Application of acetobacter orientalis T1-1 in preparation of glycyrrhetinic acid

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses application of acetobacter orientalis T1-1 in preparation of glycyrrhetinic acid. The invention discloses a high-yield strain of glycyrrhetinic acid, namely acetobacter orientalis T1-1, and the yield of the glycyrrhetinic acid can reach 1.62%-2.09% after the glycyrrhiza is fermented by the acetobacter orientalis T1-1. Compared with fermentation of a control strain, the yield of a target product can be increased by 67%-386%. On the basis, the invention provides the preparation method of the glycyrrhetinic acid, the preparation method has the advantages of short fermentation period, high target product yield, low energy consumption, mild preparation conditions and easiness in control, and can be used for green, efficient and industrial production of the glycyrrhetinic acid, and the prepared glycyrrhetinic acid can be further applied to the fields of foods, medicines, daily chemicals and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically discloses the application of Acetobacter orientalis T1-1 in the preparation of glycyrrhetinic acid. Background Art

[0002] Licorice has many pharmacological effects. Glycyrrhizic acid is a key active ingredient in licorice, and glycyrrhetinic acid is one of its derivatives. Glycyrrhetinic acid not only functions as a sweetener and food preservative, but studies have shown that it also exhibits anti-inflammatory, antiviral, hepatoprotective, anti-tumor, and immunomodulatory effects. It is clinically used to treat folliculitis, acute and chronic dermatitis, infectious hepatitis, cirrhosis, and arrhythmias. Furthermore, glycyrrhetinic acid can be used as an additive in skincare and haircare products, offering anti-inflammatory, soothing, antioxidant, anti-aging, and dermatitis-inhibiting effects. Therefore, glycyrrhetinic acid has broad application value and prospects in the food, pharmaceutical, and cosmetics industries.

[0003] Currently, the industry mainly produces glycyrrhetinic acid through two methods: (1) direct extraction using licorice as raw material: grinding the licorice root into powder, and then using solvents such as ethanol and water to obtain glycyrrhetinic acid extract through leaching, percolation, and concentration. (2) conversion using glycyrrhizic acid as raw material: extracting glycyrrhizic acid from licorice powder and then extracting glycyrrhetinic acid through hydrolysis.

[0004] However, the two aforementioned industrial extraction methods generally suffer from shortcomings such as low raw material utilization, complex and cumbersome processes, high energy consumption, and high environmental pressure. Since the natural content of glycyrrhetinic acid in licorice is relatively low, typically only 0.1% to 1%, direct extraction of glycyrrhetinic acid requires the consumption of a large amount of licorice raw material, resulting in low raw material utilization. Furthermore, traditional solvent extraction methods have poor selectivity for glycyrrhetinic acid, often accompanied by the precipitation of a large amount of impurities, requiring multiple purifications, high solvent consumption, and high equipment investment and energy costs. When glycyrrhizic acid is used as a raw material for conversion, the process chain is long and the yield is limited; and the hydrolysis method requires the hydrolysis of the glycosidic bond of glycyrrhizic acid through acid or alkali hydrolysis or acetylation and oxidation. The hydrolysis process is violent, produces many by-products, and the product purification steps are cumbersome and the waste liquid treatment is complex.

[0005] Microbial conversion methods for producing glycyrrhetinic acid have been reported in the prior art due to their mild conditions and relative environmental friendliness. However, they have not been adopted for industrial production due to low glycyrrhetinic acid yields and high production costs. There is an urgent need for a strain suitable for fermenting glycyrrhetinic acid from licorice, ideally with a short culture cycle and less demanding culture conditions, and that can significantly increase the yield of the target product, glycyrrhetinic acid, compared to other strains. Summary of the Invention

[0006] To address the low yield of the target product in existing microbial conversion processes for glycyrrhetinic acid production, the present invention screened out a high-yielding bacterium for glycyrrhetinic acid, Acetobacter orientalis T1-1. This approach lays the foundation for the green, efficient, and industrialized production of glycyrrhetinic acid, with significant social, environmental, and economic benefits.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a use of Acetobacter orientalis T1-1 in the preparation of glycyrrhetinic acid. The Acetobacter orientalis T1-1 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2015455.

