Plantaricumbacter plantarum, method for fermenting red panax notoginseng branchlets, red panax notoginseng branchlet fermentation product and application thereof

Abstract

The present application provides a plant lactobacillus plantarum and its fermentation method of red ginseng rootlet, red ginseng rootlet fermentation product and its application, relates to ginseng fermentation technical field, and the plant lactobacillus plantarum (Lactobacillus plantarum) is named KMHD-2, has been preserved in China Microorganism Culture Collection Center on August 28, 2023, and the preservation number is CGMCC No.28270.The present application utilizes the plant lactobacillus plantarum to ferment red ginseng rootlet, obtains fermentation liquor, then extracts, to obtain red ginseng rootlet fermentation product, solves the technical problems that the fermentation period of using strain in the prior art is too long, and the conversion rate of rare ginsenoside is not high, achieves the technical effects that the use value of red ginseng rootlet is effectively improved, red ginseng rootlet fermentation product can significantly promote zebra fish type I collagen gene expression, and red ginseng rootlet fermentation product can be used for cosmetics to effectively promote skin anti-wrinkle.

Description

Technical Field

[0001] This invention relates to the field of ginseng fermentation technology, and in particular to a strain of Lactobacillus plantarum and its method for fermenting red ginseng rootlets, the fermentation products of red ginseng rootlets and their applications. Background Technology

[0002] Ginseng is a traditional and precious Chinese medicinal herb, rich in chemical components and possessing a wide range of biological activities, giving it high health and medicinal value. Ginseng has specific requirements for its growing environment; it is a plant that prefers cool, moist conditions and tolerates shade. It is susceptible to waterlogging and drought, and dislikes direct sunlight, making its growing conditions quite demanding. Furthermore, ginseng cultivation is subject to crop rotation restrictions; even when transplanted, the land needs 3-4 years of rest period, typically around 7 years. Therefore, how to effectively utilize ginseng resources is a hot research topic.

[0003] Ginsenosides are the main active ingredients of ginseng. Currently, over 180 species of ginsenosides have been discovered from plants in the genus *Panax*. The basic structural composition of ginsenosides consists of a steroidal skeleton with various glycosyl groups (glucose (GLC), rhamnose (RHA), xylose (XY), and arabinose (ARA) linked at C3, C6, and C20). Ginsenosides first isolated from the root are named with an "R" prefix followed by a letter and a numerical value indicating ascending chromatographic polarity. For example, Ra is the least polar ginsenoside originating from the root, followed by Rb1, while Rg3 is more polar than Rg1. The major glycosylated ginsenosides, such as Rb1, Rb2, Rc, Rd, Re, and Rg1, account for over 80% of the total ginsenosides in wild ginseng. Rare ginsenosides, such as CK, Rh1, Rh2, Rg3, Rk1, and Rg5, have fewer glycosyl groups. The type, number, and binding sites (C-3, C-6, C-20) of glycosyl groups have been shown to affect biological activity. For example, the antitumor activity of ginsenosides with different numbers of glycosyl groups follows the order: aglycone saponins > monosaccharide saponins > disaccharide saponins > trisaccharide saponins. Deglycosylated secondary ginsenosides are present in very small amounts in Panax notoginseng plants, but deglycosylated ginsenosides have greater pharmacological activity and bioavailability than glycosylated ginsenosides.

[0004] Red ginseng is a processed form of ginseng, prepared through processes such as soaking, washing, steaming, sun-drying, and baking. The content of ginsenosides Rg3, Rg5, Rk1, and 20(S)-PPD in processed red ginseng is significantly higher than that in raw ginseng. Studies have shown that oral administration of red ginseng powder can significantly reduce the roughness of wrinkles around the eyes and the total skin damage score; other studies have demonstrated through animal experiments that topical red ginseng extract can significantly inhibit wrinkles caused by UVB exposure, and also significantly inhibit the increase in epidermal thickness induced by UVB irradiation. Furthermore, fluorescence and confocal microscopy analysis of the stereoisomer of ginsenoside Rg3 in UVB-irradiated keratinocyte experiments has shown that red ginseng possesses certain anti-aging effects. Traditional red ginseng products primarily utilize the main root, with red ginseng rootlets being a byproduct. The ginsenoside content in red ginseng rootlets is higher than that in the main root. Using red ginseng rootlets as the research subject can enhance the overall utilization value of red ginseng.

[0005] Meanwhile, studies have shown that fermented red ginseng has higher activity than regular red ginseng. The fermentation strains used mainly include molds, yeasts, and ginseng endophytes. However, the safety of these strains still needs further research. For example, the existing technology CN112980720 discloses a strain of ginseng endophyte Enterobacter Chengdui that can rapidly convert ginsenoside Rg1 into ginsenoside F1. However, this strain is not in the "List of Microbial Strains that Can Be Used in Food", so the fermented product cannot be directly used in the food industry and has certain limitations.

