A Penicillium camemberti fermentation extract and its application
The Penicillium camemberti YIM F02291 fermentation extract addresses the challenge of antibiotic-resistant E. coli by providing a cost-effective and simple method to inhibit these bacteria, achieving significant inhibition efficacy.
Patent Information
- Application Number
- CN202211417144.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-14
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2042-11-14
AI Technical Summary
Due to the widespread use of antibiotics, fecal alkali-producing bacteria has developed resistance to ampicillin, carramycin and streptomycin, resulting in the need to accelerate the development of new drugs to inhibit fecal alkali-producing bacteria. Microbial resources are of great significance in this process.
The preparation method of P.camemberti YIM F02291 fermented extract is adopted, including test tube seed culture, liquid seed culture and liquid fermentation culture. The fermented extract is obtained through shaker culture and ethanol treatment, and is used to inhibit the alkali-producing bacteria of multidrug-resistant feces.
The fermented extract of C. carmen pelvicia fermented extract has strong inhibitory activity on multidrug-resistant feces alkali-producing bacteria, with an average diameter of 41.0 mm, showing an effective antibacterial effect.
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Abstract
Description
Technical Field
[0001] The present invention relates to a fermentation extract of Penicillium camemberti YIM F02291 and its application, belonging to the technical field of microbial medicine. Background Art
[0002] Alcaligenes faecalis is a type of Gram-negative bacterium that is spherical, rod-shaped, or rod-spherical, and can parasitize on human skin, mucous membranes, and intestines, causing enteritis and urinary tract infections, etc. However, due to the widespread use of antibiotics, Alcaligenes faecalis has developed resistance to ampicillin, carramycin, and streptomycin, which has forced people to accelerate the research and development of new drugs to inhibit Alcaligenes faecalis. Microorganisms are important resources for new drug research and development. Numerous microbial-derived antibiotics such as penicillins, cephalosporins, and macrolides play important roles in the clinical treatment of infectious diseases. Screening and utilizing beneficial microorganisms that antagonize multidrug-resistant Alcaligenes faecalis (Alcaligenes faecalis that can tolerate ampicillin, carramycin, and streptomycin) is of great significance. Summary of the Invention
[0003] The purpose of the present invention is to provide a fermentation extract of Penicillium camemberti (P. camemberti) YIM F02291 and its application.
[0004] The present invention is realized as follows:
[0005] 1. The production strain of the fermentation extract of Penicillium camemberti in the present invention is Penicillium camemberti (P. camemberti) YIM F02291, which was deposited at the China Center for Type Culture Collection on October 17, 2022, at the address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20221582.
[0006] 2. The preparation method of the fermentation extract of Penicillium camemberti (P. camemberti) YIM F02291 in the present invention includes the following steps:
[0007] (1) Tube culture: Inoculate the strain YIM F02291 onto the inclined plane of a tube solid medium and culture it at 28 °C for 7 days to obtain a tube culture; the solid medium is a PDA medium, and the preparation method of the medium is to cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, filter, add 20 g of glucose and 20 g of agar to the obtained filtrate, make up the volume to 1000 ml with water, and sterilize at 121 °C for 40 minutes;
[0008] (2) Liquid seed culture: First, prepare the liquid seed medium. The liquid seed medium is PDB medium. The preparation method of PDB medium is as follows: Cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, and then filter. Add 20 g of glucose to the filtrate, and make up the volume to 1000 ml with water. Put 100 ml of the liquid seed medium into each 500-ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes. After cooling, inoculate with a 1-cm 2 large test tube culture, and culture on a shaker at 28 °C with a rotation speed of 190 rpm for 3 days to obtain the seed liquid;
[0009] (3) Preparation of Penicillium camemberti fermentation extract: First, prepare the liquid fermentation medium. The liquid fermentation medium is PDB medium, and the preparation method of PDB medium is the same as that in step (2); Put 200 ml of the liquid fermentation medium into each 1000-ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes. After cooling, inoculate with 20 ml of the seed liquid obtained in step (2), and culture on a shaker at 28 °C with a rotation speed of 190 rpm for 7 days to obtain the strain fermentation liquid; Add an equal volume of ethanol to the strain fermentation liquid, shake on a shaker for 6 hours, then filter off the mycelium, and evaporate the ethanol in the fermentation liquid to obtain the crude strain fermentation extract. Dilute the fermentation crude extract with water to the concentration of the original fermentation liquid, that is, prepare the Penicillium camemberti fermentation extract.
