A strain of Geotrichum candidum and its application

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a strain of Geotrichum candidum and its application. A strain of Geotrichum candidum was preserved in the China Center for Type Culture Collection on October 20, 2022, with a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a preservation number of CCTCC M 20221612. The present invention provides a new strain of Geotrichum candidum, whose fermentation product has high-efficiency free radical scavenging ability and anti-inflammatory ability, and has huge application potential in the fields of medicine, daily chemical industry, etc.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Geotrichum candidum strain and application thereof. Background Art

[0002] Skin aging is a process of degeneration of skin cell function and structure caused by a variety of complex factors such as aging, disease, and injury. Free radical accumulation and damage caused by various factors and skin diseases including inflammatory reactions are the most important causes of skin aging and deterioration.

[0003] Free radicals refer to active molecules or groups with unpaired electrons in the outermost orbital. The most common ones are oxygen free radicals, including superoxide anions, hydrogen peroxide, hydroxyl free radicals, carboxyl free radicals, etc. Due to their strong activity, they have a special affinity for unsaturated covalent bonds, and have a strong destructive effect on lipids, proteins, nucleic acids, etc. containing unsaturated bonds, and even directly cause cell apoptosis and necrosis, which seriously threatens skin health.

[0004] Inflammation is a response of the body to external stimuli, centered on blood vessels and involving multiple tissues, organs and their microenvironments. During the skin inflammatory response, damaged or dead cells release endogenous inflammatory molecules, which are recognized by immune cells and induce inflammatory responses in the body. With age, the ability of cells to produce damaging factors gradually increases, while the ability to clear and degrade them gradually decreases, thereby aggravating or accelerating the deterioration of the skin.

[0005] Geotrichum candidum is a fungus of the subphylum Ascomycota, class Hyphomycetes, orders Hyphomycetales, family Hyphomycetaceae, and genus Geotrichum candidum. Its morphology is between yeast and filamentous fungi. Geotrichum candidum is widely distributed, has strong adaptability, and grows rapidly. It is currently mainly used in the fields of microbial feed protein production, winemaking, and organic waste treatment. At present, there are no reports on the fermentation products of Geotrichum candidum with free radical scavenging and anti-inflammatory abilities. Summary of the invention

[0006] The invention aims to provide a Geotrichum candidum strain and application thereof.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Geotrichum candidum was preserved in the China Type Culture Collection on October 20, 2022, with the preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is: CCTCC M 20221612.

[0008] Accordingly, the use of Geotrichum candidum in anti-aging for non-diagnostic or therapeutic purposes.

[0009] Accordingly, the Geotrichum candidum is used for scavenging free radicals and / or anti-inflammation for non-diagnostic or therapeutic purposes.

[0010] Preferably, the anti-inflammatory application is the inhibition of hyaluronidase.

[0011] Preferably, the application uses a fermentation product of Geotrichum candidum.

[0012] Correspondingly, the application of Geotrichum candidum in the preparation of anti-aging products.

[0013] Accordingly, the Geotrichum candidum is used in the preparation of free radical scavenging and / or anti-inflammatory products.

[0014] Preferably, the anti-inflammatory application is the inhibition of hyaluronidase.

[0015] Preferably, the product is any one of medicine and daily chemical product.

[0016] Preferably, the application uses a fermentation product of Geotrichum candidum.

[0017] The present invention has the following beneficial effects: the present invention provides a new strain of Geotrichum candidum, the fermentation product of which has high-efficiency free radical scavenging ability and anti-inflammatory ability, and has great application potential in the fields of medicine, daily chemical industry, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the colony morphology of Geotrichum candidum strain;

[0019] Figure 2 Schematic diagram of a microbial microscope. DETAILED DESCRIPTION

[0020] The present invention provides a new strain of Geotrichum candidum, whose ITS sequence is shown in SEQ ID NO: 1. The strain is deposited in China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, zip code 430072), with a deposit date of October 20, 2022 and a deposit number of CCTCC M 20221612.

[0021] The fermented product of Geotrichum candidum has the ability of efficiently scavenging free radicals and anti-inflammatory function.

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0023] Example 1: Screening and identification of Geotrichum candidum

[0024] 1. Strain improvement. Using the wild-type Geotrichum candidum isolated from Xinjiang soil as the starting strain, after multiple rounds of UV / lithium chloride composite mutagenesis, the fermentation product's ability to scavenge DPPH free radicals and inhibit hyaluronidase activity was used as an indicator to select and obtain an improved strain Geotrichum candidum 2A9 with both high free radical scavenging activity and anti-inflammatory activity.

[0025] 2. Identification. The colony morphology of Geotrichum candidum strains is as follows: Figure 1 As shown, Figure 1 The left side is a schematic diagram of the early growth of Geotrichum candidum, where you can see the morphology of a single colony, and the right side is the overall morphology of Geotrichum candidum after it grows into a moss. Figure 2 Its ITS sequence is shown in SEQ ID NO: 1. Through ITS sequence detection, the strain has a 100% similarity with the ITS sequences of Geotrichum candidum bacteria in NCBI databases such as KF112070.1, LC054542.1, and MW092558.1, and is identified as Geotrichum candidum.

