Bacillus lactis BT810 and application thereof in preparation of rare ginsenosides from fermented ginseng flower buds

Abstract

Lactobacillus plantarum BT810 and its application in the fermentation of ginseng buds to prepare rare ginsenosides belong to the field of fermentation engineering. Lactiplantibacillus plantarum BT810 was deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2025, with accession number CCTCC NO: M20251845. This invention uses ginseng flower bud water extract as a fermentation substrate, inoculated with *Lactobacillus plantarum* BT810 for static fermentation. The β-glucosidase secreted by BT810 selectively hydrolyzes ginsenoside Re in the ginseng flower bud water extract, converting it directionally into Rg2 and Rg4. This invention has the advantages of extremely low byproducts, high conversion rate, mild reaction conditions, low preparation cost, and ease of scaling up production, providing a convenient route for the large-scale, low-cost, and efficient co-preparation of rare ginsenosides Rg2 and Rg4.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and more specifically, to a Lactiplantibacillus plantarum BT810 and its application in the fermentation of ginseng flower buds to prepare rare ginsenosides. Background Technology

[0002] Rare ginsenosides Rg2 and Rg4 both belong to the protopanaxadiol type of ginsenosides. They differ only slightly in the number and linkage of side-chain sugars, yet exhibit complementary and synergistic pharmacological activities: rare ginsenoside Rg2 has a significant advantage in rapidly improving cerebral ischemia-reperfusion injury and enhancing cognitive function; rare ginsenoside Rg4 shows significant activity in antitumor, anti-inflammatory, and neuroprotective effects. However, the natural abundance of both ginsenosides in Panax ginseng plants is extremely low (Rg2 < 0.01%, Rg4 < 0.005%, w / w).

[0003] Traditional methods for obtaining ginsenosides rely on large-scale extraction combined with repeated column chromatography, resulting in low raw material utilization and significant solvent consumption and environmental pollution. This makes it difficult to meet the large-scale demand for high-purity rare ginsenosides Rg2 and Rg4 in functional foods, special medical foods, and innovative drugs. Furthermore, while chemical methods (acid / alkali hydrolysis or high-temperature pyrolysis) can rapidly prepare ginsenosides Rg2 and Rg4 under acidic or alkaline conditions, the reaction process is violent and lacks selectivity, often accompanied by multiple side reactions such as desaccharification, dehydroxylation, and isomerization. This can easily lead to the simultaneous formation of mixed saponins such as Rd, Rg2, Rh1, and Rg4. Subsequent purification requires multiple recrystallizations combined with high-performance liquid chromatography, resulting in low yields and the generation of large amounts of saline organic wastewater, placing a heavy environmental burden on the industry. Therefore, developing green, mild, and highly selective ginsenoside biotransformation routes has become an industry consensus. However, current biotransformation research (enzyme / microbial transformation methods) mainly focuses on transformation pathways such as Rb1→Rd, Rg3→CK, and Rg1→Rh1. There are almost no reports on directional transformation systems for Re→Rg2→Rg4, meaning a complete process for simultaneously obtaining two high-value products, rare ginsenosides Rg2 and Rg4, has not yet been established. Furthermore, the enzymes or recombinant bacterial systems used in existing biotransformation research suffer from high costs and extremely unstable enzyme sources, and cannot achieve efficient co-production of rare ginsenosides Rg2 and Rg4.

[0004] Lactiplantibacillus plantarum, rich in glycoside hydrolases such as β-glucosidase and α-rhamnosidase, has been used for the conversion of Rb1 to Rd and Rg3, but there are no reports of using Lactiplantibacillus plantarum to efficiently and directionally convert ginsenoside Re in ginseng flower buds into rare ginsenosides Rg2 and Rg4. Summary of the Invention

[0005] To address the problems of low raw material utilization and yield, violent reaction process and poor selectivity, complex byproducts, environmental pollution, high cost, and inability to achieve efficient co-preparation of rare ginsenosides Rg2 and Rg4 in existing ginsenoside preparation technologies, this invention provides a *Lactiplantibacillus plantarum* BT810 and its application in the fermentation of ginseng buds to prepare rare ginsenosides. This invention achieves efficient co-preparation of rare ginsenosides Rg2 and Rg4, with advantages such as low cost, high raw material utilization and yield, mild reaction, strong selectivity, few byproducts, and environmental friendliness.

[0006] The technical solution adopted by this invention to solve the technical problem is as follows:

[0007] First, this invention provides a *Lactiplantibacillus plantarum* BT810. This *Lactiplantibacillus plantarum* BT810 was deposited at the China Center for Type Culture Collection on August 18, 2025, with accession number CCTCC NO: M20251845.