[0010] The Penicillium camemberti fermentation extract described in the present invention is applied to inhibit multi-drug resistant Alcaligenes faecalis.
[0011] The present invention provides a preparation method of a Penicillium camemberti fermentation extract, and applies the fermentation extract to inhibit multi-drug resistant Alcaligenes faecalis. The results of the plate bacteriostatic test show that the average diameter of the bacteriostatic circle of the Penicillium camemberti fermentation extract prepared by the method of the present invention against multi-drug resistant Alcaligenes faecalis is 41.0 mm, indicating that the fermentation extract has strong inhibitory activity against multi-drug resistant Alcaligenes faecalis. The Penicillium camemberti fermentation extract provided by the present invention can be effectively applied to inhibit multi-drug resistant Alcaligenes faecalis.
[0012] The present invention has the advantages of low raw material cost and simple production process, and has potential and practical application value in the medical field. Detailed implementation manners
[0013] The following further illustrates the present invention with specific embodiments.
[0014] Example 1. Identification of Penicillium camemberti (P.camemberti) YIM F02291
[0015] A fungus strain numbered YIM F02291 was screened. The DNA of the strain was extracted by the conventional CTAB method. The ITS sequence was amplified using the fungal universal primers ITS1 and ITS4. The PCR product was sent to Beijing Tsingke (Kunming) New Industry Biotechnology Co., Ltd. for sequencing. The obtained DNA sequence was searched and aligned in the NCBI database by BLAST. The similarity between the ITS sequence of the strain and the validly published species of Penicillium camemberti P. camemberti (CBS 299.48) was 100%. Therefore, this strain was identified as Penicillium camemberti (P. camemberti). Penicillium camemberti (P. camemberti) YIM F02291 was deposited in the China Center for Type Culture Collection on October 17, 2022. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20221582.
[0016] Example 2. Preparation of the fermentation extract of Penicillium camemberti inhibiting multi-drug resistant Alcaligenes faecalis
[0017] The preparation method of the fermentation extract of Penicillium camemberti inhibiting multi-drug resistant Alcaligenes faecalis includes the following steps:
[0018] (1) Tube culture: Inoculate the strain YIM F02291 onto the inclined plane of the tube solid medium and culture it at 28 °C for 7 days to obtain tube seeds; the solid medium is PDA medium. The preparation method of the medium is to cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, and then filter. Add 20 g of glucose and 20 g of agar to the obtained filtrate, make up the volume to 1000 ml with water, and sterilize at 121 °C for 40 minutes;
[0019] (2) Liquid seed culture: First, prepare the liquid seed medium. The liquid seed medium is PDB medium. The preparation method of PDB medium is: cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, and then filter. Add 20 g of glucose to the obtained filtrate, make up the volume to 1000 ml with water. Fill 100 ml of the liquid seed medium into each 500 ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes, and then inoculate a 1 cm 2 large tube seed after cooling, and culture it on a shaker at 28 °C and a rotation speed of 190 rpm for 3 days to obtain the seed liquid;
[0020] (3) Preparation of Penicillium camemberti fermentation extract: First, prepare the liquid fermentation medium. The liquid fermentation medium is PDB medium, and the preparation method of PDB medium is the same as that in step (2); put 200 ml of the liquid fermentation medium into each 1000 ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes, and after cooling, inoculate 20 ml of the seed liquid obtained in step (2), and culture on a shaker at 28 °C with a rotation speed of 190 rpm for 7 days to obtain the strain fermentation broth; add an equal volume of ethanol to the strain fermentation broth, shake on a shaker for 6 hours, then filter out the mycelium, and evaporate the ethanol in the fermentation broth to obtain the crude strain fermentation extract. Dilute the fermentation crude extract with water to the original fermentation broth concentration, and thus prepare the Penicillium camemberti fermentation extract.