[0026] Example 2: Demonstration of free radical scavenging and anti-inflammatory properties of Geotrichum candidum

[0027] This embodiment relates to a culture medium comprising:

[0028] Potato dextrose agar (PDA): 200 g / L potato, 20 g / L glucose, 20 g / L agar, natural pH.

[0029] PDA corresponding seed culture medium: PDA liquid culture medium (without agar).

[0030] PDA corresponding fermentation medium: 50g / L saccharified rice powder, 5g / L enzymatic hydrolyzed soybean powder, 10g / L sea buckthorn juice, 1g / L magnesium sulfate heptahydrate, pH=6.5.

[0031] Potato sucrose agar (PSA): 200 g / L potato, 20 g / L sucrose, 20 g / L agar, natural pH.

[0032] PSA corresponds to seed culture medium: 30 g / L sucrose, 10 g / L soluble starch, 10 g / L soy peptone, 1 g / L calcium sulfate dihydrate, pH = 5.0.

[0033] The fermentation medium corresponding to PSA is: 30 g / L sucrose, 20 g / L soluble starch, 5 g / L soy peptone, 1 g / L calcium sulfate, 10 g / L aloe vera juice, pH = 5.0.

[0034] Malt extract agar medium (MEA): 20g / L malt extract powder, 20g / L glucose, 1g / L peptone, 15g / L agar, natural pH.

[0035] MEA corresponding seed culture medium: 50g / L saccharified soluble starch, 10g / L enzymatically hydrolyzed soybean powder, 10g / L tryptone, 1g / L magnesium sulfate, pH=6.8.

[0036] MEA corresponding fermentation medium: 40g / L saccharified soluble starch, 5g / L enzymatically hydrolyzed soybean powder, 5g / L tryptone, 20g / L apple juice, 1g / L magnesium sulfate, pH = 6.8.

[0037] Oatmeal agar medium (OA): 40 g / L heated and melted instant oatmeal powder, 5 g / L agar, pH = 5.8.

[0038] OA corresponding seed culture medium: 10 g / L saccharified oatmeal powder, 50 g / L acid-hydrolyzed soybean powder, 2 g / L calcium sulfate, pH = 8.0.

[0039] OA corresponding fermentation medium: 10g / L saccharified oatmeal powder, 100g / L soy protein isolate, 10g / L wild grape juice, 1g / L calcium sulfate, pH=8.0.

[0040] Czapek yeast extract agar (CYA): 3 g / L sodium nitrate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, 5 g / L yeast extract, 30 g / L sucrose, 15 g / L agar, natural pH.

[0041] CYA corresponding seed culture medium: 20g / L saccharified corn flour, 10g / L soy peptone, 1g / L magnesium sulfate heptahydrate, 1g / L potassium dihydrogen phosphate, pH=6.8.

[0042] The corresponding fermentation medium of CYA: 80g / L saccharified corn flour, 15g / L soy peptone, 10g / L sea buckthorn juice, 1g / L magnesium sulfate heptahydrate, 1g / L potassium dihydrogen phosphate, pH=6.8.

[0043] The culture medium provided in this embodiment is exemplary. In practice, the composition of the fermentation medium preferably includes: a carbohydrate content of 2% to 10% (w / v), an amino acid and protein content of 0.1% to 2% (w / v), and a pH range of 5.0 to 8.0. The inoculation amount (v / v) of Geotrichum candidum is preferably 2% to 50%; the culture temperature is preferably 20 to 30° C.; the shaking speed is preferably 50 to 200 rpm; and the fermentation time is preferably 24 to 96 hours.

[0044] 1. Set up 5 groups, each group uses different methods to preserve the candid mold, then activates and cultures the preserved candid mold using different activation culture media, then inoculates the activated strain into the corresponding seed culture medium to culture until a seed liquid is obtained, then inoculates the seed liquid into the corresponding fermentation medium for fermentation, and collects the fermentation product supernatant / filtrate in different ways. The specific conditions of each group are shown in Table 1.

[0045] Table 1 Comparison table of setting conditions for each group

[0046]

[0047]

[0048] 2. The DPPH free radical scavenging method and the Elson-Morgan hyaluronidase inhibition method were used to detect the free radical scavenging activity and anti-inflammatory activity of the supernatant / filtrate of each group. The DPPH in vitro scavenging method test method is as follows:

[0049] ① Take 200 μL of each supernatant / filtrate obtained in Table 1, put them into 1.5 mL centrifuge tubes, add 800 μL of anhydrous ethanol, mix well and let stand at 4°C for 1 hour;

[0050] ② After standing and settling, centrifuge at 12000g for 5 min, aspirate 800 μL of supernatant, put it into a 5 mL centrifuge tube, add 2.4 mL of DPPH ethanol solution (DPPH concentration is 51.3 mg / L), and stand at 25°C in the dark for 30 min to develop color;

[0051] ③ Prepare ascorbic acid standard solutions of different concentrations and replace the supernatant in ② for color development reaction;

[0052] ④ Measure the absorbance of each sample colorimetric solution at 518 nm on a spectrophotometer and use the ascorbic acid solution concentration and OD 518nm Standard curves were drawn for the horizontal and vertical axes, respectively, and the DPPH radical scavenging potency of each fermentation sample was calculated based on ascorbic acid.

