Preparation method and application of fermentation liquor rich in rare ginsenosides

Abstract

This invention discloses a method for preparing and applying a fermentation broth rich in rare ginseng saponins. The method involves inoculating *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 into a ginseng fermentation substrate, fermenting at 25℃–40℃, and then sterilizing. The total content of rare ginseng saponins is greater than 800 mg / kg. The co-fermentation using *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 not only effectively improves the saponin conversion rate but also effectively increases the content of ginseng flavonoids and peptides. Furthermore, the method for preparing the fermentation broth rich in rare ginseng saponins is simple and easy for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a method for preparing and applying fermentation broth rich in rare ginseng saponins. Background Technology

[0002] Ginseng (Panax ginseng CAMeyer), known as the "King of Herbs," is a traditional and precious Chinese medicine with the functions of replenishing vital energy, consolidating the body, promoting body fluid production, calming the mind, and improving intelligence. Ginseng contains ginsenosides, ginseng polysaccharides, and free amino acids, among other active ingredients. Ginsenosides are its main active ingredient, accounting for approximately 4%. Ginsenosides are terpenoid compounds. The differences between different types of ginsenosides lie in the types of aglycones and sugar groups, the number of sugar groups attached, the carbon positions of the sugar groups, and the presence of acetyl or malonyl groups on the sugar groups. Ginsenosides are classified according to their aglycone structure into dammarane-type ginsenosides, octylene-type ginsenosides, and oleanolic acid ginsenosides. Dammarane-type ginsenosides include protopylene-type ginsenosides (PPD type) and protopylene-type ginsenosides (PPT type). PPD-type ginsenosides mainly include Rb1, Rb2, Rb3, Rc, Rd, Rh2, Rg3, Rg5, CK, F2, PPD, etc., while PPT-type ginsenosides mainly include Re, Rf, Rg1, Rg2, Rh1, F1, PPT, etc.

[0003] Related studies have shown that ginseng (white ginseng) generally contains mainly inherent ginsenosides such as Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, and Rg1, while rare saponins such as Rg3, Rg5, CK, F2, Rg2, and Rh1 are either absent or present in very low amounts. The absorption of inherent ginsenosides in the body mainly relies on intestinal microorganisms and enzymes to convert them into rare saponins; however, this conversion efficiency is low, resulting in low absorption efficiency of inherent ginsenosides. Among related technologies, microbial fermentation biotransformation is considered one of the safest and most efficient methods for the in vitro conversion of ginseng saponins. Lactic acid bacteria are recognized as beneficial bacteria for humans, and the enzymes produced through lactic acid bacteria fermentation can increase the content of rare ginsenosides in ginseng. However, in actual application, the conversion rate of ginsenosides using conventional lactic acid bacteria is low, and it only converts PPD-type Rg3, CK, F2, and Rh2, making it difficult to simultaneously prepare fermentation broth rich in rare ginsenoside groups Rg3, Rg5, Rh2, CK, Rg2, and Rh1.

[0004] Therefore, there is an urgent need to find a method for preparing fermentation broth rich in rare ginsenosides that can effectively improve the conversion rate of inherent ginsenosides. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for preparing a fermentation broth rich in rare saponins, which can effectively improve the conversion rate of inherent ginsenosides, and the total content of rare saponins in the obtained fermentation broth is greater than 800 mg / kg.

[0006] The present invention also proposes a fermentation broth.

[0007] This invention also proposes the application of fermentation broth in the preparation of products for the prevention of neurological diseases.

[0008] In a first aspect, the present invention provides a method for preparing a fermentation broth rich in rare ginseng saponins, comprising: inoculating the Lactobacillus paracasei B04WI2501 and the Lactobacillus paracasei B16NY2107 into a ginseng fermentation substrate, fermenting at 25°C to 40°C, and then sterilizing.

[0009] The total content of rare ginsenosides is greater than 800 mg / kg.

[0010] The fermentation broth rich in rare ginseng saponins according to embodiments of the present invention has at least the following beneficial effects: the total rare saponin content of the fermentation broth obtained by fermenting ginseng with Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 of the present invention is high, and the conversion rate of total rare saponins before and after fermentation is as high as 723%; in addition, the preparation method of the fermentation broth rich in rare ginseng saponins of the present invention is simple, does not require multi-strain stepwise fermentation, and is easy to industrialize and produce on a large scale.

[0011] In some embodiments of the present invention, the rare ginsenoside group includes Rg3, CK, Rg5, Rh2, Rg2 and Rh1.

[0012] In some embodiments of the present invention, the Lactobacillus paracasei B04WI2501 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.62875 and deposit date of October 13, 2022.

[0013] In some embodiments of the present invention, the *Lactobacillus paracasei* B04WI2501 has the 16S rDNA sequence shown in SEQ ID NO: 1.

