CN113307848A - Cyclo-serine-valine-isoleucyl-leucyl peptide with antifungal and free radical scavenging activities and preparation method thereof - Google Patents
Cyclo-serine-valine-isoleucyl-leucyl peptide with antifungal and free radical scavenging activities and preparation method thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to a cyclo-chromo-serine-valine-isoleucyl-leucyl cyclic peptide (Trp-Ser-Val-Ile-Leu cyclic peptide) with antifungal and free radical scavenging activities and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cyclo-tryptophan-serine-valine-isoleucin is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine, isoleucine and leucine through an amide bond and is white powder. This is a novel compound and has not been reported in the literature. The compound of the invention can be obtained by culturing ranunculus japonicus and then extracting and separating. The pure cyclochrome-silk-valine-isoleucin is white powder, and has half of scavenging concentration (DC) of DPPH free radical50) Comprises the following steps: 1.19mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 13.27 mm. The compound has the potential of preparing antibacterial drugs and antioxidant and anti-aging health care products.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and relates to extraction and purification of a chromo-serine-valine-isoleucinerin (Trp-Ser-Val-Ile-Leu cyclopeptide) with antifungal and free radical scavenging activities.
Background
The cyclo-chromo-serine-valine-isoleucine-leucine peptide is prepared from a species of Botrytis cinerea (Basidiobolussp.) isolating the biologically active substance from the culture with antifungal and free radical scavenging activity.
Botrytis cinerea (A. frog-dung)Basidiobolussp.) is a fungus of the genus Ranunculaceae, the family Ranunculaceae, the order entomomycetales; common in China are frog-borne frog dung mold, rana chensinensis dung mold, rana fraspo dung mold, and the like. The above strains can be separated from soil and amphibian animal feces.
Antifungal activity: many fungi are opportunistic pathogens that are susceptible to infection when people's resistance is reduced, especially in the elderly and patients. At present, the aging of the world is getting more and more serious, meanwhile, due to factors such as environmental pollution, the resistance of people is in a descending trend, the fungal infection cases are in an ascending trend, and meanwhile, the early antifungal drugs have a drug resistance phenomenon, so that the development of novel antifungal active substances is very significant.
Radical scavenging activity: free radicals (free radial), when viewed chemically, refer to groups, atoms or molecules containing unpaired electrons. Free radicals are highly chemically active. For living organisms, free radicals are intermediates of various biochemical reactions in vital activities. Under normal conditions, free radicals in the human body are in a dynamic equilibrium of generation and elimination. Free radicals are an effective defense system of the body, and if the free radicals cannot maintain a certain level, the life activities of the body are adversely affected. However, the free radicals are produced too much or removed too slowly, and by attacking vital macromolecular substances and various organelles, the free radicals can cause various damages at the molecular level, the cellular level and the tissue and organ level of the organism, accelerate the aging process of the organism and induce various diseases. Recent studies have shown that aging and many diseases in humans are associated with free radical damage. The material with the free radical scavenging activity can react with free radicals and reduce the free radicals into non-free radical compounds, can scavenge excessive free radicals generated in the metabolic process of an organism, and is an important active material capable of improving the health of a human body. The free radical scavenger has an anti-oxidation effect in a non-living system, can effectively prevent oxidation and deterioration of substances, has an important effect on prolonging the shelf life of articles, and is widely used in foods, medicines, daily chemicals and the like.
Disclosure of Invention
The invention aims to provide a cyclochrome-silk-valine-isoleucyl-leucine peptide with antifungal and free radical scavenging activities and a preparation method thereof.
In order to find a novel natural and efficient substance with antifungal activity and free radical scavenging activity, the inventor discovers that the cyclo-chromo-serine-valine-isoleucin extracted from a ranunculus japonicus has strong activity of resisting streptomyces albus and free radical scavenging activity by performing activity research on more than 100 entomopathogenic fungi metabolites in China.
The structural formula of the cyclo-chromo-serine-valine-isoleucine-leucine peptide with antifungal and free radical scavenging activities is as follows:
the cyclo-tryptophan-serine-valine-isoleucin-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine, isoleucine and leucine through amide bonds and is white powder;
the cyclo-chromo-serine-valine-isoleucyl leucine peptide has the activity of scavenging free radicals and resisting streptomyces albus;
half-scavenging concentration (DC) of the free radical of Diphenylpicrylphenylhydrazine (DPPH)50) Comprises the following steps: 1.19mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 13.27 mm.
