Pediococcus acidilactici and application of red ginseng fermented by pediococcus acidilactici in oral anti-aging effect

Abstract

The invention provides application of pediococcus acidilactici and red ginseng fermented by the pediococcus acidilactici in oral anti-aging efficacy, and relates to the technical field of biological fermentation, the pediococcus acidilactici is pediococcus acidilactici, the strain number of the pediococcus acidilactici is KMHD9-314, the registration number of the pediococcus acidilactici in the China General Microbiological Culture Collection Center is CGMCC No.32357, and the preservation number of the pediococcus acidilactici is KMHD9-314. According to the pediococcus acidilactici KMHD9-314, ginsenosides Rb1, Rb2, Rc, Rf and Re can be converted into rare ginsenosides 20 (S)-Rg3, 20 (R)-Rg3, Rk1, Rg5, Rh1, Rg6, Rh4 and Rk3, and the total amount of the rare ginsenosides is increased. The technical problem that in the prior art, the efficiency of converting rare ginsenoside through microbial fermentation is low is solved.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to the application of a strain of Pyocortisone lactis and its fermented red ginseng in oral anti-aging effects. Background Technology

[0002] Ginsenosides possess pharmacological activities such as anti-inflammatory, anti-aging, anti-tumor, antipyretic, analgesic, immune-enhancing, and cardiovascular-improving effects. The basic structural composition of ginsenosides is a steroidal skeleton with various glycosyl groups (glucose (GLC), rhamnose (RHA), xylose (XY), and arabinose (ARA) linked at C3, C6, and C20). 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. Differences in the type, number, and binding sites (C-3, C-6, C-20) of glycosyl groups have been shown to result in variations in activity. Deglycosylated secondary ginsenosides are present in very small amounts in native Panax ginseng plants, but they exhibit greater pharmacological activity and bioavailability than glycosylated ginsenosides. In processed red ginseng, the contents of rare ginsenosides Rg3, Rg5, Rk1, and 20(S)-PPD are significantly higher than those in raw ginseng (i.e., native ginseng).

[0003] Traditional Chinese medicine fermentation technology utilizes the metabolic activities of microorganisms, leveraging their rich enzyme systems to decompose macromolecules such as cellulose and proteins in medicinal materials, thereby increasing the content of rare ginsenosides and enhancing their efficacy. However, existing microbial fermentation methods primarily enhance efficacy by increasing the content of ginsenosides Rk1, Rg5, Rg3, and 20-ginsenoside Rg3, but lack a promoting effect on increasing the content of other rare ginsenosides such as Rh1, Rg6, Rh4, and Rk3.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a *Pediococcus lactis* strain to solve the technical problem of low efficiency in the microbial fermentation and conversion of rare ginsenosides in the prior art.

[0006] The second objective of this invention is to provide the application of the above-mentioned Pediococcus lactis.

[0007] The third objective of this invention is to provide a method for increasing the content of rare ginsenosides in red ginseng.

[0008] The fourth objective of this invention is to provide a fermentation agent for increasing the content of rare ginsenosides in red ginseng extract.

[0009] The fifth objective of this invention is to provide a method for preparing a fermented extract of red ginseng.

[0010] The sixth objective of this invention is to provide a fermented red ginseng extract.

[0011] The seventh objective of this invention is to provide the application of the above-mentioned red ginseng fermentation extract in the preparation of anti-aging products.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides *Pediococcus lactis*, wherein the *Pediococcus lactis* is *Pediococcus lactis* (… Pediococcus acidilactici The strain number is KMHD9-314, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32357.

[0013] Secondly, the present invention provides the use of the above-mentioned *Pediococcus lactis* in any of the following: A-1, Transformation of rare ginsenosides; A-2. Increase the content of rare ginsenosides in red ginseng; A-3. Preparation of products for increasing the content of rare ginsenosides in red ginseng; A-4. Preparation of red ginseng fermentation extract.

[0014] Thirdly, the present invention provides a method for increasing the content of rare ginsenosides in red ginseng, including fermenting red ginseng powder with the above-mentioned Pediococcus lactis KMHD9-314.