[0014] In some embodiments of the present invention, the Lactobacillus paracasei B16NY2107 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.62874 and deposit date of October 13, 2022.

[0015] In some embodiments of the present invention, the Lactobacillus paracasei B16NY2107 has the 16S rDNA sequence shown in SEQ ID NO: 2.

[0016] In some embodiments of the present invention, the inoculation amounts of Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 are each 1% to 2%.

[0017] In some embodiments of the present invention, the inoculation ratio of Lactobacillus paracasei B04WI2501 to Lactobacillus paracasei B16NY2107 is 0.8 to 1.2:1.

[0018] In some embodiments of the present invention, the inoculation ratio of Lactobacillus paracasei B04WI2501 to Lactobacillus paracasei B16NY2107 is 1:1.

[0019] In some embodiments of the present invention, the bacterial activity of *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 is 10. 7 cfu / mL ~10 9 cfu / mL.

[0020] In some embodiments of the present invention, the ginseng fermentation substrate is ginseng enzymatic hydrolysate or ginseng water extract.

[0021] In some embodiments of the present invention, the preparation method of the ginseng hydrolysate is as follows: ginseng is crushed, soaked, pulped, and then 0.8-1.2% by weight of pectinase is added for enzymatic hydrolysis. After enzymatic hydrolysis, the pH value is adjusted to 5.0-6.5, a carbon source is added, and the mixture is kept at 90℃-100℃ for 2.5-3.5 hours. After cooling, the hydrolysate is obtained.

[0022] Excessive or insufficient heat preservation time is detrimental to increasing saponin content.

[0023] In some embodiments of the present invention, the pulping time is 3-5 minutes. Pulping helps to promote the uniform mixing of ginseng powder and facilitates the dissolution of active ingredients.

[0024] In some embodiments of the present invention, the enzymatic hydrolysis temperature is 42°C to 52°C.

[0025] In some embodiments of the present invention, the enzymatic hydrolysis time is 2h to 4h.

[0026] In some embodiments of the present invention, the temperature after cooling is 37±3℃.

[0027] In some embodiments of the present invention, the carbon source is at least one of white sugar, fructooligosaccharides, concentrated apple juice, and concentrated jujube juice;

[0028] Preferably, the carbon source is white sugar.

[0029] In some embodiments of the present invention, the method for preparing the ginseng enzymatic hydrolysate specifically includes:

[0030] Step S11: Grind the ginseng into powder using a high-speed grinder and pass it through an 8-12 mesh sieve to obtain ginseng powder;

[0031] Step S12: Soak the ginseng powder in hot water with a material-to-liquid ratio of 1:15-20, then grind it into a slurry using a colloid mill, and add 0.8-1.2% pectinase by weight for enzymatic hydrolysis. After enzymatic hydrolysis, adjust the pH value to 5.0-6.5.

[0032] Step S13: Add 4.0-7.5% white sugar by weight, keep warm at 90℃-100℃ for 2.5-3.5 hours, and then use circulating cooling water to stir and cool down to 37±3℃ to obtain the product.

[0033] In some embodiments of the present invention, the preparation method of the ginseng water extract is as follows: dissolve the ginseng extract in water, adjust the pH value to 5.0-6.5, add a carbon source, keep warm at 90℃-100℃ for 0.5-1.5h, and then cool down to obtain the product.

[0034] In some embodiments of the present invention, the amount of ginseng extract added is 3% to 8%;

[0035] Preferably, the amount of ginseng extract added is 5%.

[0036] In some embodiments of the present invention, the pH-adjusting agent is sea buckthorn juice or calcium carbonate.

[0037] In some embodiments of the present invention, the carbon source is at least one of white sugar, fructooligosaccharides, concentrated apple juice, and concentrated jujube juice.

[0038] In some embodiments of the present invention, the fermentation time is 9 to 16 days;

[0039] Preferably, the fermentation time is 14 days.

[0040] In some embodiments of the present invention, the stirring speed during the fermentation process is 50 rpm to 100 rpm.

[0041] In some embodiments of the present invention, the sterilization temperature is 110°C to 125°C;

[0042] Preferably, the sterilization temperature is 115℃~121℃.

[0043] In some embodiments of the present invention, the sterilization time is 15s to 60s.

[0044] Preferably, the sterilization time is 25s to 35s.

[0045] In a second aspect, the present invention provides a fermentation broth prepared by the above-described preparation method.

[0046] A third aspect of the invention provides the use of fermentation broth rich in rare saponins in the preparation of products for the prevention of neurological diseases.

[0047] According to the application of the embodiments of the present invention, at least the following beneficial effects are achieved: the fermentation broth of the present invention, which is rich in rare saponin groups, can significantly improve the survival rate of neurons and has a significant neuroprotective effect on HD disease.

[0048] In some embodiments of the present invention, the neurological disease is Huntington's disease.