The preparation of the cyclochrome-silk-valine-isoleucyl peptide with antifungal and free radical scavenging activity comprises the following steps:
(1) strain culture
Mixing Botrytis cinerea (A.ranunculata:)Basidiobolussp.) performing slant culture, secondary liquid seed culture, tertiary liquid seed culture and quaternary culture to obtain final liquid fermentation product or final solid fermentation product;
(2) extraction and refining of effective components in the final fermentation
Extracting the effective components in the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclotomic-silk-valine-isoleucin.
The preparation technical scheme is further defined as follows:
said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of froglet frogs, froglet mould with solid spore or froglet mould with split spore.
The specific operation of the step (1) is as follows:
(1.1) slant seed culture
Inoculating the ranunculus japonicus strains to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain a first-level strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
adding a liquid culture medium with the volume of 30-50% of that of a triangular flask into a triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 slant strains into each triangular flask, and culturing for 3-7 days in a full-temperature oscillation incubator at the temperature of 25-36 ℃ and the rotating speed of 150-250 rpm to obtain a secondary strain;
the liquid shake flask culture medium comprises 25.0-50.0 g/L glucose, 10.0-30.0 g/L malt extract, 2.0-6.0 g/L peptone, 1.0-4.0 g/L yeast extract powder, and potassium chloride (KCl)2) 0.3-2.0 g/L magnesium sulfate heptahydrate (MgSO)4.7H20.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH)2PO4）0.3～2.0 g/L；
(1.3) three-stage Strain culture
Inoculating the second-level strain into a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the second-level strain is subjected to constant-temperature aeration culture for 2-5 days at the temperature of 25-36 ℃ under the condition that the aeration amount is 1: 1-2 (v/v) in volume ratio and the pressure is 0.1-0.2 MP, so as to obtain a third-level strain;
the culture medium in the fermenter is synchronized with the liquid culture medium in step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture, the third-stage strain is inoculated to a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the final fermentation product of the liquid is obtained by constant-temperature aeration culture for 5-15 days under the conditions that the aeration amount is 1: 1-2 (v/v) in volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; culture in fermenter liquid culture in step (1.2).
In the step (1.4), the fourth-stage culture is solid culture, the third-stage liquid strain is mixed into a solid culture medium, the inoculation amount is 3-10% of the solid material amount, and the solid final fermentation product is obtained after the constant-temperature culture at 25-36 ℃ for 5-15 days; the solid culture medium comprises 25.0-50.0 g/L of glucose, 10.0-30.0 g/L of malt extract, 2.0-6.0 g/L of peptone, 1.0-4.0 g/L of yeast extract powder, and potassium chloride (KCl)2) 0.3-2.0 g/L magnesium sulfate heptahydrate (MgSO)4.7H20.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH)2PO4) 0.3-2.0 g/L, 15.0-30.0 g/L agar and water.
The specific operation of the step (2) is as follows:
(2.1) pretreatment of the final fermentation
Filtering the final fermentation product of the liquid through a filter membrane of 0.20-1.0 μm or centrifuging at 12000 rpm under 2000-; freeze-drying and crushing the mycelium to obtain a pretreatment substance which is sieved by a 20-120-mesh sieve;
or drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing the solid final fermentation product into a pretreatment product of 20-120 meshes;
(2.2) extraction, separation and purification of effective components in the pretreated product
(2.2.1) extraction
Extracting the pretreated substance with methanol or ethanol; the material-liquid ratio is 1: 2-20 (W: V), the ultrasonic power is 10-100 KHz, and the extraction time is 10-200 minutes; centrifuging at a rotating speed of 2000 rpm or more, or filtering with a 0.20-1.0 μm filter membrane; taking the filtrate or centrifugal supernatant, concentrating at the temperature of less than or equal to 100 ℃ to 1/2-1/10 of the original volume to obtain concentrated dealcoholized liquid, simultaneously recovering methanol or ethanol, and using the concentrated dealcoholized liquid for further refining by chromatography;
(2.2.2) separation and purification
Purifying by adopting a reverse phase chromatography method, wherein the stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), and the particle diameter is 3-50 microns; methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and chromatographic fractions with the molecular weight of 676 on a mass spectrum detector are collected; concentrating the collected substance to viscous state at a temperature of less than or equal to 100 ℃, and drying at a temperature of less than 170 ℃; the round-silk-valine-isoleucin is obtained as a white powder.