[0008] The present invention unexpectedly discovered a high-yield strain of glycyrrhetinic acid, Acetobacter orientalis T1-1. The yield of glycyrrhetinic acid in liquorice fermented with Acetobacter orientalis T1-1 can reach as high as 1.62%-2.09%. Under the experimental scope of the present invention, under the same conditions, the yield of the target product after fermentation with Acetobacter orientalis T1-1 was increased by up to 1.15 times compared to fermentation with Lactobacillus plantarum J26, and by up to 3.86 times compared to fermentation with Acetobacter orientalis SHBCC D12928.

[0009] Acetobacter orientalis T1-1 was first disclosed in the Chinese patent application number ZL201510735042.4. In July 2015, the inventors of the invention published the Acetobacter orientalis T1-1 ( Acetobacter orientalis ) is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 2015455.

[0010] In a second aspect, the present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: fermenting licorice with the above-mentioned Acetobacter orientalis T1-1 to obtain glycyrrhetinic acid.

[0011] Preferably, the preparation method comprises the following steps: inoculating the seed liquid of the above-mentioned Acetobacter orientalis T1-1 into a licorice culture medium, and fermenting at 30° C.-40° C. to produce glycyrrhizic acid.

[0012] Preferably, the licorice culture medium comprises licorice raw material and MRS culture medium in a mass ratio of 4:1-1:1.

[0013] Preferably, the fermentation temperature is 32°C-38°C.

[0014] Preferably, the fermentation time is 18h-144h.

[0015] Further preferably, the licorice culture medium comprises licorice raw material and MRS culture medium in a mass ratio of 3:1-1:1.

[0016] Further preferably, the fermentation temperature is 35°C-38°C.

[0017] Further preferably, the fermentation time is 36h-144h.

[0018] Preferably, the licorice raw material includes licorice raw material or licorice extract.

[0019] Further preferably, the preparation method of the licorice extract comprises the following steps: taking licorice raw medicinal materials, adding 6-10 times the amount of water, decocting and extracting, combining the decoctions, and concentrating the resulting decoctions to contain 0.8g-1.2g of licorice raw medicinal materials per 1mL of concentrated solution to obtain licorice extract.

[0020] Further preferably, the decoction extraction is performed 2-3 times; The decoction and extraction time is 0.8h / time to 1.5h / time.

[0021] Illustratively, the preparation method of the licorice extract includes the following steps: taking licorice raw medicinal materials, adding 8 times the amount of water and decocting and extracting 3 times, 1 hour each time, filtering while hot after the decoction is completed, combining the decoctions, and concentrating until each 1 mL of concentrated solution contains 1.0 g of licorice raw medicinal materials to obtain licorice extract.

[0022] Preferably, the concentration of the seed solution of Acetobacter orientalis T1-1 is 4×10 9 CFU / mL-8×10 9 CFU / mL; The inoculation amount of the seed liquid of Acetobacter orientalis T1-1 is 3%-5%.

[0023] In a third aspect, the present invention provides glycyrrhetinic acid obtained by the above-mentioned preparation method of glycyrrhetinic acid. The obtained glycyrrhetinic acid can be further applied in the fields of food, medicine, daily chemicals, etc.

[0024] The present invention innovatively discovered a high-yield strain of glycyrrhetinic acid, Acetobacter orientalis T1-1. Using Acetobacter orientalis T1-1 in fermentation of licorice culture medium can increase the yield of glycyrrhetinic acid to 1.62%-2.09%. Within the experimental scope of the present invention, the yield of the target product, glycyrrhetinic acid, using Acetobacter orientalis T1-1 was 67%-386% higher than that of other strains.

