Ginseng fermentation product as well as preparation method and application thereof

Abstract

The invention discloses a ginseng fermentation product as well as a preparation method and application thereof. Aiming at the problem that the existing ginseng fermentation product has certain effects in the aspects of whitening, resisting anemia, resisting inflammation, delaying cognitive impairment, enhancing the immunity of the organism and the like, but no effect is particularly clear and prominent, the ginseng fermentation product is prepared by fermenting a ginseng fermentation substrate by using a specific probiotic composition; the composition is rich in rare ginsenoside, the content of ginsenoside Rh4 with an anti-fatigue effect is high, and the content of ginsenoside Rk1 and ginsenoside Rg5 with anti-fatigue reverse effects is low. Experimental research shows that compared with the existing ginseng fermentation product, the ginseng fermentation product disclosed by the invention has a clearer and more prominent anti-fatigue effect. The ginseng fermentation product is simple in preparation method and easy to industrialize, can be widely applied to functional food and other large health fields, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a ginseng fermentation product rich in specific rare ginsenosides, and a biological fermentation preparation method and application thereof. Background Art

[0002] Ginseng (Panax ginseng CAMey.) is the dried root and rhizome of the Araliaceae plant ginseng, which has the effects of replenishing vital energy, strengthening body fluid, promoting body fluid, calming the nerves and improving intelligence. Ginsenosides are the main active ingredients of ginseng, which are divided into natural ginsenosides and rare ginsenosides. Natural ginsenosides mainly include Rb1, Rb2, Rb3, Rc, Rg1, Re, Rf, etc., and the content is generally 4-6%. Rare ginsenosides are generally the metabolites of natural ginsenosides in the human body, including Rg3, Rk1, Rg5, CK, F2, Rh1, Rk3, Rh4, etc., and their natural content in ginseng is very low, only one in 100,000. Studies have shown that rare ginsenosides are significantly better than natural ginsenosides in anti-tumor, hypoglycemic, hypolipidemic, immune-enhancing, and anti-oxidation.

[0003] Natural ginsenosides can be absorbed in the body and converted into rare ginsenosides by intestinal microorganisms and enzymes. Ginsenosides are hydrolyzed in the acidic gastric juice environment to generate deglycosylated metabolites, which are then deglycosylated by glycoside hydrolases (such as β-glucosidase, α-rhamnosidase, xylosidase, etc.) expressed by intestinal flora to generate secondary metabolites or aglycones (deglycosylated ginsenosides), namely rare ginsenosides, which are absorbed into the systemic circulation through transmembrane transport and other methods to exert their medicinal effects.

[0004] In related technical research, the biotransformation method of ginseng fermentation by microorganisms is considered to be one of the safest and most efficient methods for the in vitro transformation of ginsenosides. In fact, technicians have used one or a combination of microorganisms to ferment ginseng fermentation matrix to produce a variety of rare ginsenosides.

[0005] However, the technical problem of these existing technologies is that the composition of various ginsenosides produced by biological fermentation may contain up to dozens of rare ginsenosides, and the biological effects of these ginsenosides are different, and the biological effects of some rare ginsenosides are even antagonistic to each other. Therefore, such ginsenoside products are more like "panacea", which may have some effects in whitening, anti-anemia, anti-inflammatory, delaying cognitive impairment, and enhancing the body's immunity, but there is no particularly clear and prominent effect in any aspect. Summary of the invention

[0006] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a ginseng fermentation product rich in rare ginsenosides with enhanced anti-fatigue effect, and a microbial fermentation preparation method and application thereof.

[0007] The inventors have found through literature research that among the rare ginsenosides, ginsenoside Rh4 is the one that can play an anti-fatigue role, while ginsenosides Rk1 and Rg5 can inhibit nerve excitement and promote sleep, that is, they have the opposite effect on fighting fatigue. Based on this, it can be considered that if the content of ginsenoside Rh4 in ginseng fermentation products can be increased while the content of ginsenosides Rk1 and Rg5 can be reduced, ginseng fermentation products with enhanced anti-fatigue effects can be prepared.