The analytical study of the present invention is illustrated below:
1. screening research discovers a frog dung mouldBasidiobolussp.) hyphal methanol extract has strong free radical scavenging activity and anti-streptococcal activity:
(1) determination of inhibitory Activity of methanol extract on Streptococcum alborum: preparing 20% DMSO solution with the concentration of the extract being 1.0 mg/ml, carrying out a white streptococcal inhibition test by using amphotericin B as a positive control and using the 20% DMSO solution as a blank control, and calculating to obtain the average inhibition zone diameter of the extract to the white streptococcal bacteria of 11.56 mm;
(2) determination of radical scavenging activity of methanol extract: the concentrations of the prepared extract samples are 0.2, 0.4, 0.8, 1.0 and 1.6 mg/mL methanol solution, the scavenging activity of the extract samples on DPPH free radicals of 0.5 mmol/L is measured at 517 nm, and the half scavenging concentration of the DPPH free radicals is calculated to be 1.52 mg/mL.
Since the methanol extract has a low content of active ingredients, further extraction and purification of the active ingredients are required.
2. Further separating and purifying the methanol extract by reverse phase chromatography to obtain a pure product with the following biological activity:
(1) determination of inhibitory Activity of Cyclochro-silk-Val-Ilexht-Bright peptide on Streptococcum albopictus: preparing 20% DMSO solution with the concentration of the extract being 0.2 mg/ml, carrying out a white streptococcal inhibition test by using amphotericin B as a positive control and using the 20% DMSO solution as a blank control, and calculating to obtain the average inhibition zone diameter of the extract to the white streptococcal bacteria of 13.27 mm;
(2) determination of the radical scavenging activity of cyclo-chromo-ser-val-iso-leucin: the concentrations of the prepared extract samples are 0.2, 0.4, 0.8, 1.0 and 1.6 mg/mL methanol solution, the scavenging activity of the extract samples on DPPH free radicals of 0.5 mmol/L is measured at 517 nm, and the half scavenging concentration of the DPPH free radicals is calculated to be 1.19 mg/mL.
3. Chemical Structure characterization of active Compounds
High resolution LC-MS analysis showed that the purified active compound was the positive ion 585.3440(M + H)+)、607.3261(M+Na+)，623.3000(M+K+) Calculated molecular formula C30H44N6O6。
Nmr data are shown in the following table:
the structure of the separated and purified active compound is the cyclochrolo-silk-valine-isoleucin-leucin which is obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis, and the active compound is shown as the chemical structural formula.
The beneficial technical effects of the invention are embodied in the following aspects:
1. the cyclo-chromo-serine-valine-isoleucin prepared by the method has the activities of inhibiting streptomyces albus and removing free radicals, and develops a new application field of the frogma faecalis. The invention discovers that the ranunculus japonicus has the function of metabolizing the active substances for the first time. On the basis of the invention, related genes can be expected to be further cloned to construct high-yield strains.
2. The cyclo-chromo-serine-valine-isoleucine-leucine peptide is a novel framework, and can be used as a drug lead compound to perform derivatization modification so as to create a compound with more and stronger activity.
3. The cyclo-chromo-serine-valine-isoleucyl peptide can be used for preparing a streptococcal albus inhibitor and a free radical scavenger. The free radical scavenger has whitening, antioxidant, antiaging, fresh keeping, antibacterial, and antiinflammatory effects, and can be used for preventing and treating fungal infection.
4. The invention adopts the microbial fermentation production, is not influenced by environment and resources, is easy to realize industrialization and automation, and is not influenced by environment and natural resources.
5. The product produced by the process method has low cost, simple and convenient process, stable process, easy regulation and control and high success rate.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1:
the preparation of the cyclochrome-silk-valine-isoleucyl peptide with antifungal and free radical scavenging activity comprises the following steps:
the A.rana used in example 1 was A.rana chensinensis.
