Preparation method and application of Morchella solid-state fermented geranium seedling extract with antibacterial effect

Abstract

The present invention belongs to the field of development and application of Chinese medicine resources, and specifically relates to a preparation method and application of a Morchella solid-state fermented geranium seedling extract with antibacterial effect. The preparation method is realized by the following steps: activating the Morchella strain by PDA solid culture medium, then inoculating it into the PDA culture medium containing geranium seedling for fermentation, extracting by 70% ethanol, centrifuging the extract, and freeze-drying after rotary evaporation to obtain a Morchella fermented geranium seedling extract (solid-state fermentation). The Morchella fermented geranium seedling extract prepared by the present invention is subjected to a broad-spectrum antibacterial experiment on pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Bacillus aerogenes. It is found that the Morchella fermented geranium seedling extract of the present invention can inhibit the growth of Staphylococcus aureus, Escherichia coli, and Bacillus aerogenes, and has a significant antibacterial effect, which provides a scientific basis for the research on the antibacterial application of Morchella fermented geranium seedling extract.

Description

Technical Field

[0001] The invention belongs to the field of development and application of traditional Chinese medicine resources, and specifically relates to a preparation method of a Morchella solid-state fermented Geranium eriophorum seedling extract with antibacterial effect and application thereof. Background Art

[0002] Morel Morchella esculenta (L.) Pers.) belongs to the kingdom Fungi, phylum Fungi, class Ceratocystis, order Pyrimorphales, family Morchellaceae, genus Morchella. It contains polysaccharides that inhibit tumors, and active ingredients that are antibacterial and antiviral, and has the effects of enhancing immunity, resisting fatigue, resisting viruses, and inhibiting tumors.

[0003] Geraniol Geraniaceae Juss .) Annual or perennial perennial herb, shrub-like. Commonly known as "long-billed geranium", it is one of the sources of the Chinese medicinal material geranium, and is widely distributed. Its main chemical components include tannins, flavonoids, organic acids, etc. Many pharmacological studies have shown that these components have anti-inflammatory, antibacterial, anti-tumor, antioxidant, antiviral and other pharmacological activities, and are therefore the material basis for the therapeutic effect of Geranium sibiricum.

[0004] Modern fermentation technology has the advantages of improving the efficacy of traditional Chinese medicine, increasing the content of effective ingredients, improving the utilization rate of traditional Chinese medicine, reducing the content of toxic ingredients in medicines, and producing new active ingredients. However, in the prior art, there is no record of using Morchella to ferment Geraniol seedlings and extracting the fermentation products to inhibit pathogenic bacteria. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for preparing a Morchella solid-state fermented Geranium oleifera extract having antibacterial effect.

[0006] The present invention also provides an application of a Morchella solid-state fermented geranium seedling extract. The Morchella fermented geranium seedling extract prepared by the present invention has a concentration of 5-10 mg / ml and can inhibit the growth of pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Clostridium perfringens.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] The present invention provides a method for preparing a Morchella solid-state fermented Geranium oleifera extract having an antibacterial effect, comprising the following steps:

[0009] (a) taking a Morchella strain, picking Morchella mycelium and inoculating it into a PDA medium, and culturing it at a constant temperature to obtain an activated Morchella;

[0010] (b) crushing the geranium seedlings, passing through a 60-mesh sieve to obtain geranium seedling powder, and then spraying the geranium seedling powder with a glycine aqueous solution and stirring evenly;

[0011] (c) adding the geranium powder to the PDA liquid culture medium, stirring while adding, and stirring evenly until the geranium powder is neither loose nor clumped, and is in a state of being tightly held in a clumping shape but dispersed when released, to obtain a geranium culture medium;

[0012] (d) sealing the Geranium culture medium with a sealing film and sterilizing it with high pressure steam;

[0013] (e) Fermenting Geranium geraniol: taking Morchella and inoculating it to the bottom of the Geranium geraniol culture medium, sealing it with a sealing film, placing it in an incubator for fermentation, and drying it to a constant weight to obtain a dried fermentation product;

[0014] (f) extracting the dried fermented product with ethanol, combining the extracts, centrifuging, taking the supernatant and performing rotary evaporation, freeze-drying after rotary evaporation, and grinding to obtain a powdered Morchella solid fermentation Geranium oleifera extract.

[0015] Furthermore, in step (a), the inoculation amount of the Morchella mycelium in the PDA solid culture medium is 5%; and the constant temperature culture is cultured at 20°C for 3 days.

[0016] Furthermore, in step (b), the mass ratio of the Geranium geranium powder to the glycine aqueous solution is 1:0.2-0.3; and the concentration of the glycine aqueous solution is 0.2 mol / L.

[0017] Furthermore, in step (c), the solid-liquid ratio of the geranium seedling powder to the PDA liquid culture medium is 1:10.