[0008] Secondly, this invention provides an application of Lactiplantibacillus plantarum BT810 in the preparation of rare ginsenosides from fermented ginseng flower buds.

[0009] Among them, the rare ginsenosides are Rg2 and Rg4.

[0010] According to the present invention, the biotransformation synthesis route of the rare ginsenosides is as follows: ginsenoside Re in the aqueous extract of ginseng flower buds is selectively hydrolyzed by β-glucosidase secreted by Lactobacillus plantarum BT810, and then one molecule of water is removed to directionally generate rare ginsenosides Rg2 and Rg4.

[0011] Finally, this invention provides a method for preparing rare ginsenosides Rg2 and Rg4 by fermenting ginseng flower buds using Lactiplantibacillus plantarum BT810, which mainly includes the following steps:

[0012] (1) After activating Lactobacillus plantarum BT810, transfer it to MRS liquid medium at an inoculation rate of 1-5% (v / v) and incubate at 37-40 ℃ for 18-22 h until the viable count reaches 2.0×10⁻⁶. 9 Centrifuge at 4–10 °C and 6000–8000 rpm for 5–10 min, collect the bacterial pellet, wash twice with sterile physiological saline, resuspend in an equal volume of sterile physiological saline, and adjust the viable count to 1.0 × 10⁻¹. 9 ~5.0×10 9 CFU·mL⁻¹ yields a suspension of Lactobacillus plantarum BT810.

[0013] (2) Take dried ginseng flower buds and add deionized water at a material-to-liquid ratio of 1:15~1:20 (w / v). Extract by hot reflux at 90℃~100℃. Filter, combine the filtrates, and concentrate under reduced pressure. Purify the ginseng flower bud extract by D101 macroporous resin column chromatography, eluenting with 0, 30%, 50% and 70% ethanol in sequence. Collect the 30% and 50% ethanol eluents, filter, combine the filtrates, recover the ethanol under reduced pressure, and freeze-dry to obtain the ginseng flower bud water extract.

[0014] (3) Add glucose and yeast extract to the water extract of ginseng flower buds, sterilize and cool, inoculate with Bacillus plantarum BT810 bacterial suspension at an inoculation rate of 1-5% (v / v), and incubate at 37-40 ℃ for 14-21 days; after fermentation, freeze dry the obtained fermentation product under vacuum to obtain the dried product.

[0015] (4) The rare ginsenosides Rg2 and Rg4 contained in the dried product were identified by high performance liquid chromatography.

[0016] The beneficial effects of this invention are:

[0017] This invention addresses the problem of efficient co-preparation of rare ginsenosides Rg2 and Rg4, providing a preparation process with mild reaction conditions, high conversion rate, environmental friendliness, strong selectivity, few byproducts, and suitability for industrial production. This invention involves screening a strain of *Lactobacillus plantarum* BT810, using ginseng flower bud water extract as a fermentation substrate, and inoculating it with *Lactobacillus plantarum* BT810 for static fermentation. During fermentation, β-glucosidase secreted by *Lactobacillus plantarum* BT810 selectively hydrolyzes ginsenoside Re in the ginseng flower bud water extract, directionally converting it into rare ginsenosides Rg2 and Rg4.

[0018] This invention utilizes a single-step static fermentation process to directionally convert ginsenoside Re in ginseng flower bud water extract into rare ginsenosides Rg2 and Rg4, with extremely low byproducts and high conversion rate.

[0019] The reaction conditions of this invention are mild, requiring no special equipment or reagents, which significantly reduces the preparation cost and makes it easy to scale up production. This provides a convenient way for the large-scale, low-cost, and efficient co-preparation of rare ginsenosides Rg2 and Rg4. Attached Figure Description

[0020] Figure 1 The HPLC chromatograms of ginseng flower bud water extract before and after fermentation with Lactobacillus plantarum BT810 are shown. Detailed Implementation

[0021] 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0023] Chinese name: Lactobacillus plantarum BT810;

[0024] Latin name: Lactiplantibacillus plantarum BT810;

[0025] Accession number: CCTCC NO: M20251845;

[0026] Deposit date: August 18, 2025;

[0027] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0028] Location of storage: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center).