[0025] The present invention provides a method for preparing glycyrrhetinic acid, which has a relatively short fermentation cycle, low energy consumption, environmental friendliness, high yield of the target product, mild preparation conditions and easy control. It can be used for the green, efficient and industrialized production of glycyrrhetinic acid, and the glycyrrhetinic acid obtained can be further applied in the fields of food, medicine and daily chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a high performance liquid chromatogram of a glycyrrhetinic acid standard with a concentration of 0.0144 mg / mL in Example 2 of the present invention; Figure 2 This is the HPLC chromatogram of fermentation broth I in Example 2 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The control strain information used in the present invention is as follows: Acetobacter orientalis SHBCC D12928 was purchased from Shanghai Collection Biotechnology Center; The strain deposit number of Lactobacillus plantarum J26 is CGMCC5448, which was purchased from Hubei JuneYao Health Beverage Co., Ltd.

[0030] The components of the MRS liquid culture medium of the present invention are as follows: 10.0 g of casein peptone, 10.0 g of beef extract, 5.0 g of yeast powder, 5.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 1.0 g of Tween 80, 2.0 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O, and 20.0 g of CaCO3. Distilled water is added to make the volume to 1.0 L, and the pH is adjusted to 6.8.

[0031] The components of the activation medium are as follows: 30 g yeast extract, 20 g glucose, 1 g KH2PO4, 1 g MgSO4 and distilled water to 1.0 L.

[0032] Preparation of Acetobacter oriental seed liquid: Select dormant Acetobacter oriental and inoculate it into activation culture medium for activation; inoculate the activated Acetobacter oriental into a shake flask containing sterilized MRS liquid culture medium (500ml Erlenmeyer flask containing 200ml culture medium), shake and culture at 180rpm and 29℃±1℃ for 16h-24h to obtain Acetobacter oriental seed liquid.

[0033] Preparation of Lactobacillus plantarum J26 seed solution: Select dormant Lactobacillus plantarum J26 and inoculate it into activation medium for activation; inoculate the activated Lactobacillus plantarum J26 into a shake flask (500 ml Erlenmeyer flask filled with 200 ml medium) filled with sterilized MRS liquid medium, and culture with shaking at 150 rpm and 37°C ± 1°C for 12-16 hours to obtain Lactobacillus plantarum J26 seed solution.

[0034] Preparation method of sterilized licorice powder: put licorice slices into a Chinese herbal medicine grinder and grind them, pass through an 80-mesh sieve to obtain licorice powder; put the licorice powder into a sterile bag and place it in a sterile bag. 60 In the Co-γ irradiation sterilization device, the irradiation dose is 5 KGy to obtain sterilized licorice herbal medicine powder.

[0035] Example 1 The present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: In a clean bench, 20 g of sterilized licorice powder was mixed with 10 g of high-pressure steam-sterilized MRS liquid culture medium and 100 mL of sterile distilled water, and the pH was adjusted to 6.8 to obtain licorice culture medium. In the clean bench, licorice culture medium was placed in a 250 mL shake flask, and the viable bacterial count was 6.8 × 10 9 cfu / mL of Acetobacter orientalis T1-1 seed solution; The inoculated shake flask was placed in a shaking incubator, wherein the shaking incubator temperature was set at 37° C. and the rotation speed was set at 150 rpm. After shaking culture for 72 hours, the fermentation liquid was obtained and recorded as fermentation liquid I.

[0036] Example 2 The present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: In a clean bench, 20 g of sterilized licorice powder was mixed with 10 g of high-pressure steam-sterilized MRS liquid culture medium and 100 mL of sterile distilled water, and the pH was adjusted to 6.8 to obtain licorice culture medium. In the clean bench, licorice culture medium was placed in a 250 mL shake flask, and the viable bacterial count was 6.8 × 10 9 cfu / mL of Acetobacter orientalis T1-1 seed solution; The inoculated shake flask was placed in a shaking incubator, wherein the shaking incubator temperature was set at 37° C. and the rotation speed was set at 180 rpm. After shaking culture for 144 h, the fermentation liquid was obtained and recorded as fermentation liquid II.