[0015] Furthermore, this includes adding Pediococcus lactis KMHD9-314 bacterial culture to red ginseng powder suspension for fermentation; Preferably, the fermentation conditions are static culture at 28~42℃ for 16~36h, and more preferably static culture at 37℃ for 24h; Preferably, the volume ratio of the *Pediococcus lactis* KMHD9-314 bacterial suspension to the red ginseng extract is 0.03~0.1:1, more preferably 0.05:1.

[0016] Furthermore, the OD of the *Pediococcus lactis* KMHD9-314 bacterial suspension... 600 The value is 1.300~1.800, preferably 1.600.

[0017] Fourthly, the present invention provides a fermentation agent for increasing the content of rare ginsenosides in red ginseng extract, comprising the aforementioned Pediococcus lactis KMHD9-314.

[0018] Fifthly, the present invention provides a method for preparing red ginseng fermentation extract, comprising taking the red ginseng fermentation extract obtained by fermenting red ginseng powder suspension with the above-mentioned Pyrococcus lactis KMHD9-314, centrifuging to obtain the supernatant, and drying to obtain red ginseng fermentation extract.

[0019] Furthermore, the process before centrifugation also includes pH adjustment and sterilization; Preferably, the pH is 2-4, and more preferably 3.0; Preferably, the sterilization includes heating at 100°C for 0.5 to 1.5 hours; Preferably, the drying process includes spray drying, vacuum drying, or freeze drying.

[0020] Sixthly, the present invention provides a red ginseng fermentation extract, which is prepared by the above-described preparation method.

[0021] In a seventh aspect, the present invention provides the application of the above-mentioned red ginseng fermentation extract in the preparation of products for anti-aging.

[0022] The *Pediococcus lactis* strain KMHD9-314 provided by this invention can convert ginsenosides Rb1, Rb2, Rc, Rf, and Re into rare ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5, Rh1, Rg6, Rh4, and Rk3, thereby increasing the total amount of rare ginsenosides. This solves the technical problem of low efficiency in the microbial fermentation conversion of rare ginsenosides in existing technologies. Attached Figure Description

[0023] 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.

[0024] Figure 1 This is a chromatogram for the detection of ginsenosides in the fermented red ginseng extract provided in Example 5 of the present invention; Figure 2 This is a chromatogram for the detection of ginsenosides in red ginseng extract provided in Comparative Example 4 of the present invention. Detailed Implementation

[0025] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0026] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0027] 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.

[0028] This invention provides a type of *Pediococcus lactis*, wherein the *Pediococcus lactis* is *Pediococcus lactis* (… Pediococcus acidilactici The strain number is KMHD9-314, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32357.

[0029] Preservation Instructions for Pediococcus lactis KMHD9-314 Strain name: Pediococcus lactis KMHD9-314; Latin name: Pediococcus acidilactici ; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: October 25, 2024; Collection Center Accession Number: CGMCC No. 32357.

[0030] Pediococcus lactis KMHD9-314 can convert ginsenosides Rb1, Rb2, and Rc20 into rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5; and convert ginsenosides Rf and Re into Rh1, Rg6, Rh4, and Rk3, thereby increasing the total amount of rare ginsenosides. This solves the technical problem of low efficiency in the microbial fermentation conversion of rare ginsenosides in existing technologies.

[0031] According to another aspect of the invention, the above-described Pediococcus lactis is also provided for use in any of the following: A-1, Transformation of rare ginsenosides; A-2. Increase the content of rare ginsenosides in red ginseng; A-3. Preparation of products for increasing the content of rare ginsenosides in red ginseng; A-4. Preparation of red ginseng fermentation extract.

[0032] In some specific embodiments, the rare ginsenosides include at least one of 20(R)-Rg3, 20(S)-Rg3, Rk1, Rg5, Rh1, Rg6, Rh4, or Rk3.

[0033] According to another aspect of the present invention, a method for increasing the content of rare ginsenosides in red ginseng is also provided, comprising fermenting red ginseng powder with the aforementioned Pediococcus lactis KMHD9-314.

[0034] Fermentation of Pediococcus lactis KMHD9-314 with red ginseng powder as a substrate can increase the content of rare ginsenosides in red ginseng powder and increase the content of effective components in red ginseng extract.

