阅读说明：本技术 具有抗真菌和清除自由基活性的环色-缬-丝-异亮-亮肽及制备方法 (Cyclo-valine-silk-isoleucin-leucin with antifungal and free radical scavenging activities and preparation method thereof ) 是由 何亚琼 于 2021-09-09 设计创作，主要内容包括：本发明涉及具有抗真菌和清除自由基活性的环色-缬-丝-异亮-亮肽(Trp-Ser-Val-Ile-Leu环肽)及制备方法,属于生物制品的提取制备技术领域。所述环色-缬-丝-异亮-亮肽是由色氨酸、缬氨酸、丝氨酸、异亮氨酸和亮氨酸,以酰胺键连接而成的十五元环状五肽。这是一个新型化合物,尚无文献报道。本发明化合物可通过培养蛙粪霉,然后提取分离得到。环色-缬-丝-异亮-亮肽纯品为白色粉末,对DPPH自由基的半数清除浓度DC-(50)为：1.32 mg/mL,在200μg/mL的浓度下对白色链珠菌的抑菌圈直径为13.25 mm。该化合物具有制备抗菌药物和抗氧化抗衰老保健品的潜力。(The invention relates to a cyclo-chromo-valine-serine-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-valine-serine-isoleucin-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, valine, serine, isoleucine 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 product of the cyclo-valine-silk-iso-bright-leucine peptide is white powder and has a concentration DC capable of removing half of DPPH free radicals 50 Comprises the following steps: 1.32mg/mL, and the diameter of the zone of inhibition for Streptococcum albopictus at a concentration of 200. mu.g/mL is 13.25 mm. The compound has the potential of preparing antibacterial drugs and antioxidant and anti-aging health care products.)

1. A cyclic-valine-silk-isoleucine-leucine peptide having antifungal and free radical scavenging activity, characterized by: the structural formula of the cyclo-chromo-valine-silk-isoleucin is as follows:

the cyclo-tryptophan-valine-serine-isoleucin-leucine peptide is white powder, and is pentadecatomic cyclic pentapeptide formed by connecting tryptophan, valine, serine, isoleucine and leucine through amide bonds;

the cyclo-chromo-valine-silk-isoleucin has the activity of scavenging free radicals and the activity of resisting streptomyces albus;

half scavenging concentration DC of p-diphenylpicrylphenylhydrazine free radical DPPH₅₀1.32mg/mL, and a zone diameter of 13.25mm at a concentration of 200. mu.g/mL for Streptococcum albopictus.

2. The process for the preparation of cyclochromene-valine-silk-isoleucin-leucin 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.) through the culture of primary slant strain, secondary liquid seed culture and tertiary liquid seed cultureCulturing and performing four-stage culture to obtain liquid final fermentation product or solid final 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 cyclol-valine-silk-isoleucin.

3. The method of claim 2, wherein: the specific operation of the step (1) is as follows:

(1.1) first order slant seed culture

Inoculating the ranunculus japonicus strains to a potato agar PDA slant culture medium, inoculating 1-3 inoculating loop original strains to each test tube, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain primary strains;

(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 by uniformly mixing 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, 0.3-2.0 g/L potassium dihydrogen phosphate and water;

(1.3) three-stage liquid seed culture

Inoculating the secondary 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, and the inoculation amount is 3-10%; carrying out constant-temperature aeration culture for 2-5 days at the aeration rate of 1L: 1-2L, the pressure of 0.1-0.2 MP and the temperature of 25-36 ℃ according to the volume ratio, 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 tank volume, and the inoculation amount is 3-10%; carrying out constant-temperature aeration culture for 5-15 days under the conditions of aeration volume of 1L: 1-2L, pressure of 0.1-0.2 MP and temperature of 25-36 ℃ according to the volume ratio, so as to obtain a final fermentation product of liquid; the medium in the fermenter was the same as the liquid medium in step (1.2).

4. 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.

5. The method according to claim 3, wherein: in the step (1.4), the fourth-stage culture is solid culture, the third-stage 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.

6. The method of claim 2, wherein: 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 the mycelium, crushing, and sieving with a 20-120-mesh sieve to obtain a pretreated substance;

(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 ratio of the material to the liquid is 1Kg to 2-20L, 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 the filtrate or centrifugal 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;

(2.2.2) separation and purification

Purifying the concentrated dealcoholized liquid by adopting a reverse phase chromatography method, wherein a stationary phase is a bonded phase filler, and the bonded phase filler is a reverse phase carbon eighteen filler (RPC 18); 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 chromatographic fraction at 100 deg.C or lower to viscous state, and drying at 170 deg.C or lower; the round-color-valine-silk-isoleucin is obtained as a white powder.

7. The method of claim 6, wherein: in the step (2.1), the solid final fermentation product is dried at the temperature of 30-130 ℃ until the water content is less than or equal to 20%, and is crushed and sieved by a sieve of 20-120 meshes to obtain a pretreatment product.

