Screening method of ginsenoside converting enzyme producing strain

Abstract

The invention belongs to the technical field of microorganisms, and relates to a method for screening a strain for producing ginsenoside converting enzyme. The method comprises the following steps: enrichment culture: obtaining an enrichment liquid from a rhizosphere soil sample of the ginseng under forest; strain domestication and primary screening: adding a supernatant of the enrichment liquid into a first screening culture medium for culture, taking a culture solution for passage to a second screening culture medium for culture, taking the culture solution for passage to a third screening culture medium for culture, and stopping passage; the concentrations of total saponin substrates in the screening culture medium are respectively 0.5 mg.mL <-1 >, 1.0 mg.mL <-1 > and 1.5 mg.mL <-1 >; carrying out gradient dilution on the domesticated strain to obtain a single colony; secondary screening of the strains: inoculating the purified strains into a liquid seed culture medium for culturing to obtain a seed solution; mixing the seed solution with a total saponin solution, culturing, and detecting and screening an enzyme-producing strain capable of converting ginsenoside through thin layer chromatography. The method disclosed by the invention is low in cost and short in time consumption, and enriches a ginsenoside transformation strain resource library.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to a method for screening strains producing ginsenoside-converting enzymes. Background Art

[0002] Ginseng (Panax ginseng C.A.Meyer) is a plant of the Araliaceae family and is a famous and precious Chinese herbal medicine well-known at home and abroad, with the effects of tonifying primordial qi, promoting fluid production and tranquilizing the mind, invigorating the spleen and benefiting the lungs, etc. Ginsenosides are the most important pharmacological active components of ginseng, having effects such as regulating blood sugar, anti-tumor, anti-inflammatory, antioxidant, and neuroprotection. Especially rare ginsenosides have characteristics such as easy absorption by the human body and high activity. However, the content of rare ginsenosides in ginseng is extremely low, lacking a wide and stable source, and the difficulty of industrial production is relatively high. Ginsenoside-converting enzymes can modify and transform the structure of ginsenosides through hydrolysis or glycosylation reactions, thereby changing the structure and activity of ginsenosides, converting certain types of ginsenosides into other rare ginsenosides with different biological activities and pharmacological effects, and thus improving the bioavailability and pharmacological activity of ginsenosides. For example, after ginsenosides (such as Rb1, Rg1, etc.) are acted upon by digestive enzymes (such as glycosidases, β-glucosidases, etc.) in vivo or in vitro, their sugar chains are hydrolyzed or modified to generate rare ginsenosides (such as Rg3, Rh2, CK, etc.). These rare saponins have significantly improved bioavailability and stronger pharmacological activity due to their smaller molecular weight and higher liposolubility.

[0003] Currently, domestic research on ginsenoside-converting enzymes focuses on enzyme resource mining, structural and functional analysis, and synthetic biology applications, while international research focuses on microbial fermentation and new conversion pathways. The current challenge of ginsenoside-converting enzymes lies in the problems of enzyme catalytic efficiency and stability. The catalytic efficiency of existing ginsenoside-converting enzymes still difficult to meet the industrial demand. It is necessary to further screen more efficient and stable strains and optimize the enzyme activity and stability through enzyme engineering technology, thereby breaking through the relevant enzyme engineering bottlenecks, developing large-scale production processes, and meeting the needs of the medical and large health markets. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a method for screening strains producing ginsenoside-converting enzymes. Using this screening method can not only enrich the strain resource library for saponin conversion, but also promote innovation and development in related fields, and thus meet the industrial production requirements.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] The present invention provides a method for screening strains producing ginsenoside-converting enzymes, comprising the following steps:

[0007] (1) Enrichment culture:

[0008] The forest ginseng rhizosphere soil sample is added to sterile water, and the supernatant is taken and added to an enrichment medium for cultivation to obtain an enrichment solution;

[0009] (2) Strain domestication and primary screening:

