Use of leuconostoc mesenteroides subsp. mesenteroides bt708 in the fermentation of ginsenoside rb1 for the preparation of rare ginsenosides

Abstract

The application of *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708 in the fermentation of ginsenoside Rb1 to prepare rare ginsenosides belongs to the field of microbial fermentation technology. *Leuconostoc mesenteroides* subsp. *mesenteroides* ( Leuconostoc mesenteroides subsp. mesenteroides The preservation number for BT708 is CCTCC NO: M20251675. Ginsenoside Rb1 is dehydrated by hydrolysis of the glucose at the C-20 position of the ginsenoside Rb1 molecule in *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708, yielding rare ginsenosides Rk1 and Rg5. This invention uses ginsenoside Rb1 as the sole substrate and *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708 as a biocatalyst, enabling the efficient conversion of ginsenoside Rb1 into rare ginsenosides Rk1 and Rg5 within a single fermentation cycle. The fermentation process is mild, green, safe, and low-cost.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to the use of Leuconostoc mesenteroides subsp. mesenteroides BT708 in the fermentation of ginsenoside Rb1 to prepare rare ginsenosides. Background Technology

[0002] Rare ginsenosides Rk1 and Rg5 are small-molecule saponins found in extremely low amounts in ginseng plants, but with significant pharmacological activity. Modern research shows that these rare ginsenosides exhibit superior bioactivity compared to the native ginsenoside Rb1 in antitumor, anti-inflammatory, neuroprotective, hypoglycemic, and immunomodulatory effects. However, the content of these rare saponins in natural ginseng or Panax notoginseng raw materials is usually less than 0.01%, and their chemical structures contain multiple chiral centers and glycosyl side chains, resulting in long chemical synthesis routes, high costs, and poor stereoselectivity, making it difficult to meet the large-scale needs of clinical and new drug development.

[0003] At present, rare ginsenosides are mainly obtained in industry through the following two methods: (1) Acid / alkali hydrolysis or high temperature degradation: Although the macromolecular prototype ginsenosides (such as Rb1) can be converted into the target product, the reaction conditions are severe and side reactions such as dehydration and epimerization are easy to occur, resulting in uncontrollable product stereoconfiguration, complex impurity spectrum, and extremely high cost of subsequent separation and purification; at the same time, a large amount of organic solvents and strong acids and bases are used, which pose environmental pollution and safety hazards. (2) Enzymatic or microbial transformation: By using β-glucosidase, α-L-arabinosidase, etc. to desugar Rb1 step by step, rare ginsenosides with a single configuration can be obtained, but the existing commercial enzymes are expensive, have a narrow substrate spectrum, and are difficult to obtain multiple active components at one time (such as Rk1 and Rg5 which need further dehydration); the reported microbial strains (such as Aspergillus and Bacillus) have problems such as low conversion efficiency, many by-products, and poor genetic stability, and cannot yet achieve industrial scale-up production.

[0004] Leuconostoc mesenteroides subsp. mesenteroides, a generally recognized safe (GRAS) lactic acid bacterium, has been widely used in the food fermentation industry. However, research on the targeted preparation of rare ginsenosides Rk1 and Rg5 by fermenting ginsenoside Rb1 using Leuconostoc mesenteroides subsp. mesenteroides has not been reported. Summary of the Invention

[0005] To achieve an efficient, green, safe, mild, and low-cost biotransformation synthesis method for rare ginsenosides Rk1 and Rg5, this invention provides a novel application of Leuconostoc mesenteroides subsp. mesenteroides BT708 and its use in the fermentation of ginsenoside Rb1 to prepare rare ginsenosides.

[0006] To achieve the above-mentioned objectives, the present invention provides the following specific technical solutions:

[0007] In a first aspect, the present invention provides a Leuconostoc mesenteroides subsp. mesenteroides BT708, which was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number CCTCC NO: M20251675.

[0008] Secondly, the present invention provides the use of Leuconostoc mesenteroides subsp. mesenteroides BT708 in the preparation of rare ginsenosides from fermented ginsenoside Rb1.

[0009] Among them, the rare ginsenosides are Rk1 and Rg5.

[0010] In this process, ginsenoside Rb1 is dehydrated by hydrolyzing the glucose at position C-20 of the ginsenoside Rb1 molecule in Leuconostoc mesenteroides subsp. mesenteroides BT708 to generate rare ginsenosides.

[0011] The beneficial effects of this invention are:

[0012] This invention utilizes a strain of *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708, isolated and extracted from the fermentation broth of naturally fermented sauerkraut from Northeast China. This strain is deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M20251675. The CCTCC is located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University Collection Center). The *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708 provided by this invention is a GRAS-grade probiotic, easily scaled up, and suitable for the large-scale production of rare ginsenosides in the pharmaceutical, food, and cosmetic fields, showing great promise for application.

