Streptomyces varioti for producing blue pigment and method for producing blue pigment by using streptomyces varioti

Abstract

The invention belongs to the technical field of pigment production, and particularly relates to streptomyces varioti for producing blue pigment and a method for producing the blue pigment by using the streptomyces varioti. The invention provides streptomyces varioti capable of producing blue pigment and a method for producing the blue pigment, the streptomyces varioti T12 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.4.7887. The streptomyces varioti T12 can be used for producing the blue pigment. The blue pigment produced by fermentation of the streptomyces varioti is a natural pigment, is high in safety and has good biological activity, meanwhile, a new way is provided for industrial production of the natural blue pigment, and the application prospect in the industries of food, dyeing and weaving, medicine and the like is good.

Description

Technical Field

[0001] The invention belongs to the technical field of pigment production, and particularly relates to a blue pigment-producing Streptomyces versicolor and a method for producing the blue pigment. Background Art

[0002] Pigments are natural or synthetic substances that give a certain color to a substance. They are widely used in the food and textile industries. According to statistics, the textile industry alone consumes about 1.3 million tons of dyes, pigments and dyeing precursors each year, with a total value of more than 23 billion US dollars. According to the source of the pigment, pigments are divided into natural pigments and synthetic pigments. Among them, synthetic pigments are mainly organic pigments extracted from coal tar or chemically synthesized using aromatic compounds as raw materials. They have strong coloring power and are cheap, but some synthetic pigments are not easy to degrade, pollute the environment, and harm human health. Therefore, there is an urgent need to develop environmentally friendly and safe natural pigments.

[0003] Natural pigments are abundant in sources, mainly from various colored substances separated or extracted from plants, animals, and microorganisms. These pigments can be used to maintain, enhance, or give color to food, medicines, or cosmetics, and have important applications in industries such as food, textile printing and dyeing, and cosmetics. Natural pigments are widely present in a variety of organisms and are divided into animal pigments, plant pigments, and microbial pigments. However, animal and plant materials are restricted by resources, the environment, and other factors, resulting in insufficient production and high prices, which limits their application development. The use of microbial fermentation to produce natural pigments overcomes many of the shortcomings of using animals and plants as raw materials to produce natural pigments, and is easy to industrialize. It has gradually gained favor among researchers.

[0004] At present, the natural pigment reported in the document is mainly based on red and yellow pigment, and blue pigment is very rare, and blue pigment, as one of the three primary colors, can be deployed into multiple pigments with red and yellow pigment for use, therefore expanding the source kind of blue pigment is of great significance, and the blue pigment used in the market is still based on synthetic pigment, and natural blue pigment is less, and comes mostly from blue vitriol mine, Polygonum indigoferum, Indigoferum sibiricum and Gardenia jasminoides etc., and there is rarely blue pigment from microbial source.Wherein, Gardenia jasminoides blue pigment has complicated manufacturing process, high cost, and limited raw materials, and has certain difficulties in large-scale industrial production.The microorganism that can produce blue pigment that has been publicly reported at present is seldom, mainly contains Streptomyces coelicolor (Streptomyces coelicolor) A3, Pseudomonas, Duganella sp. (Duganella sp.) T2013, Duganella sp. B2, Chromobacterium (Chromobacterium), Janssen bacillus (Janthinobacterium lividum) and Pseudomonas fluorescens (Pseudomonas fluorescens) B1 etc. The types, quantities and pigment production of these strains are far from meeting people's needs for natural blue research and development.

[0005] Streptomyces strains belong to the Bacteria domain, Actinomycetota, Actinomycetes, Kitasatosporales, and Streptomycetaceae. More than 700 effectively described species have been found. It is reported that more than 50% of the antibiotics currently used by humans are produced by Streptomyces. At the same time, strains of Streptomyces can produce a variety of biologically active substances, such as some strains that can produce various enzymes and secondary metabolites such as pigments and alkaloids, and some can repair heavy metals in the soil. Among them, Streptomyces coelicolor and Streptomyces lead-mutant can produce blue pigments, and their blue pigment production is not high, so it is necessary to further explore strains that can produce blue pigments.