[0008] However, the technical difficulty is that the use of microorganisms (probiotics) to transform natural ginsenosides to produce rare ginsenosides may be related to the transformation path. Among all the rare ginsenosides, ginsenoside Rh4 is difficult to produce, while ginsenosides Rk1 and Rg5 are relatively easy to produce, and often when a certain amount of ginsenoside Rh4 is produced, more ginsenosides Rk1 and Rg5 will be produced. This makes the existing ginseng fermentation products either have a very low content of ginsenoside Rh4, or have contents of ginsenoside Rk1 and Rg5 much higher than that of ginsenoside Rh4, resulting in the ginseng fermentation products being unable to exhibit the anti-fatigue effect of ginsenoside Rh4 as a whole.

[0009] Furthermore, after in-depth research, the inventors found that fermenting ginseng fermentation matrix with a specific composition of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus casei, and Bifidobacterium adolescens can produce ginseng fermentation products with a high content of ginsenoside Rh4 and a small difference in the content of ginsenosides Rk1 and Rg5, so that the ginseng fermentation product exhibits better anti-fatigue effect, thereby completing the present invention.

[0010] The technical solution of the present invention includes:

[0011] 1. A ginseng fermentation product obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5, which satisfies:

[0012] The content of ginsenoside Rh4 is ≥ 3% by weight; and

[0013] Ginsenoside Rh4 content - Ginsenoside Rk1 content - Ginsenoside Rg5 content ≥ - 5% by weight

[0014] The total amount of saponins in the ginseng fermentation product is taken as 100% by weight.

[0015] The ginseng fermentation product may be liquid or solid or a solid-liquid mixture, for example, fermented ginseng liquid or fermented ginseng powder.

[0016] 2. A bacterial composition, which is composed of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescens; wherein,

[0017] The Lactobacillus plantarum is Lactobacillus plantarum GBFG 61, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit number of GGMCC NO: 31583 and a deposit date of August 14, 2024;

[0018] The Lactobacillus paracasei is Lactobacillus paracasei GBFG 34, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of GGMCC NO: 31581 and a deposit date of August 14, 2024.

[0019] 3. The bacterial composition according to item 2, which can be used to ferment ginseng fermentation substrate to produce rare ginsenosides.

[0020] Here, the ginseng fermentation matrix refers to a fermentation matrix containing ginseng fines. The ginseng fines are obtained by reducing ginseng, preferably by physical methods such as extrusion, cutting, grinding, etc. The ginseng fines can be, for example, ginseng powder. The ginseng fermentation matrix can also contain other components commonly contained in the fermentation matrix, such as nitrogen source, carbon source, inorganic salt, pH regulator, etc.

[0021] 4. The bacterial composition according to item 2, wherein the dry weight ratio of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Bifidobacterium adolescens is: 1:(1-5):(1-5):(1-5).

[0022] 5. The bacterial composition according to item 2, wherein the Lactobacillus casei is Lactobacillus casei BL23.

[0023] 6. The bacterial composition according to item 2, wherein the Bifidobacterium adolescentis (Bifidobacterium adolescens) is Bifidobacterium adolescentis (Bifidobacterium adolescens) BH-J2356.

[0024] 7. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng fermentation substrate and fermenting at 37-42° C. for more than 30 hours to obtain a ginseng fermentation product containing rare ginsenosides;

[0025] Wherein, the bacterial composition is the bacterial composition described in any one of items 2 to 6.

[0026] 8. The preparation method according to item 7, further comprising sterilizing the ginseng fermentation product.

[0027] 9. The preparation method according to item 7, wherein the ginseng fermentation product is the ginseng fermentation product according to item 1.

[0028] 10. The preparation method according to item 7, wherein the ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.

[0029] 11. A ginseng fermentation product obtained by the preparation method of any one of items 7 to 10.

[0030] Instruction Manual

[0031] Figure 1 This is a high performance liquid chromatogram for detecting fermented ginseng powder 2.

[0032] Figure 2 This is a high performance liquid chromatogram for detecting fermented ginseng powder 5. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with specific embodiments. In the absence of conflict, the scientific terms in this specification have the meanings commonly understood by those skilled in the art. If there is a conflict, the definitions in this specification shall prevail.