[0035] In some specific embodiments, the method involves adding *Pediococcus lactis* KMHD9-314 bacterial culture to red ginseng powder suspension for fermentation, thereby increasing the content of rare ginsenosides in the red ginseng fermentation extract. In some specific embodiments, the fermentation conditions are static culture at 28-42℃ for 16-36 hours. In some specific embodiments, the volume ratio of the *Pediococcus lactis* KMHD9-314 bacterial culture to the red ginseng extract is 0.03-0.1:1. In some specific embodiments, the OD value of the *Pediococcus lactis* KMHD9-314 bacterial culture is... 600 The value is 1.300~1.800.

[0036] The fermentation temperature can be, but is not limited to, 28℃, 30℃, 32℃, 34℃, 37℃, 38℃, 40℃ or 42℃, or any value between 28℃ and 42℃, with 37℃ being the preferred temperature.

[0037] The static culture time can be, but is not limited to, 16h, 18h, 20h, 24h, 25h, 28h, 30h, 32h, 34h or 36h, or any value between 16 and 36h, with 24h being the preferred value.

[0038] The volume ratio of *Pediococcus lactis* KMHD9-314 bacterial suspension to red ginseng extract can be, but is not limited to, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1 or 0.1:1, or any value between 0.03 and 0.1:1, preferably 0.05:1.

[0039] OD of Pediococcus lactis KMHD9-314 bacterial suspension 600 The value can be, but is not limited to, 1.300, 1.400, 1.500, 1.600, 1.700 or 1.8000, or any value between 1.300 and 1.800, with 1.600 being preferred.

[0040] According to another aspect of the present invention, a fermentation agent for increasing the content of rare ginsenosides in red ginseng extract is also provided, comprising the above-mentioned Pediococcus lactis KMHD9-314.

[0041] According to another aspect of the present invention, a method for preparing red ginseng fermentation extract is also provided, comprising taking the red ginseng fermentation extract obtained by fermenting red ginseng powder suspension with the above-mentioned Pyrococcus lactis KMHD9-314, centrifuging to obtain the supernatant, and drying to obtain red ginseng fermentation extract.

[0042] Red ginseng fermentation extract obtained by fermentation with Pleurotus erythrorhizon KMHD9-314 contains rare ginsenosides and has a high content, which has excellent antioxidant and anti-aging effects.

[0043] In some specific embodiments, the process before centrifugation includes pH adjustment and sterilization; in some specific embodiments, the pH is 2-4; in some specific embodiments, the sterilization includes heating at 100°C for 0.5-1.5 hours; in some specific embodiments, the drying includes spray drying, vacuum drying, or freeze drying.

[0044] The pH value can be, but is not limited to, 2, 2.3, 2.5, 2.8, 3, 3.3, 3.5, 3.8 or 4.0, or any value between 2 and 4, with pH 3 being preferred.

[0045] The sterilization time can be, but is not limited to, 0.5h, 0.7h, 0.9h, 1h, 1.2h or 1.5h, or 0.5~1.5h, preferably 1h.

[0046] According to another aspect of the present invention, a red ginseng fermentation extract is also provided, which is prepared by the above-described preparation method.

[0047] According to another aspect of the present invention, the use of the above-mentioned red ginseng fermentation extract in the preparation of products for anti-aging is also provided.

[0048] 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.

[0049] MRS medium: Casein digest 10 g / L, beef extract 10 g / L, yeast extract 4 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, glucose 20 g / L, Tween-80 1.08 g / L, 1‰ L-cysteine ​​hydrochloride anhydrous, 2% agar powder.

[0050] Bromocresol green medium: Casein digest 10 g / L, beef extract 10 g / L, yeast extract 4 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, glucose 20 g / L, Tween-80 1.08 g / L, bromocresol green 6.5 mL / L, 1‰ L-cysteine ​​hydrochloride anhydrous, 2% agar powder.

[0051] Example 1: Isolation, Identification, and Preservation of Strains 1. Strains Isolation and Purification Weigh 1g of fermented soybean sample, add 9mL of PBS or physiological saline, and homogenize twice in a homogenizer for 2 minutes each time to prepare 10 -1 Diluent. Add 10 -1 The diluent was serially diluted 10-fold to 10. -7 A series of dilutions were plated and inverted in an anaerobic incubator at 37°C for 48 hours. Single colonies that turned bromocresol green agar yellow were picked and streaked again on MRS agar, and incubated anaerobically at 37°C. The obtained colonies were purified by streaking multiple times to obtain single bacteria.