[0010] The supernatant of the enrichment solution is added to the first screening medium for cultivation, and the culture solution is subcultured to the second screening medium for cultivation, and then the culture solution is subcultured to the third screening medium for cultivation, and the subculture is stopped; the total saponin substrate concentration of the first screening medium is 0.5 mg·mL -1 , the total saponin substrate concentration of the second screening medium is 1.0 mg·mL -1 , and the total saponin substrate concentration of the third screening medium is 1.5 mg·mL -1 ;

[0011] The domesticated strain is gradient-diluted, spread on the screening medium, different morphological strains are isolated, and purified cultivation is carried out to obtain single colonies;

[0012] (3) Strain rescreening:

[0013] The purified strain is inoculated into a liquid seed medium for cultivation to obtain a seed solution; the seed solution is mixed with the total saponin solution and cultivated, and enzyme-producing strains capable of converting ginsenosides are screened out by thin-layer chromatography detection.

[0014] In the above technical solution, further, the enrichment medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, 121 °C, sterilized for 30 min;

[0015] The screening medium includes: 2 g of (NH4)2SO4, 0.35 g of MgSO4·7H2O, 0.1 g of CaCl2, 0.5 g of K2HPO4, 0.5 g of NaH2PO4, 1 L of distilled water, 18 g of agar powder; pH 7, 121 °C, sterilized for 30 min; after sterilization, total ginsenosides are additionally added by membrane filtration method;

[0016] The seed medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, 121 °C, sterilized for 30 min;

[0017] In the above technical solution, further, in the step (2), each cultivation is carried out at 30 °C and 120 r·min -1 for 3 d of constant temperature shaking cultivation.

[0018] In the above technical solution, further, in the step (2), the dilution factor is 10 -5 ~10 -7 .

[0019] In the above technical solution, further, in the step (3), the concentration of the total saponin solution is 2 mg / mL, and the seed solution is mixed with the total saponin solution in an equal ratio.

[0020] In the above technical solution, further, in the step (3), the culture time after the seed solution and the total saponin solution are mixed is 3 to 7 days.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention solves the problem that it is difficult to screen out strains producing ginsenoside-converting enzymes by existing methods. Through three generations of domestication and using three different total saponin substrate concentrations, the acquisition of target bacteria is ensured. The screening method of the present invention is simple to operate, low in cost, small in saponin dosage, and high in screening efficiency. Screening is carried out in three steps, which can effectively prevent contamination by miscellaneous bacteria, reduce the risk of contamination, enrich the ginsenoside-converting strain resource library, and provide a new strain source for ginsenoside conversion. Brief Description of the Drawings

[0023] The following further describes the content of the present invention in conjunction with the drawings, but does not limit the present invention thereto.

[0024] Figure 1 Colony photograph of the strain producing ginsenoside-converting enzyme obtained by the screening method in Example 1 of the present invention.

[0025] Figure 2 TLC result diagram of the strain producing ginsenoside-converting enzyme obtained by the screening method in Example 1 of the present invention; in the figure, Z: refers to the ginsenoside standard; d: number of days.

[0026] Figure 3 TLC result diagram of the conversion of ginsenoside by the screened strain in Comparative Example 1; in the figure, Z: refers to the ginsenoside standard; MA, MB, and MC all refer to the screened strains. Detailed Description of the Invention

[0027] The following further describes the present invention in conjunction with specific embodiments, but does not limit the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0028] Total ginsenosides can be purchased directly or prepared by oneself. The preparation method of the total ginsenosides used in the following examples is as follows: Dry the fresh wild ginseng in the forest understory and grind it into powder. Weigh 10 g of the wild ginseng powder in the forest understory, soak it in methanol by heating, filter and collect the leachate, repeat 3 times, and distill the leachate under reduced pressure; Degrease it sufficiently with petroleum ether (60°C - 90°C), repeat 3 times, then add a small amount of deionized water to dissolve the extract, and then add an appropriate amount of water-saturated n-butanol to extract the saponins sufficiently. After combining the extraction solutions, add deionized water and wash repeatedly to remove substances such as sugars. Then, the remaining powder after distilling the extraction solution under reduced pressure is the total ginsenosides, including Re, Rg1, Rb2, Rc, Rd, Rb3, and Rh1.