[0006] Chinese patent application CN202410284862.5 discloses an engineered bacterium for synthesizing indigo, and a construction method and application thereof, wherein the engineered bacterium expresses bpsA, sfp, glnA* and gdhA; and the engineered bacterium knocks out glsK, proB, aceA and ldh. This patent application knocks out the proB and aceA genes, performs site-directed mutation on the glnA gene, replaces the glsK gene with the glnA* gene, replaces the ldh gene with the gdhA gene, and integrates the bpsA and sfp genes, which can block the generation of by-products, increase the flux of pyruvate to the tricarboxylic acid cycle and α-ketoglutarate to glutamate, increase the glutamine concentration in the organism, and promote the indigo synthase to catalyze the synthesis of indigo from glutamine, thereby increasing the yield of indigo.

[0007] Chinese patent application CN202211517065.4 discloses a method for constructing a pigment and its production engineering bacteria. A highly expressed protein was identified in the epidermis of the umbrella of a blue jellyfish by comparative proteomics. Sequence alignment and domain analysis showed that the protein was similar to the blue pigment protein precursor of the pulmonary rhizostomium jellyfish, and the protein was named jellyfish blue pigment. The jellyfish blue pigment gene expression vector was constructed, and the torotoscolemus yeast was transformed by genetic engineering methods to construct an industrial yeast strain that produces blue pigment. Summary of the invention

[0008] The current synthetic pigments have the disadvantages of being difficult to degrade, polluting the environment and damaging human health. In order to overcome the shortcomings of the above-mentioned technology, the present invention provides a blue pigment-producing Streptomyces discolor T12 and a method for producing blue pigment. The Streptomyces discolor T12 provided by the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is: CGMCC No.4.7887. The blue pigment produced by the fermentation of the Streptomyces discolor T12 is a natural pigment with high safety and good biological activity. It also provides a new way for the industrial production of natural blue pigment and has good application prospects in the food, dyeing and weaving, and medicine industries.

[0009] The present invention achieves the above technical effects through the following technical solutions:

[0010] One of the purposes of the present invention is to provide a blue pigment-producing Streptomyces versicolor T12.

[0011] The Streptomyces versicolor T12 provided by the present invention belongs to the family Streptomycetes and is a new species of the genus Streptomyces. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) in June 2024, with the deposit registration number: CGMCC No. 4.7887. On June 19, 2024, the biological material deposit certificate for patent procedures issued by the institution was received and registered.

[0012] The growth characteristics of the strain are as follows: Streptomyces versicolor T12 is an aerobic bacterium, Gram-positive, and catalase-positive. It can form well-developed aerial hyphae on ISP2 and soil extract medium, with spiral spores and thorns on the outside of the spores, resembling Xanthium sibiricum. The colony is round, convex, blue-gray or white on the front, blue on the back, and the growth temperature range is 15-42°C; NaCl tolerance is less than or equal to 6%; pH range is 4-12. No melanin is produced on ISP6 and ISP7; the strain is negative for nitrite reduction; it can hydrolyze esculin, gelatin, Tween 80 and starch, but cannot hydrolyze arginine; it does not produce urease and hydrogen sulfide. The strain is negative for the assimilation of N-acetylglucosamine, capric acid, adipic acid, citric acid and phenylacetic acid. The strain can use D-mannose, D-mannitol, D-glucose, glycerol, etc. as the sole carbon source, but cannot use octanoic acid, adipic acid, malic acid, citric acid and D-ribose as the sole carbon source. The 16S rDNA sequence of Streptomyces cyaneus T12 of the present invention is shown in SEQ.No.1. In GenBank, the model strain with the highest similarity to its 16S rDNA gene sequence is S.cyaneus NRRL B-2296. T (99.6%). The G+C content in the DNA was 70.2 mol%, which is consistent with the characteristics of Streptomyces. At the same time, the results of genome sequence analysis of Streptomyces versicolor T12 showed that Streptomyces versicolor T12 was similar to S. cyaneus NRRL B-2296 T The digital DNA-DNA hybridization value was 52.1%, which was far lower than 70%. The results of polyphasic taxonomic identification showed that the Streptomyces versicolor T12 was a new species of Streptomyces.

[0013] Another object of the present application is to provide an application of the above strain, namely, the application of the above strain in the production of blue pigment by fermentation.

[0014] Another object of the present application is to provide a method for producing blue pigment using the above-mentioned strain, which method comprises fermenting and culturing Streptomyces versicolor T12, collecting the bacteria by centrifugation after the fermentation and culturing is completed, extracting the blue pigment using an organic solvent, and concentrating and drying the extract by rotary evaporation to obtain a crude extract of the blue pigment.