[0049] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0051] Figure 1 The figure shows the neuroprotective effect of the fermentation broth samples before and after fermentation in this invention. Detailed Implementation

[0052] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0053] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] In this embodiment of the invention, the ginseng was purchased from Yanbian Changbai Mountain Ecological Park Co., Ltd., and the ginseng was 3-5 years old sun-dried ginseng with a total saponin content that met the requirements of the pharmacopoeia.

[0055] The ginseng extract was purchased from Ningbo Runwo Biotechnology Co., Ltd., and the total saponin (HPLC, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) content was ≥10%.

[0056] Both pectinase and cellulase were purchased from Nanning Pangbo Biotechnology Co., Ltd., in powder or granule form, with a total enzyme activity ≥20,000 U / g;

[0057] The hydrolyzed wheat protein powder was purchased from the Zhoushan Economic Development Zone Branch of Zhejiang Haishi Biotechnology Co., Ltd., and the content of oligopeptides (on a dry basis) is ≥25.0%.

[0058] The fructooligosaccharides were purchased from Quantum Hi-Tech (Guangdong) Biotechnology Co., Ltd., and the fructooligosaccharide content (on a dry basis) was ≥90g / 100g.

[0059] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were isolated in the laboratory and identified by the China Industrial Microbial Culture Collection Center. The test report numbers are No. 22z-0545-01215.0l-02787 and No. 22-0545-01217.01-02789, respectively.

[0061] The Lactobacillus paracasei B04WI2501 was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO.62875, on October 13, 2022.

[0062] In this embodiment of the invention, the 16S rDNA sequence of Lactobacillus paracasei B04WI2501 is as follows:

[0063]

[0064] Lactobacillus paracasei B16NY2107 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO.62874, on October 13, 2022.

[0065] The 16S rDNA sequence of Lactobacillus paracasei B16NY2107 is as follows:

[0066]

[0067] Example 1

[0068] A method for preparing a fermentation broth rich in rare ginseng saponins includes the following steps:

[0069] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng) and grind it into powder using a high-speed grinder. After passing through a 10-mesh sieve, soak it in hot water at 90-100℃ with a material-to-liquid ratio of 1:20 for 12 hours. Then grind it into a slurry using a colloid mill. Add 1.0% pectinase by weight for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the ginseng fermentation substrate is obtained. The enzymatic hydrolysis temperature is 47℃±5℃ and the enzymatic hydrolysis time is 3 hours.

[0070] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth (manufacturer: Beijing Luqiao Technology Co., Ltd.) and incubated at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture at a 2% inoculation rate, into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0071] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0072] (4) Preparation of fermentation broth rich in rare ginseng saponins: The fermentation broth obtained in step (3) was transferred to a fermenter and cooled by stirring with circulating cooling water. After the broth cooled to 37°C, the inoculum from step (2) was inoculated into a 5L fermenter at an inoculation rate of 1%. The fermentation temperature was 30°C and the rotation speed was 50 rpm. The fermentation was carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth was maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0073] Example 2

[0074] A method for preparing a fermentation broth rich in rare ginseng saponins includes the following steps:

[0075] (1) Pretreatment method of ginseng fermentation matrix: Take 50g of ginseng extract and add 950g of purified water to dissolve. After dissolution, the ginseng fermentation matrix is ​​obtained. The ginseng extract was purchased from Ningbo Runwo Biotechnology Co., Ltd., and the total saponin (HPLC, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) content is ≥10%.

[0076] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth and cultured in a constant temperature incubator at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture accounting for 2% of the total culture, and cultured for 24 h at 37℃±1℃ to ensure that the culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 3.0% edible glucose, 2.0% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0077] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng liquid in step (1) to adjust the pH value to 6, add 7.5% oligofructose of ginseng fermentation substrate by mass, stir until dissolved, heat to 90-100℃, keep warm for 1h, and obtain fermentation liquid.

[0078] (4) Preparation of fermentation broth rich in rare ginseng saponins: The fermentation broth from step (3) was transferred to a fermenter and cooled by stirring with circulating cooling water. After the broth cooled to 37°C, the inoculum from step (2) was inoculated into the fermenter at a rate of 1%. Fermentation was carried out at a constant temperature for 14 days at 40°C and a rotation speed of 100 rpm. During the fermentation process, the activity of the inoculum in the fermentation broth was maintained at 10%. 7 cfu / mL to 10 9 The concentration of cfu / mL was determined by centrifugation and filtration after fermentation. The centrifugation parameters were 4000 rpm and 5 min. The product was then passed through a 100-mesh sieve and subjected to ultra-high temperature instantaneous sterilization at 121℃ for 30 s. The product was obtained after sterilization.