(1) Culturing of bacterial strains
(1.1) slant culture of strains
Inoculating Rana chensinensis David dung mold strain to potato agar (PDA) slant culture medium, and culturing at 25 deg.C for 4 days to obtain first-stage strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: glucose 25.0g/L, malt extract 10.0g/L, peptone 2.0 g/L, yeast extract powder 1.0g/L, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, potassium dihydrogen phosphate (KH)2PO4）0.3g/L；
Adding a liquid culture medium with the volume of 30% of that of a triangular flask into a 100 ml triangular flask, inoculating 1 slant strain into each triangular flask, placing the inoculated triangular flasks in a full-temperature oscillation incubator at the temperature of 25 ℃ and the rotating speed of 150 rpm, and culturing for 3d to obtain a secondary strain;
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 5L fermentation tank, wherein the liquid loading amount is 50% of the volume of the tank, the inoculation amount is 3%, and the ventilation amount is 1:1 (v/v) in volume ratio, the pressure is 0.1MP, and the constant temperature ventilation culture is carried out for 2 days at 25 ℃ to obtain a third-level strain;
the culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
(1.4) four-stage culture
The four-stage culture adopts liquid culture: inoculating the third-level strain into a 50L fermentation tank, wherein the liquid loading amount is 50% of the tank volume, the inoculation amount is 3%, and the aeration amount is 1:1 (v/v) in volume ratio, the pressure is 0.1MP, and the final fermentation product of the liquid is obtained after the constant-temperature aeration culture at 25 ℃ for 5 days; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium;
(2) extraction and refining of effective components in the final fermentation
(2.1) pretreatment of the final fermentation
Filtering the final fermented product with 0.20 μm filter membrane to obtain mycelium; the mycelium was freeze-dried and ground to 20 mesh for use.
(2.2) extraction
Extracting the dried mycelium with methanol; the material-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; centrifuging at 2000 rpm after extraction, concentrating the supernatant at 100 deg.C to 1/2 to obtain concentrated dealcoholized liquid, recovering methanol, and purifying with chromatography.
(2.3) separation and purification
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 3 microns, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; ethanol or methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and a target peak with the molecular weight of 676 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 100 ℃, and drying at the temperature of 170 ℃; a white powder of cyclo-chromatic-silk-valine-isoleucin was obtained.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-isoleucin is as follows:
the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-isoleucin p-diphenylpicrylphenylhydrazine free radical (DPPH)50) Comprises the following steps: 1.19 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.27 mm.
Example 2
The preparation of the cyclochrome-silk-valine-isoleucyl peptide with antifungal and free radical scavenging activity comprises the following steps:
the rana faecalis used in example 2 was rana nitidissima.
(1) Culturing of bacterial strains
(1.1) slant culture of strains
Inoculating the Rana chensinensis David dung mold strain to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 36 deg.C for 8d to obtain a first-class strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: glucose 50.0 g/L, malt extract 30.0 g/L, peptone 6.0 g/L, yeast extract powder 4.0 g/L, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, potassium dihydrogen phosphate (KH)2PO4）2.0 g/L。
Adding liquid culture medium 50% of the volume of the triangular flask into 1000 ml of triangular flasks, inoculating 3 slant strains into each triangular flask, placing in a full-temperature oscillation incubator after inoculation, culturing at 36 ℃ and 250 rpm for 7d to obtain secondary strains.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 80% of the volume of the tank, the inoculation amount is 10%, and the ventilation amount is 1:2 (v/v) in volume ratio, the pressure is 0.2MP, and the constant-temperature ventilation culture is carried out for 5 days at 36 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
(1.4) four-stage culture
The fourth-stage culture is liquid culture: inoculating the third-level strain into a fermentation tank with volume of 500L or more, wherein the liquid loading amount is 80% of the tank volume, the inoculation amount is 10%, and the aeration amount is 1:2 (v/v) in volume ratio, the pressure is 0.2MP, and the constant temperature aeration culture is carried out for 15 days at 36 ℃ to obtain the final fermentation product of liquid; the culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
(2) Extraction and refining of effective components in the final fermentation
(2.1) pretreatment of the final fermentation
Filtering the final fermented product with 1.0 μm filter membrane to obtain mycelium; the mycelium was freeze-dried and ground to 120 mesh for use.
(2.2) extraction
Extracting the dried mycelium with ethanol at a ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; centrifuging at 12000 r/min, concentrating the supernatant at 40 deg.C to 1/10 to obtain concentrated dealcoholized liquid, recovering ethanol, and refining with chromatography.