[0034] Example

[0035] Example 1 Preparation of fermented ginseng powder rich in rare ginsenosides and having enhanced anti-fatigue effect

[0036] The steps include:

[0037] (1) Pretreatment of ginseng fermentation matrix: 120 g ginseng powder, 10 g glucose, 2 g yeast extract powder, 2 g citric acid, 0.05 g manganese sulfate, and 0.1 g magnesium sulfate were added to 856 g water for dissolution, and then sterilized at 121° C. for 20 min before use;

[0038] (2) Preparation of strains: Lactobacillus plantarum GBFG 61, Lactobacillus casei BL23, Lactobacillus paracasei GBFG 34, Bifidobacterium adolescentis BH-J2356, Lactobacillus plantarum Lp90, and Lactobacillus paracasei LC37 were inoculated into 5 mL of MRS broth medium and placed in a constant temperature incubator at 37°C ± 1°C for 24 h to obtain a first-level seed solution. Each bacterial solution was then inoculated into 50 mL of culture medium at a 1% inoculum volume and cultured for 24 h to obtain a second-level seed solution. The activity of each strain was 10 7 cfu / mL to 10 9 cfu / mL. The dry bacteria content in the above-mentioned second-level seed liquid was determined respectively.

[0039] It should be noted that the above-mentioned Lactobacillus plantarum GBFG 61 is a lactobacillus strain that produces β-glucosidase isolated and cultivated from the soil of the ginseng planting base in Fusong County, Baishan City, Jilin Province. The lactobacillus strain was named GBFG 61, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.30101. The deposit date is March 22, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal code of the deposit institution: 100101.

[0040] The above-mentioned Lactobacillus paracasei GBFG 34 is a lactobacillus strain that produces β-glucosidase isolated and cultivated from the soil of the ginseng planting base in Ji'an City (County) of Tonghua City, Jilin Province. The lactobacillus strain was named GBFG 34, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.31581. The deposit date is August 14, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal code of the deposit institution: 100101.

[0041] The above-mentioned Lactobacillus casei BL23 was purchased from Beijing Krisbo Biotechnology Co., Ltd.; Bifidobacterium adolescens BH-J2356 was purchased from Shanghai Bohu Biotechnology Co., Ltd.; Lactobacillus plantarum Lp90 and Lactobacillus paracasei LC37 were purchased from Weikang Probiotics (Suzhou) Co., Ltd.

[0042] (3) Fermentation: The second-level seed liquid of Lactobacillus plantarum GBFG 61, Lactobacillus casei BL23, Lactobacillus paracasei GBFG 34, and Bifidobacterium adolescentis BH-J2356 were mixed to 10 g at a dry bacteria weight ratio of 1:1:1:1 (composition 1), 1:2:3:2 (composition 2), 1:3:2:3 (composition 3), and 1:5:5:5 (composition 4), and inoculated aseptically into ginseng fermentation matrix. The fermentation temperature was set to 37° C. and the rotation speed was 50 rpm. The fermentation was carried out for 48 h, and the pH was maintained at 4-5 during the fermentation process.

[0043] As a comparison, the Lactobacillus plantarum Lp90 was used to replace the Lactobacillus plantarum GBFG 61 in the composition 2 (composition 5); and the Lactobacillus paracasei LC37 was used to replace the Lactobacillus paracasei GBFG 34 in the composition 2 (composition 6).

[0044] (4) Centrifugation, drying, and crushing: After the fermentation is completed, sterilization, centrifugation, and drying operations are performed. The sterilization parameters are: 121° C., 20 min; the centrifugation parameters are: 3000-5000 rpm, 10 min; the vacuum degree is controlled to be ≤-0.09 MPa, 60-65° C., and crushed after drying for 48 hours to obtain fermented ginseng powders 1 to 6.

[0045] Detection and analysis: The above-mentioned fermented ginseng powders were dissolved in methanol to prepare 80 mg / mL, and 1 mL of the solution was taken respectively and filtered with a 0.45 μm microporous filter membrane to obtain a clear solution, and the total ginsenoside content and the content of various rare ginsenosides therein were determined, and the difference between the content of ginsenoside Rh4 (forward) related to anti-fatigue and the content of ginsenoside Rk1 (reverse) and Rg5 (reverse) was calculated (weight %, with the total ginsenoside amount as 100 weight %). The results are shown in Table 1.

