Lactobacillus brevis and application of fermented red ginseng of lactobacillus brevis in tumor resistance

Abstract

The invention provides application of lactobacillus brevis and red ginseng fermented by the lactobacillus brevis in tumor resistance and relates to the technical field of biological fermentation, the lactobacillus brevis is lactobacillus brevis, the strain number of the lactobacillus brevis is KMHD10-222, the registration number of the lactobacillus brevis in the China General Microbiological Culture Collection Center is CGMCC No. 32358, and the number of the lactobacillus brevis is KMHD10-222. The lactobacillus brevis KMHD10-222 can produce cellulase and pectinase, can be used for fermenting red ginseng, can obviously improve the content of various rare ginsenosides in the red ginseng and the dissolution rate of the red ginseng extract, improves the effective active ingredients in the red ginseng extract, and improves the anti-tumor effect of the red ginseng extract. The technical problem that a probiotic capable of efficiently converting various rare ginsenosides is lacked in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation, and in particular to a lactobacillus brevis and application of fermented red ginseng in anti-tumor. BACKGROUND

[0002] Ginsenosides have anti-inflammatory, anti-tumor, antipyretic analgesic, immune-enhancing and cardiovascular pharmacological activities. The main ginsenosides with glycosylation, such as Rb1, Rb2, Rc, Rd, Re and Rg1, account for more than 80% of the total saponins of wild ginseng. Rare saponins such as CK, Rh1, Rh2, Rg3, Rk1 and Rg5 have fewer sugar groups. The type, number and binding site (C-3, C-6, C-20) of the sugar group have been proved to affect the biological activity. For example, the influence rule of ginsenosides with different number of sugar groups on anti-tumor activity is: aglycone saponin > monosaccharide saponin > disaccharide saponin > trisaccharide saponin. The content of deglycosylated secondary ginsenosides in the original ginseng plant is very small, but the deglycosylated ginsenosides have more pharmacological activities and bioavailability than the glycosylated ginsenosides.

[0003] Red ginseng is a processed product of ginseng. Its processing method is prepared through soaking, cleaning, steaming, airing, drying and other processes. In the processed red ginseng, the contents of rare ginsenosides Rg3, Rg5, Rk1 and 20(S)-PPD are significantly higher than those in the sun-dried ginseng (i.e. original ginseng).

[0004] Traditional Chinese medicine fermentation technology utilizes the metabolic activity of microorganisms to decompose cellulose, protein and other macromolecules in traditional Chinese medicinal materials by using their rich enzyme system, thereby improving the content of effective components and enhancing the efficacy. Previous researches on the preparation of ginseng or red ginseng fermentation extract mostly revolve around mold, yeast or double bacteria or multi-bacterial complex, and are accompanied by enzyme hydrolysis process. The mold culture time is long, or multiple bacterial strains are involved, and enzymes are extracted, resulting in complex process and low conversion efficiency of rare ginsenosides or single type of rare ginsenosides.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a lactobacillus brevis to solve the technical problem that there is no probiotic bacteria capable of efficiently converting multiple rare ginsenosides in the prior art.

[0007] The second purpose of the present application is to provide the application of the lactobacillus brevis.

[0008] The third purpose of the present application is to provide a method for increasing the content of rare ginsenosides in red ginseng.

[0009] The fourth object of the present application is to provide a fermentation agent for increasing the content of rare ginsenosides in red ginseng.

[0010] The fifth object of the present application is to provide a preparation method of a red ginseng fermentation extract.

[0011] The sixth object of the present application is to provide a red ginseng fermentation extract.

[0012] The seventh object of the present application is to provide the use of the above-mentioned red ginseng fermentation extract in the preparation of a product for anti-tumor.

[0013] In order to achieve the above-mentioned objects of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a Lactobacillus curvatus, which is Lactobacillus curvatus (Lactobacillus curvatus) Latilactobacillus curvatus , the strain number of which is KMHD10-222, and the registration number thereof in China General Microbiological Culture Collection Center is CGMCC No. 32358.

[0014] In a second aspect, the present application provides the use of the above-mentioned Lactobacillus curvatus in any one of the following aspects: A1, increasing the content of rare ginsenosides in red ginseng; A2, preparing a product for increasing the content of rare ginsenosides in red ginseng; A3, preparing a red ginseng fermentation extract. In a third aspect, the present application provides a method for increasing the content of rare ginsenosides in red ginseng, which comprises fermenting red ginseng powder using the above-mentioned Lactobacillus curvatus KMHD10-222.