[0037] Example 3 The present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: Take the raw liquorice material, add 8 times the amount of water and boil and extract 3 times, 1 hour each time. After the decoction is completed, filter while hot, combine the decoctions, and concentrate until each 1 mL of the concentrate contains 1.0 g of the raw liquorice material to obtain the liquorice extract; 40 g of licorice extract was mixed evenly with 10 g of MRS liquid medium and 100 mL of distilled water, the pH was adjusted to 6.5, and the mixture was sterilized by high-pressure steam to obtain licorice culture medium. In the clean bench, licorice culture medium was placed in a 250 mL shake flask, and the viable bacteria count was 8 × 10 9 cfu / mL of Acetobacter orientalis T1-1 seed solution; The inoculated shake flask was placed in a shaking incubator, wherein the shaking incubator temperature was set at 30°C and the rotation speed was set at 180 rpm. After shaking culture for 144 hours, the fermentation liquid was obtained and recorded as fermentation liquid III.

[0038] Example 4 The present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: In a clean bench, 30 g of sterilized licorice powder was mixed with 10 g of high-pressure steam-sterilized MRS liquid medium and 100 mL of sterile distilled water, and the pH was adjusted to 7 to obtain licorice culture medium. In the clean bench, licorice culture medium was placed in a 250 mL shake flask, and the viable bacteria count was 4 × 10 9 cfu / mL of Acetobacter orientalis T1-1 seed solution; The inoculated shake flask was placed in a water bath shaker with the water bath temperature set at 40°C and the rotation speed set at 120 rpm. After shaking culture for 36 hours, the fermentation liquid was obtained and recorded as fermentation liquid IV.

[0039] Example 5 The present invention provides a method for preparing glycyrrhetinic acid, which comprises the following steps: In a clean bench, 10 g of sterilized licorice powder was mixed with 10 g of high-pressure steam-sterilized MRS liquid medium and 100 mL of sterile distilled water, and the pH was adjusted to 6.8 to obtain licorice culture medium. In the clean bench, licorice culture medium was placed in a 250 mL shake flask, and the viable bacteria count was 5×10 9 cfu / mL of Acetobacter orientalis T1-1 seed solution; The inoculated shake flask was placed in a shaking incubator, wherein the shaking incubator temperature was set to 35° C. and the rotation speed was set to 150 rpm. After shaking culture for 18 hours, a fermentation liquid was obtained, which was recorded as fermentation liquid V.

[0040] Comparative Example 1 Comparative Example 1 of the present invention provides a method for preparing glycyrrhetinic acid. This method is essentially the same as Example 1, differing only in that the "seed solution of Acetobacter orientalis T1-1" is replaced with "seed solution of Acetobacter orientalis SHBCCD12928" having an equivalent viable bacterial concentration. The remaining steps and process parameters are the same as those in Example 1. The fermentation broth obtained by the final fermentation is designated as Fermentation Broth Comparative Example I.

[0041] Comparative Example 2 Comparative Example 2 of the present invention provides a method for preparing glycyrrhetinic acid. This method is essentially the same as Example 1, differing only in that the "Acetobacter orientalis T1-1 seed solution" is replaced with "Lactobacillus plantarum J26 seed solution" having an equivalent viable bacteria concentration. The remaining steps and process parameters are the same as those in Example 1. The fermentation broth obtained by the final fermentation is designated as Fermentation Broth Comparative II.

[0042] Effect Example 1 The OD values ​​of the fermentation broths obtained in Examples 1-5 and Comparative Examples 1-2 were measured at 600 nm, and the measurement results are shown in Table 1.