[0018] Furthermore, in step (d), the high pressure steam sterilization is performed at 115° C. for 30 min.

[0019] Furthermore, in step (e), the accession amount of the Morchella is 10%; and the fermentation culture is cultured in an incubator at 20° C. for 21 days.

[0020] Furthermore, in step (f), the volume ratio of the fermentation product to ethanol is 1:10; the concentration of the ethanol is 70% (v / v); and the extraction is performed at room temperature for 24 hours.

[0021] The present invention also provides an application of the Morchella solid-state fermented Geranium oleifera seedling extract prepared by the above preparation method in antibacterial treatment.

[0022] Furthermore, when the concentration of the Morchella solid-state fermented Geranium oleifera extract is 5-10 mg / ml, the growth of pathogenic bacteria is inhibited.

[0023] Preferably, the pathogenic bacteria are one or more of Staphylococcus aureus, Escherichia coli, and Bacillus aerogenes.

[0024] The morels used in the present invention can be commercially available, and their appearance characteristics are as follows: in the early stage, the mycelium of the morel is white or light yellow, shiny, and the mycelium tips are multi-finger-shaped or dendrite-shaped; in the middle stage, colonies are formed on the culture medium, and the older mycelium in the center of the colony secretes pigments, making the mycelium brown-yellow; when the mycelium ages, the colony changes from dark brown to dirty brown, and the luster disappears.

[0025] The beneficial effects of the present invention are as follows: the present invention utilizes Morchella to ferment Geranium seedlings for the first time, and extracts effective substances in the fermentation products to obtain Morchella fermented Geranium seedling extracts, and uses three concentrations of low, medium and high fermentation extracts to carry out antibacterial experiments on Staphylococcus aureus, Escherichia coli and Bacillus aerogenes, and compares the effects with those of unfermented Geranium seedling extracts. It is found that the Morchella solid-state fermented Geranium seedling extracts of the present invention have more significant antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison of the antibacterial effects of the two extracts on Staphylococcus aureus;

[0027] Figure 2 The effects of two extracts on the growth of Staphylococcus aureus, wherein: (a) is the effect of the Morchella fermented geranium seedling extract prepared in Comparative Example 1 on the growth of Staphylococcus aureus, and (b) is the effect of the Morchella fermented geranium seedling extract prepared in Example 1 on the growth of Staphylococcus aureus;

[0028] Figure 3 This is a comparison of the antibacterial effects of the two extracts on Escherichia coli;

[0029] Figure 4 The effects of two extracts on the growth of Escherichia coli, wherein: (a) is the effect of the extract of Morchella fermented with Geranium oleifera prepared in Comparative Example 1 on the growth of Escherichia coli, and (b) is the effect of the extract of Morchella fermented with Geranium oleifera prepared in Example 1 on the growth of Escherichia coli;

[0030] Figure 5 The comparison of the antibacterial effects of the two extracts on Bacillus aerogenes;

[0031] Figure 6 The effects of two extracts on the growth of aerobic bacteria, wherein: (a) is the effect of the extract of Morchella fermented with Geranium oleifera prepared in Comparative Example 1 on the growth of aerobic bacteria, and (b) is the effect of the extract of Morchella fermented with Geranium oleifera prepared in Example 1 on the growth of aerobic bacteria; DETAILED DESCRIPTION

[0032] In order to make the purpose, technical means and beneficial effects of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the content of the present invention is not limited to the scope described in the embodiments.

[0033] The Morchella used in the present invention is a commercially available product with a product number of HYCC231242.

[0034] Example 1

[0035] 1. Activation of Morels

[0036] (1) Preparation of PDA comprehensive culture medium: Peel the potatoes, weigh 200 g, cut into small dices, add ultrapure water and cook over low heat until it becomes viscous, filter with gauze, add other culture medium components in the PDA comprehensive culture medium listed in Table 1, and make up to 1 L with ultrapure water.

[0037] Table 1 Culture medium formula

[0038]

[0039] (2) After the prepared PDA comprehensive culture medium is sterilized by high-pressure steam (115°C, 30 min), it is placed on a clean bench, sterilized by ultraviolet light for 15 min and protected from light for 5 min, and then poured into a plate. After the culture medium solidifies, take the Morchella strain, pick the mycelium and inoculate it in the center of the plate, place it in a constant temperature incubator at 20°C and invert for 3 days to obtain the activated Morchella.

[0040] 2. Fermentation of Geranium geraniol

[0041] (1) Grind the Geranium seedlings with a grinder and pass them through a 60-mesh sieve to obtain Geranium seedling powder. Then, spray the Geranium seedling powder with a 0.2 mol / L glycine aqueous solution (mass ratio of 1:0.25) and stir evenly.

