Method for increasing coumarin content by fermenting radix glehniae based on complex microbial inoculants

Abstract

The invention relates to a method for increasing the coumarin content by fermenting radix glehniae based on a complex microbial inoculant. The method comprises the following steps: (1) pretreatment of radix glehniae: crushing and sieving radix glehniae; (2) preparing a culture medium: adding crushed and sieved radix glehniae into the culture medium to form a radix glehniae culture medium; (3) strain activation: respectively inoculating sweet wine koji powder, saccharomycetes powder and escherichia coli powder into sterile water for activation to form bacterial liquid; (4) inoculating and fermenting: proportioning the formed sweet distiller's yeast liquid, saccharomycetes liquid and escherichia coli liquid in proportion to form a composite strain, and inoculating the composite strain into a radix glehniae culture medium for fermentation; and (5) extracting coumarin: extracting coumarin from the fermentation product. According to the method, the radix glehniae is fermented by adopting the compound strains compounded by the saccharomycetes, the sweet distiller's yeast and the escherichia coli, the extraction yield of the coumarin can be remarkably increased, the strains are wide in source and simple to enrich, and the method has the advantages of being low in cost and simple, convenient and safe in process.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation and extraction of traditional Chinese medicines, and specifically to a method for increasing the coumarin content by fermenting Glehnia littoralis L. based on a composite bacterial agent. Background Art

[0002] Glehnia littoralis is the dried root of the Apiaceae plant Glehnia littoralis. It is a yin-tonifying herb, known for its lung-nourishing, stomach-boosting, and fluid-boosting properties. It is widely used to treat lung and stomach disorders. Some studies also consider Glehnia littoralis a representative pungent emollient, with dispersing and blood-activating properties. Modern research shows that Glehnia littoralis contains valuable chemical components such as coumarins, flavonoids, polyacetylenes, and polysaccharides, exhibiting anti-tumor, antioxidant, and immunomodulatory activities.

[0003] Currently, most research on Adenophora adenophora is focused on extraction, while there are fewer studies on the fermentation of Adenophora glauca. The bacterial strains are single and few in number, which greatly limits the conversion of the effective ingredients of Adenophora glauca. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for increasing the coumarin content by fermenting Glehnia littoralis based on a composite bacterial agent, aiming to broaden the fermentation research on Glehnia littoralis and thereby improve the coumarin extraction amount in Glehnia littoralis.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for increasing the coumarin content of Adenophora adenophora by fermenting it with a composite bacterial agent, comprising:

[0007] Yeast, sweet wine koji and Escherichia coli are compounded to form a composite bacterial strain, which is inoculated into a culture medium of Glehnia littoralis to achieve coumarin extraction.

[0008] In a preferred embodiment, the volume fraction of the composite bacterial strain inoculation is 2%-15%.

[0009] In a preferred embodiment, in the composite bacteria, the density ratio of yeast, sweet wine koji, and Escherichia coli is 1-4.5:1-8:2-7.

[0010] In a preferred embodiment, when the fermentation temperature is set at 35-37°C, the density ratio of the yeast, sweet wine koji, and Escherichia coli is 1-4.5:1-3.5:2-7.

[0011] In a preferred embodiment, when the fermentation temperature is set at 37-40°C, the density ratio of the yeast, sweet wine koji, and Escherichia coli is 1-4.5:3.5-8:2-5.5.

[0012] In a preferred embodiment, the method for increasing the coumarin content by fermenting Glehnia littoralis with a composite bacterial agent specifically comprises the following steps:

[0013] (1) Pretreatment of Glehnia littoralis: crush and sieve Glehnia littoralis;

[0014] (2) Preparation of culture medium: adding crushed and sieved Glehnia littoralis to the culture medium to form Glehnia littoralis culture medium;

[0015] (3) Activation of bacterial strains: Add sweet wine koji powder, yeast powder and E. coli powder into sterile water for activation to form bacterial solution;

[0016] (4) Inoculation and fermentation: The formed sweet wine koji liquid, yeast liquid, and Escherichia coli liquid are mixed in proportion to form a composite bacterial strain, and the composite bacterial strain is inoculated into a culture medium of Glehnia littoralis for fermentation;

[0017] (5) Extraction of coumarin: Extraction of coumarin from fermentation products.

