A litchi seed fermentation product and its preparation method and application in preparing products with anti-inflammatory effect

Abstract

The present invention discloses a litchi seed fermentation product, a preparation method thereof, and an application thereof in the preparation of a product with anti-inflammatory effect. The preparation method of the litchi seed fermentation product comprises the following steps: (1) mixing litchi seed powder with nutrients and water to obtain a fermentation system; (2) inoculating a fermentation strain in the fermentation system for fermentation, and after the fermentation is completed, concentrating and drying the fermentation liquid to obtain a fermentation liquid concentrated product, i.e., the litchi seed fermentation product; the fermentation strain described in step (2) is saccharomyces cerevisiae. The litchi seed fermentation product prepared by fermenting litchi seeds as raw materials by the above-mentioned method of the present invention has an anti-inflammatory effect significantly higher than that of the litchi seed extract prepared by an extraction method using litchi seeds as raw materials. Since the litchi seed fermentation product of the present invention has a good anti-inflammatory effect; therefore, using it as an active ingredient for preparing drugs, skin care products or cosmetics with anti-inflammatory effects has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation products, and in particular to a litchi seed fermentation product, a preparation method thereof, and an application of the product in preparing a product with anti-inflammatory effect. Background Art

[0002] Litchi kernel is the dried mature seed of the plant Litchi chinensis Sonn. of the Sapindaceae family; it has the effects of promoting qi and dispersing stagnation, dispelling cold and relieving pain; it is mainly used for abdominal pain caused by cold hernia and testicular swelling and pain.

[0003] Chinese invention patent CN108272977A discloses an anti-orchitis drug containing litchi kernel extract and its preparation method; it includes the following raw materials in weight: 15-25 parts of litchi kernel, 3-6 parts of ephedra, 5-8 parts of bupleurum, 6-10 parts of millettia, 2-4 parts of gentian, 6-8 parts of coix seeds, 2.5-5 parts of polygonatum, 3-6 parts of bletilla, 4-7 parts of loquat leaves and 5-8 parts of euryale. The invention uses different raw materials with different extraction processes, and various extracts can play a synergistic role through mutual support and mutual use, thereby improving the efficacy; the mutual fear and mutual killing of various extracts can eliminate toxic side effects, and the prepared finished product has the effect of safely and effectively treating orchitis.

[0004] Although the above invention discloses that litchi kernel has the effect of treating orchitis, it must be compounded with other traditional Chinese medicines to achieve a better effect. In actual research, the inventor found that the litchi kernel extract prepared by the extraction process using litchi kernel alone as raw material has poor anti-inflammatory effect, which needs to be further improved. Summary of the invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention first provides a method for preparing a litchi seed fermentation product.

[0006] The present invention aims to solve the above technical problems, which are achieved through the following technical solutions:

[0007] The present invention first provides a method for preparing a litchi seed fermentation product, which comprises the following steps:

[0008] (1) mixing litchi seed powder, nutrients and water to obtain a fermentation system;

[0009] (2) inoculating a fermentation strain into a fermentation system for fermentation, and after the fermentation is completed, concentrating and drying the fermentation liquid to obtain a fermentation liquid concentrated product, i.e., the litchi seed fermentation product;

[0010] The fermentation strain described in step (2) is Saccharomyces cerevisiae.

[0011] The inventors surprisingly found in their research that the anti-inflammatory effect of the litchi seed fermentation product prepared by fermenting litchi seeds as raw materials through the above-mentioned method of the present invention is significantly higher than that of the litchi seed extract prepared by the extraction method using litchi seeds as raw materials.

[0012] Preferably, the nutrients in step (1) include yeast powder, peptone and glucose.

[0013] Further preferably, the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water in the fermentation system is 10-20:1-2:2-4:2-4:100-200.

[0014] Most preferably, the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water in the fermentation system is 15:1.5:3:3:150;

[0015] Preferably, the inoculated weight of the fermentation bacteria in step (2) is 4-6% of the weight of the fermentation system.

[0016] Most preferably, the inoculated weight of the fermentation bacteria in step (2) is 5% of the weight of the fermentation system.

[0017] Preferably, the fermentation bacteria in step (2) are composed of Saccharomyces cerevisiae and Lactobacillus bulgaricus.

[0018] The inventors found in further research that in the present invention, the composition of the fermented bacteria plays an important role in the anti-inflammatory effect of the prepared litchi seed fermentation product. The inventors surprisingly found in a large number of experiments that the litchi seed fermentation product prepared by adding Lactobacillus bulgaricus during the fermentation process and fermenting with Saccharomyces cerevisiae and Lactobacillus bulgaricus has a significantly higher anti-inflammatory effect than the litchi seed fermentation product prepared by fermenting with Saccharomyces cerevisiae alone.