[0079] Comparative Example 1

[0080] A method for preparing ginseng fermentation liquid includes the following steps:

[0081] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng) and grind it into powder using a high-speed grinder. After passing through a 10-mesh sieve, soak it in hot water with a material-to-liquid ratio of 1:20 and then grind it into a slurry using a colloid mill to obtain ginseng fermentation substrate.

[0082] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth (manufacturer: Beijing Luqiao Technology Co., Ltd.) and incubated at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture at a 2% inoculation rate, into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0083] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0084] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0085] Comparative Example 2

[0086] A method for preparing ginseng fermentation liquid includes the following steps:

[0087] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng), soak it in hot water with a material-to-liquid ratio of 1:20, cut it into small pieces, and then grind it into a pulp using a colloid mill. Add 1.0% by weight of cellulase for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the ginseng fermentation substrate is obtained. The enzymatic hydrolysis temperature is 47℃±5℃ and the enzymatic hydrolysis time is 3h.

[0088] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth (manufacturer: Beijing Luqiao Technology Co., Ltd.) and incubated at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture at a 2% inoculation rate, into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0089] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0090] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0091] Comparative Example 3

[0092] A method for preparing ginseng fermentation liquid includes the following steps:

[0093] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng), soak it in hot water with a material-to-liquid ratio of 1:20, cut it into small pieces and grind it into a pulp using a colloid mill to obtain ginseng fermentation substrate.

[0094] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth (manufacturer: Beijing Luqiao Technology Co., Ltd.) and incubated at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture at a 2% inoculation rate, into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0095] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0096] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0097] Comparative Example 4

[0098] A method for preparing ginseng fermentation liquid includes the following steps:

[0099] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng), soak it in hot water with a material-to-liquid ratio of 1:20, cut it into small pieces and grind it into a pulp using a colloid mill to obtain ginseng fermentation substrate.

[0100] (2) Preparation of fermentation strains: Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMR broth (manufacturer: Beijing Luqiao Technology Co., Ltd.) and incubated at 37℃±1℃ for 24 h. Then, the two bacterial cultures were inoculated at a 1:1 ratio, with each culture at a 2% inoculation rate, into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0101] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 0.5h to obtain fermentation liquid.

[0102] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0103] Comparative Example 5

[0104] A method for preparing ginseng fermentation liquid includes the following steps:

[0105] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng) and grind it into powder using a high-speed grinder. After passing through a 10-mesh sieve, soak it in hot water at 90-100℃ with a material-to-liquid ratio of 1:20 for 12 hours. Then grind it into a slurry using a colloid mill. Add 1.0% pectinase by weight for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the ginseng fermentation substrate is obtained. The enzymatic hydrolysis temperature is 47℃±5℃ and the enzymatic hydrolysis time is 3 hours.

[0106] (2) Preparation of fermentation strain: Lactobacillus paracasei B04WI2501 was inoculated into 100 mL of LMR broth and cultured in a constant temperature incubator at 37℃±1℃ for 24 h. Then, 4% of the bacterial culture was inoculated into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 10 9 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0107] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0108] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0109] Comparative Example 6

[0110] A method for preparing ginseng fermentation liquid includes the following steps:

[0111] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng) and grind it into powder using a high-speed grinder. After passing through a 10-mesh sieve, soak it in hot water with a material-to-liquid ratio of 1:20 and then grind it into a slurry using a colloid mill. Add 1.0% by weight of pectinase for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the ginseng fermentation substrate is obtained. The enzymatic hydrolysis temperature is 47℃±5℃ and the enzymatic hydrolysis time is 3h.

[0112] (2) Preparation of fermentation strain: Lactobacillus paracasei B16NY2107 was inoculated into 100 mL of LMR broth and cultured in a constant temperature incubator at 37℃±1℃ for 24 h. Then, 4% of the bacterial culture was inoculated into 500 mL of culture medium for expansion culture for 24 h at 37℃±1℃. The culture medium contained 10% active bacteria. 7 cfu / mL to 109 The culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0113] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0114] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0115] Comparative Example 7

[0116] A method for preparing ginseng fermentation broth using compound bacteria and ginseng as raw materials includes the following steps:

[0117] (1) Pretreatment method of ginseng fermentation substrate: Take 200g of ginseng (sun-dried ginseng) and grind it into powder using a high-speed grinder. After passing through a 10-mesh sieve, soak it in hot water with a material-to-liquid ratio of 1:20 and then grind it into a slurry using a colloid mill. Add 1.0% by weight of pectinase for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the ginseng fermentation substrate is obtained. The enzymatic hydrolysis temperature is 47℃±5℃ and the enzymatic hydrolysis time is 3h.