[0042] (2) Add the geranium powder to the PDA liquid culture medium, stirring while adding, and stir evenly until the geranium powder is neither loose nor clumping, and is in a state of being tightly clumped but dispersed when released, thereby obtaining the geranium culture medium.

[0043] (3) Seal the Geraniol culture medium with sealing film and sterilize it with high-pressure steam (115 ℃, 30 min).

[0044] (4) Fermentation of Geranium chinense: Morchella edulis is inoculated into the bottom of the Geranium chinense culture medium (inoculation amount 10%), sealed with a sealing film, and placed in an incubator for fermentation. The fermentation product is dried to constant weight to obtain a dried fermentation product.

[0045] 3. Extraction of fermentation products

[0046] The dried fermented product was extracted with 70% ethanol (V / V) (solid-liquid ratio 1:10), centrifuged, the supernatant was put into a rotary evaporator for rotary evaporation, and then freeze-dried with a freeze dryer, and ground to obtain a powdered Morchella fermented Geranium seedling extract.

[0047] 4. Drug preparation

[0048] Weigh the fermented Geranium edulis seedling extract and the unfermented Geranium edulis seedling extract separately, add 25% ethanol to each, and ultrasonically shake until fully dissolved. Prepare three drug concentrations of low, medium and high, namely 5 mg / ml, 10 mg / ml and 15 mg / ml, and place in a 4°C refrigerator for use.

[0049] Comparative Example 1

[0050] 1. The activation of Morchella is the same as in Example 1;

[0051] 2. Fermentation of Geranium geraniol

[0052] (1) Grind the Geranium geraniol with a grinder and pass it through a 60-mesh sieve to obtain Geranium geraniol powder.

[0053] (2) Add the geranium powder to the PDA liquid culture medium, stirring while adding, and stir evenly until the geranium powder is neither loose nor clumping, and is in a state of being tightly clumped but dispersed when released, thereby obtaining the geranium culture medium.

[0054] (3) Seal the Geraniol culture medium with sealing film and sterilize it with high-pressure steam (115 ℃, 30 min).

[0055] (4) Fermentation of Geranium chinense: Morchella edulis is inoculated into the bottom of the Geranium chinense culture medium (inoculation amount 10%), sealed with a sealing film, and placed in an incubator for fermentation. The fermentation product is dried to constant weight to obtain a dried fermentation product.

[0056] The other steps are the same as in Example 1.

[0057] Effect Example

[0058] 1. Strain culture

[0059] (1) Strain activation: Staphylococcus aureus, Escherichia coli, and Bacillus aeruginosa

[0060] The frozen strains were thawed at room temperature, and the thawed strains were inoculated into LB solid medium (as shown in Table 1) by the three-zone streak method, cultured at 37°C for 1 day, and a single colony was picked and inoculated into LB liquid medium, and cultured at 37°C and 220 rpm on a shaker for 1 day to prepare the seed solution.

[0061] (2) Bacterial strain preservation: Staphylococcus aureus, Escherichia coli, and Bacillus aeruginosa

[0062] Sterilize 80% glycerol with high pressure steam for later use. Prepare a high-concentration bacterial suspension with sterile water from the cultured slant strains. Take 1 ml of sterilized glycerol and mix it thoroughly with the bacterial solution to make the glycerol concentration about 10%-30%, and freeze it at -80℃.

[0063] 2. Antibacterial experiment

[0064] 100 μL of seed liquid was taken and inoculated in LB solid culture medium respectively, and the inhibitory activity against Staphylococcus aureus, Escherichia coli and Bacillus aerogenes was tested by Oxford cup method. Method: The Oxford cup was placed on the LB solid culture medium coated with Staphylococcus aureus, Escherichia coli and Bacillus aerogenes, and 200 μL of the prepared drug was added to each Oxford cup. The mixture was cultured at 37°C for 1 day, and the diameter of the inhibition zone was calculated by the cross method. The results showed that the extract of fermented geranium seedlings of Morchella had a more obvious inhibitory effect on Staphylococcus aureus, Escherichia coli and Bacillus aerogenes than the extract of unfermented geranium seedlings, and the inhibitory effect increased with the increase of drug concentration. 3. Effect of the antibacterial activity of fermented geranium seedlings of Morchella on Staphylococcus aureus

[0065] As shown in Table 2 and Figure 1 , Figure 2 As shown, the extracts of Morchella fermented with Geraniol seedlings (Example 1 and Comparative Example 1) at low, medium and high drug concentrations of 5 mg / ml, 10 mg / ml and 15 mg / ml all had inhibitory effects on Staphylococcus aureus, and the antibacterial activity was enhanced in a concentration-dependent manner. The antibacterial effect of the extract prepared in Example 1 was more obvious. The antibacterial rates of the three groups with low, medium and high concentrations increased by 23%, 24% and 27% respectively.