[0052] 2. Identification of strains The morphology of single bacterial colonies after multiple purifications was observed. Selected strains were round, milky white or grayish white, with a raised center, smooth surface, and regular edges. Gram staining was purple, enzyme contact reaction was negative, and microscopic examination revealed spherical shapes. Using colony DNA as a template, the 16S rRNA gene fragment was amplified using universal primers 27F and 1492R for bacterial sequencing. The PCR amplification reaction system and amplification program are shown in Tables 1 and 2.

[0053] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0054] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0055] Table 1 PCR reaction system

[0056] Table 2 PCR reaction procedure

[0057] Amplicon sequence alignment was performed, and the PCR products were sent to the company for 16S rRNA gene amplicon sequencing. The 16S sequence is as follows:

[0058] The base sequence from the assay results was entered into the NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for comparison, and strain KMHD9-314 was identified as Pediococcus lactis. Pediococcus acidilactici The strain KMHD9-314 was inoculated into glycerol tubes and cryopreserved. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with the registration number CGMCC No. 32357.

[0059] Example 2: Preparation of bacterial culture The *Pediococcus lactis* KMHD9-314 strain selected in Example 1 was used. After thawing the glycerol tubes of the strain, single colonies were isolated by streaking on agar plates. These single colonies were then inoculated into MRS liquid medium and incubated statically at 37°C until OD500 was achieved. 600 The bacterial solution is obtained when the value reaches 1.300~1.800, with a value closer to 1.600 being ideal.

[0060] The red ginseng rootlets in the following examples are from Kangmei Xinkaihe (Jilin) ​​Pharmaceutical Co., Ltd.

[0061] Example 3 Red ginseng rootlets were crushed and passed through a 20-mesh sieve to obtain red ginseng powder. The red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 3% (V / V) of Pediococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The suspension was allowed to ferment at 37℃ for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100℃ for 60 minutes. The red ginseng fermentation broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermentation broth was frozen at -80℃ overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0062] Example 4 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 10% (V / V) of Pyrococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0063] Example 5 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pyrococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0064] Example 6 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pyrococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 16 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0065] Example 7 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Porphyromonas lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 36 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0066] Example 8 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Porphyromonas lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 28°C for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0067] Example 9 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pleurotus erythrorhizon KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 42℃ for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100℃ for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80℃ overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0068] Example 10 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pleurotus erythrorhizon KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 30 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0069] Example 11 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pleurotus erythrorhizon KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 90 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0070] Example 12 The difference from Example 3 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pediococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 2.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0071] Example 13 The difference from Example 5 is: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, 5% (V / V) of Pyrococcus lactis KMHD9-314 bacterial solution was added to the red ginseng powder suspension. The mixture was allowed to ferment at 37°C for 24 hours. After fermentation, the pH was adjusted to 4.0 with citric acid, and then sterilized at 100°C for 60 minutes. The fermented ginseng broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermented broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0072] Comparative Example 1 Unlike Example 5, *Pediococcus lactis* strain FSB-S7-10 (isolated strain from the microbiology laboratory of Kangmei Huada Gene Technology Co., Ltd.) was selected, and bacterial culture was prepared according to the method in Example 2. OD 600 The value was 1.600, and *Pediococcus lactis* FSBB-S7-10 bacterial suspension was used instead of *Pediococcus lactis* KMHD9-314 bacterial suspension.

[0073] Comparative Example 2 Unlike Example 5, the pH value was not adjusted: 20 times the amount of pure water was added to the red ginseng powder, and the suspension was pasteurized for 30 minutes. Then, the bacterial culture of Porphyromonas lactis KMHD9-314 was added to the red ginseng powder suspension at a ratio of 5% (V / V). The suspension was allowed to ferment at 37°C for 24 hours, and then sterilized at 100°C for 60 minutes. The red ginseng fermentation broth was obtained by centrifugation and filtration at 10,000 rpm for 10 minutes. The fermentation broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0074] Comparative Example 3 Unlike Example 5, the red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, the bacterial suspension of *Pediococcus lactis* KMHD9-314 was added to the red ginseng powder suspension at a ratio of 5% (V / V). The suspension was allowed to ferment at 37°C for 24 hours, and then matured at 25°C for 24 hours. The matured culture broth was centrifuged and filtered at 10,000 rpm for 10 minutes to obtain the red ginseng fermentation broth. The fermentation broth was frozen at -80°C overnight and then freeze-dried for 24 hours to obtain the red ginseng fermentation extract.