[0006] Lactic acid bacteria are widely found in nature and can be isolated from dairy products, kimchi, pickles, beer, and wine in daily life. They are widely recognized probiotics, and numerous studies have shown that they have multiple functions, such as regulating gut microbiota, controlling the growth of putrefactive bacteria in the gut, and improving food digestibility and bioavailability. *Lactobacillus plantarum* is a type of lactic acid bacteria and is also listed in the "List of Microbial Strains that Can Be Used in Food," indicating a high level of safety.

[0007] The prior art CN109536560 discloses a method for increasing the content of rare saponins in ginseng water extract, including the following steps: (1) Grinding ginseng into powder and sieving it to obtain ginseng powder, then dissolving the ginseng powder in water and treating it with ultrasound and microwave to filter out the ginseng powder residue, thereby obtaining ginseng water extract; (2) Taking the ginseng water extract obtained in step (1), adding compound bacteria and sugar source, and fermenting at 28-40℃ for 4-8 days, wherein the compound bacteria are lactic acid bacteria and acetic acid bacteria; (3) Raising the fermentation temperature to 42-55℃ and continuing fermentation for 10-20 days, and after the fermentation is completed, filtering to leave the fermentation liquid; the concentration of ginsenoside Rg3 after fermentation by this method is 0.043 mg / L, an increase of 224.87%; however, this method has the problems of excessively long fermentation cycle and low conversion rate.

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] One of the objectives of this invention is to provide a strain of Lactobacillus plantarum that possesses in vitro antioxidant capacity. This strain was deposited on August 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28270.

[0010] The second objective of this invention is to provide a method for fermenting red ginseng rootlets with Lactobacillus plantarum, which not only has a short fermentation cycle, but also effectively increases the yield of rare ginsenosides Rg3, Rg5, Rk1 and Rh1, thereby effectively enhancing the use value of red ginseng rootlets.

[0011] The third objective of this invention is to provide a fermented product of red ginseng rootlets, which has a high content of rare ginsenosides and can be applied in the food and cosmetic fields.

[0012] The fourth objective of this invention is to provide an application of fermented red ginseng rootlets that can effectively achieve anti-wrinkle and firming effects on the skin, resulting in outstanding application effects.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0014] Firstly, a strain of Lactobacillus plantarum, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28270, is a living cell form and possesses antioxidant functions in vitro.

[0015] Furthermore, the *Lactobacillus plantarum* is derived from sauerkraut.

[0016] Secondly, a method for fermenting red ginseng rootlets with Lactobacillus plantarum includes the following steps:

[0017] Using red ginseng rootlets as a substrate, fermentation is carried out using any of the above-mentioned *Lactobacillus plantarum* to obtain a fermentation broth. The fermentation broth is then extracted to obtain the red ginseng rootlet fermentation product.

[0018] Furthermore, the addition of red ginseng rootlets as a substrate includes the addition of a suspension made from red ginseng rootlet powder and / or a red ginseng rootlet extract;

[0019] Preferably, the red ginseng root powder has a mesh size of 40-80.

[0020] Furthermore, the amount of *Lactobacillus plantarum* added is 3-10%;

[0021] Preferably, the solvent of the suspension includes water;

[0022] Preferably, the ratio of red ginseng root powder to water in the suspension is 1:10;

[0023] Preferably, the fermentation temperature is 30–37°C and the fermentation time is 24–72 h.

[0024] Furthermore, the preparation method of the red ginseng root extract includes the following steps:

[0025] The red ginseng rootlets were mixed with water and extracted at 100°C, then concentrated to obtain the red ginseng rootlet extract.

[0026] Furthermore, the fermentation speed is 0-150 rpm;

[0027] Preferably, the fermentation process further includes a centrifugation step to obtain a fermentation broth;

[0028] Preferably, the extraction conditions for the fermentation broth include extraction at a temperature of 70-100℃ for 2-3 hours;

[0029] Preferably, the fermentation broth further includes a freeze-drying step after extraction to obtain the red ginseng rootlet fermentation product.

[0030] Thirdly, a fermented product of red ginseng rootlets prepared by the method described in any of the above-mentioned methods.

[0031] Furthermore, the content of rare ginsenoside Rg3 in the fermented product of the red ginseng rootlets is 21.94-27.12 mg / g;

[0032] Preferably, the content of rare ginsenoside Rg5 in the fermented red ginseng rootlets is 17.31-23.79 mg / g;

[0033] Preferably, the content of rare ginsenoside Rk1 in the fermented product of red ginseng rootlets is 5.90-8.58 mg / g;

[0034] Preferably, the content of rare ginsenoside Rh1 in the fermented red ginseng rootlets is 4.59-5.23 mg / g.