[0015] Further, the Lactobacillus curvatus KMHD10-222 bacterial liquid is added to a red ginseng powder suspension for fermentation; Preferably, the fermentation conditions are static culture at 28-42℃ for 12-36h, preferably static culture at 37℃ for 24h. Preferably, the volume ratio of the Lactobacillus curvatus KMHD10-222 bacterial liquid to the red ginseng extract liquid is 0.03-0.1:1, preferably 0.05:1.

[0016] Further, the OD 600 value of the Lactobacillus curvatus KMHD10-222 bacterial liquid is 1.200-1.800, preferably 1.500.

[0017] In a fourth aspect, the present application provides a fermentation agent for increasing the content of rare ginsenosides in red ginseng, comprising the above-mentioned Lactobacillus curvatus KMHD10-222.

[0018] In a fifth aspect, the present application provides a preparation method of the red ginseng fermentation extract, comprising obtaining the red ginseng fermentation extract from the fermentation red ginseng powder suspension of the Lactobacillus curvatus KMHD10-222, centrifuging to obtain the supernatant, and drying to obtain the red ginseng fermentation extract.

[0019] Further, the centrifugation further comprises adjusting pH and sterilization before the centrifugation. Preferably, the pH is 2.0-4.0, preferably 3.0. Preferably, the sterilization comprises heating at 100℃ for 0.5-1.5h. Preferably, the drying comprises any one of spray drying, vacuum drying or freeze drying.

[0020] In a sixth aspect, the present application provides a red ginseng fermentation extract prepared by the above preparation method.

[0021] In a seventh aspect, the present application provides the use of the above red ginseng fermentation extract in the preparation of a product for anti-tumor.

[0022] The Lactobacillus curvatus provided by the present application can produce cellulase and pectinase, can be used for fermenting red ginseng, can significantly increase the content of various rare ginsenosides in red ginseng and the dissolution rate of red ginseng extract, can increase the effective active ingredients in the red ginseng extract, and can improve the anti-tumor effect. The present application solves the technical problem in the prior art that there is a lack of probiotics capable of efficiently converting various rare ginsenosides. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 The results of the influence of the red ginseng fermentation extract provided by the present application on tumor volume in the anti-tumor animal experiment; Figure 2 The results of the influence of the red ginseng fermentation extract provided by the present application on tumor growth in the anti-tumor animal experiment; Figure 3 The results of the influence of the red ginseng fermentation extract provided by the present application on IFN-γ, TNF-α and GZMB indexes in the anti-tumor animal experiment. DETAILED DESCRIPTION

[0025] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprising A or B" means "comprising A or B or both". Also, the use of "comprising" or "including" or other forms for "comprise" or "including" is intended to be non- limiting.

[0026] Unless otherwise indicated, the methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated.

[0027] The technical solutions of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0028] In one aspect, the present application provides a Lactobacillus curvatus, which is Lactobacillus curvatus (Lactobacillus curvatus) Latilactobacillus curvatus , the strain number of which is KMHD10-222, and the registration number of which in China General Microbiological Culture Collection Center is CGMCC No. 32358.

[0029] Latilactobacillus curvatus Strain name: Lactobacillus curvatus KMHD10-222; Latin name: Latilactobacillus curvatus ; Preservation agency: China General Microbiological Culture Collection Center; Preservation agency abbreviation: CGMCC; Address: No. 3, Beichen West Road, Beijing City, Chaoyang District; Preservation date: October 25, 2024; Preservation center preservation number: CGMCC No. 32358.

[0030] Lactobacillus curvatus KMHD10-222 can produce cellulase and pectinase, and can be used for fermenting red ginseng. It can significantly increase the content of various rare ginsenosides in red ginseng and the dissolution rate of red ginseng extract, increase the effective active ingredients in red ginseng extract, and improve the anti-tumor effect of red ginseng extract. It solves the technical problem that there is a lack of probiotics that can efficiently convert various rare ginsenosides in the prior art.

