CN111018954B - Cyclo-serine-valine-leucine peptide with antifungal and free radical scavenging activities and preparation method thereof - Google Patents
Cyclo-serine-valine-leucine peptide with antifungal and free radical scavenging activities and preparation method thereof Download PDFInfo
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to a cyclo-chromo-serine-valine-leucine peptide (Trp-Ser-Val-Val-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-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine and leucine through an amide bond. 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 Cyclo-Ser-Val-Leu peptide is white powder, and has half of scavenging concentration (DC) for DPPH free radical 50 ) Comprises the following steps: 1.17mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 14.62 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-leucine peptide (Trp-Ser-Val-Val-Leu cyclic peptide) with antifungal and free radical scavenging activities.
Background
The Cyclochromene-serine-valine-leucine peptide is extracted from a wood frog mould (A)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 order entomomycetales(ii) a 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 becoming more and more serious, and meanwhile, due to factors such as environmental pollution, the resistance of people tends to decline, the cases of fungal infection tend to rise, and meanwhile, the drug resistance phenomenon of early antifungal drugs appears, 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 organism, and if the free radicals cannot maintain a certain level, the life activities of the organism 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 cyclo-chromo-serine-valine-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-leucine peptide extracted from a froglea faecalis has strong activity against 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-leucine peptide with antifungal and free radical scavenging activities is as follows:
the cyclo-tryptophan-serine-valine-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine and leucine through an amide bond;
the cyclo-chromo-serine-valine-leucine peptide has the activity of scavenging free radicals and the activity of resisting streptomyces albus;
half-scavenging concentration (DC) of the free radical of Diphenylpicrylphenylhydrazine (DPPH) 50 ) Comprises the following steps: 1.17mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 14.62 mm.
The preparation of the cyclochrome-silk-valine-leucine peptide with antifungal and free radical scavenging activities comprises the following steps:
(1) culture of bacterial species
Mixing all the wood frogs of Rana NigromaculataBasidiobolussp.) 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;
said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of Rana chensinensis David dung mold, Rana chensinensis David dung mold or Rana torulosa diels.
(2) Extraction and refining of effective components in the final fermented product
Extracting the effective components of the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclocarya-silk-valine-leucine peptide.
The preparation technical scheme is further defined as follows:
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 liquid seed 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 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ）0.3～2.0 g/L；
(1.3) three-stage liquid seed 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 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ) 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 mu m or centrifuging the final fermentation product under the condition of 2000-12000 r/min to obtain mycelium; freeze-drying and crushing the mycelium to obtain a pretreatment substance which is sieved by a sieve of 20-120 meshes;
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 more than or equal to 2000 rpm, 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: performing gradient elution at the ratio of 100-0, and collecting chromatographic fractions with the molecular weight of 676 on a mass spectrum detector; concentrating the collected substance to be viscous at a temperature of less than or equal to 100 ℃, and drying at a temperature of less than 170 ℃; the round-color-silk-valine-leucine peptide is obtained in white powder.
The analytical study of the present invention is illustrated below:
1. screening research discovers a frog dung mold (A)Basidiobolussp.) 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 on the white streptococcal bacteria of 11.58 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 1.51 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-leucine on Streptococcum albus: 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 14.62 mm.
(2) Determination of the radical scavenging activity of cyclochro-silk-valine-leucine peptide: 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 1.17 mg/mL.
3. Chemical Structure characterization of active Compounds
High resolution LC-MS analysis showed the purified active compound cation 585.3440(M + H) + )、607.3261(M+Na + )，623.3000(M+K + ) Calculated molecular formula C 30 H 44 N 6 O 6 。
Nmr data are shown in the following table:
the structure of the separated and purified active compound is the cyclochrolo-silk-valine-leucine peptide obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis, and the active compound is shown in the chemical structural formula.
The beneficial technical effects of the invention are embodied in the following aspects:
1. the cyclo-chromo-serine-valine-leucine peptide 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-leucine peptide is a novel framework, can be used as a medicinal lead compound for derivatization modification, and can create a compound with more and stronger activity.
3. The cyclo-chromo-serine-valine-leucine peptide can be used for preparing a streptomyces 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-leucine peptide with antifungal and free radical scavenging activities comprises the following steps:
1.1 Rana Nigromaculata Strain selection
The Rana chensinensis mould is Rana chensinensis mould;
1.2 Strain culture
The culture method is liquid culture and comprises the following specific steps:
1.2.1 slant seed culture
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 25 deg.C for 4 days to obtain first-class strain;
1.2.2 Secondary liquid seed 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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）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;
1.2.3 three-stage liquid seed 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.