[0013] This invention provides a complete biotransformation synthesis method for rare ginsenosides Rk1 and Rg5. Using ginsenoside Rb1 as the sole substrate and Leuconostoc mesenteroides subsp. mesenteroides BT708 as the biocatalyst, the method simultaneously enriches high-value rare ginsenosides Rk1 and Rg5 through a single fermentation step. The fermentation process is mild, requiring no organic solvents or exogenous enzymes, making it green, safe, and energy-efficient, significantly reducing production costs and safety risks. Furthermore, this invention can efficiently convert ginsenoside Rb1 into rare ginsenosides Rk1 and Rg5 within a single fermentation cycle, with a total molar yield ≥85%, controllable stereoconfiguration, and the resulting products (Rk1 and Rg5) exhibiting high purity and a simple impurity profile. They can be directly freeze-dried for use, significantly reducing subsequent separation and purification costs. Attached Figure Description

[0014] Figure 1 This is an HPLC chromatogram of Leuconostoc mesenteroides subsp. mesenteroides BT708 before (0d) fermentation of ginsenoside Rb1 and after (40d) fermentation, provided by the present invention. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention; any strains, culture conditions and extraction processes based on equivalent substitutions or improvements fall within the protection scope of the claims of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0016] I. Separation

[0017] Take the naturally fermented Northeastern sauerkraut fermentation liquid and perform serial dilutions (10⁻¹, 10⁻², 10⁻³, 10⁻¹). 4 10⁻ 5Afterward, the cultures were spread onto sucrose-thiamine solid medium (10 g sucrose, 0.01 g thiamine (vitamin B1), 10 g peptone, 5 g yeast extract, 5 g NaCl, 15 g agar, 1 L distilled water, pH 7.0) and incubated at 25°C for 48 h. Single colonies of different morphologies were then streaked onto bromocresol purple-sucrose-thiamine medium (with 5-10 mL of 0.1% bromocresol purple added). The cultures were purified twice. Strains producing acid and exhibiting distinct colony morphology were subjected to Gram staining, microscopic examination, and H2O2 enzyme assay. Gram-positive, H2O2-negative strains with spherical or ellipsoidal colonies were selected for preservation. The purified cultures were inoculated into liquid MRS medium and cultured further. After adding 60% glycerol, the cultures were stored at -80°C. One strain was named TB708.

[0018] II. Identification

[0019] First, genomic DNA of the selected strains was extracted using a bacterial genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal 16S rRNA primers.

[0020] The forward primer is 7F: 5'-CAGAGTTTGATCCTGGCT-3';

[0021] The reverse primer is 540R: 5'-AGGAGGTGATCCAG CCGCA-3'.

[0022] The PCR amplification conditions were as follows: pre-denaturation: 95℃ for 5 min; denaturation: 95℃ for 15 s, annealing: 55℃ for 15 s, extension: 72℃ for 1.5 min, for a total of 35 cycles; final extension: 72℃ for 7 min, and storage at 4℃.

[0023] The amplified products were then sequenced, and the 16S rDNA sequence is shown in SEQ ID NO:1. Homology analysis of the 16S rDNA sequence was performed in the GenBank database using the BLAST program. The results showed that the 16S rDNA sequence of strain TB708 was highly homologous to the 16S rRNA gene sequence of *Leuconostoc mesenteroides* subsp. *mesenteroides* ATCC 8293 in the NCBI database (GenBank accession number NR_113353.1). Based on these results, strain TB70814 was identified as *Leuconostoc mesenteroides* subsp. *mesenteroides*.

[0024] The 16S rDNA sequence is as follows:

[0025]

[0026] III. Preservation

[0027] The identified *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708 has been deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M20251675. The China Center for Type Culture Collection (CCTCC) is located at Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University Collection Center).

[0028] IV. Preparation of rare ginsenosides using *Leuconostoc mesenteroides* subsp. *mesenteroides* BT708

[0029] (1) Preparation of BT708 bacterial suspension

[0030] Strawberry strain BT708 was streaked onto MRS agar plates and incubated at 37 °C for 24 h to activate it. A single colony was picked and inoculated into 50 mL of MRS liquid medium, incubated at 37 °C for 20 h, and then transferred to 200 mL of MRS liquid medium at a 3% (v / v) inoculation rate and incubated at 28 °C for 18 h. The culture was centrifuged at 4 °C, 6000 × g for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile phosphate-buffered saline (PBS, pH 6.8). The viable cell count was then adjusted to 1.0 × 10⁻⁶ cells / mL with sterile physiological saline. 9 The BT708 bacterial suspension was obtained by measuring CFU m / L and stored at 4 ℃.

[0031] (2) Pretreatment of ginsenoside Rb1

[0032] Weigh commercially available ginsenoside Rb1 (Shanghai Yuanye Biotechnology Co., Ltd., HPLC purity ≥98%), add it to sterile deionized water at 70 ℃ to prepare a 100 g / L ginsenoside Rb1 stock solution, sterilize with a 0.22 µm filter membrane, and store in the dark.

