Application of total saponins containing ginsenoside Rh2 prepared by fermentation conversion of plant lactobacillus GLP40 in cosmetics for resisting skin aging

Abstract

The invention relates to the technical field of cosmetics, in particular to lactobacillus plantarum GLP40 and application of the lactobacillus plantarum GLP40 in preparation of ginsenoside Rh2 through fermentation conversion in the cosmetics, the lactobacillus plantarum GLP40 is preserved in China Center for Type Culture Collection on March 9, 2022, and the preservation number of the lactobacillus plantarum GLP40 is CCTCC (China Center for Type Culture Collection) NO: M2022222. The total saponins containing the rare ginsenoside Rh2 are applied to cosmetics, and the functions of resisting oxidation, scavenging free radicals and resisting skin aging of products can be remarkably improved. The cosmetic has good stability, safety and efficacy, and can be widely applied to the fields of high-end cosmetics for wrinkle resistance, repairing, moisturizing and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an application of ginsenoside Rh2 prepared by fermentation and conversion of Lactobacillus plantarum GLP40 in cosmetics. Background Art

[0002] Ginseng, a traditional Chinese beauty and skincare product, features in prescriptions in classic medical texts such as Qianjin Fang and Shengji Zonglu. Since the 1980s, it has been widely used in cosmetics and is currently one of the most widely used botanical cosmetic ingredients. Its whitening, anti-wrinkle, and anti-aging properties are widely recognized. Modern pharmacological studies have shown that ginsenosides can activate the expression of type I collagen synthase by increasing the phosphorylation of Smad2, a key transcription factor involved in type I collagen synthesis. Furthermore, ginsenosides can also antagonize aging-induced oxidative stress by reducing malondialdehyde (MDA) activity and increasing superoxide dismutase (SOD) levels, thereby exerting anti-aging effects. However, cosmetics are applied externally, and the skin's absorption of most ingredients is limited. Studies have shown that the skin permeability of ginseng's active ingredients is low, with significant variations among different ingredients. Saponins with smaller molecular weight and lower polarity have higher skin permeability, for example, ginsenoside Rg1 > notoginsenoside R1 > ginsenoside Rb1. Therefore, developing rare ginsenosides with higher skin permeability, such as ginsenoside Rh2, is of great significance for enhancing the efficacy of ginseng cosmetics.

[0003] At present, the preparation methods of rare ginsenosides mainly include acidic or alkaline hydrolysis, but these methods have problems such as low specificity, harsh reaction conditions, and difficulty in recycling waste liquids, resulting in environmental pollution and increased production costs. In recent years, microbial fermentation has gradually attracted attention due to its advantages such as high efficiency, environmental protection, and safety. Studies have shown that certain lactic acid bacteria (such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, etc.) can transform ginsenosides through fermentation to produce rare ginsenosides such as Rg3, F2, and CK. However, most of these studies focus on the conversion of a few saponins and lack an efficient preparation process for ginsenoside Rh2.

[0004] This invention, for the first time, utilizes Lactobacillus plantarum GLP40 to ferment and convert total ginsenosides, efficiently producing a rare saponin containing ginsenoside Rh2. This process not only addresses the shortcomings of traditional methods but also significantly increases the yield and purity of ginsenoside Rh2 by optimizing fermentation conditions. Furthermore, the invention further utilizes total saponins containing ginsenoside Rh2 in anti-aging cosmetics, leveraging its high skin permeability and anti-aging activity, providing a novel, highly efficient, environmentally friendly, and safe raw material for the cosmetics industry. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for preparing total saponins containing ginsenoside Rh2 by fermentation and conversion by Lactobacillus plantarum GLP40 and applying the same in cosmetics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The Lactobacillus plantarum GLP40 of the present invention has been deposited in the China Center for Type Culture Collection on March 9, 2022, with a deposit number of CCTCC NO: M2022222.

[0008] The invention discloses an application of Lactiplantibacilus plantarum GLP40 in the fermentation and conversion preparation of total saponins containing ginsenoside Rh2.