[0118] (2) Preparation of fermentation strains: *Lactobacillus paracasei* B04WI2501, *Lactobacillus fermentum* (CECT5716), *Lactobacillus plantarum* (B01WI2301), and *Lactobacillus acidophilus* (La-14) were inoculated into 100 mL of MRS broth medium and cultured at 37℃±1℃ for 24 h. Then, 1% of each of the four bacterial cultures was transferred to the culture medium at a 1:1:1:1 ratio and inoculated for 24 h at 37℃±1℃ to achieve a bacterial activity level of 10⁻⁶. 7 cfu / mL to 10 9The concentration of cfu / mL in the culture medium contains 2% fructooligosaccharides, 1.25% hydrolyzed wheat protein powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80, and the remainder purified water.

[0119] (3) Sample preparation before ginseng fermentation: Add calcium carbonate to the ginseng fermentation matrix obtained in step (1) to adjust the pH value to 6.0, and add white sugar at a mass ratio of 5% of the ginseng fermentation matrix. Heat to 90-100℃ and keep warm for 3 hours to obtain fermentation liquid.

[0120] (4) Preparation of ginseng fermentation broth: The fermentation broth obtained in step (3) is transferred to a fermentation tank and cooled by stirring with circulating cooling water. After the broth cools to 37°C, the inoculum from step (2) is inoculated into a 5L fermentation tank at an inoculation rate of 1%. The fermentation temperature is 30°C and the rotation speed is 50 rpm. The fermentation is carried out at a constant temperature for 14 days. During the fermentation process, the activity of the inoculum in the fermentation broth is maintained at 10%. 7 cfu / mL to 10 9 cfu / mL. After fermentation, centrifugation and filtration were performed at 3000 rpm for 10 min, followed by passing through an 80-mesh vibrating sieve. Then, ultra-high temperature instantaneous sterilization was carried out at 115℃ for 30 s. The product was obtained after sterilization.

[0121] Example 1: Detection of ginseng fermentation liquid content

[0122] 1. Test materials

[0123] The ginseng samples before fermentation in this experiment were the ginseng samples obtained in step (3) of Examples 1-2 and Comparative Examples 1-7 above, which were subjected to centrifugation (3000 rpm, 10 min), passing through a vibrating sieve (80 mesh), and ultra-high temperature instantaneous sterilization. The ultra-high temperature instantaneous sterilization temperature was 115℃ and the sterilization time was 30 s.

[0124] The fermented ginseng sample in this experiment is the ginseng fermentation liquid prepared in step (4) of Examples 1-2 and Comparative Examples 1-7 above.

[0125] 2. Test methods

[0126] (1) HPLC method for detecting saponins

[0127] Sample preparation: Take 5.00 mL of the same batch of sample, weigh it in a 20.00 mL volumetric flask, add about 10 mL of methanol and shake gently. Continue to add methanol to make up to 20.00 mL, sonicate for 5 min, cool to room temperature, and centrifuge at 4000 r / min for 5 min. Take 8.00 mL of the supernatant, evaporate the solvent using nitrogen, add 1.000 mL of methanol to reconstitute, vortex for 1 min, sonicate for 10 min, centrifuge at 4000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane.

[0128] HPLC conditions: Column: Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm); mobile phase: (A) acetonitrile, (B) water; flow rate: 0.6 mL / min; detection wavelength: 203 nm; injection volume: 2 μL; column temperature: 40 °C.

[0129] Using ginsenoside standards as a reference, the results were qualitatively determined by retention time and quantitatively calculated using the external standard method.

[0130] Conversion rate % = (content after fermentation mg / kg - content before fermentation mg / kg) / content before fermentation mg / kg × 100.

[0131] (2) Flavonoid detection methods

[0132] The flavonoid content in this experiment was determined according to Method 2, Part 2, Section 15 of the "Technical Guidelines for Testing and Evaluation of Physicochemical and Hygienic Indicators of Health Foods" (2020 Edition), and was calculated as rutin.

[0133] (3) Peptide detection methods

[0134] The peptide detection method in this experiment refers to Appendix B of GB / T 22492-2008.

[0135] (4) Lactic acid detection method

[0136] The lactic acid detection in this test case was performed in accordance with GB 5009.157-2016.

[0137] 3. Test Results

[0138] (1) The content and conversion rate of rare saponins in the ginseng fermentation liquid prepared in Examples 1 and 2 were tested before and after fermentation. The specific results are shown in Table 1.

[0139] Table 1: Content and conversion rate of rare saponins before and after fermentation

[0140]

[0141] The results in Table 1 show that fermenting ginseng with *Lactobacillus paracasei* B04WI2501 and B16NY2107 significantly increased the content of rare ginsenosides Rg3, CK, Rg5, Rh2, Rg1, and total rare ginsenosides. Specifically, compared to before fermentation, in Example 1, the content of Rg3 increased by 972%, CK by 417%, Rg5 by 800%, Rh2 by 277%, Rg1 by 633%, Rh1 by 518%, and total rare ginsenosides by 723%. In Example 2, compared to before fermentation, the content of Rg3 increased by 1136%, CK by 914%, Rg5 by 1179%, Rh2 by 402%, Rg1 by 305%, Rh1 by 402%, and total rare ginsenosides by 799%.