(2.3) separation and purification
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 50 microns, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and a target peak with the molecular weight of 676 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 30 ℃, and drying at the temperature of-30 ℃; the round-silk-valine-isoleucin is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-isoleucin is as follows:
the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-isoleucin p-diphenylpicrylphenylhydrazine free radical (DPPH)50) Comprises the following steps: 1.19 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.27 mm.
Example 3
The preparation of the cyclochrome-silk-valine-isoleucyl peptide with antifungal and free radical scavenging activity comprises the following steps:
the strain selected in this example 3 is Botrytis cinerea.
(1) Culturing of bacterial strains
(1.1) slant culture of strains
Inoculating the ranunculus mucronatus strain to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 30 ℃ for 6 days to obtain a first-level strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 35.0 g/L glucose, 20.0 g/L malt extract, 4.0 g/L peptone, 2.0 g/L yeast extract powder, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, potassium dihydrogen phosphate (KH)2PO4）1.0 g/L。
Adding liquid culture medium 40% of the volume of the triangular flask into 500 ml of triangular flasks, inoculating 2 slant strains into each triangular flask, placing in a full-temperature oscillation incubator after inoculation, culturing at 30 ℃ and 200 rpm for 5d to obtain secondary strains.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 60% of the volume of the tank, the inoculation amount is 6%, and the ventilation amount is 1:1.5 (v/v) in volume ratio, the pressure is 0.15MP, and the constant-temperature ventilation culture is carried out for 3 days at 30 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
(1.4) four-stage culture
The fourth-stage culture is solid culture: mixing the third-stage liquid strain with solid culture medium, inoculating 3% of the solid material, and performing constant temperature aeration culture at 25 deg.C for 10 days to obtain solid final fermented product; the solid culture medium is as follows: glucose 25.0g/L, malt extract 10.0g/L, peptone 2.0 g/L, yeast extract powder 1.0g/L, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, potassium dihydrogen phosphate (KH)2PO4) 0.3 g/L; agar 15.0 g/L.
(2) Extraction and refining of effective components in the final fermentation
(2.1) pretreatment of the final fermentation
The solid final fermentation was dried at 30 ℃ to a moisture content of 20% and then ground to 20 mesh for use.
(2.2) extraction
Extracting the dried and pulverized final fermented product with ethanol; the material-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; filtering with 0.20 μm filter membrane after extraction; concentrating the filtrate at 30 deg.C to 1/2 of original volume to obtain concentrated dealcoholized liquid, recovering ethanol, and refining with chromatography;
(2.3) separation and purification
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 25 micrometers, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and a target peak with the molecular weight of 676 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 40 ℃, and drying at the temperature of 10 ℃; the round-silk-valine-isoleucin is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-isoleucin is as follows:
the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-isoleucin p-diphenylpicrylphenylhydrazine free radical (DPPH)50) Comprises the following steps: 1.19 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.27 mm.
Example 4:
the preparation of the cyclochrome-silk-valine-isoleucyl peptide with antifungal and free radical scavenging activity comprises the following steps:
the mold for Rana chensinensis used in this example 4 produces mold for Rana chensinensis.
(1) Culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
(1.1) slant culture of strains
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 29 deg.C for 5d to obtain first-stage strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: glucose 40.0 g/L, malt extract 25.0g/L, peptone 3.0 g/L, yeast extract powder 3.0 g/L, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, potassium dihydrogen phosphate (KH)2PO4）1.2 g/L。
Adding liquid culture medium 40% of the volume of the triangular flask into 500 ml of triangular flasks, inoculating 1 slant strain into each triangular flask, placing in a full-temperature oscillation incubator after inoculation, culturing at 30 ℃ and 180 rpm for 4d to obtain the secondary strain.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 30L fermentation tank, wherein the liquid loading amount is 65% of the volume of the tank, the inoculation amount is 6%, and the ventilation amount is 1:1.2 (v/v) in volume ratio, the pressure is 0.12MP, and the constant temperature ventilation culture is carried out for 3 days at 32 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
(1.4) four-stage culture
The fourth-stage culture adopts solid culture: mixing the third-stage liquid strain with solid culture medium, inoculating 10% of the solid material, and performing constant temperature aeration culture at 36 deg.C for 8 days to obtain solid final fermented product;
the solid culture medium is: glucose 50.0 g/L, malt extract 30.0 g/L, peptone 6.0 g/L, yeast extract powder 4.0 g/L, potassium chloride (KCl)2) 0.3g/L, magnesium sulfate heptahydrate (MgSO)4.7H2O) 0.3g/L, phosphoric acid bisPotassium hydrogen (KH)2PO4) 2.0 g/L agar and 30.0 g/L agar.