[0009] As a preferred embodiment, the method comprises the following steps:

[0010] A Lactobacillus plantarum GLP40 bacterial suspension was prepared and inoculated into a sterilized liquid fermentation medium. A filter-sterilized ginsenoside extract was added for co-fermentation. The resulting fermentation product was sequentially concentrated under reduced pressure at low temperature and freeze-dried to obtain a dry product containing ginsenoside Rh2. The dry product was identified by HPLC.

[0011] As a preferred embodiment, the preparation method of the Lactobacillus plantarum GLP40 bacterial suspension is as follows:

[0012] Lactobacillus plantarum GLP40 was inoculated into liquid MRS medium and cultured at 37-45°C for 16-25 h. The culture was centrifuged at 4000-8000 rpm for 5-10 min at 4-10°C to collect the bacterial precipitate. The viable bacterial count was adjusted to 1.0×10 9 ~1.0×10 10 CFU / ml, and obtain the Lactiplantibacilus plantarum GLP40 bacterial suspension.

[0013] As a preferred embodiment, the preparation method of the ginseng total saponin extract is as follows:

[0014] Ginseng root powder was extracted with hot water at 80-100°C, and the obtained extract was concentrated under reduced pressure and freeze-dried to obtain a crude saponin extract. The crude extract was then chromatographed on a D101 macroporous resin column and eluted with different concentrations of ethanol. The eluate was collected, the solvent was recovered, and the extract was freeze-dried to obtain a ginsenoside extract.

[0015] As a preferred embodiment, the liquid fermentation medium comprises: 2-5 g / L glucose, 1-3 g / L soybean oligopeptide, pH 6.0-7.0, and is sterilized at 121° C. for 15 min.

[0016] As a preferred embodiment, in the liquid fermentation medium, the addition amount of the ginsenoside total saponin extract is 10-20 g / L, and the inoculation amount of the Lactobacillus plantarum GLP40 bacterial suspension is 10-30 ml / L.

[0017] As a preferred embodiment, the temperature of the co-fermentation culture is 37-42° C., and the fermentation is allowed to proceed statically for 7-14 days.

[0018] As a preferred embodiment, the fermentation product is freeze-dried to obtain total saponins containing ginsenoside Rh2.

[0019] As a preferred embodiment, the specific process of identifying the dried product by HPLC is as follows:

[0020] The HPLC analysis conditions for ginsenosides were as follows: an Agilent pursuit5 SB-C18 column, an injection volume of 20 μL, a flow rate of 1 mL / min, a column temperature of 30°C, and a detection wavelength of 203 nm; the mobile phases were A: water, B: acetonitrile, and the concentrations of B at 0-40 min were 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); and 88-89.80 min. 59.6~65.0%(B); 89.80~97min65%(B); 97~102min 65~75%(B); 102~110min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min 18.0%(B).

[0021] Accurately weigh the ginsenoside Rh2 standard to prepare a 0.5 mg / mL methanol solution as the standard stock solution, and dilute it to 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / mL for HPLC analysis. Record the chromatographic peak area corresponding to the standard at different concentrations. With the concentration of the reference substance (mg / mL) as the abscissa (X) and the corresponding peak area (mAU*S) as the ordinate (Y), draw the standard curve of the ginsenoside Rh2 reference substance: y=80.81x+58.2, R 2 =0.9998.

[0022] As a preferred embodiment, the specific process of using animal experiments to test the anti-skin aging ability of ginsenoside Rh2 cosmetics is as follows:

[0023] The total saponins containing ginsenoside Rh2 obtained by fermentation were dispersed in a cosmetic base to create a ginsenoside ointment containing 3-5% ginsenosides. SPF mice were randomly divided into four groups, each consisting of eight mice: a blank group, a model group, a blank matrix group, and a ginsenoside group. The mice were subcutaneously injected with 1 g / kg of D-galactose daily for 45 days, while the blank control group received an equal volume of normal saline. Before the experiment, the dorsal area of the aged mice was shaved with electric clippers (2 cm × 2 cm in all cases) and the following day, the medication was applied. The model group received no ointment, the blank matrix group received a base ointment without the active ingredient, and the ginsenoside group received a total saponin ointment containing ginsenoside Rh2 twice daily for 50 days. During the experiment, mice in each experimental group had free access to food and water.