8. The method of claim 6, wherein: in the step (2.2.2), the particle diameter of the reverse carbon eighteen-filler RPC18 is 3-50 microns.

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-valine-serine-isoleucine-leucine peptide (Trp-Ser-Val-Ile-Leu cyclopeptide) with antifungal and free radical scavenging activities.

Background

The cyclo-chromo-valine-filament-isoleucin is extracted from a species of Botrytis farinosa (Vasella farinosa) (Vasella)Basidiobolussp.) culture medium and the product thereof with antifungal and free radical scavenging activityA biologically active substance.

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

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-valine-serine-isoleucin-leucin extracted from ranunculus japonicus 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, and aims to provide the cyclo-chromo-valine-serine-isoleucin-leucin with antifungal and free radical scavenging activity and a preparation method thereof.

A cyclic-valine-silk-isoleucin having antifungal and free radical scavenging activity has the following structural formula:

the cyclo-tryptophan-valine-serine-isoleucin-leucine peptide is white powder, and is pentadecatomic cyclic pentapeptide formed by connecting tryptophan, valine, serine, isoleucine and leucine through amide bonds;

the cyclo-chromo-valine-silk-isoleucin 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) 1.32mg/mL, and a zone diameter of 13.25mm at a concentration of 200. mu.g/mL for Streptococcum albopictus.

The preparation operation steps of the cyclo-chromo-valine-silk-isoleucyl-leucyl peptide with the antifungal activity and the free radical scavenging activity are as follows:

(1) strain culture

Mixing Botrytis cinerea (A.ranunculata:)Basidiobolussp.) performing primary 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;

(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 cyclol-valine-silk-isoleucin.

The preparation method comprises the following specific steps:

(1.1) slant seed culture

Inoculating a frog dung mold strain to a potato agar (PDA) slant culture medium, inoculating 1-3 inoculating loops of original strains to each test tube, and culturing at a constant temperature of 25-36 ℃ for 4-8 days 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 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 and water are mixed evenly to prepare the water-based paint;

(1.3) three-stage liquid seed culture

Inoculating the secondary 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, and the inoculation amount is 3-10%; carrying out constant-temperature aeration culture for 2-5 days under the conditions of aeration volume of 1L: 1-2L (v/v), pressure of 0.1-0.2 MP and temperature of 25-36 ℃ according to the volume ratio, 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 tank volume, and the inoculation amount is 3-10%; carrying out constant-temperature aeration culture for 5-15 days at the conditions of aeration volume of 1L: 1-2L (v/v), pressure of 0.1-0.2 MP and temperature of 25-36 ℃ according to the volume ratio, so as to obtain a final liquid fermented product; the medium in the fermenter was the same as the liquid medium in step (1.2).

The specific technical scheme is as follows:

in the step (1.4), the fourth-stage culture is solid culture, the third-stage 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 the mycelium, crushing, and sieving with a 20-120-mesh sieve to obtain a pretreated substance;

(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 ratio of the material to the liquid is 1Kg to 2-20L (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 the filtrate or centrifugal 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;

(2.2.2) separation and purification

Purifying the concentrated dealcoholized liquid by adopting a reverse phase chromatography method, wherein a stationary phase is a bonded phase filler, and the bonded phase filler is a reverse phase carbon eighteen filler (RPC 18); 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 chromatographic fraction at 100 deg.C or lower to viscous state, and drying at 170 deg.C or lower; the round-color-valine-silk-isoleucin is obtained as a white powder.

In the step (2.1), the solid final fermentation product is dried at the temperature of 30-130 ℃ until the water content is less than or equal to 20%, and is crushed and sieved by a sieve of 20-120 meshes to obtain a pretreatment product.

In the step (2.2.2), the particle diameter of the reverse carbon eighteen-filler RPC18 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-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-Val-Si-Allen-Leucin 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 13.25 mm;

(2) determination of the radical scavenging activity of cyclo-chromo-valine-silk-isoleucin: 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 1.32 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 cyclo-chromo-valine-serine-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-valine-serine-isoleucin-leucin prepared by the method has the activities of inhibiting streptomyces albus and removing free radicals, and develops a new application field of the frogma fargesii. 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-valine-silk-iso-leucine peptide is a novel framework, can be used as a medicine lead compound for derivatization modification, and can create a compound with more and stronger activity.

3. The cyclo-chromo-valine-serine-isoleucin 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 cyclo-chromo-valine-silk-isoleucyl-leucyl peptide 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 first order slant seed culture

Inoculating the preserved Rana Nigromaculata strains to a potato agar (PDA) slant culture medium, inoculating 1 inoculating loop of original strains to each test tube, and culturing at constant temperature of 25 deg.C for 4d to obtain primary strains;

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)₄.7H₂O) 0.3g/L, potassium dihydrogen phosphate (KH)₂PO₄) 0.3g/L and water are mixed evenly to prepare the water-based paint.

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 1L:1L (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 1L:1L (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.