[0015] As a preferred technical solution, the liquid culture medium used for fermentation of Streptomyces versicolor T12 contains: sucrose 103g, glucose 10g, MgCl 2 6H 2 O 10.12g, K 2 SO 4 0.25g, TES 5.73g, yeast extract 3-5g, casamino acid 0.1g, trace elements 2mL, distilled water to 1000mL, after sterilization, add the following before use: potassium dihydrogen phosphate (0.5%) 10mL, L-proline (20%) 15mL, calcium chloride dihydrate (5M) 4mL, sodium hydroxide (1M) 7mL (adjust pH 7.0-7.2). S. discolor T12 was inoculated on the surface of solid culture medium by plate streak method for proliferation culture and detection of miscellaneous bacteria.

[0016] As a preferred technical solution, the fermentation culture conditions of the Streptomyces versicolor T12 are 8-28° C. for 3-7 days. As a further preferred technical solution, the fermentation culture conditions of the Streptomyces versicolor T12 are 28° C. for 6 days.

[0017] As a preferred technical solution, the shake flask fermentation culture conditions of the Streptomyces versicolor T12 are: fermentation for 3-7 days, liquid volume 50-100mL / 500mL, inoculation amount 1-5%, pH 7.0-7.4, temperature 25-28°C, and shaking culture.

[0018] As a further preferred technical solution, the organic solvent is one of ethyl acetate, tetrahydrofuran or acetone.

[0019] The present invention studies the crude extract of blue pigment and finds that the solution in which the blue pigment is dissolved is blue when the pH value is ≥ 9, and is red when the pH value is ≤ 5.5.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0021] 1. The Streptomyces T12 of the present invention is a newly discovered strain of the genus Streptomyces, which is significantly different from existing strains of the same genus. The results of genome sequence analysis of the Streptomyces T12 of the present invention show that the digital DNA-DNA hybridization value of the Streptomyces T12 of the color change and S. cyaneus NRRL B-2296T is 52.1%, which is far lower than 70%. The results of polyphasic taxonomic identification show that the Streptomyces T12 of the color change is a new species strain of the genus Streptomyces.

[0022] 2. The pigment production of Streptomyces chromophore T12 of the present invention is significantly higher than that of Streptomyces coelicolor. The blue pigment production of Streptomyces T12 of the present invention is more than 3 times that of Streptomyces coelicolor M145.

[0023] 3. The pigment produced by Streptomyces T12 of the present invention has good thermal stability. When treated at 70-100°C for 2 hours, the absorbance value of its aqueous solution at 584nm remains basically unchanged, and the pigment stability is significantly higher than that of pigments produced by other reported strains. The pigment loss rate is 2.55% when treated at 100°C for 2 hours; while the blue pigment produced by Pseudomonas fluorescens B1 CGMCC No.1194 has a loss rate of 60.57% when treated at 100°C for 4 hours; the blue pigment produced by Xanthomonas B29 CGMCC No.3699 has a pigment loss rate of 18.8% when treated at 100°C for 2 hours, so the pigment loss rate in 4 hours is greater than 18.8%.

[0024] Collection information:

[0025] Bacteria name: Streptomyces versicolor

[0026] Latin: Streptomyces discolor

[0027] Strain number: T12.

[0028] Classification and nomenclature: Streptomyces discolor T12.

[0029] Depository: General Microbiology Center, China National Microbiological Culture Collection Administration.

[0030] The abbreviation of the collection agency is: CGMCC.

[0031] Address of the preservation institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Deposit number: CGMCC No.4.7887.

[0033] Date of preservation: June 19, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a phylogenetic tree constructed based on the 16S rRNA gene sequences of Streptomyces versicolor T12 and its reference strain.

[0035] Figure 2 This is a colony photo of Streptomyces versicolor T12 of the present invention.

[0036] Figure 3 This is an optical microscope photograph of mycelium of Streptomyces versicolor T12 of the present invention.

[0037] Figure 4 The following are scanning electron microscope photos of spores and spores of Streptomyces versicolor T12 of the present invention.

[0038] Figure 5The results are thin layer chromatography test results of the blue pigment crude extract of Streptomyces versicolor T12 and Streptomyces coelicolor of the present invention.