[0035] Fourthly, the application of any of the above-mentioned red ginseng root fermentation products in the preparation of anti-wrinkle skin products.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects:

[0037] The *Lactobacillus plantarum* provided by this invention is in live cell form and has antioxidant function in vitro. Fermenting red ginseng rootlets with this *Lactobacillus plantarum* can improve the anti-wrinkle effect of the red ginseng rootlets.

[0038] The present invention provides a method for fermenting red ginseng rootlets with *Lactobacillus plantarum*. Using red ginseng rootlets as a substrate, the method involves fermentation with a specific *Lactobacillus plantarum*, followed by extraction, thereby obtaining high-value fermented red ginseng rootlet products. This significantly increases the value of the whole red ginseng plant and solves the technical problems of excessively long fermentation cycles and low conversion rates of rare ginsenosides in existing technologies. The method achieves the technical effect of effectively increasing the yield of rare ginsenosides Rg3, Rg5, Rk1, and Rh1 within a shorter fermentation cycle, significantly enhancing the use value of red ginseng rootlets, and improving the utilization rate of ginseng resources.

[0039] The fermented red ginseng rootlets provided by this invention have a high content of rare ginsenosides and can be used in the food and cosmetic fields. They have a high anti-wrinkle effect on the skin and also provide data for the use of fermented red ginseng as a cosmetic raw material.

[0040] The application of the red ginseng root fermentation product provided by this invention can effectively achieve anti-wrinkle and firming effects on the skin, achieving outstanding application results. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is a colony morphology diagram of strain KMHD-2 provided in Example 1 of the present invention;

[0043] Figure 2 This is a microscopic image of strain KMHD-2 provided in Example 1 of the present invention;

[0044] Figure 3 This is a schematic diagram showing the change in the relative expression level of zebrafish type I collagen gene obtained in Experiment Example 2 of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] According to a first aspect of the present invention, a strain of Lactobacillus plantarum is provided, which has in vitro antioxidant capacity and was deposited on August 28, 2023 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28270.

[0047] The *Lactobacillus plantarum* provided by this invention is in live cell form and has antioxidant function in vitro. Fermenting red ginseng rootlets with this *Lactobacillus plantarum* can improve the anti-wrinkle effect of the red ginseng rootlets.

[0048] In a preferred embodiment, the *Lactobacillus plantarum* of the present invention can be derived from sauerkraut, and the method for obtaining it through isolation from sauerkraut includes the following steps:

[0049] Potential *Lactobacillus plantarum* were screened, isolated, and purified from sauerkraut samples using MRS medium containing bromocresol green indicator. Strains with good tolerance were obtained through resistance evaluation using simulated gastric acid and simulated intestinal fluid. Molecular identification of the strains confirmed *Lactobacillus plantarum*. In vitro antioxidant capacity evaluation of *Lactobacillus plantarum* revealed *Lactobacillus plantarum* with antioxidant capabilities.

[0050] The antioxidant capacity evaluation includes the ability to scavenge DPPH free radicals, the ability to scavenge hydroxyl free radicals, and the reducing capacity.

[0051] According to a second aspect of the present invention, a method for fermenting red ginseng rootlets with *Lactobacillus plantarum* is provided, comprising the following steps:

[0052] Using red ginseng rootlets as a substrate, fermentation is carried out using any of the above-mentioned *Lactobacillus plantarum* to obtain a fermentation broth. The fermentation broth is then extracted to obtain the red ginseng rootlet fermentation product.

[0053] In a preferred embodiment, the method for fermenting red ginseng rootlets with *Lactobacillus plantarum* provided by the present invention includes the following steps:

[0054] A suspension of red ginseng root powder and / or red ginseng root extract is mixed with *Lactobacillus plantarum* and fermented to obtain a fermentation broth. The fermentation broth is then extracted to obtain the red ginseng root fermentation product.

[0055] In a preferred embodiment, the mesh size of the red ginseng root powder can be 40-80 mesh, with typical but non-limiting mesh sizes such as 40 mesh, 60 mesh, and 80 mesh being more conducive to improving the fermentation effect of the red ginseng root, allowing it to be fully fermented and effectively increasing the yield of rare ginsenosides.

[0056] In a preferred embodiment, the solvent of the suspension includes, but is not limited to, water; wherein, the ratio of red ginseng root powder to water in the suspension can be 1:10, but is not limited to this, which is more conducive to improving the fermentation effect of red ginseng root.

[0057] In a preferred embodiment, the suspension includes, but is not limited to, sterilized suspension, which is more conducive to improving the fermentation of Lactobacillus plantarum; wherein, the sterilization method includes, but is not limited to, pasteurization.