[0029] Example 1: Screening of Lactobacillus plantarum BT810

[0030] 1. Isolation and initial screening of bacterial strains;

[0031] Take the naturally fermented Northeastern sauerkraut fermentation liquid, shake it thoroughly, and then make 10⁻¹~10⁻ 5Serial dilutions were performed, with 100 μL of the diluted solution spread onto MRS solid medium (pH 5.7) supplemented with 0.5% CaCO3, and incubated anaerobically at 37 °C for 48 h. Single, milky-white colonies with distinct calcium dissolution zones and irregular edges were picked and streaked three times onto MRS plates containing 0.02% natamycin for purification. The purified colonies were subjected to Gram staining, microscopic examination, and catalase assay. Gram-positive, catalase-negative, rod-shaped, or short rod-shaped strains were selected for preservation. The obtained pure cultures were inoculated into liquid MRS medium, incubated statically at 37 °C overnight, then 30% glycerol was added, and the culture was stored at -80 °C. One of these strains was named BT810.

[0032] 2. Identification of the strain;

[0033] Genomic DNA was extracted from strain BT810 using a bacterial genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal primers for 16S rRNA. The amplified products were analyzed by 1% agarose gel electrophoresis and then sequenced. The obtained 16S rDNA sequence, compared with the 16S rRNA gene sequence of *Lactiplantibacillus plantarum* ATCC14917 (GenBank accession number: AF080101.1) by NCBI BLAST, showed 99.93% homology. Therefore, based on these identification results, strain BT810 was identified as *Lactiplantibacillus plantarum*. Strain BT810 was then biopreserved.

[0034] In this embodiment, the sequence information of the universal primer for 16S rRNA is as follows:

[0035] The forward primer is 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0036] The reverse primer is 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0037] In this embodiment, the PCR amplification conditions are as follows:

[0038] The PCR reaction system was 50 μL. The amplification conditions were: 95 ℃ for 5 min; 95 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 90 s, for a total of 35 cycles; and 72 ℃ for 10 min.

[0039] In this embodiment, the 16S rDNA sequence information of strain BT810 is as follows:

[0040]

[0041] Example 2: Preparation of Lactobacillus plantarum BT810 bacterial suspension

[0042] Lyophilized *Lactobacillus plantarum* BT810 was streaked onto MRS agar plates and activated by static incubation at 37 ℃ for 18 h. A single colony was picked and inoculated into 50 mL of MRS liquid medium and incubated at 37 ℃ for 12 h to obtain primary seed culture. The primary seed culture was then transferred to 300 mL of MRS liquid medium at a 1% (v / v) inoculation rate and incubated at 37 ℃ for 18 h until the viable count reached 2.0 × 10⁻⁶. 9 At CFU·mL⁻¹, the bacterial pellet was collected by centrifugation at 4 ℃ and 8000 rpm for 5 min. The pellet was washed twice with sterile physiological saline and then resuspended in an equal volume of sterile physiological saline to adjust the viable count to 1.0 × 10⁻¹. 9 -5.0×10 9 CFU·mL⁻¹ yields a suspension of *Lactobacillus plantarum* BT810, which is stored at 4 °C for later use.

[0043] Example 3: Preparation of water extract of ginseng flower buds

[0044] Take 500g of dried ginseng flower buds, add deionized water at a material-to-liquid ratio of 1:15 (w / v), and extract twice by hot reflux at 100 ℃, 1.5 h each time; filter, combine the filtrates, and concentrate under reduced pressure to 5000 mL. Purify the ginseng flower bud extract by D101 macroporous resin column chromatography, eluting successively with 0%, 30%, 50%, and 70% ethanol, collecting the 30% and 50% ethanol eluates, filtering, combining the filtrates, recovering the ethanol under reduced pressure, and freeze-drying to obtain the ginseng flower bud aqueous extract.

[0045] Example 4: Targeted preparation of rare ginsenosides Rg2 and Rg4 from ginseng flower bud water extract using *Lactobacillus plantarum* BT810 bacterial suspension fermentation.

[0046] Glucose and yeast extract (3 g / L glucose, 2 g / L yeast extract) were added to the ginseng flower bud water extract obtained in Example 3 as a liquid fermentation medium, and the mixture was sterilized at 121 °C for 15 min. After cooling to room temperature, a *Lactobacillus plantarum* BT810 bacterial suspension obtained in Example 2 was inoculated at a 3% (v / v) inoculum and cultured statically at 37 °C for 14 days. After fermentation, the fermentation product was freeze-dried under vacuum (-80 °C, 5 Pa) to obtain the dried product.

[0047] Example 5: Identification of rare ginsenosides in the dried product

[0048] 1. Sample preparation;

[0049] Accurately weigh 5.0 mg each of ginsenoside Re, Rg2 and Rg4 standards (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%) and the dried product obtained in Example 4, and use the uninoculated ginseng flower bud water extract obtained in Example 3 as a blank control. Dissolve in chromatographic grade methanol by sonication (40 kHz, 25 ℃, 10 min) and dilute to 5 mL. Filter through a 0.22 µm organic microporous membrane and keep the filtrate for later use.