[0046] The detection method of total ginsenosides was carried out according to the method for determining the content of total ginsenosides in the Pharmacopoeia of the People's Republic of China (2020 edition). 48 H 82 O 18 )count.

[0047] The detection method of rare ginsenosides is carried out according to the method for determining the content of rare ginsenosides in T / CNHFA 001-2021.

[0048] As an example, the HPLC analysis of the fermented ginseng powder 2 obtained by using the above composition 2 is as follows: Figure 1 As shown, the high performance liquid chromatography of the fermented ginseng powder 5 obtained by using the composition 5 is as shown in Figure 2 shown.

[0049] Table 1: Detection of rare ginsenoside content in fermented ginseng powder obtained using different bacterial strain compositions

[0050]

[0051] Difference = Ginsenoside Rh4 content - (Ginsenoside Rk1 content + Ginsenoside Rg5 content)

[0052] As shown in Table 1, the content of ginsenoside Rh4 with anti-fatigue effect in fermented ginseng powders 1 to 4 obtained by using Lactobacillus plantarum GBFG 61 and Lactobacillus paracasei GBFG 34 is more than 3% by weight, and the difference between the content of ginsenoside Rh4 and (the content of ginsenoside Rk1 + the content of ginsenoside Rg5) is more than -5% by weight; on the contrary, the content of ginsenoside Rh4 with anti-fatigue effect in fermented ginseng powders 5 and 6 obtained by using other Lactobacillus plantarum strains and Lactobacillus paracasei strains is less than 3% by weight, and the difference between the content of ginsenoside Rh4 and (the content of ginsenoside Rk1 + the content of ginsenoside Rg5) is less than -5% by weight. It can be expected that fermented ginseng powders 1 to 4 have better anti-fatigue effect than fermented ginseng powders 5 and 6.

[0053] It should also be noted that, in addition to the above-mentioned Lactobacillus plantarum Lp90 and Lactobacillus paracasei LC37 used as comparisons, the inventors also tried a variety of Lactobacillus plantarum and Lactobacillus paracasei strains isolated by themselves, but none of them could achieve the technical effect of "the content of ginsenoside Rh4 is 3 weight % and the difference is greater than -0.5%".

[0054] Example 2 Verification of the Anti-fatigue Effect of Fermented Ginseng Powder

[0055] With reference to Example 6 of Chinese Patent CN 114762529 B, the anti-fatigue effect test of the fermented ginseng powder was slightly modified to confirm that the anti-fatigue effect of the fermented ginseng powder 2 is better than that of the fermented ginseng powder 5. It should be noted that the content of ginsenoside Rh4 in the fermented ginseng powder 2 and the fermented ginseng powder 5 is similar, but only the fermented ginseng powder 2 meets the difference of more than -5% by weight.

[0056] Experimental animals: 60 SPF grade Kunming mice (30 male mice, 30 female mice), weighing 18-22g, were randomly divided into 3 groups, 20 mice / group (10 male mice, 10 female mice in each group), and adaptive feeding was carried out for 7 days. Experimental conditions: SPF animal room, temperature 20-26℃, humidity 40%-70%, light and dark alternation time of 12h / 12h. Dosage grouping and test sample administration time: After the mouse adaptation period, the 3 groups were divided into blank control group, 2 groups of fermented ginseng powder, and 5 groups of fermented ginseng powder; according to the recommended dose of 10 times of human body, the oral administration (250mg / kg) was given for 4 weeks. Experimental method: ① 30min after the last administration of the test sample, the mice with 5% body weight lead at the base of the tail were placed in a swimming box for swimming. The water depth was not less than 20cm, and the water temperature was 25℃±2℃. The time from the start of swimming to death of each group of mice (10 mice in each group) was detected, that is, the weight-bearing swimming time of the mice. ② After the last administration, the mice in each group swam in 30℃ water without weight, and blood was collected from the eye sockets to detect the blood urea nitrogen (BUN), blood lactate (LA), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) in the serum of each group of mice (10 mice in each group); the liver was separated to detect the content of liver glycogen (LG) in the liver. The test results are shown in Table 2.