[0142] Furthermore, the content of protopanaxadione before and after fermentation in Examples 1 and 2 was detected, as shown in Table 2.

[0143] Table 2: Content and Conversion Rate of Progeny Ginsenosides

[0144]

[0145] As shown in Table 2, the total saponin conversion rate in Example 1 reached 96.4%, with Rb1, Re, Rc, Rg1, and Rb3 achieving a conversion rate of 100%. The total saponin conversion rate in Example 2 reached 85.9%, with Rb1, Re, Rc, Rb2, Rg1, and Rb3 all exceeding 80%. Compared to Example 1, increasing the total saponin content before fermentation in Example 2 increased the total rare saponin content after fermentation. The total rare saponin content in Example 2 after fermentation reached 3537.30 mg / kg, indicating that the *Lactobacillus paracasei* used in this invention has a very strong saponin conversion ability.

[0146] Furthermore, comparing the total amount of protopanaxadione before fermentation and the total amount of rare saponins after fermentation in Examples 1 and 2, it can be seen that the total rare saponin content in Example 2 after fermentation is 4.35 times that of Example 1, while it is 3.98 times before fermentation. The main reason is that Example 1, using ginseng as raw material, increased the protopanaxadione content and promoted the conversion of some rare saponins through methods such as powdering, pulping, and enzymatic hydrolysis. This is also why the conversion factor of rare saponins in Example 1 is lower than that in Example 2. Before fermentation, the total amount of protopanaxadione in Example 2 is 7.46 times that of Example 1, while after fermentation, the rare saponin content is 4.35 times higher. From an economic perspective, Example 1 is superior.

[0147] (2) The content of ginsenosides in comparative examples 1 to 4 before fermentation was detected, and the results are shown in Table 3.

[0148] Table 3: Ginsenoside content before fermentation in Comparative Examples 1–3

[0149]

[0150]

[0151] As shown in Table 3, Example 1, using ginseng as raw material, can increase the content of original ginsenosides after powdering, pulping, enzymatic hydrolysis, and heat treatment, with the total saponin content reaching 780 mg / kg. Compared with Comparative Example 1 (without enzymatic hydrolysis), the total saponin content before fermentation increased by 6.08%; compared with Comparative Example 2 (without powdering), the total saponin content before fermentation increased by 17.96%; compared with Comparative Example 3 (without enzymatic hydrolysis and powdering), the total saponin content before fermentation increased by 33.13%; and compared with Comparative Example 4 (without powdering, enzymatic hydrolysis, and heat treatment), the total saponin content before fermentation increased by 47.91%. In summary, using ginseng as raw material and processing it through powdering, pulping, enzymatic hydrolysis, and heat treatment can increase the content of original ginsenosides.

[0152] (3) The content of rare saponins in comparative examples 5-7 after fermentation was tested, and the test results are shown in Table 4.

[0153] Table 4: Content and conversion rate of rare saponins after fermentation in Comparative Examples 5–7

[0154]

[0155] As shown in Tables 1 and 4, compared with single-strain fermentation (Comparative Examples 5 and 6), the total rare saponin content of Example 1, prepared by co-fermentation of *Lactobacillus paracasei* B04WI2501 and B16NY2107 (Example 1), was 813.00 mg / kg, which was 2.70 times that of Comparative Example 5 and 2.78 times that of Comparative Example 6. This indicates that the two strains of *Lactobacillus paracasei* B04WI2501 and B16NY2107 had a synergistic effect on increasing the total rare saponin content. Comparative Example 7 was prepared by co-fermentation of *Lactobacillus paracasei* B04WI2501 with *Lactobacillus plantarum*, *Lactobacillus plantarum*, and *Lactobacillus acidophilus*, and the total rare saponin content was 309.78 mg / kg. Compared with Comparative Example 5, which was prepared by single-strain *Lactobacillus paracasei* B04WI2501, the total rare saponin content was not significantly increased. The complex bacteria could transform rare saponins, but no synergistic effect was achieved.

[0156] (4) The contents of active ingredients (lactic acid, total flavonoids and ginseng peptides) before and after fermentation in Examples 1-2 and Comparative Examples 5-7 were detected, and the results are shown in Table 5.

[0157] Table 5: Content and Increase Ratio of Active Ingredients Before and After Fermentation

[0158]

[0159] As shown in Table 5, the preparation method of Example 1 can increase the total flavonoid and peptide content in ginseng, and the content is higher than that of single bacteria (Comparative Examples 5 and 6) and compound bacteria preparation group (Comparative Example 7).