[0039] Figure 6 This is the solubility characteristics of pigments in different solvents.

[0040] Figure 7 The color change of blue pigment solution at different pH values ​​and the full wavelength scanning absorption curve.

[0041] Figure 8 This is the effect of ultraviolet light on the stability of crude blue pigment.

[0042] Fig. 9 The effect of food additives on the stability of crude blue pigment.

[0043] Fig.10 This is the effect of metal ions on crude blue pigment. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0045] A method for producing blue pigment of the present invention, and the related experiments for preparing the blue pigment are as follows:

[0046] Example 1: Isolation, screening and identification of Streptomyces versicolor T12 strain of the present invention

[0047] 1. Sample collection:

[0048] The soil was collected from Nongcui Garden of Anhui Agricultural University in Hefei, Anhui Province.

[0049] 2. Isolation and screening of strains

[0050] Soil extract medium: Weigh 500 g of soil, add it to 2 L of sterilized water, stir to mix, let it stand for 10-20 min, take the supernatant, dilute it 3 times and 10 times respectively, add 2% agar, pH 7.0-7.2, prepare soil extract medium, and pour it into plates.

[0051] Use the dilution method to separate, weigh 10.0g of soil sample, pour it into a triangular flask containing 100mL of sterile water, shake it well, dilute it in sequence, and make a soil suspension; take 100μL of soil suspension and spread it on the above-mentioned separation medium plate, culture it at 28℃ for 2-3 days, select blue single colonies, and purify them by repeated streaking. Vigorous blue single colonies can be grown on the soil extract medium; through microscopic examination, until the microscopic examination is a pure strain (consistent cell morphology), this strain is named T12, that is, Streptomyces (S.discolor) T12. For short-term storage, place it in a 4℃ refrigerator and activate it before the experiment. It can also be stored for a long time at -80℃ in a mixture of R2A and 40% glycerol.

[0052] Prepare ISP2 solid culture medium to activate the strain: 0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 20.0 g agar, add 1000 mL water, and adjust the final pH to 7.0-7.4.

[0053] 3. Identification of strains

[0054] The strain samples were taken after culturing in R2A medium at 28°C for 3 days, and the morphological characteristics of mycelium and spores of the strain were observed under optical microscope and electron microscope. Gram staining experiment was performed using standard Gram staining method. The culture characteristics of Streptomyces versicolor T12 under different temperatures (8, 20, 28, 30, 33°C), different NaCl concentrations (0, 0.3, 0.5, 0.7, 1, 1.5, 2%) and different initial pH of the medium (5.0, 6.0, 7.0, 8.0, 9.0, 10.0) were analyzed. 3% (v / v) H 2 O 2 The catalase activity of freshly grown cells was determined by adding 2% ethanol and 1% ethanol. The oxidase activity was detected by using the oxidase reagent bioMérieux according to the standard procedure. Indole and hydrogen sulfide experiments were performed according to the Tindall experimental method. The ability to utilize different carbon and nitrogen sources and the analysis of cell wall amino acids and sugar components were also studied.

[0055] The results show that the front of the colony of S. discolor T12 on the soil extract is white or blue. The colony photo of the Streptomyces discolor T12 of the present invention is as follows: Figure 2 As shown, the colony diameter is 3-4 mm, and it is radiating and circular; the surface is covered with dense creeping aerial hyphae ( Figure 3 ). Compared with S. cyaneus NRRLB-2296 T The aerial hyphae of Streptomyces versicolor T12 are more developed, thicker in diameter and more numerous. The spores are spiral, with a small number of branches, gathered into dense masses and diffused around. Under the scanning electron microscope, the spores are arranged in a spiral shape, with thorns on the outer surface of the spores, and the shape is similar to that of Xanthium sibiricum. The spores are about 0.9-1.1 microns long and 0.4-0.5 microns wide ( Figure 4). Thin layer chromatography and high performance liquid chromatography were used to analyze the cell wall amino acids, whole cell sugars, polar lipids and respiratory quinones of Streptomyces versicolor T12. The main component of the cell wall amino acid of strain T12 is LL-DAP (LL-diaminopimelic acid,), the cell wall mainly contains ribose and glucose, the main polar lipids are phosphatidylethanolamine (PE), phosphatidylinositol (PI) and diphosphatidylglycerol (DPG), and it also contains phosphatidylinositol mannoside (PIM), 5 unidentified phospholipids (PL1-5), 2 unidentified amino lipids (AL1-2) and 1 unidentified glycolipid, which is consistent with the composition characteristics of polar lipids of Actinomycetes strains, indicating that strain T12 belongs to the genus Actinomycetes.

