CN111018953B - Cyclocasein-isoleucyl-leucyl-tryptophyl-threo-peptide with antifungal and free radical scavenging activities and preparation method thereof - Google Patents
Cyclocasein-isoleucyl-leucyl-tryptophyl-threo-peptide with antifungal and free radical scavenging activities and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a cyclocasein-isoleucyl-leucyl-chromo-threonine (Tyr-Ile-Leu-Trp-Thr cyclopeptide) with antifungal activity and free radical scavenging activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cycloparaffin-isoleu-leu-tryptophan-threonine peptide is a pentadecentricular pentapeptide formed by connecting tyrosine, isoleucine, leucine, tryptophan and threonine by amido bonds. This is a new compound and has not been reported in the literature. The compound can be obtained by culturing Rana chensinensis mould, and extracting and separating. The pure product of Cyclocasein-isoleum-leum-cromogen-threonine is white powder, and has half scavenging concentration (DC) for DPPH free radical 50 ) Comprises the following steps: 0.82mg/mL, and the diameter of the zone of inhibition of Streptococcum albopictus at a concentration of 200. mu.g/mL is 11.36 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 cyclocasein-isoleucine-leucine-chromo-threonine (Tyr-Ile-Leu-Trp-Thr cyclopeptide) with antifungal and free radical scavenging activities.
Background
The cyclosulfame-isophoto-leu-chromo-sapeptide is prepared from a wood frog mould (A)Basidiobolussp.) isolated from cultureBiologically active substances with antifungal and free radical scavenging activity.
Botrytis cinerea (A. frog-dung)Basidiobolussp.) fungi of the genus Rana of the family Ranunculaceae of the order Entomophthorales; 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 excessive production or slow elimination of free radicals can cause various damages at the molecular level, the cellular level and the tissue and organ level of the organism by attacking the living macromolecular substances and various organelles, 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
In order to find a novel natural and efficient substance with antifungal activity and free radical scavenging activity, the inventor discovers that the cycloparaffin-isoleucyl-leucyl-chromo-threonine extracted from a frogspawpaw copromoter has strong activity of resisting streptomyces albus and free radical scavenging activity by performing activity research on more than 100 strains of entomopathogenic fungi metabolites in China.
The formula of the cycloparaffin-isoleu-leu-chromo-threitol with antifungal and free radical scavenging activity is as follows:
the cycloparaffin-isoleu-leu-tryptophan-threonine peptide is a pentadecenoic cyclic pentapeptide formed by connecting tyrosine, isoleucine, leucine, tryptophan and threonine by an amido bond;
the cycloparasticin-isoleun-leu-chromo-threonine has free radical scavenging activity and activity against streptococci albus; half-scavenging concentration (DC) of the free radical of Diphenylpicrylphenylhydrazine (DPPH) 50 ) Comprises the following steps: 0.82 mg/mL; the diameter of the inhibition zone for the white streptococci under the concentration of 200 mug/mL is 11.36 mm.
The preparation method of the cyclocasein-isoleucyl-leucyl-tryptophorin with the antifungal and free radical scavenging activities 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;
the frog dung mould (A), (B)Basidiobolussp.) is one of froglet frogs, froglet mould with solid spore or froglet mould with split spore.
(2) Extraction and purification of effective components from culture
Extracting the effective components from the culture with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclocasein-isoleucin-leucin-ghrelin.
The preparation technical scheme is further defined as follows:
the specific preparation operation steps of the step (1) are as follows:
(1.1) slant seed culture
Inoculating a frog dung mold strain to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain a primary strain;
the frog dung mould (A), (B)Basidiobolussp.) is one of froglet mould, froglet mould with solid spore or froglet mould with split spore;
(1.2) Secondary liquid seed 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 rotation speed of 150-250 rpm to obtain a secondary strain;
the liquid 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 and water are mixed evenly to prepare the water-based paint;
(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 third-level strain is obtained by ventilating and culturing for 2-5 days under the constant temperature conditions of the ventilation volume according to the volume ratio of 1: 1-2 (v/v), the pressure of 0.1-0.2 MP and the temperature of 25-36 ℃;
medium in fermenter the liquid medium of step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture or solid culture;
(1.4.1) liquid culture: inoculating the third-level strain into a fermentation tank with the volume of 50-80% and the inoculation amount of 3-10% of the volume of the tank, and performing aeration culture for 5-15 days under the constant temperature conditions of the aeration amount according to the volume ratio of 1: 1-2 (v/v), the pressure of 0.1-0.2 MP and the temperature of 25-36 ℃ to obtain a final fermentation product of liquid;
the medium in the fermenter was synchronized with the liquid medium of step (1.2).