(2) Extraction and refining of effective components in the final fermentation
(2.1) pretreatment of the final fermentation
The solid final fermentation product was dried at 130 ℃ to a water content of 10% and then pulverized to 120 mesh for use.
(2.2) extraction
Extracting the dried and pulverized final fermented product with methanol at a ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; filtering with 1.0 μm filter membrane after extraction; concentrating the filtrate at 40 deg.C to 1/10 volume to obtain concentrated dealcoholized liquid, recovering methanol, and further refining with chromatography;
(2.3) separation and purification
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 10 microns, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and a target peak with the molecular weight of 676 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 100 ℃, and drying at the temperature of 170 ℃; the round-silk-valine-isoleucin is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-isoleucin is as follows:
the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-isoleucin p-diphenylpicrylphenylhydrazine free radical (DPPH)50) Comprises the following steps: 1.19 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.27 mm.
Claims (6)
1. A chromo-silk-valine-isoleucyl-leucyl peptide having antifungal and free radical scavenging activity, characterized by: the structural formula of the cyclo-chromo-serine-valine-isoleucine-leucine peptide is as follows:
the cyclo-tryptophan-serine-valine-isoleucin-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine, isoleucine and leucine through amide bonds and is white powder;
the cyclo-chromo-serine-valine-isoleucyl leucine peptide has the activity of scavenging free radicals and resisting streptomyces albus;
half-scavenging concentration (DC) of the free radical of Diphenylpicrylphenylhydrazine (DPPH)50) Comprises the following steps: 1.19mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 13.27 mm.
2. A process for the preparation of cyclochromene-silk-valine-isoleucin with antifungal and free radical scavenging activity according to claim 1, characterized by the following operating steps:
(1) strain culture
Mixing Botrytis cinerea (A.ranunculata:)Basidiobolussp.) performing slant culture, secondary liquid seed culture, tertiary liquid seed culture and quaternary culture to obtain final liquid fermentation product or final solid fermentation product;
(2) extraction and refining of effective components in the final fermentation
Extracting the effective components in the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclotomic-silk-valine-isoleucin.
3. The method of claim 2, wherein: said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of froglet frogs, froglet mould with solid spore or froglet mould with split spore.
4. The method for preparing the peptide of claim 2, wherein the specific operation of step (1) is as follows:
(1.1) slant seed culture
Inoculating the ranunculus japonicus strains to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain a first-level strain;
(1.2) Secondary Strain culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
adding a liquid culture medium with the volume of 30-50% of that of a triangular flask into a triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 slant strains into each triangular flask, and culturing for 3-7 days in a full-temperature oscillation incubator at the temperature of 25-36 ℃ and the rotating speed of 150-250 rpm to obtain a secondary strain;
the liquid shake flask culture medium comprises 25.0-50.0 g/L glucose, 10.0-30.0 g/L malt extract, 2.0-6.0 g/L peptone, 1.0-4.0 g/L yeast extract powder, and potassium chloride (KCl)2) 0.3-2.0 g/L magnesium sulfate heptahydrate (MgSO)4.7H20.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH)2PO4）0.3～2.0 g/L；
(1.3) three-stage Strain culture
Inoculating the second-level strain into a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the second-level strain is subjected to constant-temperature aeration culture for 2-5 days at the temperature of 25-36 ℃ under the condition that the aeration amount is 1: 1-2 (v/v) in volume ratio and the pressure is 0.1-0.2 MP, so as to obtain a third-level strain;
the culture medium in the fermenter is synchronized with the liquid culture medium in step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture, the third-stage strain is inoculated to a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the final fermentation product of the liquid is obtained by constant-temperature aeration culture for 5-15 days under the conditions that the aeration amount is 1: 1-2 (v/v) in volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; culture in fermenter liquid culture in step (1.2).