[0053] The Elson-Morgan hyaluronidase inhibition assay is as follows:

[0054] ① Reagent preparation:

[0055] Acetic acid solution: Measure 11.55 mL of glacial acetic acid and dilute to 1 L with water.

[0056] Sodium acetate solution: Weigh 16.4 g of anhydrous sodium acetate, dissolve it in water and make up to 1 L.

[0057] Sodium carbonate solution: Weigh 105.99 g of anhydrous sodium carbonate, dissolve it in water and make up to 1 L.

[0058] Calcium chloride solution: Weigh 277.45 g of calcium chloride, dissolve it in water and make up to 1 L.

[0059] Sodium hydroxide solution: Weigh 200g of sodium hydroxide, dissolve it in water and make up to 1L.

[0060] Acetate buffer (0.1 M, pH = 5.6): Measure 452 mL of acetic acid solution and 48 mL of sodium acetate solution, and make up to 1 L with water.

[0061] Sodium hyaluronate solution: Weigh 0.5 g of sodium hyaluronate, dissolve it in acetate buffer (0.1 M, pH 5.6) and make up to 1 L.

[0062] Hyaluronidase solution: Weigh 5000U hyaluronidase (extracted from bovine testicles) and dissolve it in 10mL acetate buffer (0.1M, pH=5.6), i.e. 500U / mL enzyme solution, which is prepared and used immediately.

[0063] Acetylacetone solution: Measure 3.5 mL of acetylacetone and dissolve it in 50 mL of sodium carbonate solution. Prepare and use immediately.

[0064] p-Diaminobenzaldehyde colorimetric agent (P-DAB colorimetric agent): Weigh 0.8 g of p-diaminobenzaldehyde, dissolve it in 15 mL of concentrated hydrochloric acid (38%, w / v), add 15 mL of anhydrous ethanol, and mix well.

[0065] ②Hyaluronidase activity inhibition reaction:

[0066] Take 4 10mL colorimetric tubes, numbered A, B, C, and D, and add 0.5mL of the sample solution to be tested (the supernatant / filtrate in Table 1) to A and B respectively; add 0.5mL of pure water to C and D respectively; add 0.5mL of hyaluronidase solution to A and C respectively; add 0.5mL of acetate buffer (0.1M, pH=5.6) to B and D respectively, mix well, and incubate in a 37℃ water bath for 20min. Add 0.1mL of calcium chloride solution to each colorimetric tube, mix well, and incubate in a 37℃ water bath for 20min.

[0067] ③Hyaluronic acid hydrolysis reaction:

[0068] Add 0.5 mL of sodium hyaluronate solution to each of the above colorimetric tubes A and C, and add 0.5 mL of acetate buffer (0.1 M, pH = 5.6) to each of B and D. After mixing evenly, incubate in a 37°C water bath for 40 min, and then place at room temperature for 10 min.

[0069] ④P-DAB color reaction:

[0070] Add 0.5 mL of pure water, 0.1 mL of sodium hydroxide solution, and 0.5 mL of acetylacetone solution to each colorimetric tube, mix well, heat in a boiling water bath for 15 min, cool in an ice bath for 10 min, and place at room temperature for 10 min. Add 1 mL of P-DAB color developer, mix well, add anhydrous ethanol to a total volume of 8 mL, mix again, and place at room temperature for 30 min to develop color. Measure the absorbance of the solution in each colorimetric tube at 530 nm, and count them as A, B, C, and D respectively. The hyaluronidase inhibition rate of the corresponding sample (Group A) is calculated according to the following formula:

[0071] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) × 100%.

[0072] The test results of each group are shown in Table 2. The supernatant / filtrate used in each group in Table 2 corresponds to the number in Table 1.

[0073] Table 2 Comparison of results of each group

[0074] Group DPPH free radical scavenging activity Inhibition rate of hyaluronidase Group 1 164mg Vc / L 52％ Group 2 135mg Vc / L 61％ Group 3 162mg Vc / L 58％ Group 4 148mg Vc / L 66％ Group 5 166mg Vc / L 70％

[0075] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Geotrichum candidum ( Geotrichum candidum ) strain 2A9, characterized in that: The deposit number of the Geotrichum candidum strain 2A9 is: CCTCC M 20221612.

2. Use of the Geotrichum candidum strain 2A9 according to claim 1 in scavenging DPPH free radicals for non-diagnostic or therapeutic purposes.

3. Use of the Geotrichum candidum strain 2A9 according to claim 1 in inhibiting hyaluronidase for non-diagnostic or therapeutic purposes.

Images