(1.4.2) solid culture: 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 for 5-15 days at a constant temperature of 25-36 ℃ 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 .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 preparation operation steps of the step (2) are as follows:
(2.1) pretreatment
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, and crushing to 20-120 meshes to obtain a powdery culture;
and drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and crushing the solid final fermentation product to 20-120 meshes to obtain the powdery culture product.
(2.2) extraction
Extracting the powdered culture with methanol or ethanol; extracting for 10-200 minutes under the conditions that the material-liquid ratio is 1: 2-20 (W: V) and the ultrasonic power is 10-100 KHz; 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 supernatant through filtrate or centrifugation, concentrating the supernatant to 1/2-1/10 of the original volume at the temperature of less than or equal to 100 ℃ to obtain a concentrated dealcoholized liquid, and simultaneously recovering methanol or ethanol, wherein the concentrated liquid is used for further refining through chromatography;
(2.3) separation and purification
Purifying by reverse phase chromatography; the stationary phase is bonding phase filler reversed phase carbon eighteen; methanol and water are added according to the ratio of 0-100: 100-0, performing gradient elution, and collecting chromatographic fractions with the molecular weight of 676 on a mass spectrometer; concentrating the collected substance at a temperature of 100 deg.C or lower to viscous state, and drying at a temperature of 170 deg.C or lower to obtain white powdered cyclocasein-isoleucin-leucin-vie-sapeptide.
In the step (2.3), the bonding phase filler is reverse carbon eighteen filler RPC18, and the particle diameter is 3-50 microns.
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-Streptococcum albus 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 10.21 mm;
(2) determination of radical scavenging activity of methanol extract: preparing methanol solutions with the concentration of the extract samples of 0.2, 0.4, 0.8, 1.0 and 1.6 mg/mL, measuring the scavenging activity of the methanol solutions to DPPH free radicals of 0.5 mmol/L at 517 nm, and calculating to obtain the half scavenging concentration of the DPPH free radicals of 1.25 mg/mL;
since the content of the effective components of the methanol extract is low, further extraction and purification of the effective components 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) inhibition activity of cyclosulfame-isoleun-leu-cro-threo peptide on streptomyces albus was determined: 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 11.36 mm;
(2) determination of scavenging free radical activity of cyclocasein-isoleun-leu-chromo-threo peptide: preparing methanol solutions with the concentration of the extract samples of 0.2, 0.4, 0.8, 1.0 and 1.6 mg/mL, measuring the scavenging activity of the methanol solutions to DPPH free radicals of 0.5 mmol/L at 517 nm, and calculating to obtain the half scavenging concentration of the DPPH free radicals of 0.82 mg/mL;
3. chemical Structure characterization of active Compounds
High resolution LC-MS analysis showed the purified active compound cation 677.3677 (M + H) + )、699.3500 (M+Na + )、715.3220(M+K + ) Calculated molecular formula C 36 H 48 N 6 O 7 。
Nmr data are shown in the following table:
the structural formula of the separated and purified active cyclocasein-isoleucyl-leucyl-tryptophane-peripeptide is obtained by integrating liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis, and is as follows:
the beneficial technical effects of the invention are embodied in the following aspects:
1. the cyclosulfame-isolucan-lucan-chromo-threonine prepared by the method has the activities of inhibiting streptomyces albus and removing free radicals, and develops a new application field of the frogspawn. The invention discovers that the ranunculus japonicus has the functions of generating active substances for inhibiting the streptomyces albus and removing free radicals 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 cycloparaffin-isoleum-leum-chromo-threonine is a novel framework, can be used as a drug lead compound for derivatization modification, and creates a compound with more and stronger activity.
3. The streptomyces albus inhibitor and the free radical scavenger prepared by the invention have the potential of further utilization. The free radical scavenger has whitening, antioxidant, antiaging, fresh keeping, antibacterial, and antiinflammatory effects, and the white streptococcal inhibitor can be used for preventing and treating fungal infection, and can be used for medicine production.