[0019] Preferably, the weight ratio of Saccharomyces cerevisiae to Lactobacillus bulgaricus is 2-4:1.

[0020] Most preferably, the weight ratio of Saccharomyces cerevisiae to Lactobacillus bulgaricus is 3:1.

[0021] Preferably, the fermentation is performed at 25-30° C. for 40-60 hours.

[0022] Most preferably, the fermentation is performed at 28°C for 48 hours.

[0023] Preferably, the concentrated fermentation liquid product is further loaded onto a D101 macroporous resin column, and firstly eluted with 3 to 6 times the column volume of an ethanol aqueous solution with a volume fraction of 32 to 35% to remove impurities; then eluted with 3 to 6 times the column volume of an ethanol aqueous solution with a volume fraction of 57 to 60%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 57 to 60% is collected, concentrated and dried to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

[0024] Most preferably, the concentrated product of the fermentation broth is further loaded onto a D101 macroporous resin column, and first eluted with 5 times the column volume of an ethanol aqueous solution with a volume fraction of 33% to remove impurities; then eluted with 5 times the column volume of an ethanol aqueous solution with a volume fraction of 58%, and the eluate eluted with the 58% ethanol aqueous solution is collected, concentrated and dried to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

[0025] The inventors found in further research that eluting the concentrated fermentation broth product through the macroporous resin column described in the present invention can further enrich more anti-inflammatory active ingredients; the anti-inflammatory effect of the prepared litchi kernel fermentation product is further significantly improved compared with the concentrated fermentation broth product.

[0026] The inventors also found in their research that the elution conditions of the macroporous resin column play a decisive role in whether the further prepared lychee seed fermentation product can further significantly improve the anti-inflammatory effect compared with the concentrated fermentation broth product. The inventors surprisingly found in a large number of experiments that only the lychee seed fermentation product prepared under the macroporous resin elution conditions of the present invention can enrich a large number of anti-inflammatory active ingredients, and its anti-inflammatory effect can be further significantly improved compared with the concentrated fermentation broth product; while the lychee seed fermentation product prepared under other macroporous resin elution conditions cannot enrich a large number of anti-inflammatory active ingredients, and its anti-inflammatory effect cannot be improved or significantly improved compared with the concentrated fermentation broth product.

[0027] The present invention also provides a litchi seed fermentation product prepared by the above preparation method.

[0028] The present invention also provides an application of the litchi seed fermentation product in preparing a product with anti-inflammatory effect.

[0029] Preferably, the product is a medicine, a skin care product or a cosmetic.

[0030] Beneficial effects: The present invention provides a new method for preparing a lychee seed fermentation product. The lychee seed fermentation product prepared by fermenting lychee seeds as raw materials through the above method of the present invention has a significantly higher anti-inflammatory effect than the lychee seed extract prepared by an extraction method using lychee seeds as raw materials. Since the lychee seed fermentation product of the present invention has a good anti-inflammatory effect; therefore, it has important application value to use it as an active ingredient in the preparation of drugs, skin care products or cosmetics with anti-inflammatory effects. DETAILED DESCRIPTION

[0031] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0032] The brewer's yeast in the following examples is Angel Liquor King brewer's highly active dry yeast; the Lactobacillus bulgaricus used is 10 billion CFU / g of Lactobacillus bulgaricus freeze-dried powder.

[0033] Example 1 Preparation of Litchi Seed Fermentation Product

[0034] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 15:1.5:3:3:150;

[0035] (2) Inoculate the fermentation strain into the fermentation system and place it in a shaker (shaker speed is 200 rpm) for

[0036] Fermenting at 28° C. for 48 hours, and after the fermentation is completed, the fermentation liquid is concentrated and dried to obtain a concentrated fermentation liquid product, namely the litchi seed fermentation product;

[0037] The inoculated weight of the fermentation bacteria in step (2) is 5% of the weight of the fermentation system; the fermentation bacteria is brewer's yeast.

[0038] Example 2 Preparation of Litchi Seed Fermentation Product

[0039] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 10:2:4:2:100;

[0040] (2) inoculating the fermentation bacteria in the fermentation system, placing the fermentation bacteria in a shaker (shaking speed is 200 rpm) at 25° C. for fermentation for 60 hours, and after the fermentation is completed, concentrating and drying the fermentation liquid to obtain a fermentation liquid concentrated product, i.e., the litchi seed fermentation product;

[0041] The inoculated weight of the fermentation bacteria in step (2) is 6% of the weight of the fermentation system; the fermentation bacteria is brewer's yeast.