[0075] Comparative Example 4 The preparation method of red ginseng extract involves using red ginseng powder (crushed and passed through a 20-mesh sieve) as raw material. The specific steps are as follows: Add 20 times the volume of pure water to the red ginseng powder, then extract at 100℃ for 60 minutes. Centrifuge and filter to obtain the red ginseng liquid. Freeze-dry the red ginseng liquid and collect the red ginseng extract.

[0076] Comparative Example 5 The preparation method of red ginseng extract involves using red ginseng powder (crushed and passed through a 20-mesh sieve) as raw material. The specific steps are as follows: Add 20 times the volume of pure water to the red ginseng powder, adjust the pH to 3.0 with citric acid, and then extract at 100℃ for 60 minutes. Centrifuge and filter to obtain the red ginseng solution. Freeze-dry the red ginseng solution and collect the red ginseng extract.

[0077] Experiment 1: Detection of Ginsenosides in Red Ginseng Fermentation Extract The detection methods for ginsenosides are as follows: Gradient elution was performed using acetonitrile containing 0.1% formic acid as mobile phase B and water containing 0.1% formic acid as mobile phase C. The chromatographic column was an ACQUITY UPLC BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm); the flow rate was 0.4 mL / min; the column temperature was 30℃; the injection volume was 3 μL; and the autosampler temperature was 4℃. The gradient elution program was as follows: 0–5 min, 20%–30% B; 5–12 min, 30%–33% B; 12–13 min, 33%–40% B; 13–23 min, 40%–47% B; 23–24 min, 47%–60% B; 24–26 min, 60%–65% B; 26–33 min, 65%–78% B; 33–35 min, 78%–80% B; 35–36 min, 80%–20% B; 36–41 min, 20% B.

[0078] The ultra-high performance liquid chromatography (UHPLC) conditions are shown in Table 3.

[0079] Table 3

[0080] The red ginseng fermentation extracts prepared in Examples 3-13 and Comparative Examples 1-3, as well as the red ginseng extracts prepared in Comparative Examples 4 and 5, were tested according to the ginsenoside detection method. The results are shown in Table 4, which contains the ginsenoside detection results in mg / g.

[0081] Table 4

[0082] As shown in Table 4, the content of rare ginsenosides in the fermented red ginseng extracts of Examples 3 to 13 was significantly higher than that in Comparative Examples 4 and 5, indicating that direct extraction or acid extraction without fermentation could not increase the content of rare ginsenosides.

[0083] The content of rare ginsenosides in Comparative Example 1 was significantly lower than that in Example 5. Compared with Comparative Example 5, Rh1 and Rk3 in Comparative Example 1 were actually lower, while Rg6h was less than half the same, indicating that *Pediococcus lactis* FSB-S7-10 could only convert a portion of the rare ginsenosides, and the conversion efficiency was significantly lower than that of *Pediococcus lactis* KMHD9-314. The content of rare ginsenosides in Comparative Example 2 was significantly lower than that in Example 5, indicating that acid treatment to adjust the pH after fermentation had the effect of increasing the content of rare ginsenosides. The content of rare ginsenosides in Comparative Example 3 was lower than that in Example 5, indicating that acid treatment to adjust the pH after fermentation was more effective in increasing the content of rare ginsenosides than the ripening process.

[0084] Chromatograms of ginsenosides in the fermented red ginseng extract of Example 5 and the red ginseng extract of Comparative Example 4 are shown below. Figure 1 and Figure 2 As shown, compared with unfermented red ginseng extract, the contents of Rb1, Rb2, Rf and Re in fermented red ginseng extract decreased, while the contents of 20(R)-Rg3, 20(S)-Rg3, Rk1, Rg5, Rh1, Rg6, Rh4 and Rk3 increased to varying degrees.

[0085] Experiment 2: Anti-aging animal experiment of red ginseng fermentation extract 1. Experimental animals: C57BL / 6J mice were purchased and acclimatized for one week before modeling was performed.