[0058] In a preferred embodiment, the preparation method of red ginseng root extract includes the following steps:

[0059] Red ginseng rootlets are mixed with water and extracted at 100°C, then concentrated to obtain red ginseng rootlet extract. The extraction can be performed twice, but is not limited to this. Each extraction can last for 2 hours, and the filtrate is then concentrated to obtain the extract.

[0060] In a preferred embodiment, the amount of *Lactobacillus plantarum* added can be 3-10%, with typical but non-limiting addition amounts such as 3%, 5%, and 10%. A suitable amount of *Lactobacillus plantarum* is more conducive to improving the fermentation effect of red ginseng rootlets, allowing them to be fully fermented and effectively increasing the yield of rare ginsenosides. If the amount of *Lactobacillus plantarum* is too high, it will lead to excessive degradation of total saponins. If the amount of *Lactobacillus plantarum* is too low, it will lead to a lower conversion of Rg3, Rg5, Rk1, and Rh1.

[0061] In a preferred embodiment, the fermentation temperature can be 30-37°C, with typical but non-limiting fermentation temperatures such as 30°C, 33°C, 35°C, and 37°C. A suitable fermentation temperature is more conducive to improving the fermentation effect of red ginseng rootlets, allowing them to be fully fermented and effectively increasing the yield of rare ginsenosides. If the fermentation temperature is too high, it will lead to poor growth of the strain and affect the fermentation effect. If the fermentation temperature is too low, it will lead to insufficient strain activity.

[0062] In a preferred embodiment, the fermentation time can be 24 to 72 hours, with typical but non-limiting fermentation times being 24 hours, 36 hours, 48 ​​hours, 60 hours, and 72 hours. A suitable fermentation time is more conducive to improving the fermentation effect of red ginseng rootlets, allowing them to be fully fermented and effectively increasing the yield of rare ginsenosides. If the fermentation time is too long, it will lead to an excessively long production cycle; if the fermentation time is too short, it will lead to poor saponin conversion.

[0063] In a preferred embodiment, the fermentation speed can be 0-150 rpm, with typical but non-limiting speeds such as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 rpm, which is more conducive to further improving the fermentation effect of *Lactobacillus plantarum* on red ginseng rootlets.

[0064] The method for fermenting red ginseng rootlets with Lactobacillus plantarum provided by this invention ferments red ginseng rootlets with Lactobacillus plantarum. With the synergistic effect of various process parameters, not only is the fermentation cycle short, but the yield of rare ginsenosides Rg3, Rg5, Rk1 and Rh1 can also be effectively increased, thereby significantly improving the use value of red ginseng rootlets and increasing the utilization rate of ginseng resources.

[0065] In a preferred embodiment, the present invention further includes a centrifugation step after fermentation to obtain a fermentation broth.

[0066] After fermentation, impurities are removed by centrifugation and filtration to obtain fermentation broth, which is more conducive to improving the extraction effect of subsequent fermentation broth.

[0067] In a preferred embodiment, the extraction conditions of the fermentation broth include, but are not limited to, extraction at a temperature of 70-100°C for 2-3 hours, and the extracted fermentation broth is filtered through filter paper to obtain the extract of fermented red ginseng rootlets.

[0068] In a preferred embodiment, the fermentation broth further includes a freeze-drying step after extraction to obtain red ginseng root fermentation product. For example, the obtained red ginseng root fermentation extract can be placed at -80°C for 24 to 48 hours for freeze-drying, the freeze-dried powder can be collected, the red ginseng root fermentation product can be obtained, and its yield can be calculated.

[0069] The method for fermenting red ginseng rootlets using *Lactobacillus plantarum* provided in this invention, through the synergistic coordination of each step and its process parameters, not only shortens the fermentation cycle compared to traditional red ginseng extraction processes but also significantly increases the content of rare saponins. The resulting fermented red ginseng rootlets exhibit superior anti-wrinkle activity, and the fermented product can be applied in the food and cosmetic fields. Therefore, the method of this invention effectively enhances the utilization value of red ginseng rootlets and improves the utilization rate of ginseng resources.

[0070] According to a third aspect of the present invention, a fermented product of red ginseng rootlets prepared by any of the methods described above is provided.

[0071] The fermented red ginseng rootlets provided by this invention have a high content of rare ginsenosides and can be used in the food and cosmetic fields. They have a high anti-wrinkle effect on the skin and also provide data for the use of fermented red ginseng as a cosmetic raw material.

[0072] In a preferred embodiment, the content of rare ginsenoside Rg3 in the fermented red ginseng rootlets can be 21.94-27.12 mg / g, the content of rare ginsenoside Rg5 can be 17.31-23.79 mg / g, the content of rare ginsenoside Rk1 can be 5.90-8.58 mg / g, and the content of rare ginsenoside Rh1 can be 4.59-5.23 mg / g.