[0042] Example 3 Preparation of Litchi Seed Fermentation Product

[0043] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 20:1:2:4:200;

[0044] (2) inoculating the fermentation bacteria in the fermentation system, placing the fermentation bacteria in a shaker (shaking speed is 200 rpm) at 30° C. for fermentation for 40 hours, and after the fermentation is completed, concentrating and drying the fermentation liquid to obtain a concentrated fermentation liquid product, i.e., the litchi seed fermentation product;

[0045] The inoculated weight of the fermentation bacteria in step (2) is 4% of the weight of the fermentation system; the fermentation bacteria is brewer's yeast.

[0046] Example 4 Preparation of Litchi Seed Fermentation Product

[0047] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 15:1.5:3:3:150;

[0048] (2) Inoculate the fermentation strain into the fermentation system and place it in a shaker (shaker speed is 200 rpm) for

[0049] Fermenting at 28° C. for 48 hours, and after the fermentation is completed, the fermentation liquid is concentrated and dried to obtain a concentrated fermentation liquid product, namely the litchi seed fermentation product;

[0050] The inoculated weight of the fermentation bacteria in step (2) is 5% of the weight of the fermentation system; the fermentation bacteria is composed of saccharomyces cerevisiae and Lactobacillus bulgaricus in a weight ratio of 3:1.

[0051] Example 5 Preparation of Litchi Seed Fermentation Product

[0052] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 15:1.5:3:3:150;

[0053] (2) inoculating a fermentation strain in a fermentation system, placing the fermentation strain in a shaker (shaking speed is 200 rpm) at 28° C. for fermentation for 48 hours, and concentrating and drying the fermentation liquid after the fermentation to obtain a fermentation liquid concentrated product; wherein the inoculation weight of the fermentation strain is 5% of the weight of the fermentation system; and the fermentation strain is composed of saccharomyces cerevisiae and Lactobacillus bulgaricus in a weight ratio of 3:1;

[0054] (3) placing the concentrated fermentation product on a D101 macroporous resin column (loaded with 30 times the weight of the concentrated fermentation product), first using 5 times the column volume of a 33% ethanol aqueous solution for elution and impurity removal; then using 5 times the column volume of a 58% ethanol aqueous solution for elution, collecting the eluate eluted with the 58% ethanol aqueous solution, concentrating and drying to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

[0055] Comparative Example 1 Preparation of Litchi Seed Extract

[0056] Take litchi kernel powder, add 10 times the weight of 95% ethanol aqueous solution, then heat to 75°C for reflux extraction for 1.5 hours. After the extraction is completed, concentrate and dry the extract to obtain the litchi kernel extract.

[0057] Comparative Example 2 Preparation of Litchi Kernel Fermentation Products

[0058] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 15:1.5:3:3:150;

[0059] (2) inoculating a fermentation strain in a fermentation system, placing the fermentation strain in a shaker (shaking speed is 200 rpm) at 28° C. for fermentation for 48 hours, and concentrating and drying the fermentation liquid after the fermentation to obtain a fermentation liquid concentrated product; wherein the inoculation weight of the fermentation strain is 5% of the weight of the fermentation system; and the fermentation strain is composed of saccharomyces cerevisiae and Lactobacillus bulgaricus in a weight ratio of 3:1;

[0060] (3) The concentrated fermentation product is loaded onto a D101 macroporous resin column (loaded with 30 times the weight of the concentrated fermentation product), and is first eluted with 10% ethanol aqueous solution with a volume fraction of 5 times the column volume to remove impurities; then, it is eluted with 40% ethanol aqueous solution with a volume fraction of 5 times the column volume, and the eluate eluted with the 40% ethanol aqueous solution is collected, concentrated and dried to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

[0061] Comparative Example 3 Preparation of Litchi Kernel Fermentation Product

[0062] (1) mixing litchi seed powder with yeast powder, peptone, glucose and water to obtain a fermentation system; wherein the weight ratio of litchi seed powder to yeast powder, peptone, glucose and water is 15:1.5:3:3:150;

[0063] (2) inoculating a fermentation strain in a fermentation system, placing the fermentation strain in a shaker (shaking speed is 200 rpm) at 28° C. for fermentation for 48 hours, and concentrating and drying the fermentation liquid after the fermentation to obtain a fermentation liquid concentrated product; wherein the inoculation weight of the fermentation strain is 5% of the weight of the fermentation system; and the fermentation strain is composed of saccharomyces cerevisiae and Lactobacillus bulgaricus in a weight ratio of 3:1;