[0056] The 16s rDNA sequence of S. discolor T12 was determined. The 16s rDNA sequence of S. discolor T12 of the present invention is shown in SEQ. No. 1, and its sequence is registered in Genbank as OQ154868. A phylogenetic analysis was performed based on the 16S rDNA gene sequences of S. discolor T12 and the model strain and reference strain with the closest relationship thereto in GenBank. A phylogenetic tree was constructed using MEGA6 software using the neighbor-joining method, the maximum likelihood method, and the maximum parsimony method. Figure 1 The draft genome of the strain was determined, and the results showed that the strain was similar to the reference strain S. cyaneus NRRLB-2296 T The dDDH value was 51.2%, which was much lower than 70%, indicating that strain T12 was different from S. cyaneus NRRLB-2296. T The new strain has a G+C content of 70.2 mol%.

[0057] Table 1 Differences in physiological characteristics between Streptomyces versicolor T12 and existing model strains of Streptomyces

[0058]

[0059]

[0060] Note: “+” means positive reaction, “-” means negative reaction.

[0061] Example 2 Preparation of blue pigment using S. discolor T12 strain

[0062] 1. Strain activation

[0063] Prepare solid ISP2 medium. Sterilize the prepared medium at 121°C for 20-30 minutes, pour into a plate, inoculate S. discolor T12 after cooling, and culture at 28°C for 4-7 days.

[0064] Prepare liquid R5 medium for strain fermentation: sucrose 103g, glucose 10g, magnesium chloride hexahydrate 10.12g, potassium sulfate 0.25g, TES 5.73g, yeast extract 5g, casamino acids 0.1g, trace elements 2mL, distilled water to 1000mL. For solid culture, add agar with a final concentration of 2.0%. After sterilization, add potassium dihydrogen phosphate (0.5%) 10mL, L-proline (20%) 15mL, calcium chloride dihydrate (5M) 4mL, sodium hydroxide (1M) 7mL, and adjust the pH to 7.0. Pick an appropriate amount of single colonies and inoculate them in liquid culture medium, and shake and culture at 28℃ for 4-7d.

[0065] 2. Extraction of blue pigment

[0066] The pigment produced by Streptomyces versicolor T12 turns blue under alkaline conditions and red under acidic conditions, and can only be extracted by ethyl acetate under acidic conditions, so ethyl acetate is selected as the best pigment extraction solvent. Add dilute hydrochloric acid to the fermented bacterial liquid, and when the solution changes from blue to red, add ethyl acetate and other organic solvents to extract the blue pigment, shake at 16℃ for 2h, and after standing and stratifying, transfer the upper ethyl acetate phase pigment solution to a new bottle. If there is still pigment remaining in the lower bacterial liquid, repeat the above steps until no more pigment is extracted. The obtained supernatant is evaporated and dried by a rotary evaporator to obtain the crude extract of blue pigment.

[0067] 3. Thin layer chromatography analysis of blue pigment crude extract

[0068] A 50×100G silica gel plate was used, and the developing solvent was benzene:acetic acid (9:1). The results were as follows: Figure 5 As shown, the blue pigment contains at least 5 components, while the blue pigment extract of Streptomyces coelicolor has only two components, and the blue pigment components produced by the two strains are obviously different. The blue pigment yield of Streptomyces versicolor T12 of the present invention is significantly higher than that of Streptomyces coelicolor, and the blue pigment yield of Streptomyces T12 of the present invention is more than 3 times that of Streptomyces coelicolor.