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; freeze drying mycelium, pulverizing, and sieving with 20 mesh sieve to obtain pretreated product.

Extraction of

Extracting the pretreated substance with methanol; the material-liquid ratio is 1Kg to 2L (W to V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; centrifuging at 2000 rpm, 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 cyclo-chromo-valine-silk-isoleucin was obtained.

The structural identification result shows that the structural formula of the cyclo-chromo-valine-serine-isoleucine-leucine peptide is as follows:

the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-valine-serine-isoleucin-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH)₅₀) Comprises the following steps: 1.32 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.25 mm.

Example 2

The preparation of the cyclo-chromo-valine-silk-isoleucyl-leucyl peptide 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 first order slant seed culture

Inoculating the preserved Rana chensinensis David dung mold strain to a potato agar (PDA) slant culture medium, inoculating 3 inoculating rings of original strains to each test tube, and culturing at constant temperature of 36 deg.C for 8d to obtain primary 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)₄.7H₂O) 0.3g/L, potassium dihydrogen phosphate (KH)₂PO₄) 2.0 g/L and water are mixed evenly to prepare the water-based paint.

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 1L:2L (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-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; freeze drying mycelium, pulverizing, and sieving with 120 mesh sieve to obtain pretreated product.

Extraction of

Extracting the pretreated substance with ethanol at a ratio of 1Kg to 20L (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 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-color-valine-silk-isoleucin is obtained as a white powder.

The structural identification result shows that the structural formula of the cyclo-chromo-valine-serine-isoleucine-leucine peptide is as follows:

the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-valine-serine-isoleucin-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH)₅₀) Comprises the following steps: 1.32 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.25 mm.

Example 3:

the preparation of the cyclo-chromo-valine-silk-isoleucyl-leucyl peptide 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 first order slant seed culture

Inoculating the preserved Rana chensinensis David dung mold strain to a potato agar (PDA) slant culture medium, inoculating 2 inoculating rings of original strains to each test tube, and culturing at constant temperature of 30 deg.C for 6d to obtain primary 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)₄.7H₂O) 0.3g/L, potassium dihydrogen phosphate (KH)₂PO₄) 1.0g/L and water are mixed evenly to prepare the water-based paint.

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 aeration amount is 1L:1.5L (v/v) in volume ratio, the pressure is 0.15MP, and the constant-temperature aeration 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: 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.

Extraction and refining of effective components in the final fermentation

3.3.1 pretreatment of the final fermentate

Drying the solid final fermented product at 30 deg.C until the water content is 20%, pulverizing, and sieving with 20 mesh sieve to obtain pretreated product.

Extraction of

Extracting the pretreated substance with ethanol; the material-liquid ratio is 1Kg to 2L (W to 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: 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-color-valine-silk-isoleucin is obtained as a white powder.

The structural identification result shows that the structural formula of the cyclo-chromo-valine-serine-isoleucine-leucine peptide is as follows:

the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-valine-serine-isoleucin-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH)₅₀) Comprises the following steps: 1.32 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.25 mm.

Example 4:

the preparation of the cyclo-chromo-valine-silk-isoleucyl-leucyl peptide with antifungal and free radical scavenging activity comprises the following steps:

4.1 Rana Nigromaculata strains selection

The Rana chensinensis mould is Rana chensinensis mould;

4.2 Strain culture

The culture method is liquid-solid mixed culture and comprises the following specific steps:

4.2.1 first order slant seed culture

Inoculating the preserved Rana Nigromaculata Botrytis strains to a potato agar (PDA) slant culture medium, inoculating 2 inoculating rings of original strains to each test tube, and culturing at 29 deg.C for 5d to obtain first-stage strains;

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)₄.7H₂O) 0.3g/L, potassium dihydrogen phosphate (KH)₂PO₄) 1.2 g/L and water are mixed evenly to prepare the water-based paint.

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-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 aeration amount is 1L:1.2L (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-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 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, potassium dihydrogen phosphate (KH)₂PO₄) 2.0 g/L agar and 30.0 g/L agar.

Extraction and refining of effective components in the final fermentation

4.3.1 pretreatment of the final fermentate

Drying the solid final fermented product at 130 deg.C to water content of 10%, pulverizing, and sieving with 120 mesh sieve to obtain pretreated product.

Extraction of

Extracting the pretreated substance with methanol at a ratio of 1Kg to 20L (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-valine-silk-isoleucin is obtained as a white powder.

The structural identification result shows that the structural formula of the cyclo-chromo-valine-serine-isoleucine-leucine peptide is as follows:

the activity assay results show that the median scavenging concentration (DC) of the cyclo-chromo-valine-serine-isoleucin-leupeptin p-diphenylpicrylphenylhydrazine free radical (DPPH)₅₀) Comprises the following steps: 1.32 mg/mL; the diameter of the zone of inhibition for Streptococcum albus at a concentration of 200. mu.g/mL was 13.25 mm.

It will be understood by those skilled in the art that the foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.