[0064] (3) placing the concentrated fermentation product on a D101 macroporous resin column (loaded with 30 times the weight of the concentrated fermentation product), first using 5 times the column volume of a 55% ethanol aqueous solution for elution and impurity removal; then using 5 times the column volume of an 80% ethanol aqueous solution for elution, collecting the eluate eluted with the 80% ethanol aqueous solution, concentrating and drying to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

[0065] Experimental Example 1 Anti-inflammatory Experimental Results of Litchi Seed Fermentation Products of the Present Invention

[0066] (1) Zebrafish embryos were treated with test substances

[0067] Zebrafish embryos were randomly divided into 9 groups, with 24 in each group; 8 experimental groups and 1 model control group; the zebrafish embryos in the model control group were placed in zebrafish embryo culture medium containing 10 μmol / L copper sulfate, and the zebrafish embryos in the experimental group were placed in zebrafish embryo culture medium containing 10 μmol / L copper sulfate and 1 g / L of the test substance, and cultured in a constant temperature incubator at 28±1℃ for 45 min.

[0068] Among them, the test substance of experimental group 1 is the litchi kernel fermentation product prepared in Example 1; the test substance of experimental group 2 is the litchi kernel fermentation product prepared in Example 2; the test substance of experimental group 3 is the litchi kernel fermentation product prepared in Example 3; the test substance of experimental group 4 is the litchi kernel fermentation product prepared in Example 4; the test substance of experimental group 5 is the litchi kernel fermentation product prepared in Example 5; the test substance of experimental group 6 is the litchi kernel extract prepared in Comparative Example 1; the test substance of experimental group 7 is the litchi kernel fermentation product prepared in Comparative Example 2; the test substance of experimental group 8 is the litchi kernel fermentation product prepared in Comparative Example 3.

[0069] (2) Zebrafish embryo fixation and staining

[0070] 2.1 After the culture, the zebrafish embryos in each group were fixed in paraformaldehyde for 1 hour, and then treated with PBST for 3 times, each time for 5 minutes, and then treated with 50% ethanol for 3 minutes;

[0071] 2.2 After the zebrafish embryos were stained with Sudan black staining solution at room temperature for 1 hour, they were washed with 70% ethanol 4 times, each time for 5 minutes, and then treated with PBST 2 times, each time for 5 minutes;

[0072] 2.3 Treat zebrafish embryos with bleach solution (an aqueous solution containing 1% KOH and 3% H2O2 by mass) for 10 min, then treat with 70% ethanol by volume for 5 min; treat with PBST for 1 min, treat with cleaning solution 1 (an aqueous solution containing 20% ​​glycerol and 0.25% KOH by mass) for 15 min, treat with cleaning solution 2 (an aqueous solution containing 50% glycerol and 0.25% KOH by mass) for 10 min, and then treat with PBST for 3 min.

[0073] (3) Microscopic analysis of samples

[0074] The fixed and stained zebrafish embryos were placed sideways and then placed under a stereo microscope to take pictures of the tail of the zebrafish embryo.

[0075] (4) Data and result calculation

[0076] The number of neutrophils in the lateral line region three quarters of the caudal from the anus of each zebrafish embryo was counted.

[0077] Calculate the neutrophil aggregation inhibition rate:

[0078] Inhibition rate = [(CS) / C] x 100%; where S is the average number of neutrophils in zebrafish embryos in the experimental group; C is the average number of neutrophils in zebrafish embryos in the model control group. The higher the inhibition rate of neutrophil aggregation, the better the anti-inflammatory effect; the experimental results are shown in Table 1.

[0079] Table 1. Anti-inflammatory experimental results of the invented lychee seed fermentation products

[0080] Neutrophil aggregation inhibition rate Example 1 Litchi seed fermentation product prepared 40％ Example 2 Litchi seed fermentation product prepared 34％ Example 3 Litchi seed fermentation product prepared 35％ Example 4 Litchi seed fermentation product prepared 58％ Example 5 Litchi seed fermentation product prepared 83％ Litchi seed extract prepared in Comparative Example 1 18％ Comparative Example 2: Litchi seed fermentation product prepared 41％ Comparative Example 3: Litchi seed fermentation product prepared 62％

[0081] It can be seen from the experimental results in Table 1 that the inhibition rate of the neutrophil aggregation of zebrafish embryos by the litchi seed fermentation product prepared in Example 1 is 40%, which is significantly higher than that of the litchi seed extract prepared in Comparative Example 1; this indicates that the litchi seed fermentation product prepared by fermentation using litchi seed as a raw material by the method of the present invention has a significantly higher anti-inflammatory effect than the litchi seed extract prepared by the extraction method using litchi seed as a raw material.