[0018] In a preferred embodiment, the culture medium in step (2) comprises glucose, peptone, sodium chloride, magnesium sulfate, potassium chloride, magnesium chloride and purified water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The method provided by the present invention for increasing the coumarin content by fermenting Glehnia littoralis with a composite bacterial agent uses a composite bacterial strain of yeast, sweet wine koji, and Escherichia coli to ferment Glehnia littoralis, which can significantly increase the extraction yield of coumarin. Specifically, yeast can secrete a variety of extracellular enzymes, including pectinase and cellulase, which can effectively degrade pectin and cellulose in the cell wall of Glehnia littoralis, and is conducive to the release of secondary metabolites such as coumarins in the cell. The oligosaccharides and monosaccharides produced by yeast decomposition of complex polysaccharides provide an easily accessible carbon source for the mold in the sweet wine koji, stimulating mold growth and enzyme production. The mold in the sweet wine koji can produce amylase and saccharifying enzyme, which can efficiently hydrolyze starch into small molecular sugars such as glucose and maltose. At the same time, yeast converts fermentable sugars (such as glucose) in Glehnia littoralis into ethanol through fermentation. Escherichia coli can produce β-glucosidase, which can specifically hydrolyze the β-glycosidic bond between coumarin aglycones (free coumarins) and glucose (or other sugars), converting bound coumarin glycosides into free coumarin aglycones, which are generally soluble in ethanol. Based on this, yeast decomposes the plasmodium and provides a solvent, sweet wine koji provides a carbon source, and Escherichia coli carries out biotransformation, thereby releasing free coumarins. At the same time, the strains of the present invention are widely available, easy to enrich, and have the advantages of low cost, simple process and safety. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] The present invention discloses a method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent, comprising the following steps:

[0023] (1) Pretreatment of Glehnia littoralis: crush and sieve Glehnia littoralis;

[0024] (2) Preparation of culture medium: adding crushed and sieved Glehnia littoralis to the culture medium to form Glehnia littoralis culture medium;

[0025] The culture medium includes glucose, peptone, sodium chloride, magnesium sulfate, potassium chloride, magnesium chloride and purified water;

[0026] (3) Activation of bacterial strains: Add sweet wine koji powder, yeast powder and E. coli powder into sterile water for activation, respectively, to form sweet wine koji liquid, yeast liquid and E. coli liquid;

[0027] (4) Inoculation and fermentation: The formed sweet wine koji liquid, yeast liquid, and Escherichia coli liquid are mixed in proportion to form a composite strain, and the composite strain is inoculated into a culture medium of Glehnia littoralis for fermentation. The fermentation takes 4-8 days to obtain a Glehnia littoralis fermentation product;

[0028] (5) Extraction of coumarin: Extract coumarin from the fermentation product by performing 80% alcohol extraction (material-liquid ratio 1:250) and measuring the coumarin content.

[0029] Specifically, in step (4), the volume fraction of the composite bacteria inoculation is 2%-15%; and the density ratio of yeast, sweet wine koji, and Escherichia coli in the composite bacteria is 1-4.5:1-8:2-7.

[0030] More specifically, when the fermentation temperature is set at 35-37°C, the density ratio of yeast, sweet wine koji, and Escherichia coli is 1-4.5:1-3.5:2-7;

[0031] When the fermentation temperature is set at 37-40℃, the density ratio of yeast, sweet wine koji and Escherichia coli is 1-4.5:3.5-8:2-5.5.

[0032] The present invention will be described in detail below with reference to the examples. It should also be understood that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range based on the description herein, and are not to be limited to the specific values ​​exemplified below.

[0033] Example 1

[0034] 1) Experimental group:

[0035] Experimental group 1: The fermentation temperature was 35-37°C, the solid-liquid ratio of the Northern Glehnia littoralis fermentation system (Northern Glehnia littoralis and liquid culture medium) was 1:20, the volume fraction of the composite bacterial inoculation was 5%-15%, the density ratio of yeast, sweet wine koji, and Escherichia coli was 1-3:1-3:5-7, and the fermentation time was 6-8 days.