[0160] Example 2: Evaluation of the neuroprotective efficacy of ginseng fermentation liquid in treating HD (high-grade angina pectoris)

[0161] 1. Experimental Principle

[0162] Caenorhabditis elegans is a complete living organism with a simple yet sophisticated nervous system containing more than 300 neurons. It contains a variety of neurotransmitters that are relevant to humans. Its processes of neuronal synthesis, storage, and metabolism are highly similar to those of mammals, and it has a high degree of homology with human genes. It has become a major model for studying the mechanisms of neurodegenerative diseases.

[0163] The Caenorhabditis elegans (CHE) model of Huntington's disease, HA759, expresses HtnQ150 in four types of neurons: head sensory neurons (ASH, ASI), and tail sensory neurons (PHA, PHB). Simultaneously, it expresses green fluorescent protein (GFP) in these neurons. This facilitates neuronal localization and allows for the assessment of neuronal survival status through GFP expression. Since HtnQ150 expression is most intense in head sensory neurons (ASH), and the pqe-1 gene modification further accelerates its aggregation toxicity on ASH neurons, leading to rapid and progressive death of ASH neurons and the disappearance of GFP fluorescence spots, this study used C. elegans HA759 as a model for Huntington's disease neurological injury. The survival rate of head sensory neurons (ASH) was used to evaluate the neuroprotective efficacy of the test substance against Huntington's disease.

[0164] 2. Test methods

[0165] (1) Dip a single Escherichia coli NA22 monoclonal strain into an inoculation loop and place it in 80 mL of liquid. Incubate overnight at 37°C and 150 rpm on a shaker. Collect a certain number of oviposition-stage HA759 elegans lysed into fragments, collect the eggs and culture for 24 hours to obtain a large number of uniform L1 stage elegans lysates for later use.

[0166] (2) After centrifuging and concentrating the cultured E. coli NA22, the contents were added to a 96-well plate, and the absorbance was measured up to the OD value. 570 The concentration was 0–0.5. The prepared L1 stage nematodes were transferred to a 24-well plate, and 100 μL of concentrated Escherichia coli NA22 and an appropriate amount of L1 stage nematodes were added to each well. The plate was then incubated at 20°C and 150 rpm in a shaker.

[0167] (3) On day 5 of the experiment, live-cell imaging was used to photograph *C. elegans*. A 384-well plate was selected, using a 4x microscope, FITC channel, and bright field channel. The exposure time was adjusted to 500 ms. The number of fluorescent dots on the heads of all *C. elegans* in each well was counted (nematodes without fluorescent dots on their heads were considered invalid). Among them:

[0168] Neuron survival rate = number of nematodes with 2 fluorescent dots in their head / (number of nematodes with 2 fluorescent dots in their head + number of nematodes with 1 fluorescent dot in their head).

[0169] (4) Compared with the control group, the proportion of surviving neurons was significantly increased (P<0.05), indicating that the test substance had neuroprotective effect at this concentration.

[0170] 3. Test Results

[0171] The neuroprotective efficacy of the ginseng fermentation liquids prepared in Example 1 and Comparative Examples 5-7 was tested, and the results are shown in Table 6. Figure 1 As shown;

[0172] Table 6: Effects of ginseng fermentation broth on neuronal survival rate before and after fermentation

[0173]

[0174] From Table 6 and Figure 1 As can be seen, the fermented samples showed highly significant differences compared to the control group in all three concentration groups (P < 0.001), while there was no significant difference between the unfermented samples and the control group. The fermented samples, after being concentrated 4 times, showed a significant difference compared to the positive control group (P < 0.05). These results indicate that the fermentation broth containing rare saponins prepared using this technique has a significant neuroprotective effect against HD disease, and its effect is superior to the positive control group. The data from the three concentration groups after fermentation also showed a dose-trend, with the 4-fold concentration showing better results than the 2-fold concentration and the unconcentrated groups.

[0175] Furthermore, Table 6 shows that Comparative Examples 5, 6, and 7 also have neuroprotective effects against HD, but the effects are lower than those of Example 1. There is no significant difference compared with the positive group. This is mainly because the contents of rare saponin groups Rg3, CK, Rg5, Rh2, and Rg2 in Comparative Examples 5 to 7 are significantly lower than those in Example 1.

[0176] In summary, this invention provides a method for preparing a fermentation broth containing rare ginseng saponin groups Rg3, Rg5, Rh2, CK, Rg2, and Rh1 using ginseng enzymatic hydrolysate or ginseng water extract as raw materials and *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 as inoculum. The method for preparing this fermentation broth rich in rare ginseng saponins has the following advantages:

[0177] (1) This invention uses lactic acid bacteria for fermentation. Lactic acid bacteria are recognized probiotics. Compared with other inventions that use molds, yeasts, etc., it is safer and contains probiotic metabolites, which are more nutritious and healthy.