[0029] Example 1

[0030] The screening method for strains producing ginsenoside-converting enzyme is as follows:

[0031] (1) Enrichment culture:

[0032] Take 5 g of the rhizosphere soil sample of wild ginseng in the forest understory in the ginseng planting areas of Liaoning and Heilongjiang regions, add it to 10 mL of sterile water with glass beads, place it in a constant temperature culture oscillator at 30°C and 120 r·min -1 Shake well and let it stand for a while. Use a pipette to suck 1 mL of the supernatant and add it to 100 mL / 250 mL of enrichment medium, and place it in a constant temperature culture oscillator at 30°C and 120 r·min -1 Cultivate for 24 h to obtain the enrichment solution;

[0033] The enrichment medium used is: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, sterilize at 121°C for 30 min.

[0034] (2) Strain domestication and primary screening:

[0035] Suck 200 μL of the supernatant of the enrichment solution obtained in the above step and add it to 20 ml / 50 ml of the screening medium with the substrate concentration of total ginsenosides (including Re, Rg1, Rb2, Rc, Rd, Rb3, Rh1) being 0.5 mg·mL -1 Place it in a constant temperature culture oscillator at 30°C and 120 r·min -1 Cultivate for 3 d. After 3 d, take 200 μL of the previous culture solution and transfer it to a new screening medium, and increase the total saponin substrate concentration to 1.0 mg·mL -1 And cultivate under the same conditions. Repeat the operation and increase the substrate concentration until the highest total saponin concentration reaches 1.5 mg·mL -1 That's it; namely, domesticate for 3 generations, and the total saponin substrate concentrations are 0.5 mg·mL -1 、1.0 mg·mL -1 And 1.5 mg·mL-1 , the screening medium used is: 2 g of (NH4)2SO4, 0.35 g of MgSO4·7H2O, 0.1 g of CaCl2, 0.5 g of K2HPO4, 0.5 g of NaH2PO4, 1 L of distilled water, and 18 g of agar powder is added more for the solid medium. pH 7, sterilized at 121 °C for 30 min. After sterilization, total ginsenosides are added additionally by membrane filtration method.

[0036] Perform gradient dilution on the above-mentioned bacterial liquid of the last passage (take 10 test tubes filled with 9 mL of sterile water, and label them as No. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 respectively. Aspirate 1 mL of the bacterial liquid and inject it into the No. 1 test tube, and shake the test tube thoroughly to make the bacterial liquid and sterile water fully mixed evenly. This is the bacterial suspension with a dilution factor of 10 -1 . Then aspirate 1 mL of the bacterial suspension from the No. 1 test tube and inject it into the 9 mL of sterile water in the No. 2 test tube, and mix well to prepare a bacterial suspension with a dilution factor of 10-2. According to the above method, bacterial suspensions with dilution factors of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 are obtained in sequence. Aspirate 100 μl of the bacterial suspension of each gradient and coat it on the screening medium, with 3 parallels for each gradient. Incubate it in an inverted position at 30 °C for 3 - 5 days, and observe its growth status every day. Select the gradient with growing bacteria and non-dense and non-overlapping colonies. In this example, 10 -5 , 10 -6 , 10 -7 are selected, pick strains with different morphologies, and perform isolation streaking purification culture. Incubate it in an inverted position at 30 °C until single colonies are obtained.

[0037] (3) Rescreening of strains:

[0038] Take 2 - 3 inoculation loops of the strains purified in the above steps and inoculate them into 100 mL / 250 mL of liquid seed medium, place them in a shaker, and culture them in a constant temperature culture oscillator at 30 °C and 120 r·min -1 for 24 h to obtain seed liquid; the liquid seed medium is 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, sterilized at 121 °C for 30 min.

[0039] Take 200 μL of the total saponin solution at 2 mg / mL and mix it with an equal volume of the seed liquid mixture in a 1.5 mL EP tube. Incubate at 30 °C and 120 rpm. Every day, take 50 μL and mix it with the same volume of water-saturated n-butanol to terminate the reaction, and perform thin-layer chromatography detection. Select the original enzyme-producing strain that can convert ginsenosides and preserve it using the slant preservation method and the glycerol preservation method.