[0043] Table 1 OD values ​​of different fermentation broths (mean ± SD)

[0044] As shown in Table 1, in Examples 1-2, 4-5 and Comparative Examples 1-2, which used licorice powder in the licorice culture medium, the OD 600 The values ​​were significantly higher than those of the comparative example group. Therefore, the viable cell count of Acetobacter orientalis T1-1 selected in the embodiment was significantly higher than that of Acetobacter orientalis SHBCC D12928 or Lactobacillus plantarum J26 in the comparative example. The reason for this may be that: Acetobacter orientalis T1-1 has significantly better nutrient utilization efficiency, environmental adaptability and metabolic characteristics on licorice culture medium than the comparative example strains Acetobacter orientalis SHBCC D12928 and Lactobacillus plantarum J26, which makes it grow more vigorously under the same culture conditions.

[0045] Effect Example 2 The present invention measured the glycyrrhetinic acid content in the fermentation broth obtained in Examples 1-5 and Comparative Examples 1-2, and further calculated the glycyrrhetinic acid yield in different Examples or Comparative Examples.

[0046] 1. The present invention adopts high performance liquid chromatography to detect the content of glycyrrhetinic acid, and the specific determination method is as follows: (1) Chromatographic conditions Chromatographic column: Discovery C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile-water-methanol (volume ratio 7:2:1); Flow rate: 0.8 mL / min; Detection wavelength: 268nm; Column temperature: 15°C; Injection volume: 10 μL.

[0047] (2) Preparation of test solution and standard solution Preparation of the test solution: Take 20 mL of fermentation broth and centrifuge at 3000 rpm for 15 min. Take the supernatant and adjust the pH value of the fermentation broth to 3 with 10% hydrochloric acid. Extract with ethyl acetate three times, separate the organic phase, and then evaporate the ethyl acetate on a rotary evaporator. Add an appropriate amount of methanol to re-dissolve, filter with a 0.22 μm organic filter membrane to obtain the test solution, and place it into a liquid phase sampling bottle.

[0048] Preparation of standard solution: Accurately weigh 2.3 mg of glycyrrhetinic acid standard into a 10 mL volumetric flask. Dilute to the mark with chromatographic-grade methanol to obtain a stock solution with a mass concentration of 0.23 mg / mL. Accurately pipette 1 mL of the stock solution and dilute it with chromatographic-grade methanol to obtain a series of standard solutions with concentrations of 0.115, 0.0575, 0.0288, 0.0144, 0.00719, and 0.00360 mg / mL, respectively. Pass the solution through a 0.22 μm filter and transfer to a liquid chromatography vial.

[0049] (3) Detection According to the preset chromatographic conditions, the series of concentration standard solutions, the test solutions prepared from the fermentation broths obtained in Examples 1-5 and Comparative Examples 1-2 were injected into the HPLC instrument for detection. Figure 1 As shown in the figure, the HPLC chromatogram of fermentation broth I is as follows: Figure 2 shown.

[0050] Based on the concentration and peak area of ​​the series of concentration standard solutions, a standard curve was drawn to calculate the concentration of glycyrrhetinic acid in different fermentation broths. Then, based on the input amount of licorice raw medicinal materials, the yield of glycyrrhetinic acid in different glycyrrhetinic acid preparation methods was calculated. The specific measurement results are shown in Table 2 below.

[0051] Table 2 Glycyrrhetinic acid yields from different glycyrrhetinic acid preparation methods (mean ± SD)

[0052] As shown in Table 4, the glycyrrhetinic acid yields achieved by the glycyrrhetinic acid preparation methods provided by the present invention ranged from 1.623% to 2.086%, significantly higher than those achieved by the comparative examples using Acetobacter orientalis SHBCC D12928 (0.429%) or Lactobacillus plantarum J26 (0.974%). Furthermore, under the same experimental conditions, it was found that, after 72 hours of fermentation, the glycyrrhetinic acid yield decreased with increasing fermentation time.