[0082] It can be seen from the experimental results in Table 1 that the lychee seed fermentation product prepared in Example 4 has an inhibition rate of 58% on the aggregation of neutrophils of zebrafish embryos, which is significantly higher than the lychee seed fermentation product prepared in Example 1; this shows that: in the present invention, the composition of the fermentation bacteria plays an important role in the anti-inflammatory effect of the prepared lychee seed fermentation product; the above experiments show that the lychee seed fermentation product prepared by adding Lactobacillus bulgaricus during the fermentation process and fermenting Saccharomyces cerevisiae and Lactobacillus bulgaricus together has a significantly higher antibacterial effect than the lychee seed fermentation product prepared by fermenting Saccharomyces cerevisiae alone.

[0083] It can be seen from the experimental results in Table 1 that the lychee seed fermentation product prepared in Example 5 has an inhibition rate of 83% on the aggregation of neutrophils of zebrafish embryos, which is further significantly higher than the lychee seed fermentation product prepared in Example 4; this shows that: the concentrated fermentation broth product can be further enriched to obtain more anti-inflammatory active ingredients by eluting it through the macroporous resin column described in the present invention; the lychee seed fermentation product prepared by further eluting the concentrated fermentation broth product through the macroporous resin elution conditions described in the present invention can further significantly improve its anti-inflammatory effect compared with the concentrated fermentation broth product.

[0084] It can be seen from the experimental results in Table 1 that the inhibition rate of the neutrophil aggregation of zebrafish embryos by the lychee seed fermentation product prepared in Comparative Example 2 is 41%, which is lower than 58% of Example 4; the inhibition rate of the neutrophil aggregation of zebrafish embryos by the lychee seed fermentation product prepared in Comparative Example 3 is 62%, which is higher than that in Example 4, but the increase is not large; this shows that the elution conditions of the macroporous resin column of the present invention play a decisive role in whether the lychee seed fermentation product prepared further can further significantly improve the anti-inflammatory effect compared with the concentrated product of the fermentation broth; the above-mentioned examples Experiments have shown that only under the macroporous resin elution conditions of the present invention (i.e., first eluting with an ethanol aqueous solution with a volume fraction of 55% for impurities; then eluting with an ethanol aqueous solution with a volume fraction of 80%) can the lychee seed fermentation product prepared be enriched with a large amount of anti-inflammatory active ingredients, and its anti-inflammatory effect can be further greatly improved compared with the concentrated product of the fermentation broth; while the lychee seed fermentation product prepared under other macroporous resin elution conditions cannot be enriched with a large amount of anti-inflammatory active ingredients, and its anti-inflammatory effect cannot be improved or greatly improved compared with the concentrated product of the fermentation broth.

Claims

1. A use of a litchi seed fermentation product in preparing a product with anti-inflammatory effect, characterized in that: The method for preparing the lychee seed fermentation product comprises the following steps: (1) mixing litchi seed powder, nutrients and water to obtain a fermentation system; (2) Inoculate the fermentation bacteria in the fermentation system for fermentation, and after the fermentation is completed, take the fermentation liquid and concentrate and dry it Then the fermentation liquid is concentrated to obtain a product; The fermentation bacteria in step (2) are composed of saccharomyces cerevisiae and Lactobacillus bulgaricus, wherein the weight ratio of saccharomyces cerevisiae to Lactobacillus bulgaricus is 2-4:1; The concentrated product of the fermentation liquid is further loaded onto a D101 macroporous resin column, and is first eluted and removed with an ethanol aqueous solution with a volume fraction of 33% for 5 times the volume of the column; then it is eluted with an ethanol aqueous solution with a volume fraction of 58% for 5 times the volume of the column, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 58% is collected, concentrated and dried to obtain the active part of the macroporous resin column, i.e., the litchi seed fermentation product.

2. The use according to claim 1, characterized in that: The nutrients described in step (1) include yeast powder, peptone and glucose.

3. The use according to claim 2, characterized in that: The weight ratio of litchi seed powder to yeast powder, peptone, glucose and water in the fermentation system is 10-20:1-2:2-4:2-4:100-200.

4. The use according to claim 1, characterized in that: The inoculation weight of the fermentation bacteria in step (2) is 4-6% of the weight of the fermentation system.

5. The use according to claim 1, characterized in that: The weight ratio of Saccharomyces cerevisiae to Lactobacillus bulgaricus was 3:

1.

6. The use according to claim 1, characterized in that: The fermentation refers to fermentation at 25-30°C for 40-60 hours.

7. The use according to claim 1, characterized in that: The product is a medicine or a cosmetic.