[0021] Example 3. Plate antibacterial test
[0022] The plate antibacterial test includes the following steps:
[0023] (1) Preparation of the fermentation extract of Penicillium camemberti for inhibiting multidrug-resistant Alcaligenes faecalis in the test
[0024] The fermentation extract of the microorganism for inhibiting multidrug-resistant Alcaligenes faecalis of the present invention is prepared by a liquid fermentation method using the strain YIM F02291, and the specific preparation method is the same as that in Example 2.
[0025] (2) Culture of multidrug-resistant Alcaligenes faecalis
[0026] Inoculate multidrug-resistant Alcaligenes faecalis on a test tube slant medium and culture at 37 °C for 5 days until the bacterial cells grow well for standby. The test tube slant medium is a conventional LB medium, and its formula is: tryptone 10 g; yeast powder 5 g; sodium chloride 10 g; agar powder 15 g; water 1000 ml, sterilize at 121 °C for 40 minutes.
[0027] (3) Plate antibacterial test method
[0028] The plate antibacterial experiment adopts the conventional filter paper method:
[0029] Take a 0.5 cm 2 large test tube slant of the cultured multidrug-resistant Alcaligenes faecalis, mix it evenly with 20 ml of LB medium, pour it into a sterilized petri dish with an inner diameter of 9 cm to make a bacteria-containing plate. Place filter papers on each bacteria-containing plate, add 0.2 ml of the prepared Penicillium camemberti fermentation extract on the filter papers, use sterile water as a blank control, with 3 replicates for each treatment, place it in an incubator at 37 °C for 48 hours, and then measure the diameter of the antibacterial circle by the cross method. The results are shown in Table 1.
[0030] Table 1. Antibacterial results of Penicillium camemberti fermentation extract against multidrug-resistant Alcaligenes faecalis
[0031]
[0032] The experimental results show that the average diameter of the inhibition zone of the fermentation extract of Penicillium camemberti against multi-drug resistant Alcaligenes faecalis is 41.0 mm.
Claims
1. A Penicillium camemberti fermentation extract, characterized in that, The production strain of the Penicillium camemberti fermentation extract is Penicillium camemberti( Penicillium camemberti ) YIM F02291, which was deposited at the China Center for Type Culture Collection on October 17, 2022. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20221582; The preparation method of the fermentation extract of Penicillium camemberti comprises the following steps: (1) Tube culture: Inoculate the strain YIM F02291 onto the slant of a tube solid medium and culture it at 28 °C for 7 days to obtain tube culture; the solid medium is PDA medium, and the preparation method of the medium is to cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, filter, add 20 g of glucose and 20 g of agar to the obtained filtrate, make up the volume to 1000 ml with water, and sterilize at 121 °C for 40 minutes; (2) Liquid seed culture: First, prepare the liquid seed medium. The liquid seed medium is PDB medium. The preparation method of PDB medium is as follows: Cut 200 g of peeled potatoes into pieces, add 1000 ml of water, boil for 20 minutes, and then filter. Add 20 g of glucose to the obtained filtrate, and make up the volume to 1000 ml with water. Fill 100 ml of the liquid seed medium into each 500-ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes, and after cooling, inoculate with a test tube seed of 1 cm 2 in size, and culture on a shaker at 28 °C with a rotation speed of 190 rpm for 3 days to obtain the seed liquid; (3) Prepare the fermentation extract of Penicillium camemberti: First, prepare a liquid fermentation medium, and the liquid fermentation medium is PDB medium, and the preparation method of PDB medium is the same as that in step (2); put 200 ml of the liquid fermentation medium into each 1000 ml Erlenmeyer flask, sterilize at 121 °C for 40 minutes, after cooling, inoculate 20 ml of the seed liquid obtained in step (2), and culture it on a shaker at 28 °C and a rotation speed of 190 rpm for 7 days to obtain the strain fermentation broth; add an equal volume of ethanol to the strain fermentation broth, filter off the mycelium after shaking on a shaker for 6 hours, evaporate the ethanol in the fermentation broth to obtain the crude strain fermentation extract, and dilute the fermentation crude extract with water to the concentration of the original fermentation broth, thus preparing the fermentation extract of Penicillium camemberti.
Citation Information
Patent Citations
Penicillium camemberti and application thereof
CN101892163A
Novel strains of penicillium camemberti
US20220017854A1