[0066] Table 2 Determination of the antibacterial effect of geranium extract on Staphylococcus aureus

[0067]

[0068] 4. Effect of the antibacterial activity of Morchella edulis fermented with Geranium sphaerocephala extract on Escherichia coli

[0069] As shown in Table 3 and Figure 3 , Figure 4 As shown, the three low, medium and high drug concentrations of 5 mg / ml, 10 mg / ml and 15 mg / ml of Morchella fermented geranium seedling extract prepared in Example 1 all have an inhibitory effect on Escherichia coli, while the fermented geranium seedling extract prepared in Comparative Example 1 has no antibacterial effect at low concentrations, but has an antibacterial effect at medium and high concentrations. Moreover, the geranium seedling extract prepared in Example 1 has a better antibacterial effect than the fermented geranium seedling extract prepared in Comparative Example 1, and the antibacterial rates of the medium and high concentration groups are increased by about 34% and 38%, respectively.

[0070] Table 3 Determination of the antibacterial effect of geranium extract on Escherichia coli

[0071]

[0072] 5. Effect of Morchella Fermentation Geraniol Extract on Antibacterial Activity of Aerobacterium

[0073] As shown in Table 4 and Figure 5 , Figure 6 As shown, the extracts of Morchella fermented with Geranium seedlings at low, medium and high drug concentrations of 5 mg / ml, 10 mg / ml and 15 mg / ml (Example 1 and Comparative Example 1) all have an inhibitory effect on Bacillus aerogenes, but the antibacterial effect of the fermented Geranium seedlings extract prepared in Example 1 is more significant, and the antibacterial rates of the low, medium and high concentration groups are increased by about 24%, 28% and 20%, respectively.

[0074] Table 4 Determination of the antibacterial effect of the extract of Geraniol on aerogenes

[0075]

[0076] In summary, through the comparison of the Morchella fermented geranium seedling extract in Example 1 and Comparative Example 1, the present invention found that the Morchella fermented geranium seedling extract prepared by the preparation method provided by the present invention has a more excellent antibacterial effect on three pathogenic bacteria: Staphylococcus aureus, Escherichia coli, and Clostridium perfringens, providing a valuable reference for the subsequent development of antibacterial drugs.

[0077] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Morchella solid-state fermented Geranium oleifera extract having antibacterial effect, characterized in that: The following steps are involved: (a) taking a Morchella strain, picking Morchella mycelium and inoculating it in a PDA solid medium, and culturing it at a constant temperature to obtain an activated Morchella; The inoculation amount of the Morchella mycelium in the PDA solid culture medium is 5%; the constant temperature culture is cultured at 20° C. for 3 days; (b) crushing the geranium seedlings, passing through a 60-mesh sieve to obtain geranium seedling powder, and then spraying the geranium seedling powder with a glycine aqueous solution and stirring evenly; The mass ratio of the geranium powder to the glycine aqueous solution is 1:0.2-0.3; the concentration of the glycine aqueous solution is 0.2 mol / L; (c) adding the geranium powder to the PDA liquid culture medium, stirring while adding, and stirring evenly until the geranium powder is neither loose nor clumped, and is in a state of being tightly held in a clumping shape but dispersed when released, to obtain a geranium culture medium; The solid-liquid ratio of the geranium seedling powder to the PDA liquid culture medium is 1:10; (d) sealing the geranium culture medium with a sealing film and sterilizing with high pressure steam; (e) Fermenting Geranium geraniol: taking Morchella and inoculating it to the bottom of the Geranium geraniol culture medium, sealing it with a sealing film, placing it in an incubator for fermentation, and drying it to constant weight to obtain a dried fermentation product; The accession amount of the Morchella is 10%; the fermentation culture is cultured in an incubator at 20° C. for 21 days; (f) extracting the dried fermented product with ethanol, combining the extracts, centrifuging, taking the supernatant and performing rotary evaporation, freeze-drying after rotary evaporation, and grinding to obtain a powdered Morchella solid fermentation Geranium geranium extract.

2. The preparation method according to claim 1, characterized in that: In step (d), the high pressure steam sterilization is performed at 115° C. for 30 min.

3. The preparation method according to claim 1 or 2, characterized in that: In step (f), the volume ratio of the fermentation product to ethanol is 1:10; the concentration of the ethanol is 70%, v / v; and the extraction is performed at room temperature for 24 hours.

4. Use of the Morchella solid-state fermentation Geranium oleifera extract prepared by the preparation method according to any one of claims 1 to 3 in the preparation of antibacterial drugs.

5. The use according to claim 4, characterized in that: When the concentration of the Morchella solid-state fermented Geranium erinaceus extract is 5-10 mg / ml, the growth of pathogenic bacteria is inhibited.

6. The use according to claim 5, characterized in that: The pathogenic bacteria are one or more of Staphylococcus aureus, Escherichia coli and Bacillus aerogenes.

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