[0031] According to another aspect of the present application, there is further provided the use of the above-mentioned Lactobacillus curvatus in any one of the following: A1, increasing the content of rare ginsenosides in red ginseng; A2, preparing a product for increasing the content of rare ginsenosides in red ginseng; A3, preparing a red ginseng fermentation extract. According to another aspect of the present application, there is further provided a method for increasing the content of rare ginsenosides in red ginseng, comprising fermenting red ginseng powder with the above-mentioned Lactobacillus curvatus KMHD10-222.

[0032] The Lactobacillus curvatus KMHD10-222 can ferment red ginseng powder as a substrate to increase the content of rare ginsenosides in the red ginseng powder and improve the anti-tumor effect of the red ginseng extract.

[0033] In some specific embodiments, the Lactobacillus curvatus KMHD10-222 bacterial solution is added to a red ginseng powder suspension for fermentation; in some specific embodiments, the fermentation conditions are static culture at 28-42℃ for 12-36h; in some specific embodiments, the volume ratio of the Lactobacillus curvatus KMHD10-222 bacterial solution to the red ginseng extract is 0.03-0.1:1.

[0034] The fermentation temperature can be, but is not limited to, 28℃, 30℃, 32℃, 34℃, 37℃, 38℃, 40℃ or 42℃, or any value between 28-42℃, and is preferably 37℃.

[0035] The static culture time can be, but is not limited to, 12h, 14h, 16h, 18h, 20h, 24h, 25h, 28h, 30h, 32h, 34h or 36h, or any value between 12-36h, and is preferably 24h.

[0036] The volume ratio of the Lactobacillus curvatus KMHD10-222 bacterial solution to the 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 0.03-0.1:1, and is preferably 0.05:1.

[0037] In some specific embodiments, the OD 600 value of the Lactobacillus curvatus KMHD10-222 bacterial solution is 1.200-1.800.

[0038] The OD 600The value can be, but is not limited to, 1.200, 1.300, 1.400, 1.500, 1.600, 1.700 or 1.800, can also be any value between 1.200 and 1.800, and is preferably 1.500.

[0039] According to another aspect of the present application, there is also provided a fermentation agent for increasing the content of rare ginsenosides in red ginseng, comprising the above-mentioned Lactobacillus brevis KMHD10-222.

[0040] According to another aspect of the present application, there is also provided a preparation method of a red ginseng fermentation extract, comprising taking a red ginseng fermentation extract liquid obtained by fermenting a red ginseng powder suspension with the above-mentioned Lactobacillus brevis KMHD10-222, centrifuging to take supernatant, and drying to obtain a red ginseng fermentation extract.

[0041] The red ginseng fermentation extract prepared by fermentation with the Lactobacillus brevis KMHD10-222 significantly increases the content of various rare ginsenosides and enhances the anti-tumor effect of red ginseng.

[0042] The rare ginsenosides include at least one of Rh1, Rg3, Rk1, Rg5, Rh4, Rg6 or Rk3.

[0043] In some specific embodiments, the centrifuging further comprises adjusting pH and sterilization before the centrifuging; in some specific embodiments, the pH is 2.0-4.0; in some specific embodiments, the sterilization comprises heating at 100°C for 0.5-1.5 h; in some specific embodiments, the drying comprises any one of spray drying, vacuum drying or freeze drying.

[0044] The pH can be, but is not limited to, 2, 2.3, 2.5, 2.8, 3, 3.3, 3.5, 3.8 or 4.0, can also be any value between 2.0 and 4.0, and is preferably 3.0.

[0045] The sterilization time can be, but is not limited to, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.2 h or 1.5 h, can also be 0.5-1.5 h, and is preferably 1 h.

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

[0047] According to another aspect of the present application, there is also provided the use of the above-mentioned red ginseng fermentation extract in the preparation of a product for anti-tumor.

[0048] The present application is further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0049] MRS medium: casein enzymatic digest 10 g / L, beef extract powder 10 g / L, yeast extract powder 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 enzymatic digest 10 g / L, beef extract powder 10 g / L, yeast extract powder 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] Isolation, identification and preservation of the strain of Example 1 1. Isolation and purification of the strain 1 g of the sample was weighed into a sterile bag, 9 mL of sterile normal saline was added, and the mixture was beaten twice in a homogenizer for 2 min each time to prepare a 10 -1 dilution. The 10 -1 dilution was diluted by 10 times in gradient to 10 -7 , and a series of dilutions were plated and inverted plates were cultured in an anaerobic incubator at 37℃ for 48 h. Single colonies that made the bromocresol green medium yellow were picked and streaked on MRS medium again, and cultured in an anaerobic incubator at 37℃. The obtained colonies were purified by streaking multiple times to obtain single bacteria.