[0073] According to a fourth aspect of the present invention, the use of the red ginseng root fermentation product described in any of the above claims in the preparation of anti-wrinkle skin products is provided.

[0074] The application of the red ginseng root fermentation product provided by this invention can effectively achieve anti-wrinkle and firming effects on the skin, and can achieve outstanding application results.

[0075] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0076] Example 1

[0077] A strain of *Lactobacillus plantarum* and its method for fermenting red ginseng rootlets, comprising the following steps:

[0078] A. Isolation of lactic acid bacteria with antioxidant activity from fermented foods:

[0079] (1) Take 2.5g of sample from sauerkraut, add it to a sterile bag containing 22.5mL of sterile physiological saline, tap for 2min, prepare MRS medium containing bromocresol green, obtain single colonies by 10-fold serial dilution, anaerobic culture at 37℃ for 36-48h, pick the single colonies that turn the bromocresol green medium yellow and streak them on the MRS medium again, anaerobic culture at 37℃, and purify the obtained colonies by streaking multiple times to obtain single bacteria;

[0080] (2) Experiments on the resistance of Lactobacillus plantarum to artificial gastric acid / intestinal fluid yielded strains with good tolerance;

[0081] The artificial gastric acid resistance test involved weighing 2.0g NaCl and 3.2g pepsin, adding 7.0ml concentrated HCl, and adjusting the volume to 1000ml with ddH2O. 50ml of this solution was taken separately, and the pH was adjusted to 2.0 and 3.0, then filtered for sterilization. The glycerol culture was activated and passaged twice. 1ml of the overnight culture was placed in a 1.5ml centrifuge tube and centrifuged (5000 rpm for 5 min) to remove the supernatant. The bacterial cells were resuspended in an equal volume of simulated gastric fluid, mixed, and immediately 100μl of the bacterial solution was aspirated and added to 900μl of sterile physiological saline. Serial dilutions were performed, generally 10 μl was taken at 0h. -6 10 -7 The bacterial culture was plated; after sampling, the bacterial culture was placed in an incubator at 37°C and incubated. Samples were taken at 2 hours (simulating the original experimental timeframe) to simulate gastric fluid concentration and then diluted and plated. At each sampling time point, the bacterial culture was removed from the centrifuge tube, mixed, and then serially diluted. Two parallel samples were prepared for each strain, and two replicates were performed for each parallel sample. The plated plates were incubated at 37°C for 36–48 hours, and colony counts were performed and the data were recorded.

[0082] Artificial intestinal fluid tolerance test: Weigh 6.8g potassium dihydrogen phosphate (kH2O) and dilute to 250ml; measure 77ml of 0.2M NaOH and dilute to 500ml with kH2O; mix 250ml of kH2O with 500ml of NaOH solution, add 10.0g of trypsin and 0.3% bile salts, adjust pH to 6.8±0, and dilute to 1000ml. Filter and sterilize for later use; activate and subculture the bacterial culture with glycerol twice, take 1ml of overnight culture in a 1.5ml centrifuge tube, centrifuge (5000 rpm for 5 min), and discard the supernatant; resuspend the bacterial cells in an equal volume of simulated intestinal fluid, mix well, and immediately aspirate 100μl of the above bacterial solution, mix it with 900μl of sterile physiological saline, and perform serial dilutions. Generally, take 10 μl of the solution at 0h. -6 10 -7 The bacterial suspension was plated; after sampling, the bacterial suspension was placed in an incubator at 37°C and incubated. Sampling was performed at time points 3 and 6 hours to simulate intestinal fluid, according to the original experimental time length. At each sampling time point, the bacterial suspension was taken from the centrifuge tube, mixed, and then sampled for serial dilution. Two parallel samples were prepared for each strain, and two replicates were made for each parallel sample. After the plated plates were incubated in an incubator at 37°C for 36–48 hours, colony counts were performed and the data were recorded. See Table 1.

[0083] Table 1. Experimental data of strains simulating gastric acid and intestinal fluid.

[0084]

[0085] (3) Identification of Lactobacillus plantarum KMHD-2:

[0086] KMHD-2 colonies were observed by streaking multiple times. They were round, white or off-white, 1-2 μm in diameter, with a smooth, moist surface, an arc-shaped central protrusion, and were opaque with neat edges and no halo. Gram staining was purple, enzyme contact reaction was negative, and microscopic examination revealed rod-shaped colonies. The colony morphology of strain KMHD-2 is shown in the figure below. Figure 1 Microscopic examination revealed Figure 2 ;

[0087] Molecular identification:

[0088] DNA was extracted from strain KMHD-2 according to the steps of the bacterial genome extraction kit. Using it as a template, its 16S rDNA sequence was amplified by PCR (polymerase chain reaction) using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The amplification products were detected by electrophoresis on a 1% agarose gel. The PCR amplification products were then sent for sequencing. After sequencing, the sequencing results were analyzed by NCBI-BLAST in GenBank, identifying strain KMHD-2 as *Lactobacillus plantarum*.