[0053] In summary, the present invention creatively discovered a high-yield strain of glycyrrhetinic acid - Acetobacter orientalis T1-1. Therefore, the present invention uses Acetobacter orientalis T1-1 as a fermentation strain to ferment a culture medium containing licorice under specific culture conditions for the preparation of glycyrrhetinic acid. The yield of glycyrrhetinic acid in the preparation method of glycyrrhetinic acid provided by the present invention can reach 1.62%-2.09%. Within the experimental scope of the present invention, under the same conditions, compared with the fermentation of Lactobacillus plantarum J26, the yield of the target product after fermentation by Acetobacter orientalis T1-1 was increased by up to 1.15 times; compared with the fermentation of Acetobacter orientalis SHBCC D12928, the yield of the target product after fermentation by Acetobacter orientalis T1-1 was increased by up to 3.86 times. The reason for this may be that the nutrient utilization efficiency, environmental adaptability and metabolic characteristics of Acetobacter orientalis T1-1 on licorice culture medium are significantly better than those of the comparative strains Acetobacter orientalis SHBCCD12928 and Lactobacillus plantarum J26, making it grow more vigorously under the same culture conditions.

[0054] The method for preparing glycyrrhetinic acid provided by this invention offers advantages such as a short fermentation cycle, high yield of the target product, and easily controllable culture conditions, providing an innovative approach for green and efficient industrial production. The fermentation broth obtained can be further purified according to actual needs, and has potential for industrial application in a variety of fields, including food, medicine, and daily chemicals.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of Acetobacter orientalis T1-1 in the preparation of glycyrrhetinic acid, characterized in that: The Acetobacter orientalis T1-1 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2015455.

2. A method for preparing glycyrrhetinic acid, characterized in that: The preparation method comprises the following steps: fermenting licorice with the Acetobacter orientalis T1-1 described in claim 1 to produce glycyrrhizic acid.

3. The method for preparing glycyrrhetinic acid according to claim 2, wherein: The specific steps include: The seed liquid of Acetobacter orientalis T1-1 is inoculated into a liquorice culture medium and fermented at 30° C.-40° C. to produce glycyrrhetinic acid.

4. The method for preparing glycyrrhetinic acid according to claim 3, wherein: The licorice culture medium comprises a licorice raw material and an MRS culture medium in a mass ratio of 4:1-1:1; and / or The fermentation temperature is 32°C-38°C; and / or The fermentation time is 18h-144h.

5. The method for preparing glycyrrhetinic acid according to claim 4, wherein: The licorice culture medium comprises a licorice raw material and an MRS culture medium in a mass ratio of 3:1-1:1; and / or The fermentation temperature is 35°C-38°C; and / or The fermentation time is 36h-144h.

6. The method for preparing glycyrrhetinic acid according to claim 4 or 5, wherein: The licorice raw material includes licorice raw material or licorice extract.

7. The method for preparing glycyrrhetinic acid according to claim 6, wherein: The preparation method of the licorice extract comprises the following steps: taking licorice raw medicinal materials, adding 6-10 times the amount of water, decocting and extracting, and concentrating the obtained decoction until each 1 mL of concentrated solution contains 0.8g-1.2g of licorice raw medicinal materials to obtain the licorice extract.

8. The method for preparing glycyrrhetinic acid according to claim 7, wherein: The preparation method of the liquorice extract comprises the following steps: the decoction is performed 2 to 3 times; and / or The decoction and extraction time is 0.8h / time to 1.5h / time.

9. The method for preparing glycyrrhetinic acid according to claim 3, wherein: The concentration of the seed solution of Acetobacter orientalis T1-1 is 4×10 9 CFU / mL-8×10 9 CFU / mL; and / or The inoculation amount of the seed liquid of Acetobacter orientalis T1-1 is 3%-5%.

10. A glycyrrhetinic acid, characterized in that: The glycyrrhetinic acid is prepared by the preparation method of any one of claims 2 to 9.

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