[0052] 2. Identification of the strain: The colony morphology of the single bacteria purified multiple times was observed. The strain Lactobacillus curvatus KMHD10-222 had a round, milky white colony, the outer circle of the colony was slightly transparent, the surface was wet and smooth, the edge was neat, and the colony was purple after Gram staining, negative after enzyme contact reaction, and rod-shaped under microscope.

[0053] The 16S rRNA gene fragment was amplified using the universal primers 27F and 1492R for bacterial sequencing with the colony DNA as the template. The PCR amplification reaction system and amplification program are shown in Tables 1 and 2.

[0054] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0055] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0056] Table 1 PCR reaction system

[0057] Table 2 PCR reaction procedure

[0058] 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:

[0059] 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 KMHD10-222 was identified as *Lactobacillus curvilinearis*. Latilactobacillus curvatus Figure 1 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. 32358.

[0060] Example 2: Preparation of Lactobacillus curvilinearis KMHD10-222 bacterial culture After thawing the glycerol tubes of Lactobacillus curvatureus strain KMHD10-222, the bacterial solution was streaked on a plate on a clean bench to obtain a single colony. The single colony was then inoculated into MRS liquid medium and incubated at 37°C to obtain the fermentation seed liquid of the strain.

[0061] Experiment 1: Enzyme activity assay of Lactobacillus curvaturei KMHD10-222 Cellulase screening medium: 10g bacteriological peptone, 10g beef extract, 4g yeast extract, 1g anhydrous citric acid, 5g sodium acetate, 3g glucose, 0.2g anhydrous magnesium sulfate, 0.05g manganese sulfate monohydrate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 20g agar, 2g Congo red, 10g CMC-Na, and 1L purified water. Fermentation medium: 10g bacteriological peptone, 4g yeast extract, 1g triammonium citrate, 5g sodium acetate, 0.2g anhydrous magnesium sulfate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 10g CMC-Na, and 1L purified water.

[0062] Pectinase screening medium: 10g bacteriological peptone, 10g beef extract, 4g yeast extract, 1g anhydrous citric acid, 5g sodium acetate, 3g glucose, 0.2g anhydrous magnesium sulfate, 0.05g manganese sulfate monohydrate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 20g agar, 2g Congo red, 4g pectin, and 1L purified water. Fermentation medium: 10g bacteriological peptone, 4g yeast extract, 1g triammonium citrate, 5g sodium acetate, 0.2g anhydrous magnesium sulfate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 4g pectin, and 1L purified water.

[0063] The formula for calculating enzyme activity is: ; In the formula: U is the enzyme activity (ug / min / mL), S is the glucose content (mg) corresponding to the average absorbance value of the sample on the standard curve, 1000 is the conversion factor between mg and ug, T is the enzyme reaction time (min), and V is the volume of enzyme solution added to the reaction system (mL).

[0064] 1. Cellulase activity assay: 2 μL of bacterial suspension (prepared according to the method in Example 2, bacterial suspension OD) was measured. 600 A cellulase concentration of 1.500 was inoculated onto filter paper discs of cellulase selection medium and cultured for 36–72 h. The appearance of a clear zone around the colony was observed to assess the cellulase activity of the strain. A 2% inoculum was added to fermentation medium and fermented at 37°C for 2 days. The bacterial culture was centrifuged at 10,000 rpm for 5 min, and the supernatant was the crude enzyme solution. 120 μL of the crude enzyme solution was added to 80 μL of 1.0% CMC-Na, and reacted in a 50°C water bath for 30 min. Immediately afterwards, 100 μL of DNS reagent was added, the mixture was shaken to mix, and the reaction was terminated. The mixture was then placed in a boiling water bath for 10 min for color development. After cooling to room temperature, 700 μL of water was added and mixed thoroughly. The mixture was centrifuged at 10,000 rpm for 5 min, and the absorbance was measured at 540 nm. The blank control was the crude enzyme solution inactivated in boiling water at 100°C for 10 min.