[0086] 2. Experimental Procedure: Except for the blank control group, the remaining mice were intraperitoneally injected with D-galactose (100 mg / (kg.bw)), at a dose of 10 mL / kg, once daily for 6 consecutive weeks. MDA was measured in orbital blood samples compared to the blank control group. The MDA of the model group was significantly higher than that of the blank control group, indicating successful modeling. The model groups were randomly divided into an aging model group, a fermented red ginseng group (administered with the fermented red ginseng extract from Example 5), and an unfermented red ginseng group (administered with the red ginseng extract from Comparative Example 4). During the experiment, the fermented and unfermented red ginseng groups were administered the corresponding samples orally via gavage, while the aging model group received the same volume of pure water. The fermented, unfermented, and aging model groups continued to receive the same amount of D-galactose intraperitoneally for 42 days. The blank control group received the same volume of physiological saline by injection and the same volume of pure water by gavage for 42 days. After the experiment, the mice were fasted for 12 hours, and their body weight, visceral specific gravity, and the levels of MDA, SOD, and GSH-Px in the spleen and liver were measured and recorded.

[0087] 3. Results (1) The organ index of mice is shown in Table 5.

[0088] Table 5

[0089] As shown in Table 5, the organ indices of the model group were significantly lower than those of the blank group, and the improvement effect of the fermented red ginseng group was significantly better than that of the unfermented red ginseng group. The fermented red ginseng extract can improve the changes in organ indices.

[0090] (2) The liver and spleen indicators are shown in Table 6.

[0091] Table 6

[0092] MDA, SOD, and GSH-Px are important indicators of antioxidant and anti-aging effects. Table 6 shows that, compared to the model group, both the fermented and unfermented red ginseng groups reduced malondialdehyde (MDA) in mouse liver, but the fermented red ginseng group showed a better effect. Compared to the model group, both the fermented and unfermented red ginseng groups significantly increased SOD and GSH-Px levels, but the fermented red ginseng group showed a better effect, indicating that fermentation can enhance the antioxidant and anti-aging effects of red ginseng.

[0093] 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. Pediococcus acidilactici, characterized in that, The lactic acid cocci are *Pediococcus lactis* ( Pediococcus acidilactici The strain number is KMHD9-314, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32357.

2. The use of the Pediococcus acidilactici of claim 1 in any one of: A-1, transforming rare ginsenosides; A-2, increasing the content of rare ginsenosides in red ginseng; A-3, preparing a product for increasing the content of rare ginsenosides in red ginseng; A-4, preparing a red ginseng fermented extract.

3. A method for increasing the content of rare ginsenosides in red ginseng, characterized in that, comprising fermenting red ginseng powder using the Pediococcus acidilactici KMHD9-314 of claim 1.

4. The method of claim 3, wherein, comprising adding Pediococcus acidilactici KMHD9-314 bacterial solution to a red ginseng powder suspension for fermentation; Preferably, the fermentation condition is static culture at 28-42℃ for 16-36h, preferably static culture at 37℃ for 24h. Preferably, the volume ratio of the Pediococcus acidilactici KMHD9-314 bacterial solution to the red ginseng extract is 0.03-0.1:1, preferably 0.05:

1.

5. The method of claim 4, wherein, The OD of the *Pediococcus lactis* KMHD9-314 bacterial suspension 600 The value is 1.300~1.800, preferably 1.

600.

6. A fermentation agent for improving the content of rare ginsenosides in red ginseng extract, characterized in that, comprising the Pediococcus acidilactici KMHD9-314 of claim 1.

7. A method for preparing a red ginseng fermentation extract, characterized by, comprising fermenting a red ginseng powder suspension using the Pediococcus acidilactici KMHD9-314 of claim 1 to obtain a red ginseng fermented extract, centrifuging to obtain supernatant, and drying to obtain a red ginseng fermented extract.

8. The preparation method according to claim 7, characterized in that, Preferably, the pH is 2-4, preferably 3.

0. Preferably, the sterilization comprises heating at 100℃ for 0.5-1.5h. Preferably, the drying comprises spray drying, vacuum drying or freeze drying. obtained by the preparation method of claim 7 or 8.

9. A red ginseng fermentation extract, characterized by, 10. The use of the red ginseng fermented extract of claim 9 in the preparation of a product for anti-aging. ​

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