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 cycloparaffin-isoleu-leu-chromo-threitol with antifungal and free radical scavenging activity 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.
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-stage 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 by aeration culture at the constant temperature of 25 ℃ for 5 days. 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 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 solution, recovering methanol, and refining by chromatography;
1.3.3 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 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 ℃; the cyclocasein-isoleucin-leu-chromo-threonine is obtained as a white powder.
The structural identification result shows that the structural formula of the cycloparaffin-isoleucyl-leucyl-chromo-threonine is as follows:
the activity measurement result shows that the half clearing concentration (DC) of the free radical (DPPH) of the paracasei-isoleucyl-leucyl-chromo-sapeptide p-diphenylpicryl phenylhydrazine 50 ) Comprises the following steps: 0.82 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 11.36 mm.
Example 2
The preparation of the cycloparaffin-isoleu-leu-chromo-threitol 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-stage strain into a 50L fermentation tank, wherein the liquid filling amount is 80% of the tank volume, 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-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 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.
Extraction and refining of effective components in the final fermentation
2.3.1 pretreatment of the final fermentate
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.
Extraction of
Extracting the dried mycelium or culture with ethanol; the material-liquid ratio is 1:20 (W: V), the ultrasonic power is 100KHz, and the extraction time is 200 minutes; 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;
2.3.3 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 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 cyclocasein-isoleucin-leu-chromo-threonine is obtained as a white powder.
The structural identification result shows that the structural formula of the cycloparaffin-isoleucyl-leucyl-chromo-threonine is as follows:
the activity measurement result shows that the half clearing concentration (DC) of the free radical (DPPH) of the paracasei-isoleucyl-leucyl-chromo-sapeptide p-diphenylpicryl phenylhydrazine 50 ) Comprises the following steps: 0.82 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 11.36 mm.
Example 3
The preparation of the cycloparaffin-isoleu-leu-chromo-threitol with antifungal and free radical scavenging activity 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 Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 30 deg.C for 6 days to obtain first-stage 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 a liquid culture medium with the volume of 40% of that of a triangular flask into a 500 ml triangular flask, inoculating 2 slant strains into each triangular flask, placing the triangular flasks in a full-temperature oscillation incubator after inoculation, and culturing for 5d at the temperature of 30 ℃ and the rotating speed of 200 rpm to obtain a secondary strain;
3.2.3 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;
3.2.4 Quaternary cultures
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 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.3g/L agar and 15.0 g/L agar.
Extraction and refining of effective components in the final fermentation
3.3.1 pretreatment of the final fermentate
The solid final fermentation product was dried at 30 ℃ until the water content was 20%, and then pulverized to 20 mesh for use.
Extraction of
Extracting the dried culture 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; the filtrate was concentrated to 1/2 of the original volume at 30 ℃ to give a concentrated dealcoholized liquid with recovery of ethanol, which was used for further purification by 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 25 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 40 ℃, and drying at the temperature of 10 ℃; the obtained cyclized casein-isolucent-leu-chromo-threonine is white powder.
The structural identification result shows that the structural formula of the cycloparaffin-isoleucyl-leucyl-chromo-threonine is as follows:
the activity measurement result shows that the half clearing concentration (DC) of the free radical (DPPH) of the paracasei-isoleucyl-leucyl-chromo-sapeptide p-diphenylpicryl phenylhydrazine 50 ) Respectively as follows: 0.82 mg/mL; the diameter of the inhibition zone of the streptomyces albus at the concentration of 200 mug/mL is 11.36 mm;
example 4
The preparation of the cycloparaffin-isoleu-leu-chromo-threitol with antifungal and free radical scavenging activity comprises the following steps:
4.1 Rana Nigromaculata strains selection
The used frogmatophagoides farinae generates frogmatophagoides farinae;
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 5d 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 4 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;
4.2.4 Quaternary cultures
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 as follows: 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 30.0 g/L;
4.3 extraction and purification of active ingredients 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 mycelium or culture with methanol; the material-liquid ratio is 1:20 (W: V), the ultrasonic power is 100KHz, and the extraction time is 200 minutes; filtering with 1.0 μm filter membrane after extraction; the filtrate was concentrated to 1/10 of the original volume at 40 ℃ to give a concentrated dealcoholized solution with recovery of methanol, which was used for further purification by 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 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 ℃; obtaining white powdery cyclo-casein-iso-bright-color-threonine;
the structural identification result shows that the structural formula of the cycloparaffin-isoleucyl-leucyl-chromo-threonine is as follows:
the activity measurement result shows that the half scavenging concentration (DC) of the free radical (DPPH) of the cyclo-casein-isoleum-leum-cromoglycide p-diphenylpicrylphenylhydrazine 50 ) Comprises the following steps: 0.82mg/mL, and the diameter of the zone of inhibition of Streptococcum albopictus at a concentration of 200. mu.g/mL is 11.36 mm.