[0065] An enzyme activity unit is defined as the amount of enzyme required to catalyze the degradation of a substrate to produce 1 μg of reducing sugar per minute under the above experimental conditions.

[0066] The cellulase activity of Lactobacillus curvaturei KMHD10-222 was measured to be 0.085 ug / min / mL.

[0067] 2. Pectinase activity assay: 2 μL of bacterial suspension (prepared according to the method in Example 2, bacterial suspension OD) was measured. 600 A 1.500 value was inoculated onto filter paper discs of the selection medium and cultured for 36–72 h. A clear zone was observed around the colonies to assess the pectinase activity of the strain. A 2% inoculum was added to fermentation medium and fermented at 37°C for 2 days. The bacterial culture was centrifuged at 10,000 rpm for 5 min, and the supernatant was the crude enzyme solution. 0.2 mL of the crude enzyme solution was transferred to an EP tube, and 0.2 mL of pectin solution was added, mixed well, and reacted in a 46°C water bath for 1 h. Immediately afterward, 0.8 mL of DNS reagent was added to the EP tube and mixed thoroughly. After heating at 100°C for 10 min, 0.1 mL was transferred to another EP tube, diluted 10-fold with 0.9 mL of pure water, centrifuged at 10,000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. An inactivated enzyme solution was used as a blank control. Each sample was measured in triplicate, and the average value was taken.

[0068] Pectinase activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of galacturonic acid from pectin per minute in 1.0 mL of pectinase solution at 46℃ and pH 6.5, which is defined as 1 unit of enzyme activity (U / mL).

[0069] The pectinase activity of Lactobacillus curvaturei KMHD10-222 was measured to be 0.479 ug / min / mL.

[0070] Example 3 The preparation method of red ginseng fermentation extract involves crushing red ginseng rootlets and passing them through a 20-mesh sieve to obtain red ginseng powder as raw material. The method provided in Example 2 is used to prepare Lactobacillus curvaturei KMHD10-222 bacterial suspension with an OD600 value of 1.500. The specific steps are as follows: Red ginseng powder was added to 20 times its volume of pure water, and the suspension was pasteurized for 30 minutes. Then, Lactobacillus curvaturei KMHD10-222 bacterial suspension was added to the red ginseng powder suspension at a ratio of 3% (V / V). The suspension 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 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.

[0071] 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension was added to the red ginseng powder suspension at a ratio of 10% (V / V). The suspension 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 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.

[0072] 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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. After fermentation, the pH was adjusted to 3.0 with citric acid, 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.

[0073] Example 6 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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 16 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, 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] Example 7 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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 36 hours. After fermentation, the pH was adjusted to 3.0 with citric acid, 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.

[0075] Example 8 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension was added to the red ginseng powder suspension at a ratio of 5% (V / V). The suspension was allowed to ferment at 28℃ 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.

[0076] 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension was added to the red ginseng powder suspension at a ratio of 5% (V / V). The suspension 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 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.

[0077] Example 10 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 30 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.

[0078] 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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. After fermentation, the pH was adjusted to 3.0 with citric acid, and then sterilized at 100°C for 90 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.

[0079] Example 12 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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. After fermentation, the pH was adjusted to 2.0 with citric acid, 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.

[0080] 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, Lactobacillus curvaturei KMHD10-222 bacterial suspension 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. After fermentation, the pH was adjusted to 4.0 with citric acid, 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.

[0081] Comparative Example 1 Unlike Example 5, OD was selected. 600 The same value of Lactobacillus curvilinearis FBC-3-2 bacterial solution was used instead of Lactobacillus curvilinearis KMHD10-222 bacterial solution.

[0082] Comparative Example 2 Unlike Example 5, OD was selected. 600 The same value of Lactobacillus curvilinearis FBC-1-8 bacterial solution was used instead of Lactobacillus curvilinearis KMHD10-222 bacterial solution.

[0083] Comparative Example 3 Red ginseng powder was added to 20 times its volume of pure water and then extracted at 100℃ for 60 minutes. The mixture was then centrifuged at 10,000 rpm for 10 minutes to obtain red ginseng liquid. The red ginseng liquid was frozen at -80℃ overnight and then freeze-dried for 24 hours to collect the red ginseng extract.