[0033] (3) Fermentation and conversion to prepare rare ginsenosides

[0034] Prepare the conversion medium: glucose 30 g / L, yeast extract 10 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L; sterilize at 121 ℃ for 15 min.

[0035] Fermentation and transformation: Under aseptic conditions, ginsenoside Rb1 stock solution was added to the sterilized transformation medium to achieve a final concentration of 10 g / L. Subsequently, BT708 bacterial suspension was inoculated at a rate of 10 ml / L, mixed thoroughly, and dispensed into sterile anaerobic bottles (60% full). Fermentation was carried out at 28 ℃ for 40 days. After fermentation, the resulting fermentation product was freeze-dried under vacuum (-80 ℃, 5 Pa) to obtain a dried product containing rare ginsenosides Rk1 and Rg5.

[0036] V. Identification of Rare Ginsenosides

[0037] (1) Sample preparation

[0038] Accurately weigh 5.0 mg each of commercially available ginsenoside Rb1, rare ginsenoside Rk1, and rare ginsenoside Rg5 standards (all purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity ≥98%) and the dried product obtained above. Dissolve them in chromatographic grade methanol by sonication (40 kHz, 25 ℃, 10 min) and bring the volume to 5 mL. Filter the solution through a 0.22 µm organic microporous membrane and keep the filtrate for later use.

[0039] (2) HPLC chromatographic conditions

[0040] Instrument: Agilent 1260 Infinity II high performance liquid chromatograph with evaporative light scattering detector (ELSD).

[0041] Column: Agilent Pursuit 5 SB-C18 (4.6 × 250 mm, 5 µm).

[0042] Column temperature: 30 ℃; flow rate: 1.0 mL min⁻¹; detection wavelength: 203 nm; injection volume: 20 µL.

[0043] The moving phases are: phase A is water, and phase B is acetonitrile.

[0044] Gradient elution program: 0~40 min 18~21% (B); 40~42 min 21~26% (B); 42~46 min 26~32% (B); 46~66 min 32~34% (B); 66~71 min 34~38% (B); 71~77.70 min 38.0~49.1% (B); 77.70~82 min 49.1% (B); 82~83 min 49.1~50.6% (B); 83~88 min 50.6~59.6% (B); 88~89.80 min 59.6~65.0% (B); 89.80~97 min 65% (B); 97~102 min 65~75% (B); 102~110 min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min 18.0%(B).

[0045] (3) Result determination

[0046] HPLC chromatogram as shown Figure 1 As shown, under the above HPLC chromatographic conditions, the standards of rare ginsenosides Rk1 and Rg5 exhibited single, symmetrical peaks at 89.725 min and 90.691 min, respectively, with peak purity factors greater than 990. The dried product obtained above showed chromatographic peaks consistent with the standards at the corresponding retention times (deviation < 0.2 min); the blank control (without BT708 bacterial suspension) showed no significant absorption in the range of 85–95 min. This confirms that ginsenoside Rb1 has been directionally converted into rare ginsenosides Rk1 and Rg5 after fermentation with BT708 bacterial suspension.

[0047] (4) Biochemical synthesis route

[0048] This invention achieves simultaneous epimerization, desaccharification, and dehydration of ginsenoside Rb1 at the C-20 position using a one-step method. Specifically, after the glucose at the C-20 position of ginsenoside Rb1 is hydrolyzed by strain BT708, ginsenoside Rb1 is dehydrated to generate rare ginsenosides Rk1 and Rg5.

[0049] (5) Conclusion

[0050] The method of this invention enables ginsenoside Rb1 to be efficiently and directionally converted into rare ginsenosides Rk1 and Rg5 through fermentation by strain BT708, providing a green, safe, and controllable biotransformation synthesis route for the industrial production of rare ginsenosides Rk1 and Rg5.

[0051] This invention provides a new method for the green and large-scale preparation of rare ginsenosides, which is of great significance for filling the existing technological gap and promoting the high-value utilization of ginsenosides.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Leuconostoc mesenteroides subsp. enterica ( Leuconostoc mesenteroides subsp. mesenteroides The use of BT708 in the preparation of rare ginsenosides from fermented ginsenoside Rb1 is characterized by, The enteromembranous subsp. *Leuconostoc mesenteroides* ( Leuconostoc mesenteroides subsp. mesenteroides BT708 was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number CCTCC NO: M20251675; the rare ginsenosides mentioned are Rk1 and Rg5.

2. The *Leuconostoc mesenteroides* subsp. *enteroides* according to claim 1 ( Leuconostoc mesenteroides subsp. mesenteroides The use of BT708 in the preparation of rare ginsenosides from fermented ginsenoside Rb1 is characterized by, The ginsenoside Rb1 in Leuconostoc mesenteroides subsp. enteromembranosus ( Leuconostoc mesenteroides subsp. mesenteroides BT708 hydrolyzes the glucose at the C-20 position of ginsenoside Rb1 molecule and then dehydrates it to generate rare ginsenosides.

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