[0050] 2. HPLC chromatographic conditions;

[0051] Instrument: Agilent 1260 Infinity II high performance liquid chromatograph with evaporative light scattering detector (ELSD).

[0052] Column: Agilent Pursuit 5 SB-C18 (4.6 × 250 mm, 5 µm).

[0053] Column temperature: 30 ℃; flow rate: 1.0 mL min⁻¹; detection wavelength: 203 nm; injection volume: 20 µL.

[0054] Dynamic phase: Phase A is water, Phase B is acetonitrile; 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); 102~110min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min 18.0%(B).

[0055] 3. Result determination;

[0056] like Figure 1 As shown, under the above HPLC chromatographic conditions, the retention times of the reference standards were as follows: ginsenoside Re 36.764 min, ginsenoside 20-(S) Rg2 54.90 min, 20-(R) Rg2 55.12 min, and ginsenoside Rg4 78.10 min.

[0057] Qualitative and quantitative analyses of the ginsenoside components in the dried products before and after fermentation showed that the dried products obtained in Example 2 had retention times consistent with the standards for rare ginsenosides Rg2 and Rg4, while the blank control (uninoculated ginseng flower bud water extract) showed no significant absorption peaks in the 40-42 min range. These results indicate that the present invention successfully prepared rare ginsenosides Rg2 and Rg4 from ginseng flower bud water extract using *Lactobacillus plantarum* BT810 suspension fermentation, thus successfully converting Re in the ginseng flower bud water extract into rare ginsenosides Rg2 and Rg4. In summary, after fermentation and conversion by *Lactobacillus plantarum* BT810, the content of ginsenoside Re in the ginseng flower bud water extract was significantly reduced, and ginsenoside Re was efficiently converted into rare ginsenosides Rg2 and Rg4. Furthermore, calculations using the standard curve showed that the contents of rare ginsenosides 20-(S / R)Rg2 and Rg4 were 126.7 mg / g and 87.5 mg / g, respectively.

[0058] In this invention, the biotransformation synthesis route for rare ginsenosides Rg2 and Rg4 is as follows:

[0059] Ginsenoside Re in the aqueous extract of ginseng flower buds is selectively hydrolyzed by β-glucosidase secreted by Lactobacillus plantarum BT810, which then removes one molecule of water to generate rare ginsenosides Rg2 and Rg4.

[0060] 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. A method for preparing rare ginsenosides Rg2 and Rg4 by fermenting ginseng flower buds using Lactiplantibacillus plantarum BT810, characterized in that, The Lactiplantibacillus plantarum BT810 was deposited at the China Center for Type Culture Collection on August 18, 2025, with accession number CCTCC NO: M20251845; the method includes the following steps: (1) After activating Lactobacillus plantarum BT810, transfer it to MRS liquid medium at an inoculation rate of 1-5% (v / v) and incubate at 37-40℃ for 18-22 h until the viable count reaches 2.0×10⁻⁶. 9 Centrifuge at 4–10 °C and 6000–8000 rpm for 5–10 min, collect the bacterial pellet, wash twice with sterile physiological saline, resuspend in an equal volume of sterile physiological saline, and adjust the viable count to 1.0 × 10⁻¹. 9 ~5.0×10 9 CFU·mL⁻¹ yields a suspension of Lactobacillus plantarum BT810. (2) Take dried ginseng flower buds and add deionized water at a material-to-liquid ratio of 1:15~1:20 (w / v). Extract by hot reflux at 90℃~100℃. Filter, combine the filtrates, and concentrate under reduced pressure. Purify the ginseng flower bud extract by D101 macroporous resin column chromatography, eluenting with 0, 30%, 50% and 70% ethanol in sequence. Collect the 30% and 50% ethanol eluents, filter, combine the filtrates, recover the ethanol under reduced pressure, and freeze-dry to obtain the ginseng flower bud water extract. (3) Add glucose and yeast extract to the water extract of ginseng flower buds, sterilize and cool, inoculate with Bacillus plantarum BT810 bacterial suspension at an inoculation rate of 1-5% (v / v), and incubate at 37-40 ℃ for 14-21 days; after fermentation, freeze dry the obtained fermentation product under vacuum to obtain the dried product. (4) The rare ginsenosides Rg2 and Rg4 contained in the dried product were identified by high performance liquid chromatography.

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