[0084] Comparative Example 4 Unlike Comparative Example 3, the red ginseng powder was added to 20 times its volume of pure water, and the pH was adjusted to 3.0 with citric acid. Extraction was then performed at 100℃ for 60 minutes, followed by centrifugation at 10,000 rpm for 10 minutes to obtain the red ginseng solution. This solution was then frozen at -80℃ overnight and freeze-dried for 24 hours to collect the red ginseng extract.

[0085] Comparative Example 5 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, Lactobacillus curvularis KMHD10-222 bacterial suspension 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.

[0086] Experiment 2: Detection of effective components in red ginseng fermentation extract 1. Detection methods for rare ginsenosides Liquid chromatography conditions: 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.

[0087] The ultra-high performance liquid chromatography (UHPLC) conditions are shown in Table 3.

[0088] Table 3

[0089] The results of the rare ginsenoside detection are shown in Table 4.

[0090] Dissolution rate of red ginseng extract: Dissolution rate of red ginseng extract = red ginseng powder / red ginseng raw material * 100%. The results are shown in Table 4.

[0091] Table 4

[0092] Table 4 shows that, compared with Comparative Example 3, the dissolution rate and the content of each rare ginsenoside in Examples 3 to 13 were improved. This indicates that fermentation of red ginseng with *Lactobacillus curvularis* KMHD10-222 can increase the content and dissolution rate of rare ginsenosides. Comparative Example 4, based on Comparative Example 3, adjusted the pH to 3.0 with citric acid, resulting in a slight increase in the content of rare ginsenosides. Compared with Example 5, the dissolution rate and the content of each rare ginsenoside in Comparative Examples 1 and 2 were reduced. This indicates that not all *Lactobacillus curvularis* species can achieve the same efficiency as *Lactobacillus curvularis* KMHD10-222 in converting rare ginsenosides. Comparative Example 5, without adjusting the pH with citric acid, had a higher content of rare ginsenosides than the other comparative examples, but it was significantly lower than that in Example 5.

[0093] Experiment 3: Antitumor cell assay of red ginseng fermentation extract This experiment used the fermented red ginseng extract prepared in Example 5 and the red ginseng extract prepared in Comparative Example 3 for anti-tumor cell experiments.

[0094] 1. Experimental Methods: Red ginseng powder and fermented red ginseng powder were prepared into a high-concentration stock solution of 5 mg / mL using PBS, and diluted to the required concentration with culture medium before the experiment. Each cell line (lung cancer cell A549, human liver cancer cell HepG2, mouse liver cancer cell Hepa1-6, cervical cancer cell Hela, human glioma cell U87, and colorectal cancer CT-26) was cultured in its corresponding complete culture medium, passaged every 2-3 days to ensure the cells were in the logarithmic growth phase. Cells in the logarithmic growth phase were collected, counted, and the density was adjusted to 5000 cells per well. After culturing for 24 hours, the following day, drug treatment was performed by adding 100 μL of culture medium containing different drug concentrations to each well via medium exchange. The control group received an equal volume of culture medium. The cells were incubated for 24 hours, followed by incubation with 10 μL / well of CCK8 solution for another 2 hours. The absorbance was measured at 450 nm. Cell viability at each concentration was calculated, and IC50 values ​​were calculated by plotting curves.

[0095] 2. The results of the IC50 test for tumor cells are shown in Table 5.

[0096] Table 5

[0097] As shown in Table 5, the IC50 values ​​of Example 5 are 13.73% to 49.19% of those of Comparative Example 3, indicating that the fermented red ginseng extract has a better effect on inhibiting tumor cells than the unfermented red ginseng extract.

[0098] Experiment 4: Antitumor animal experiment of red ginseng fermentation extract 1. Test Methods Male BALB / c mice, 7-8 weeks old and weighing 20-26 g, were purchased. After one week of acclimatization feeding, they were randomly divided into 4 groups according to their weight. Except for the control group, all mice in the other groups were injected with approximately 1×10⁻⁶ mg / L of vaccine under the armpit. 6 5 × 10⁶ CT-26 cells (diluted to 5 × 10⁶ with 6 Tumor modeling was initiated by injecting 200 μL of the sample at a rate of 1 tumor per mL. After continuing feeding for 7-10 days, the tumor size was observed, and tumors larger than 40 mm were selected. 3 The mice were then used in subsequent experiments.