[0178] (2) The present invention uses Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 for fermentation. Compared with single-strain or multi-strain fermentation, the Rg3, Rg5, Rh2, CK, Rg2, and Rh1 are increased by a higher multiple. The total rare saponin content of ginseng fermentation liquid obtained by the fermentation method of the present invention is more than 2.7 times that of single-strain or multi-strain fermentation.

[0179] (3) The ginseng fermentation substrate of the present invention is used for fermentation and transformation, which preserves the nutritional components and bioactive substances of ginseng to the maximum extent, promotes the dissolution of ginsenosides, and increases the content of ginseng flavonoids, peptides and other substances.

[0180] (4) The ginseng fermentation liquid preparation method of the present invention combines ginseng powdering, colloid milling, enzymatic hydrolysis and heat treatment to decompose the large molecular components such as pectin and fiber in ginseng into small molecules, increasing the content of disaccharides and monosaccharides, providing a carbon source for subsequent lactic acid bacteria fermentation, and promoting microbial growth; effectively increasing the yield of ginsenosides and increasing the total saponin content of the fermentation matrix.

[0181] (5) The present invention adopts a one-step fermentation method, which has a high conversion rate of rare saponins. Compared with other inventions that use multi-strain step fermentation, the preparation process is simpler and easier to industrialize and produce on a large scale.

[0182] (6) The ginseng fermentation liquid containing rare saponin groups Rg3, Rg5, Rh2, CK, Rg2 and Rh1 prepared by the present invention can effectively improve the neuronal survival rate of Huntington's disease and play a role in preventing Huntington's disease.

[0183] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a fermentation broth rich in rare ginseng saponins, characterized in that, include: The Lactobacillus paracasei ( Lacticaseibacillus paracasei B04WI2501 and the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei B16NY2107 is inoculated into the ginseng fermentation substrate, fermented at 25℃~40℃, and then sterilized. The total content of rare ginsenosides is greater than 800 mg / kg; The Lactobacillus paracasei B04WI2501 was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO.62875, on October 13, 2022. The Lactobacillus paracasei B16NY2107 was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO.62874, on October 13, 2022.

2. The preparation method according to claim 1, characterized in that, The Lactobacillus paracasei B04WI2501 has the 16S rDNA sequence shown in SEQ ID NO:

1.

3. The preparation method according to claim 2, characterized in that, The Lactobacillus paracasei B16NY2107 has the 16S rDNA sequence shown in SEQ ID NO:

2.

4. The preparation method according to claim 1, characterized in that, The inoculation amounts of *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 are each 1% to 2%.

5. The preparation method according to claim 4, characterized in that, The inoculation ratio of *Lactobacillus paracasei* B04WI2501 to *Lactobacillus paracasei* B16NY2107 is 0.8~1.2:

1.

6. The preparation method according to claim 5, characterized in that, The bacterial activity of *Lactobacillus paracasei* B04WI2501 and *Lactobacillus paracasei* B16NY2107 was 10. 7 cfu / mL ~10 9 cfu / mL.

7. The preparation method according to claim 5, characterized in that, The ginseng fermentation substrate is ginseng enzymatic hydrolysate or ginseng water extract.

8. The preparation method according to claim 7, characterized in that, The preparation method of the ginseng enzymatic hydrolysate is as follows: after ginseng is crushed, soaked and pulped, 0.8-1.2% by weight of pectinase is added for enzymatic hydrolysis. After enzymatic hydrolysis, the pH value is adjusted to 5.0-6.5, a carbon source is added, and the mixture is kept at 90℃-100℃ for 2.5-3.5 hours. After cooling, the hydrolysate is obtained.

9. The preparation method according to claim 8, characterized in that, The enzymatic hydrolysis temperature is 42℃~52℃.

10. The preparation method according to claim 8, characterized in that, The enzymatic hydrolysis time is 2h~4h.

11. The preparation method according to claim 7, characterized in that, The method for preparing the ginseng water extract is as follows: dissolve the ginseng extract in water, adjust the pH value to 5.0~6.5, add a carbon source, keep warm at 90℃~100℃ for 0.5~1.5h, and then cool down to obtain the product.

12. The preparation method according to any one of claims 3 to 11, characterized in that, The fermentation time is 9 to 16 days.

13. The preparation method according to claim 12, characterized in that, The stirring speed during the fermentation process is 40 rpm to 60 rpm.

14. The preparation method according to claim 12, characterized in that, The sterilization temperature is 110℃~125℃.

15. The preparation method according to claim 12, characterized in that, The sterilization time is 15s to 60s.

16. A fermentation broth, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 15.

17. The use of the fermentation broth as described in claim 16 in the preparation of a product for the prevention of neurological diseases, wherein the neurological disease is Huntington's disease.

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