[0040] Further, the steps of the thin-layer chromatography detection method include:

[0041] Prepare a suitable silica gel plate. Pour the prepared developing agent into the chromatography tank and wait for 30 min to allow the developing agent to diffuse and fill the chromatography tank with gas [development system: n-butanol - ethyl acetate - water (1:4:5)]. Take samples using a capillary needle. The distance between the sample spots is 0.5 cm, and the number of sample applications is 20 - 30 times. After each sample application, quickly dry it with a hair dryer. After the solvent is dried, place it in the chromatography tank, and the immersion depth in the developing agent is 0.5 - 1 cm from the bottom edge of the thin-layer plate (do not submerge the sample application position). When the migration band of the developing agent approaches the upper edge of the silica gel plate, take out the silica gel plate from the developing system, and the development work is completed. Volatilize the developing agent absorbed by the silica gel plate, spray the coloring solution (20% sulfuric acid ethanol solution) in the vertical direction of the plate, and finally dry it for coloring.

[0042] Compare the colored position of the product with the reference ginsenoside standard. It is found that the band changes and the depth change is obvious, indicating that the strain screened by this method has the ability to convert ginsenosides.

[0043] Comparative Example 1

[0044] The same screening method as in Example 1 is used, except that in step (2) strain domestication and primary screening, the total saponin substrate concentrations are 0.25 mg·mL -1 、0.5 mg·mL -1 and 0.75 mg·mL -1 , and the rest is the same as in Example 1. After thin-layer chromatography detection, from the 1st day to the 7th day, the detection results are the same as those of the standard product, proving that the screened strain does not have the ability to convert ginsenosides.

Claims

1. A method for screening a strain producing ginsenoside converting enzyme, characterized in that: The following steps are involved: (1) Enrichment culture: The rhizosphere soil samples of the forest ginseng were added into sterile water, and the supernatant was added into the enrichment medium for cultivation to obtain the enrichment solution; (2) Strain domestication and initial screening: The supernatant of the enriched solution was added to the first screening medium for culture, the culture medium was subcultured to the second screening medium for culture, and then the culture medium was subcultured to the third screening medium for culture, and the subculture was stopped; the total saponin substrate concentration of the first screening medium was 0.5 mg·mL -1 , the total saponin substrate concentration of the second screening medium is 1.0 mg mL -1 The total saponin substrate concentration of the third screening medium is 1.5 mg·mL -1 ; The domesticated strains were diluted in gradients and spread on screening medium to separate strains with different morphologies and purified to obtain single colonies; (3) Strain rescreening: The purified strain is inoculated into a liquid seed culture medium for cultivation to obtain a seed liquid; The seed liquid is mixed with the total saponin solution and then cultured, and the enzyme-producing strain capable of transforming ginsenosides is screened out by thin layer chromatography.

2. The screening method according to claim 1, characterized in that The enrichment medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, 121° C., sterilized for 30 min; The screening medium comprises: (NH4)2SO4 2g, MgSO4·7H2O 0.35g, CaCl2 0.1g, K2HPO4 0.5g, NaH2PO4 0.5g, distilled water 1L, agar powder 18g; pH 7, 121°C, sterilization for 30min; after sterilization, total ginsenosides are additionally added by membrane filtration method; The seed culture medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, pH 7, 121° C., and sterilization for 30 min.

3. The screening method according to claim 1, characterized in that In the step (2), each culture is carried out at 30°C and 120 r·min -1 The cells were cultured at constant temperature with shaking for 3 days.

4. The screening method according to claim 1, characterized in that In the step (2), the dilution is 10 -5 ~10 -7 .

5. The screening method according to claim 1, characterized in that In the step (3), the concentration of the total saponin solution is 2 mg / mL, and the seed solution and the total saponin solution are mixed in equal proportions.

6. The screening method according to claim 1, characterized in that In the step (3), the seed solution and the total saponin solution are mixed and cultured for 3 to 7 days.

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