[0099] Mice that successfully developed the tumor model were randomly divided into a tumor model group, a fermented red ginseng group (red ginseng fermentation extract from Example 5), and an unfermented red ginseng group (red ginseng extract from Comparative Example 3). The tumor model group received 0.5 mL of physiological saline via gavage daily. The fermented / unfermented red ginseng groups received 0.5 mL of diluted red ginseng fermentation extract via gavage daily. The concentration of the lyophilized red ginseng fermentation extract was 5460 mg / kg, and the dosage was 10 mL / kg. Dosage was administered once daily for 22 consecutive days. Tumor volume was measured and calculated on days 0, 3, 6, 9, 12, 15, 17, and 21 after the start of administration (formula: V = 1 / 2ab², where a is the tumor length and b is the tumor width). On day 22, after measuring the weight of the mice, blood samples were collected from the mice using the ocular blood sampling method for subsequent blood index detection (IFN-γ, TNF-α, and GZMB). The mice were euthanized and their tumors were dissected and separated. Non-tumor tissues were removed, and the mice were weighed, photographed, and the tumor growth inhibition rate was calculated (tumor inhibition rate (%) = 1 - (average tumor weight of experimental group mice / average tumor weight of model group mice) × 100%).

[0100] 2. Results (1) Changes in tumor volume, such as Figure 2 As shown, the tumor volume of the tumor model mice in the fermented red ginseng group was smaller than that in the unfermented red ginseng group and smaller than that in the model group, indicating that the fermented red ginseng extract enhanced the anti-tumor effect of red ginseng.

[0101] (2) Tumor inhibition rate, such as Figure 3 As shown, fermented red ginseng extract has a better effect on inhibiting tumor growth than red ginseng extract.

[0102] (3) Blood index test results as follows ​ As shown, the levels of IFN-γ, TNF-α, and GZMB in the fermented red ginseng group were all higher than those in the unfermented red ginseng group, indicating that the fermented red ginseng extract improved the anti-tumor effect of red ginseng.

[0103] 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. *Lactobacillus curvilinearus*, characterized in that, The *Lactobacillus curvilinearus* is *Lactobacillus curvilinearus* (… Latilactobacillus curvatus The strain number is KMHD10-222, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32358.

2. The use of the *Lactobacillus curvilinearus* according to claim 1 in any of the following species: A1. Increase the content of rare ginsenosides in red ginseng; A2. Preparation of products for increasing the content of rare ginsenosides in red ginseng; A3. Preparation of red ginseng fermentation extract.

3. A method for increasing the content of rare ginsenosides in red ginseng, characterized in that, This includes fermented red ginseng powder using Lactobacillus curvatureus KMHD10-222 as described in claim 1.

4. The method according to claim 3, characterized in that, This includes adding Lactobacillus curvaturei KMHD10-222 bacterial culture to red ginseng powder suspension for fermentation; Preferably, the fermentation conditions are static culture at 28~42℃ for 12~36h, and more preferably static culture at 37℃ for 24h; Preferably, the volume ratio of the Lactobacillus curvaturei KMHD10-222 bacterial suspension to the red ginseng extract is 0.03~0.1:1, more preferably 0.05:

1.

5. The method according to claim 4, characterized in that, The OD of the Lactobacillus curvaturei KMHD10-222 bacterial suspension 600 The value is 1.200~1.800, preferably 1.

500.

6. A fermentation agent for increasing the content of rare ginsenosides in red ginseng, characterized in that, Includes *Lactobacillus curvilinearis* KMHD10-222 as described in claim 1.

7. A method for preparing a fermented red ginseng extract, characterized in that, The method includes taking the red ginseng fermentation extract obtained by fermenting red ginseng powder suspension with Lactobacillus curvularis KMHD10-222 as described in claim 1, centrifuging to collect the supernatant, and drying to obtain the red ginseng fermentation extract.

8. The preparation method according to claim 7, characterized in that, The process of centrifugation also includes pH adjustment and sterilization. Preferably, the pH is 2.0 to 4.0, 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 any one of spray drying, vacuum drying, or freeze drying.

9. A fermented red ginseng extract, characterized in that, It is prepared by the preparation method described in claim 7 or 8.

10. The use of the red ginseng fermentation extract according to claim 9 in the preparation of products for antitumor purposes.

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