[0024] After mice were sacrificed, dorsal skin tissue was obtained and homogenized at low temperature for later use. The activities of SOD, CAT, and GSH, as well as the MDA content, were measured using test kits to assess changes in the antioxidant capacity of each group of mice after treatment with the test drug. Elisa assays were used to measure the levels of TNF-α, IL-6, and IL-10 in the dorsal skin homogenate to evaluate the inhibitory effects of the test drug on inflammatory factors in mouse skin tissue.

[0025] Mouse skin tissue was stained with hematoxylin and eosin (H&E). After the mice were sacrificed, 1 cm × 1 cm skin tissue was obtained, fixed with 4% paraformaldehyde for 48 hours, and embedded in paraffin. The wax block was placed in a paraffin slicer for serial sectioning and dried in a 40°C constant temperature oven before use. The sections were stained with hematoxylin solution (60°C) for 20 minutes and eosin solution for 8 minutes. The sections were dried, sealed with neutral gum, and histological changes were observed under an optical microscope.

[0026] The beneficial effects of the present invention are:

[0027] The present invention screens and obtains total saponins containing ginsenoside Rh2, produced through biotransformation with Lactobacillus plantarum GLP40. Anti-skin aging cosmetics are prepared using the total saponins containing ginsenoside Rh2 obtained through fermentation and transformation as raw materials. This process offers the advantages of safety, high efficiency, and low cost, and is suitable for large-scale industrial production. The resulting ginsenoside cosmetics exhibit anti-skin aging, free radical scavenging, and antioxidant properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the HPLC chromatogram of the dry product containing ginsenoside Rh2 prepared in step (3) of Example 2 after fermentation.

[0029] Figure 2 H&E staining of mouse skin tissue (100×) DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1 Isolation, identification and preservation of strains

[0032] 1. Isolation of strains

[0033] Samples and sources used for isolated strains: Samples of traditionally fermented Northeastern sauerkraut in Yanji City, Jilin Province were collected in September 2021. The collected samples were ground and gradiently diluted, then spread on Lactobacillus selective solid culture medium plates and cultured at 37°C for 48 hours. Typical colonies on the plates (milky white, round, with neat edges, raised, opaque, and moist and smooth surfaces) were picked and streaked on MRS agar plates to isolate pure colonies. The pure cultured strains were inoculated into liquid MRS culture medium for culture, and then 60% glycerol was added and stored in a -80°C refrigerator. One of the Lactobacillus strains was named GLP40.

[0034] 2. Identification of strains

[0035] The physiological and biochemical identification results of GLP40 are as follows: Gram staining positive, catalase test negative, benzidine test negative, indole test negative, and acetylmethyl carbinol test positive; it does not hydrolyze starch, liquefy gelatin, or produce hydrogen sulfide, and ferments glucose to produce acid but not gas; it is a non-motile bacillus; it can grow at 15°C and 45°C, with the optimal growth temperature being 37-42°C; its suitable pH is 5.0-7.0; it tolerates 6.5% NaCl; GLP40 grows uniformly turbidly in liquid MRS culture medium, and the bacteria form a white precipitate after prolonged storage.

[0036] Table 1. Identification results of API 50CH (Mérieux, France) for GLP40

[0037]

[0038]

[0039] In the above table, “+” indicates that the strain has a positive reaction; “-” indicates that the strain has a negative reaction.

[0040] 16S rDNA and pheS gene sequence amplification and alignment. Genomic DNA was extracted using the CTAB method. 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACT T-3') and pheS primers (pF: 5'-CCGTGAAGAAC TGGAACA-3', pR: 5'-CCTAACCCAAAGGCAAAA-3') were designed based on conserved regions of the gene sequence. PCR amplification and product sequencing were performed. The obtained 16S rDNA and pheS sequences were then compared with sequences of known strains in the GenBank database for homology analysis using BLAST to identify the strains. Genus and species classification was performed using a criterion of greater than 99% homology between the 16S rDNA and pheS sequences. The obtained 16S rDNA sequence is shown in SEQ ID NO:1 in the sequence listing, and the obtained pheS sequence is shown in SEQ ID NO:2 in the sequence listing. The 16srDNA sequence and pheS sequence were compared and analyzed in the GenBank database using the BLAST program. It was found that the homology between GLP40 and Lactobacillus plantarum reached 99%.