[0057] Table 2: Anti-fatigue animal test index detection of fermentation products of different bacterial species

[0058]

[0059] Note: Compared with the blank control group, ***P<0.001, **P<0.01, *P<0.05; compared with composition 3, ###P<0.001, ##P<0.01, #P<0.05

[0060] As can be seen from the above table, compared with the blank control group, the weighted swimming time of the mice in the fermented ginseng powder 2 group was significantly increased, indicating that the ginseng fermentation product of the present invention has the anti-fatigue activity of prolonging the weighted swimming time of mice. Among them, the weighted swimming time of the mice in the fermented ginseng powder 2 group was significantly different from that in the fermented ginseng powder 5 group, which shows that, compared with the fermented ginseng powder 5 group, the fermented ginseng powder 2 group has a stronger anti-fatigue activity in prolonging the weighted swimming time of mice.

[0061] In terms of serum and liver test indicators, the blood lactic acid (LA), urea nitrogen (BUN), and lactate dehydrogenase (LDH) contents of mice in the blank control group, fermented ginseng powder 2, and fermented ginseng powder 5 groups were significantly reduced, and the liver glycogen (LG) content and creatine phosphokinase (CPK) were significantly increased, indicating that both fermented ginseng powder samples have anti-fatigue effects. It is thus proved that the fermented ginseng powder of the embodiment of the present invention may be through reducing the content of LA and BUN, reducing the activity of LDH, and increasing the content of LG and CPK, thereby exerting anti-fatigue activity. Moreover, compared with fermented ginseng powder 5, fermented ginseng powder 2 has significant differences in BUN, LDH, and CPK content.

[0062] Based on the above test results, it can be seen that compared with the comparative fermented ginseng powder 5 (difference less than -0.5 weight %), the fermented ginseng powder 2 of the present invention (difference greater than -0.5 weight %) has significantly stronger anti-fatigue effect.

[0063] It should be noted that, under the premise that it is feasible and does not obviously violate the main purpose of the present invention, any technical feature or combination of technical features described as a component of a technical solution in this specification can also be applied to other technical solutions; and, under the premise that it is feasible and does not obviously violate the main purpose of the present invention, the technical features described as components of different technical solutions can also be combined in any way to form other technical solutions. The present invention also includes technical solutions obtained by combination under the above circumstances, and these technical solutions are equivalent to those recorded in this specification.

[0064] The present invention is described above through specific implementation modes and examples, but those skilled in the art should understand that these are not intended to limit the scope of the present invention, and the scope of the present invention should be determined by the claims.

[0065] Industrial Applicability

[0066] The present invention provides a ginseng fermentation product containing rare ginsenosides and having enhanced anti-fatigue effect, and a preparation method and application thereof.

Claims

1. A ginseng fermentation product obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5, which satisfies: The content of ginsenoside Rh4 is ≥ 3% by weight; and Ginsenoside Rh4 content - Ginsenoside Rk1 content - Ginsenoside Rg5 content ≥ - 5% by weight The total amount of saponins in the ginseng fermentation product is taken as 100% by weight.

2. A bacterial composition, which is composed of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescentis; wherein, The plant lactobacillus is plant lactobacillus (Lactobacillus plantarum) GBFG 61, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of GGMCCNO: 31583 and a deposit date of August 14, 2024; The Lactobacillus paracasei is Lactobacillus paracasei GBFG 34, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of GGMCC NO: 31581 and a deposit date of August 14, 2024; The Lactobacillus casei is Lactobacillus casei BL23; and The bifidobacterium adolescentis Bifidobacterium adolescens BH-J2356.

3. The bacterial composition according to claim 2, which can be used to ferment ginseng fermentation matrix to produce rare ginsenosides.

4. The bacterial composition according to claim 2, wherein The dry weight ratio of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescens is 1:(1-5):(1-5):(1-5).

5. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng fermentation substrate, and fermenting at 37-42°C, preferably at 37°C for more than 30 hours, preferably 35-60 hours, more preferably 45-55 hours, to obtain a ginseng fermentation product containing rare ginsenosides; in, The bacterial composition is the bacterial composition according to any one of claims 2 to 4.

6. The preparation method according to claim 5, wherein: The ginseng fermentation product is the ginseng fermentation product according to claim 1.

7. The preparation method according to claim 5, wherein: The ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.

8. A ginseng fermentation product obtained by the preparation method according to any one of claims 5 to 7.

Images