-06 for fermenting and converting ginsenoside Re to prepare rare ginsenosides. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve this. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately alter and combine the products described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

Claims

1. Lactobacillus putrefaciens ( Loigolactobacillus coryniformis )CY-06, characterized in that This strain was deposited in the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025299.

2. The rod-shaped putrefactive lactobacillus according to claim 1 ( Loigolactobacillus coryniformis ) Application of CY-06 fermentation and conversion of ginsenoside Re in the preparation of rare ginsenosides, wherein the rare ginsenosides are Rg2, Rg6, F4 and Rh1.

3. The use according to claim 2, characterized in that The biotransformation synthesis route of the rare ginsenosides Rg2, Rg6, F4 and Rh1 is as follows: ginsenoside Re is synthesized in the presence of coryneform Lactobacillus putrefaciens ( Loigolactobacillus coryniformis ) Under the action of CY-06, the glucose at the C-20 position was hydrolyzed to produce the rare ginsenoside Rg2. Furthermore, the rare ginsenoside Rg2 was dehydrated to produce the rare ginsenosides F4 and Rg6. At the same time, the rare ginsenoside Rg2 removed a rhamnose at the C-6 position to produce the rare ginsenoside Rh1.

4. The use according to claim 2, characterized in that The steps include: dissolving ginsenoside Re in 2 mL of dimethyl sulfoxide, bathing in a 50°C water bath for 10 min, adding 0.5 g / L of ginsenoside Re to a sterilized liquid fermentation medium, sterilizing the medium through a microporous filter membrane, and separating the rod-shaped putrefactive Lactobacillus ( Loigolactobacillus coryniformis )CY-06 was prepared into bacterial suspension, and the bacterial suspension was diluted to 2×10 7 The inoculum size of CFU / ml was inoculated into a liquid fermentation medium supplemented with ginsenoside Re, and anaerobically fermented at 37±0.5°C for 7 to 14 days. After the fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50°C and freeze-dried at -80°C and 5PA. The residue was dissolved in methanol and centrifuged at 10,000 rpm and 4°C for 15 minutes. The supernatant was decompressed and the solvent was recovered, and then vacuum freeze-dried at -80°C and 5PA to obtain a dry product, which contained rare ginsenosides Rg2, Rh1, Rg6 and F4.

5. The use according to claim 4, characterized in that Lactobacillus putrefaciens ( Loigolactobacillus coryniformis )CY-06 is prepared into a bacterial suspension as follows: the rod-shaped putrefactive lactobacillus ( Loigolactobacillus coryniformis CY-06 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24–48 h. The bacterial precipitate was collected by centrifugation at 4000–8000 rpm for 5–10 min at 4°C and then suspended in sterile PBS to adjust the viable bacterial count to 1.0 × 10 7 ~1.0×10 8 CFU / ml, obtained rod-shaped putrefactive lactobacillus ( Loigolactobacillus coryniformis )CY-06 bacterial suspension.

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