[0069] Example 3 Detection of the physicochemical properties of the blue pigment obtained by fermentation of the S. discolor T12 strain of the present invention

[0070] 1. Solubility characteristics: Weigh 5 mg of crude blue-purple pigment extract and dissolve it in 1 mL of water, acetonitrile, methanol, ethanol, isopropanol, acetone, tetrahydrofuran, ethyl acetate, chloroform, n-hexane, petroleum ether (60-90) (the order of the above reagents is the same as Figure 6 The pigment was dissolved in each solvent in the same order as in the above examples. The pigment was shaken and dissolved at room temperature for 1 hour. The solution was allowed to stand and the dissolution of the pigment in each solvent was observed. The results were as follows: Figure 6 As shown, the blue pigment is soluble in acetonitrile, methanol, ethanol, acetone, tetrahydrofuran, and ethyl acetate, slightly soluble in water, and difficult to dissolve in n-hexane and petroleum ether, among which the solubility is the highest in methanol, acetone, and tetrahydrofuran.

[0071] 2. Thermal stability of pigments

[0072] The blue pigment extract obtained by the present invention was prepared into an aqueous solution with a concentration of 3 mg / mL, adjusted to pH 9, and placed in a water bath at 28°C, 40°C, 60°C, 80°C, and 100°C for 2 hours, respectively. The absorbance at 584nm was measured to calculate the residual amount of the pigment. The results showed that after the pigment was treated at high temperature for 2 hours, except for a loss rate of 2.55% at 100°C, the other losses were less than 2%, indicating that the pigment of the present invention has strong heat resistance.

[0073] Table 2 Pigment loss rate at different temperatures

[0074] Temperature(℃) Processing time (h) Absorbance (584nm) Loss rate (%) 70 2 0.953 0.7％ 80 2 0.945 1.65％ 90 2 0.944 1.67％ 100 2 0.938 2.55％

[0075] 3. Acid-base stability of pigments

[0076] like Figure 7 , HCl or NaOH solution is added to the pigment dissolved by ethyl hexanoate in 10mL, and the pH value of the solution is measured when the color of the pigment solution changes. It is found that when the pigment solution pH≥9 is blue, the solution turns blue, and the color deepens as the pH increases. When pH=7.0, the pigment solution is purple, and when pH≤5.5, the pigment solution is red. Full wavelength scanning is performed using an ultraviolet-visible spectrophotometer, and the results show that the pigment solution has specific absorption peaks at wavelengths of 370nm and 570nm respectively; when the pigment solution pH≤5.5, the secondary peak (570nm) disappears. Moreover, the pigment obtained by the present invention and the acid-base reaction discoloration belong to a reversible reaction, and alkali is added to the pigment solution that turns red by adding acid, and the pigment turns blue again, and it will turn red again by adding acid again, and the color of the pigment solution depends on the pH value.

[0077] 4. Photostability of pigments

[0078] The blue pigment extract obtained by the present invention is prepared into a 3 mg / mL aqueous solution, adjusted to pH 9, added into a quartz cuvette, and irradiated under ultraviolet light. After a certain period of time, the pigment loss rate is as follows: Figure 8 The results show that the pigment solution OD 584 nm The absorbance value decreased slightly, and the pigment solution did not change significantly, indicating that it has good tolerance to ultraviolet rays.

[0079] 5. Effect of food additives on pigment stability

[0080] The blue pigment extract obtained by the present invention is prepared into a 3mg / mL aqueous solution, adjusted to pH 9 for later use, 2.5mL of the pigment solution is taken, 0.5mL and 1mL of 10% sucrose and salt (sodium chloride) solution are added respectively, and then 5mL is supplemented with distilled water, and 2.5mL of the pigment alcohol solution plus 2.5mL of deionized water is used as a control. After mixing and standing for a period of time, the OD of each is measured. 584 The results showed that additives such as sucrose and salt had no effect on the stability of the pigment ( Fig. 9 ).

[0081] 6. The influence of metal ions on crude blue pigment

[0082] The blue pigment extract obtained by the present invention is prepared into a 3 mg / mL aqueous solution, adjusted to pH 9 for standby use, 5 test tubes are taken and 3 mL of the pigment solution is added respectively, and a certain amount of potassium chloride, sodium chloride, magnesium chloride hexahydrate, and anhydrous aluminum chloride are added to 4 of them respectively so that the concentration in the solution is 1 mol / L, and the other test tube is used as a control. After mixing and standing for a period of time, the OD values ​​of each test tube are measured. 584 The results show that metal ion K + 、Na + Mg 2+ and Al 3+ The addition of will increase the OD of T12 pigment solution 584 The value increases, but the pigment stability is not affected much (such as Fig.10 shown).