Claims (3)
1. Cyclocaseino-iso-leu-chromeo-threo-peptide having antifungal and free radical scavenging activity, characterized in that: the structural formula of the cycloparaffin-isoleu-leu-chromo-threonine is as follows:
the cycloparaffin-isoleu-leu-tryptophan-threonine peptide is a pentadecenoic cyclic pentapeptide formed by connecting tyrosine, isoleucine, leucine, tryptophan and threonine by an amido bond;
the cycloparasticin-isoleun-leu-chromo-threonine has free radical scavenging activity and activity against streptococci albus; the half clearing concentration of the free radical of the p-diphenylpicrylphenylhydrazine is as follows: 0.82 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 11.36 mm.
2. The process for the preparation of cyclocasein-isoleu-leu-chromo-threo peptides 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;
said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of froglet frogs, froglets or froglets;
the specific operation steps of strain culture are 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;
said Botrytis cinerea (A), (B), (CBasidiobolussp.) is one of froglet mould, froglet mould with solid spore or froglet mould with split spore;
(1.2) Secondary liquid seed 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 rotation speed of 150-250 rpm to obtain a secondary strain;
the liquid culture medium is prepared by adding water into 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 and mixing uniformly;
(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 third-level strain is obtained by ventilating and culturing for 2-5 days under the constant temperature conditions of the ventilation volume according to the volume ratio of 1: 1-2 (v/v), the pressure of 0.1-0.2 MP and the temperature of 25-36 ℃;
medium in fermenter the liquid medium of step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture;
inoculating the third-level strain into a fermentation tank with the volume of 50-80% and the inoculation amount of 3-10% of the volume of the tank, and performing aeration culture for 5-15 days under the constant temperature conditions of the aeration amount according to the volume ratio of 1: 1-2 (v/v), the pressure of 0.1-0.2 MP and the temperature of 25-36 ℃ to obtain a final fermentation product of liquid;
medium in fermenter the liquid medium of step (1.2);
(2) extraction and purification of effective components from culture
Extracting effective components from the culture with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclocasein-isoleucin-leucin-ghrelin;
the specific operation steps are as follows:
(2.1) pretreatment
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 powdery culture;
or drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, crushing and sieving with a 20-120-mesh sieve to obtain a powdery culture;
(2.2) extraction
Extracting the powdered culture with methanol or ethanol; extracting for 10-200 minutes under the conditions that the material-liquid ratio is 1: 2-20 (W: V) and the ultrasonic power is 10-100 KHz; centrifuging at a rotating speed of 2000 rpm or more, or filtering with a 0.20-1.0 μm filter membrane; concentrating the centrifugal supernatant or filtrate to 1/2-1/10 of the original volume at the temperature of less than or equal to 100 ℃ to obtain a concentrated dealcoholized liquid, and simultaneously recovering methanol or ethanol, wherein the concentrated liquid is used for further refining by chromatography;
(2.3) separation and purification
Purifying by reverse phase chromatography; the stationary phase is bonding phase filler reversed phase carbon eighteen; methanol and water are used according to the weight ratio of 0-100: 100-0, performing gradient elution, and collecting chromatographic fractions with the molecular weight of 676 Dalton on a mass spectrum detector; concentrating the collected substance at 100 deg.C or lower to viscous state, and drying at 170 deg.C or lower to obtain white powdered cyclocasein-isoleucin-leucin-tryptophane-threonine;
the bonding phase filler is reverse carbon eighteen filler RPC18, and the particle diameter is 3-50 microns.
3. The method of claim 2, wherein: the fourth-stage culture in the step (1.4) is solid culture;
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 for 5-15 days at a constant temperature of 25-36 ℃ to obtain a solid final fermentation product;
the solid culture medium is prepared by adding 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 and 15.0-30.0 g/L of agar and uniformly mixing the components by adding water.
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