5. The method according to claim 4, wherein: in the step (1.4), the quaternary culture is solidCulturing, namely mixing the third-stage liquid strain into a solid culture medium, wherein the inoculation amount is 3-10% of the solid material amount, and culturing at the constant temperature of 25-36 ℃ for 5-15 days to obtain a solid final fermentation product; the solid culture medium comprises 25.0-50.0 g/L of glucose, 10.0-30.0 g/L of malt extract, 2.0-6.0 g/L of peptone, 1.0-4.0 g/L of yeast extract powder, and potassium chloride (KCl)2) 0.3-2.0 g/L magnesium sulfate heptahydrate (MgSO)4.7H20.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH)2PO4) 0.3-2.0 g/L, 15.0-30.0 g/L agar and water.
6. The method according to claim 3, wherein the step (2) is specifically performed by:
(2.1) pretreatment of the final fermentation
Filtering the final fermentation product of the liquid through a filter membrane of 0.20-1.0 μm or centrifuging at 12000 rpm under 2000-; freeze-drying and crushing the mycelium to obtain a pretreatment substance which is sieved by a 20-120-mesh sieve;
or drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing the solid final fermentation product into a pretreatment product of 20-120 meshes;
(2.2) extraction, separation and purification of effective components in the pretreated product
(2.2.1) extraction
Extracting the pretreated substance with methanol or ethanol; the material-liquid ratio is 1: 2-20 (W: V), the ultrasonic power is 10-100 KHz, and the extraction time is 10-200 minutes; centrifuging at a rotating speed of 2000 rpm or more, or filtering with a 0.20-1.0 μm filter membrane; taking the filtrate or centrifugal supernatant, concentrating at the temperature of less than or equal to 100 ℃ to 1/2-1/10 of the original volume to obtain concentrated dealcoholized liquid, simultaneously recovering methanol or ethanol, and using the concentrated dealcoholized liquid for further refining by chromatography;
(2.2.2) separation and purification
Purifying by adopting a reverse phase chromatography method, wherein the stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), and the particle diameter is 3-50 microns; methanol and water are used according to the weight ratio of 0-100: gradient elution is carried out according to the proportion of 100-0, and chromatographic fractions with the molecular weight of 676 on a mass spectrum detector are collected; concentrating the collected substance to viscous state at a temperature of less than or equal to 100 ℃, and drying at a temperature of less than 170 ℃; the round-silk-valine-isoleucin is obtained as a white powder.
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| CN113583094A (en) * | 2021-09-09 | 2021-11-02 | 皖北卫生职业学院 | Cyclo-valine-silk-isoleucin-leucin with antifungal and free radical scavenging activities and preparation method thereof |
| CN115572324A (en) * | 2022-11-24 | 2023-01-06 | 安徽农业大学 | Cyclochromo-valine-propyl-leucine-phenylpropyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115636869A (en) * | 2022-11-24 | 2023-01-24 | 安徽农业大学 | Cyclo threo casein iso-brilliant phenylpropanoid with hepatoma carcinoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115636871A (en) * | 2022-11-24 | 2023-01-24 | 安徽农业大学 | Cyclochrolo-threo-casein-isoleucin-phenylpropyl peptide with hepatoma carcinoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115636870A (en) * | 2022-11-24 | 2023-01-24 | 安徽农业大学 | Cyclochromo-threo-valine-leucine peptide with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115651068A (en) * | 2022-11-24 | 2023-01-31 | 安徽农业大学 | Cyclo-C-casein-isoleucin-phenylpropanoid with hepatoma carcinoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115651069A (en) * | 2022-11-24 | 2023-01-31 | 安徽农业大学 | Cyclochromo-threo-casein-valine-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115651067A (en) * | 2022-11-24 | 2023-01-31 | 安徽农业大学 | Cyclo-C-Val-isoleu-leucin with hepatotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115716864A (en) * | 2022-11-24 | 2023-02-28 | 安徽农业大学 | Cyclo-valine-leucine peptide with hepatotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
| CN116023443A (en) * | 2022-11-24 | 2023-04-28 | 安徽农业大学 | Cyclochrome-silk-valyl-leucinyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
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| CN115572324A (en) * | 2022-11-24 | 2023-01-06 | 安徽农业大学 | Cyclochromo-valine-propyl-leucine-phenylpropyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
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| CN115716864A (en) * | 2022-11-24 | 2023-02-28 | 安徽农业大学 | Cyclo-valine-leucine peptide with hepatotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
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| CN115651067B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
| CN115651068B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-propyl-casein-isopropyl-phenyl propyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
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| CN115636871B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-threo-tyrosol-isoleucyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
| CN115716864B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-valyl-leucinyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
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