[0036] Experimental group 2: The fermentation temperature was 35-37°C, the solid-liquid ratio of the Northern Adenophora fermentation system was 1:16, the volume fraction of the composite strain inoculation was 2%-10%, the density ratio of yeast, sweet wine koji, and Escherichia coli was 2-4.5:1-3.5:2-6.5, and the fermentation time was 4-6 days.

[0037] Experimental group 3: The fermentation temperature was 37-40℃, the solid-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:20, the volume fraction of the composite strain inoculation was 5%-15%, the density ratio of yeast, sweet wine koji, and Escherichia coli was 1-3:4-8:2-5.5, and the fermentation time was 6-8 days.

[0038] Experimental Group 4: The fermentation temperature was 37-40°C, the solid-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:16, the volume fraction of the composite bacterial inoculation was 2%-10%, the density ratio of yeast, sweet wine koji, and Escherichia coli was 2-4.5:3.5-7:2-5.5, and the fermentation time was 4-6 days.

[0039] 2) Control group:

[0040] Control group 1: The fermentation temperature was 35-37°C, the material-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:20, and the fermentation was carried out using sterile culture.

[0041] Control group 2: The fermentation temperature was 35-37°C, the material-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:16, and the fermentation was carried out using sterile culture.

[0042] Control group 3: The fermentation temperature was 37-40°C, the material-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:20, and the fermentation was carried out using sterile culture.

[0043] Control group 4: the fermentation temperature was 37-40°C, the material-liquid ratio of the Northern Glehnia littoralis fermentation system was 1:16, and the fermentation was carried out using sterile culture.

[0044] The experimental results are shown in Table 1:

[0045] Table 1 Comparison of coumarin extraction content between experimental group and control group at different temperatures

[0046]

[0047]

[0048] From the table above we can see that:

[0049] 1) Under different fermentation temperatures, the density ratio of the composite strains changed. This is because different temperatures will affect the growth and metabolism of the strains, resulting in changes in their relative advantages in a specific environment. As the material-liquid ratio of the Adenophora adenophora fermentation system changes, the density ratio of the composite strains will also change, which indicates that the Adenophora adenophora fermentation system has an impact on the strain density ratio, and the fermentation time changes with changes in the fermentation temperature and the Adenophora adenophora fermentation system.

[0050] 2) Compared with the sterile fermentation control group 1, the coumarin extract content of experimental group 1 increased by 5-9 times; compared with the sterile fermentation control group 2, the coumarin extract content of experimental group 2 increased by 4-6 times; compared with the sterile fermentation control group 3, the coumarin extract content of experimental group 3 increased by 5-8 times; and compared with the sterile fermentation control group 4, the coumarin extract content of experimental group 4 increased by 4-8 times. By inoculating the culture medium of Adenophora australis with a composite strain composed of yeast, sweet wine koji, and Escherichia coli, the coumarin extract content of Adenophora australis can be significantly increased compared with sterile fermentation.

[0051] Example 2

[0052] This example uses the same experimental group 1 as Example 1, and the control group is set up as follows:

[0053] Control group 1 used a single yeast strain with the same biomass as the composite strain in experimental group 1, that is, the sweet wine koji and E. coli in experimental group 1 were replaced with yeast with the same biomass, and other culture and fermentation conditions were the same as those in experimental group 1;

[0054] Control group 2 used a single strain of sweet wine koji with the same biomass as the composite strain in experimental group 1, that is, the yeast and E. coli in experimental group 1 were replaced with sweet wine koji with the same biomass, and other culture and fermentation conditions were the same as those in experimental group 1;

[0055] Control group 3 used a single strain of Escherichia coli with the same biomass as the composite strain in experimental group 1, that is, the yeast and sweet wine koji in experimental group 1 were replaced with the same biomass of Escherichia coli, and other culture and fermentation conditions were the same as those in experimental group 1;

[0056] The experimental results are shown in Table 2:

[0057] Table 2 Comparison of coumarin extraction content in experimental and control groups of different strains

[0058]

[0059] It can be seen from the above table that the coumarin extraction content of the experimental group 1 using the composite strain is significantly improved compared with the coumarin extraction content of the control group 1, control group 2 and control group 3 using a single strain. By inoculating the composite strain composed of yeast, sweet wine koji and Escherichia coli into the Northern Glehnia littoralis culture medium, the coumarin extraction content in the Northern Glehnia littoralis can be significantly improved compared with the fermentation of the same amount of single strain.