[0089] 16S sequencing sequence:

[0090]

[0091]

[0092]

[0093] The inventor deposited Lactobacillus plantarum KMHD-2 at the China General Microbiological Culture Collection Center (CGMCC) on August 28, 2023, with accession number CGMCC No. 28270.

[0094] (4) In vitro antioxidant capacity of Lactobacillus plantarum KMHD-2:

[0095] Scavenging ability of DPPH free radicals: Take 0.2 mL of physiological saline suspension of the test strain, add 0.2 mL of DPPH solution, prepare with anhydrous ethanol to a final concentration of 0.2 mmol / L, mix well, and react in the dark at room temperature for 30 min. Then centrifuge at 6000 rpm for 10 min, take the supernatant and measure the absorbance value at 517 nm (sample A). Measure three parallel values. For the blank group sample, replace the DPPH anhydrous ethanol solution with an equal volume of anhydrous ethanol. For the control group sample, replace the sample with an equal volume of distilled water. Zero the blank with an equal volume of distilled water and anhydrous ethanol mixture. Scavenging rate = (A control - A sample) / A control × 100. The patented strain CGMCC NO.18094 was used as a control. The test results are shown in Table 2.

[0096] Hydroxyl radical scavenging ability: 1 mL of 1.25 mmol / L o-phenanthroline was placed in a test tube, followed by 2 mL of PBS buffer solution (0.2 mol / L, pH = 7.4). After mixing, 1 mL of 1.25 mmol / L FeSO4 was added, and then 1 mL of 1.5% H2O2 was added. The mixture was incubated at 37℃ for 90 min, and the absorbance at 536 nm was measured as A. 1 mL of distilled water was used instead of 1 mL of H2O2 as the blank group, denoted as B. 1 mL of the test sample was used instead of 1 mL of distilled water as the sample group, denoted as C. Hydroxyl radical scavenging rate (%) = (CA) / (BA) × 100. The results are shown in Table 2, using the patented strain CGMCC NO.18094 as a control.

[0097] Determination of reducing activity: Take 0.2 mL of the sample to be tested, add 0.2 mL of PBS buffer solution (0.2 mol / L, pH=6.6) and 0.2 mL of potassium ferricyanide (mass fraction 1%), mix well, and place in a 50℃ constant temperature water bath for 20 min, then cool in ice water; add 0.2 mL of 5% trichloroacetic acid, centrifuge at 6000 r / min for 5 min, take 0.1 mL of the supernatant, add 0.1 mL of distilled water and 0.025 mL of 0.1% ferric chloride, shake well, let stand for 10 min, and then measure the absorbance at 700 nm; the larger the A700 nm value, the stronger the reducing ability; L-cysteine ​​was used as a standard to represent the reducing power of lactic acid bacteria; standard solutions of L-cysteine ​​(0-400 μmol / L) of different concentrations were prepared, and the above steps were followed to determine the standard curve; the patented strain CGMCCNO.18094 was used as a control, and the detection results are shown in Table 2;

[0098] Table 2: In vitro antioxidant capacity test of strains

[0099]

[0100] (5) Preparation of fermentation broth: The bacterial broth is streaked on a plate to separate single colonies, and the single colonies are inoculated into liquid culture medium and incubated statically to obtain fermentation broth;

[0101] B. Fermentation extraction:

[0102] (a) Fermentation of red ginseng rootlets: After the red ginseng rootlets are crushed and passed through an 80-mesh sieve, 10 times the mass of pure water is added to make a suspension of red ginseng rootlet powder.

[0103] After pasteurization, the suspension of red ginseng root powder was mixed with the above-mentioned Lactobacillus plantarum and fermented at 37°C and 120 rpm for 72 hours. After fermentation, the mixture was centrifuged and filtered to obtain the fermentation broth.

[0104] The amount of *Lactobacillus plantarum* added was 10%.

[0105] (b) Fermentation broth extraction: The fermentation broth was extracted at 100℃ for 2-3 hours. The extracted fermentation broth was filtered through filter paper to obtain the extract of fermented red ginseng rootlets.

[0106] (c) Freeze-drying: The extract obtained after fermentation of red ginseng rootlets was placed in a refrigerator at -80℃ for 12 to 36 hours, and then freeze-dried. The freeze-dried powder of fermented red ginseng rootlets was collected to obtain the fermented red ginseng rootlet product, and the extract yield was calculated.