[0041] Based on the above results, strain GLP40 was identified as Lactiplantibacilus plantarum.

[0042] 3. Preservation of strains

[0043] The plant lactobacillus (Lactiplantibacilus plantarum) GLP40 of the present invention has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on March 9, 2022. 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: M2022222.

[0044] Example 2: Preparation of total saponins containing ginsenoside Rh2 by fermentation and conversion with Lactiplantibacilus plantarum GLP40

[0045] 1. Preparation of Lactiplantibacilus plantarum GLP40 bacterial suspension

[0046] Lactobacillus plantarum GLP40 (hereinafter referred to as GLP40) was inoculated into liquid MRS medium and incubated at 37°C for 16 h. The culture was centrifuged at 4°C and 6000 rpm for 8 min, and the bacterial precipitate was collected. The culture was suspended in an aqueous solution of fermentation sterilization medium and the viable cell count was adjusted to 1.0 × 10 9 CFU / mL to obtain GLP40 bacterial suspension.

[0047] 2. Preparation of ginsenoside extract

[0048] Ginseng root powder (purchased from Shaanxi Yunling Biotechnology Co., Ltd.) was extracted with hot water (80°C), and the resulting extract was concentrated under reduced pressure and freeze-dried to obtain a crude saponin extract. The crude extract was then chromatographed on a D101 macroporous resin column and eluted with different concentrations of ethanol (0-90%). The eluate was collected, the solvent was recovered, and the extract was freeze-dried to obtain a ginsenoside extract.

[0049] 3. Fermentation conversion

[0050] The liquid fermentation medium contained 3 g / L glucose and 1 g / L soybean oligopeptide, maintained at pH 6.6, and sterilized at 121°C for 15 minutes. The liquid fermentation medium was supplemented with 10 g / L of ginsenoside extract and inoculated with a 10 ml / L suspension of GLP40 bacteria. The culture was then incubated at 37°C for 14 days. After fermentation, the resulting product was concentrated under reduced pressure and freeze-dried to obtain total saponins containing ginsenoside Rh2.

[0051] 4. The synthetic route of ginsenoside conversion by Lactiplantibacilus plantarum GLP40 fermentation is as follows: ginsenosides Rb1 and Rd are hydrolyzed by β-D-glucosidase produced by Lactiplantibacilus plantarum GLP40 to produce ginsenoside 20(S)-Rg3, which is further hydrolyzed by β-D-glucosidase to produce ginsenoside Rh2.

[0052] Example 3 Identification and Quantification of Ginsenoside Rh2

[0053] 1. High performance liquid chromatography (HPLC) analysis

[0054] Accurately weigh the ginsenoside Rh2 standard to prepare a 0.5 mg / mL methanol solution as the standard stock solution, and dilute it to 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / mL for HPLC analysis. Record the chromatographic peak area corresponding to the standard at different concentrations. With the concentration of the reference substance (mg / mL) as the abscissa (X) and the corresponding peak area (mAU*S) as the ordinate (Y), draw the standard curve of the ginsenoside Rh2 reference substance: y=80.81x+58.2, R 2 =0.9998.

[0055] The ginsenoside extract prepared in step (2) of Example 2 and the dry product containing ginsenoside Rh2 prepared in step (3) of Example 2 were dissolved in methanol, filtered through a 0.22 μm microporous filter membrane, and then used for HPLC chromatography analysis.

[0056] HPLC chromatographic analysis method: The chromatographic column was Agilent pursuit5 SB-C18 column, the injection volume was 20 μL, the flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 203 nm. Mobile phase: A: water, B: acetonitrile, 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).

[0057] 2. Identification and Quantification Results

[0058] Analysis of changes in ginsenoside content in the product before and after fermentation revealed that the retention time of ginsenoside Rh2 under HPLC conditions was consistent with that of a ginsenoside standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., HPLC ≥98%). This demonstrates that ginsenosides Rb1 and Rd in the total ginsenoside extract can be converted into ginsenoside Rh2 through fermentation with Lactobacillus plantarum GLP40. Furthermore, calculation using the standard curve revealed a ginsenoside Rh2 content of 62.37 mg / g.