[0083] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0084] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0085] The present invention is not limited to the above description of the embodiments. Any improvement or modification made by those skilled in the art based on the contents disclosed by the present invention without creative work shall be within the protection scope of the present invention.

[0086] DTD version: V1_3

[0087] File name: A blue pigment-producing Streptomyces chromophore and a method for producing blue pigment.xml

[0088] Software Name: WIPOSequence

[0089] Software version: 2.3.0

[0090] Date of creation: 2024-07-01

[0091] Basic Information:

[0092] Current application / applicant profile name: Anhui Agricultural University

[0093] Applicant's name: Anhui Agricultural University

[0094] Applicant's name / language:zh

[0095] Applicant's name or title / Latin name:Anhui Agricultural University

[0096] Inventor Name: Mei Fengmin

[0097] Inventor Name / Language:zh

[0098] Inventor Name / Latin Name:Mei fengmin

[0099] Invention title: A blue pigment-producing Streptomyces chromophore and a method for producing blue pigment (zh)

[0100] Total number of sequences: 1

[0101] sequence:

[0102] Serial number (ID): 1

[0103] Length:1492

[0104] Molecule type: DNA

[0105] Feature Position / Qualifier:

[0106] -source,1..1492

[0107] >mol_type,genomic DNA

[0108] >organism,unidentified

[0109] Residue:

[0110]

[0111]

Claims

1. A blue pigment-producing Streptomyces T12, the depository of which is the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit registration number: CGMCC No.4.7887.

2. The Streptomyces versicolor T12 according to claim 1, characterized in that The growth characteristics of the Streptomyces versicolor T12 are: aerobic, Gram-positive, catalase-positive, able to form well-developed aerial hyphae on ISP2 and soil extract medium, with spiral spores and thorns on the outside of the spores, similar to Xanthium sibiricum. The colony is round, convex, blue-gray or white on the front, blue on the back, with a growth temperature range of 15-42°C, NaCl tolerance of less than or equal to 6%, and a pH range of 4-12.

3. Use of Streptomyces versicolor T12 according to claim 1 in producing blue pigment by fermentation.

4. A method for producing blue pigment using the Streptomyces versicolor T12 according to claim 1, characterized in that: The Streptomyces versicolor T12 was fermented and cultured, and the cells were collected by centrifugation after the fermentation was completed. The blue pigment was extracted with an organic solvent, and the extract was concentrated and dried by rotary evaporation to obtain a crude blue pigment extract.

5. The method for producing blue pigment by using Streptomyces versicolor T12 according to claim 1 according to claim 4, characterized in that: The liquid culture medium used for fermentation of Streptomyces versicolor T12 contains per liter: sucrose 103g, glucose 10g, MgCl2·6H2O 10.12g, K2SO4 0.25g, TES 5.73g, yeast extract 3-5g, casamino acid 0.1g, trace elements 2mL, distilled water to 1000mL, and after sterilization before use, it is necessary to add 0.5% potassium dihydrogen phosphate solution 10mL, 20% L-proline 15mL, 5M calcium chloride dihydrate 4mL, and 1M sodium hydroxide 7mL.

6. The method for producing blue pigment by using Streptomyces versicolor T12 according to claim 1 according to claim 4, characterized in that: The fermentation culture conditions of the Streptomyces versicolor T12 are as follows: fermentation culture at 8-28° C. for 3-8 days.

7. The method for producing blue pigment by utilizing Streptomyces versicolor T12 according to claim 1 according to claim 6, characterized in that The fermentation culture conditions of the Streptomyces versicolor T12 are 28° C. for 6 days.

8. The method for producing blue pigment by using Streptomyces versicolor T12 according to claim 1 according to claim 4, characterized in that: The shake flask fermentation culture conditions of the Streptomyces versicolor T12 are: fermentation for 3-7 days, liquid volume 50-100mL / 500mL, inoculation amount 1-5%, pH 7.0-7.4, temperature 25-28°C, and shaking culture.

9. The method for producing blue pigment by using Streptomyces versicolor T12 according to claim 1 according to claim 4, characterized in that: The organic solvent is one of ethyl acetate, tetrahydrofuran or acetone.

10. The method for producing blue pigment using Streptomyces versicolor T12 according to claim 4, characterized in that: When the pH of the solution containing the blue pigment is ≥9, the pigment solution is blue, and when the pH is ≤5.5, the pigment solution is red.

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