[0107] Example 2

[0108] The difference between this embodiment and Embodiment 1 is that in this embodiment, red ginseng root extract concentrate is used instead of red ginseng root powder suspension in step (a) of fermentation extraction;

[0109] The extraction method of red ginseng root extract concentrate includes: adding 10 times the amount of water to red ginseng root extract, extracting twice at 100℃, mixing the two extracts, and then concentrating under reduced pressure by 1 time to obtain red ginseng root extract concentrate;

[0110] The remaining steps and process parameters are the same as in Example 1, and the fermented product of red ginseng rootlets is obtained.

[0111] Example 3

[0112] The difference between this embodiment and Embodiment 1 is that in step (a) of fermentation extraction in this embodiment, the red ginseng rootlets are pulverized and sieved through a 60-mesh screen. The remaining steps and process parameters are the same as in Embodiment 1, and the fermented red ginseng rootlets are obtained.

[0113] Example 4

[0114] The difference between this embodiment and Embodiment 1 is that in step (a) of fermentation extraction in this embodiment, the red ginseng rootlets are pulverized and sieved through a 40-mesh screen. The remaining steps and process parameters are the same as in Embodiment 1, and the fermented red ginseng rootlets are obtained.

[0115] Example 5

[0116] The difference between this embodiment and Embodiment 1 is that in this embodiment, the amount of *Lactobacillus plantarum* added in step (a) of fermentation extraction is 5%, while the remaining steps and process parameters are the same as in Embodiment 1, to obtain the fermented product of red ginseng rootlets.

[0117] Example 6

[0118] The difference between this embodiment and Embodiment 1 is that in this embodiment, the amount of *Lactobacillus plantarum* added in step (a) of fermentation extraction is 3%, while the remaining steps and process parameters are the same as in Embodiment 1, to obtain the fermented product of red ginseng rootlets.

[0119] Example 7

[0120] The difference between this embodiment and Embodiment 1 is that the fermentation temperature in step (a) of the fermentation extraction in this embodiment is 30°C, while the remaining steps and process parameters are the same as in Embodiment 1, to obtain the fermented product of red ginseng rootlets.

[0121] Example 8

[0122] The difference between this embodiment and Embodiment 1 is that the fermentation time in step (a) of fermentation extraction in this embodiment is 48 hours, while the remaining steps and process parameters are the same as in Embodiment 1, to obtain the fermented product of red ginseng rootlets.

[0123] Example 9

[0124] The difference between this embodiment and Embodiment 1 is that the fermentation time in step (a) of fermentation extraction in this embodiment is 24 hours, while the remaining steps and process parameters are the same as in Embodiment 1, to obtain the fermented product of red ginseng rootlets.

[0125] Comparative Example 1

[0126] The difference between this comparative example and Example 1 is that in this comparative example, the amount of *Lactobacillus plantarum* added in step (a) of fermentation extraction is 15%, while the remaining steps and process parameters are the same as in Example 1, to obtain the fermented product of red ginseng rootlets.

[0127] Compared with Example 1, the drawback or deficiency of this comparative method is that although the amount of microbial agent added was increased, the content of the target saponin was not significantly increased.

[0128] Comparative Example 2

[0129] The difference between this comparative example and Example 1 is that the amount of *Lactobacillus plantarum* added in step (a) of fermentation extraction in this comparative example is 1%, while the remaining steps and process parameters are the same as in Example 1, to obtain the fermented product of red ginseng rootlets.

[0130] Compared with Example 1, the drawback or deficiency of this comparative method is that the low concentration of the starting bacteria leads to slower fermentation and poor conversion of rare saponins.

[0131] Comparative Example 3

[0132] The difference between this comparative example and Example 1 is that the fermentation temperature in step (a) of the fermentation extraction in this comparative example is 25°C, while the remaining steps and process parameters are the same as in Example 1, resulting in the fermented product of red ginseng rootlets.

[0133] Compared with Example 1, the drawback or deficiency of this comparative method is that the conversion rate of the target saponin is lower.

[0134] Comparative Example 4

[0135] The difference between this comparative example and Example 1 is that the fermentation temperature in step (a) of the fermentation extraction in this comparative example is 45°C, while the remaining steps and process parameters are the same as in Example 1, to obtain the fermented product of red ginseng rootlets.

[0136] Compared with Example 1, the defect or deficiency of this comparative method is that the growth of *Lactobacillus plantarum* is poor, resulting in low efficiency of target saponin conversion.

[0137] Comparative Example 5

[0138] The difference between this comparative example and Example 1 is that the fermentation time in step (a) of the fermentation extraction in this comparative example is 18 hours, while the remaining steps and process parameters are the same as in Example 1, to obtain the fermented product of red ginseng rootlets.

[0139] Compared with Example 1, the drawback or deficiency of this comparative method is that the fermentation time is short, resulting in poor conversion of the target saponins.