[0060] Example 3

[0061] This example uses the same experimental group 1 as Example 1, and the control group is set up as follows:

[0062] Control group 1 was the same as that of experimental group 1 except that the yeast in experimental group 1 was replaced with an equal amount of Bacillus subtilis, the sweet wine koji and Escherichia coli remained unchanged, and other culture and fermentation conditions were the same as those of experimental group 1;

[0063] Control group 2 was the same as that of experimental group 1, except that the sweet wine koji in experimental group 1 was replaced with an equal amount of Aspergillus oryzae, while the yeast and Escherichia coli remained unchanged. Other culture and fermentation conditions were the same as those of experimental group 1.

[0064] Control group 3 was the same as that of experimental group 1 except that the Escherichia coli in experimental group 1 was replaced with an equal amount of Lactobacillus plantarum, the yeast and sweet wine koji remained unchanged, and other culture and fermentation conditions were the same as those of experimental group 1;

[0065] The experimental results are shown in Table 3:

[0066] Table 3 Comparison of coumarin extraction content in experimental and control groups of different strains

[0067]

[0068] Note: The above results support the technical feature of the present invention that the composite bacterial agent is composed of yeast, sweet wine koji, and Escherichia coli, and exclude the simple replacement of functionally similar bacterial species.

[0069] It can be seen from the above table that the coumarin extraction content of experimental group 1 using a composite bacterial strain composed of yeast, sweet wine koji and Escherichia coli was significantly increased compared with control group 1, control group 2 and control group 3, that is, the composite bacterial strain composed of yeast, sweet wine koji and Escherichia coli can synergize with Northern Glehnia littoralis, thereby significantly improving the coumarin extraction yield in Northern Glehnia littoralis.

[0070] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent, characterized in that: Yeast, sweet wine koji and Escherichia coli are compounded to form a composite bacterial strain, which is inoculated into a culture medium of Glehnia littoralis to achieve coumarin extraction.

2. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 1, characterized in that: The volume fraction of the composite bacterial strain inoculation is 2%-15%.

3. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 2, characterized in that: In the composite bacteria, the density ratio of yeast, sweet wine koji and Escherichia coli is 1-4.5:1-8:2-7.

4. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 3, characterized in that: When the fermentation temperature is set at 35-37° C., the density ratio of the yeast, sweet wine koji, and Escherichia coli is 1-4.5:1-3.5:2-7.

5. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 3, characterized in that: When the fermentation temperature is set at 37-40° C., the density ratio of the yeast, sweet wine koji, and Escherichia coli is 1-4.5:3.5-8:2-5.

5.

6. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 1, characterized in that: The specific steps include: (1) Pretreatment of Glehnia littoralis: crush and sieve Glehnia littoralis; (2) Preparation of culture medium: adding crushed and sieved Glehnia littoralis to the culture medium to form Glehnia littoralis culture medium; (3) Activation of bacterial strains: Add sweet wine koji powder, yeast powder and E. coli powder into sterile water for activation to form bacterial solution; (4) Inoculation and fermentation: The formed sweet wine koji liquid, yeast liquid, and Escherichia coli liquid are mixed in proportion to form a composite bacterial strain, and the composite bacterial strain is inoculated into a culture medium of Glehnia littoralis for fermentation; (5) Extraction of coumarin: Extraction of coumarin from fermentation products.

7. The method for increasing the coumarin content of Glehnia littoralis by fermenting Glehnia littoralis with a composite bacterial agent according to claim 6, characterized in that: The culture medium in step (2) includes glucose, peptone, sodium chloride, magnesium sulfate, potassium chloride, magnesium chloride and purified water.

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