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;
1.3 extraction and purification of the active principles in the final fermentation
1.3.1 pretreatment of the final fermentate
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.
Extraction of
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.
Separating and purifying
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 cyclochol-silk-valine-leucine peptide was obtained.
The structural identification result shows that the structural formula of the cyclo-chromo-serine-valine-leucine peptide is as follows:
the activity assay result shows that the ring color is-silk-valine-Median scavenging concentration (DC) of leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH) 50 ) Comprises the following steps: 1.17 mg/mL; the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 14.62mm in diameter.
Example 2
The preparation of the cyclo-chromo-ser-val-leucin with antifungal and free radical scavenging activity comprises the following steps:
2.1 selection of Botrytis frog-dung mould Strain
The Rana chensinensis mould is Rana chensinensis mould;
2.2 Strain culture
The culture method is liquid culture and comprises the following specific steps:
2.2.1 slant seed culture
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 36 deg.C for 8 days to obtain first-class strain;
2.2.2 Secondary liquid seed 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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）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.
Three stage liquid seed 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.
Four stage culture
The fourth-stage culture is liquid culture: inoculating the third-stage strain into a fermentation tank with volume of more than or equal to 500L, 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 liquid is subjected to constant-temperature aeration culture at 36 ℃ for 15 days to obtain a final fermentation product of the liquid; the culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
Extraction and refining of effective components in the final fermented product
2.3.1 pretreatment of the final fermentate
Filtering the final fermentation product of the liquid through a 1.0 mu m filter membrane to obtain mycelium; the mycelium was freeze-dried and ground to 120 mesh for use.
Extraction of
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.
Separating and purifying
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 micrometers, and the diameter of the chromatographic column 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-color-silk-valine-leucine peptide is obtained in white powder.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-leucine peptide is as follows:
the activity assay result shows that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH) 50 ) Comprises the following steps: 1.17 mg/mL; inhibition of Streptococcum albus at a concentration of 200. mu.g/mLThe diameter of the fungus ring is 14.62 mm.
Example 3:
the preparation of the cyclochrome-silk-valine-leucine peptide with antifungal and free radical scavenging activities comprises the following steps:
3.1 Rana Nigromaculata Strain selection
The selected strain is ranunculus glaucocalyx;
3.2 Strain culture
The culture method is liquid-solid mixed culture and comprises the following specific steps:
3.2.1 slant seed culture
Inoculating the preserved Rana chensinensis Mucor strain to potato agar (PDA) slant culture medium, and culturing at constant temperature of 30 deg.C for 6d to obtain first-class strain;
3.2.2 Secondary liquid seed 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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）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.
Three stage liquid seed 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.
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: 25.0g/L glucose, 10.0g/L malt extract, 2.0 g/L peptone, 1.0g/L yeast extract powder, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.3 g/L; agar 15.0 g/L.
Extraction and refining of effective components in the final fermentation
3.3.1 pretreatment of the final fermentate
The solid final fermentation was dried at 30 ℃ to a moisture content of 20% and then ground to 20 mesh for use.
Extraction of
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;
3.3.3 isolation 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: performing gradient elution at the ratio of 100-0, and collecting a target peak with the molecular weight of 676 on a mass spectrum detector; concentrating the collected substance to be viscous at the temperature of below 40 ℃, and drying at the temperature of 10 ℃; the round-color-silk-valine-leucine peptide is obtained in white powder.
The structural identification result shows that the structural formula of the cyclo-chromatic-silk-valine-leucine peptide is as follows:
the activity assay result shows that the median scavenging concentration (DC) of the cyclo-chromo-serine-valine-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH) 50 ) Comprises the following steps: 1.17 mg/mL; inhibition zone for streptococci albus under concentration of 200 mug/mLThe diameter is 14.62 mm.
Example 4:
the preparation of the cyclochrome-silk-valine-leucine peptide with antifungal and free radical scavenging activities comprises the following steps:
4.1 Rana Nigromaculata strains selection
The used frog dung mold generates frog dung mold;
4.2 Strain culture
The culture method is liquid-solid mixed culture and comprises the following specific steps:
4.2.1 slant seed culture
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;
4.2.2 Secondary liquid seed 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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）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.
Three stage liquid seed culture
Inoculating the second-stage strain into a 30L fermentation tank, wherein the liquid filling amount is 65% of the tank volume, the inoculation amount is 6%, and the aeration amount is 1:1.2 (v/v) in volume ratio, the pressure is 0.12MP, and the constant temperature aeration culture is carried out for 3 days at 32 ℃ to obtain a third-stage strain.
The culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ) 2.0 g/L agar and 30.0 g/L agar.
Extraction and refining of effective components in the final fermented product
4.3.1 pretreatment of the final fermentate
The solid final fermentation product was dried at 130 ℃ to a water content of 10% and then pulverized to 120 mesh for use.
Extraction of
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;
4.3.3 isolation 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-color-silk-valine-leucine peptide is obtained in white powder.
The structural identification result shows that the structural formula of the cyclo-chromo-serine-valine-leucine peptide is as follows:
the activity measurement result shows that the median scavenging concentration (DC) of the cyclo-S-V-L-P-diphenylpicrylphenylhydrazine free radical (DPPH) 50 ) Comprises the following steps: 1.17 mg/mL; white at a concentration of 200. mu.g/mLThe diameter of the inhibition zone of the color streptococci is 14.62 mm.
Claims (3)
1. Cyclochromen-serine-valine-leupeptin having antifungal and free radical scavenging activity, characterized by: the structural formula of the cyclo-chromo-serine-valine-leucine peptide is as follows:
the cyclo-tryptophan-serine-valine-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, serine, valine and leucine through an amide bond;
the cyclo-chromo-serine-valine-leucine peptide has the activity of scavenging free radicals and the activity of resisting streptomyces albus;
the half clearing concentration of the free radical of the p-diphenylpicrylphenylhydrazine is as follows: 1.17mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 14.62 mm.
2. A process for the preparation of cyclochromene-silk-valine-leucine peptides having antifungal and free radical scavenging activity according to claim 1, characterized by the following operating steps:
(1) culture of bacterial species
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;
said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of froglet frogs, froglets or froglets;
the specific strain culture operation is as follows:
(1.1) slant seed culture
Inoculating the frog manure mould strain to a potato agar slant culture medium, and culturing for 4-8 days at a constant temperature of 25-36 ℃ to obtain a primary strain;
(1.2) Secondary liquid seed 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 is prepared from 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, 0.3-2.0 g/L potassium chloride, 0.3-2.0 g/L magnesium sulfate heptahydrate and 0.3-2.0 g/L potassium dihydrogen phosphate;
(1.3) three-stage liquid seed 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 medium in the fermenter is synchronized with the liquid 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 ℃; the culture medium in the fermenter is synchronized with the liquid culture medium in step (1.2);
(2) extraction and refining of effective components in the final fermented product
Extracting effective components in the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclochromata-silk-valine-leucine peptide;
the specific operation 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 the mycelium, crushing, and sieving with a 20-120-mesh sieve to obtain a pretreated substance;
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 dried solid final fermentation product through a sieve of 20-120 meshes to obtain a pretreatment product;
(2.2) extraction, separation and purification of effective ingredients 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; concentrating the centrifuged supernatant or filtrate at a temperature of 100 ℃ or lower to 1/2-1/10 of the original volume to obtain a concentrated dealcoholized liquid, and simultaneously recovering methanol or ethanol, wherein the concentrated dealcoholized liquid is used 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 mixture ratio of 100-0, and chromatographic fractions with the molecular weight of 676 Dalton (Dalton) 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-color-silk-valine-leucine peptide is obtained in white powder.
3. The method of claim 2, wherein: in the step (1.4), the fourth-stage culture or the solid culture is carried out, 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 is prepared by uniformly mixing 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, 0.3-2.0 g/L of potassium chloride, 0.3-2.0 g/L of magnesium sulfate heptahydrate, 0.3-2.0 g/L of potassium dihydrogen phosphate, 15.0-30.0 g/L of agar and water.
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| CN113072458B (en) * | 2021-04-12 | 2023-04-14 | 安徽农业大学 | Fumonisin A with anti-aging activity and preparation method thereof |
| CN113264987B (en) * | 2021-05-31 | 2023-11-17 | 安徽农业大学 | Cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113307848B (en) * | 2021-05-31 | 2023-11-17 | 安徽农业大学 | Cyclic color-silk-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
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| CN115636870B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-threo-valyl-leucinyl peptide 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 |
| CN115636869B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic stachyose isoleucyl-propyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof |
| CN115651069B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
| CN115536734B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-propyl-valyl-leucins with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
| 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 |
| CN115572324B (en) * | 2022-11-24 | 2024-04-30 | 安徽农业大学 | Cyclic color-valyl-leu-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
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