[0140] Comparative Example 6

[0141] The difference between this comparative example and Example 1 is that the fermentation time in step (a) of the fermentation extraction in this comparative example is 84 hours, while the remaining steps and process parameters are the same as in Example 1, to obtain the fermented product of red ginseng rootlets.

[0142] Compared with Example 1, the drawback or deficiency of this comparative method is that although the fermentation time is extended, the content of the target saponin is not significantly increased.

[0143] Comparative Example 7

[0144] The difference between this comparative example and Example 1 is that this comparative example uses a traditional extraction method, including the following steps:

[0145] Red ginseng rootlets were added to 10 times their weight of purified water and extracted twice at 100°C. The extracts from the two extractions were then mixed and freeze-dried to obtain freeze-dried red ginseng rootlets powder obtained through traditional extraction.

[0146] Comparative Example 8

[0147] This comparative example provides a commercially available red ginseng powder 1.

[0148] Comparative Example 9

[0149] This comparative example provides a commercially available red ginseng powder 2.

[0150] Experimental Example 1

[0151] The ginsenoside content of the fermented red ginseng root freeze-dried powder obtained in Example 1, the traditionally extracted red ginseng root freeze-dried powder obtained in Comparative Example 7, and the commercially available red ginseng powder provided in Comparative Examples 8-9 was detected by ultra-high performance liquid chromatography. The results are shown in Table 3.

[0152] Table 3 Comparison of Rare Ginsenoside Content

[0153]

[0154] The ginsenoside content of the fermented red ginseng root freeze-dried powder obtained in Examples 2-9 was detected using the same method as described above, and the results are shown in Table 4.

[0155] Table 4

[0156]

[0157] The ginsenoside content of the fermented red ginseng root freeze-dried powders obtained in Comparative Examples 1-6 was detected using the same method described above, and the results are shown in Table 5.

[0158] Table 5

[0159]

[0160] Experimental Example 2

[0161] Methods for Promoting Type I Collagen Gene Expression in Zebrafish: Collagen is the most abundant extracellular matrix protein in the human body, with type I collagen being the most abundant protein in the skin. The distribution of type I collagen in zebrafish is similar to that in humans and shows a high degree of conservation. Their innate expression significantly decreases at 6 days post-fertilization and becomes very low between 10 and 12 days post-fertilization. The expression of zebrafish type I collagen genes (col1a1a, col1a1b, and col1a2) was tested, and the relative expression levels of zebrafish type I collagen genes were compared between fermented red ginseng root freeze-dried powder and red ginseng root freeze-dried powder. The results are shown in [Figure number missing]. Figure 3 Calculating the type I collagen gene expression promotion rate can evaluate the anti-wrinkle and firming effects of raw materials or products.

[0162] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactobacillus plantarum strain, characterized in that, Lactobacillus plantarum, preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC No.28270.

2. The Lactiplantibacillus plantarum of claim 1, characterized in that, The Lactobacillus plantarum is derived from sauerkraut.

3. A method for fermenting red ginseng roots by Lactobacillus plantarum, characterized by, The method comprises the following steps: The red ginseng rootlet is used as a substrate, and the Lactobacillus plantarum in claim 1 or 2 is used for fermentation to obtain a fermentation liquor, and the fermentation liquor is extracted to obtain a red ginseng rootlet fermentation product.

4. The method of claim 3, wherein, The red ginseng rootlet is added in the form of a suspension liquid prepared from red ginseng rootlet powder and / or a red ginseng rootlet extract.

5. The method of claim 4, wherein, The red ginseng rootlet powder has a mesh size of 40-80 mesh.

6. The method of claim 4, wherein, The Lactobacillus plantarum is added in an amount of 3-10%.

7. The method of claim 4, wherein, The solvent of the suspension liquid comprises water.

8. The method of claim 4, wherein, The ratio of the red ginseng rootlet powder to water in the suspension liquid is 1:

10.

9. The method of claim 4, wherein, The fermentation temperature is 30-37℃, and the fermentation time is 24-72h.

10. The method of claim 4, wherein, The preparation method of the red ginseng rootlet extract comprises the following steps: The red ginseng rootlet is mixed with water, and then extracted at a temperature of 100℃, and concentrated to obtain the red ginseng rootlet extract.

11. The method according to any one of claims 3-10, characterized in that, The rotation speed of the fermentation is 0-150rpm.

12. The method of claim 11, wherein, The fermentation further comprises a centrifugation step to obtain a fermentation liquor. The extraction condition of the fermentation liquor comprises extraction at a temperature of 70-100℃ for 2-3h. The fermentation liquor further comprises a freeze-drying step after extraction to obtain a red ginseng rootlet fermentation product.

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