[0059] Example 4 Evaluation of the anti-aging effect of ointment containing ginsenoside Rh2 total saponin

[0060] 1. Experimental Animal Handling

[0061] The total saponins containing ginsenoside Rh2 prepared in step (3) of Example 2 were dispersed in a cosmetic matrix to prepare a ginsenoside ointment, wherein the content of ginsenoside was 1%. SPF mice were randomly divided into 4 groups, each with 8 mice, namely a blank group, a model group, a blank matrix group, and a ginsenoside group. The mice were subcutaneously injected with 1g / kg of D-galactose every day for 45 consecutive days, and the blank control group was injected with an equal volume of normal saline. Before the experiment, the back area of the mice was shaved with electric scissors (area 2cm×2cm), and the medicine was applied the next day. Among them, the model group was not applied with ointment, the blank matrix group was applied with a matrix ointment without ginsenoside Rh2, and the ginsenoside group was applied with a matrix ointment containing ginsenoside Rh2, twice a day, for 50 consecutive days. During the experiment, mice in each experimental group were free to eat and drink water.

[0062] 2. Detection of anti-aging indicators in mouse skin tissue

[0063] Table 2. Effects of ginsenosides on oxidative stress indicators in mouse skin

[0064]

[0065] To determine the effects of total ginsenosides on oxidative stress markers in mouse skin tissue, kits were used to measure the activities of SOD, CAT, and GSH, as well as the MDA content, in mouse dorsal skin tissue homogenates. The results (Table 2) showed that, under the treatment with ginsenoside Rh2, the activities of SOD, CAT, and GSH-Px in the skin tissue of mice in the ginsenoside group were significantly increased compared with those in the model group and the blank matrix group. Furthermore, the ginsenoside group significantly inhibited the increase in MDA caused by D-galactose. Elisa assays were used to determine the levels of TNF-α, IL-6, and IL-10 in mouse dorsal skin tissue homogenates. The results (Table 3) showed that ginsenosides effectively reduced the expression of inflammatory factors in mouse skin tissue, suggesting that ginsenosides can achieve anti-skin aging effects by improving oxidative stress and suppressing inflammation.

[0066] Table 3. Effects of ginsenosides on inflammatory cytokine levels in mouse skin

[0067]

[0068]

[0069] 3. Pathological changes of mouse skin tissue

[0070] The mouse skin tissue was stained with hematoxylin and eosin (H&E) to investigate the pathological changes of mouse skin tissue. Figure 2It can be seen that the back skin of the blank group mice showed a complete skin structure with normal cell nuclear morphology; the skin appendages (hair follicles, sebaceous glands, sweat glands, etc.) had normal morphology and were large in number. The stratum corneum of the model group mice was significantly shed, and the structural degradation of each layer was more serious; the skin appendages degenerated, had abnormal morphology, and decreased in number. The blank matrix group did not have a significant improvement effect, but after treatment with ginsenoside ointment, the thickness of the mouse skin epidermis increased, the morphology of the skin appendages was significantly improved, and the number also increased. This shows that ginsenosides have a good effect on improving the changes in skin tissue morphology caused by aging.

[0071] The present invention discloses a plant lactobacillus GLP40 and its application in the fermentation conversion preparation of ginsenoside Rh2. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. Lactobacillus plantarum GLP40, characterized in that This strain was deposited in the China Center for Type Culture Collection on March 9, 2022, with the deposit number: CCTCC NO: M2022222.

2. Use of ginsenoside Rh2 prepared by fermentation and conversion by Lactobacillus plantarum GLP40 as claimed in claim 1 in cosmetics.

3. The use according to claim 2, characterized in that The following steps are involved: A Lactobacillus plantarum GLP40 bacterial suspension was prepared and inoculated into a sterilized liquid fermentation medium. A ginsenoside total saponin extract that had been sterilized by filtration was added for co-fermentation and culture. The resulting fermentation product was sequentially subjected to low-temperature reduced-pressure concentration, freeze-dried, extracted, and centrifuged. The resulting supernatant was then subjected to reduced-pressure solvent recovery and freeze-dried to obtain a dry product containing ginsenoside Rh2. The content of the dry product was determined using HPLC. The total saponins containing ginsenoside Rh2 obtained by fermentation were configured in a cosmetic base, and its anti-skin aging effect was detected using an animal model.

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