A pigment extraction method and application of a red pigment-producing streptomyces spectabilis s-13

Abstract

This invention discloses a red pigment-producing Streptomyces spectabilis ( Streptomyces spectabilis This invention discloses a method and application for pigment extraction from *Streptomyces spectinomyces* S-13. The method involves fermenting *Streptomyces spectinomyces* S-13 with the addition of a surfactant to extract the pigment. This method releases intracellular pigments into the fermentation broth, significantly increasing pigment yield and simplifying the extraction and purification process. The red pigment produced by *Streptomyces spectinomyces* S-13 exhibits excellent stability, antioxidant activity, and antibacterial effects, making it suitable for applications in the printing and dyeing industry. The pigment extraction method described in this invention offers advantages such as short production cycle, low cost, safety, and environmental friendliness, overcoming the limitations of traditional microbial pigment production processes, including high extraction difficulty, low yield, and limited applicability.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and more specifically, to a red pigment-producing Streptomyces speciosa (…). Streptomyces spectabilis Pigment extraction methods and applications of S-13. Background Technology

[0002] Textiles are an important part of human life, but the pigments and dyes produced and used in the textile industry are almost entirely chemically synthesized, and the discharge of dyeing wastewater causes great harm to the ecological environment. The development and utilization of natural textile dyes is of great significance for solving the environmental pollution problems caused by traditional chemical dyes. As people pay more and more attention to environmental protection and health, natural pigments from different sources are gradually becoming a focus of attention in various industries due to their good safety, environmental compatibility, and biodegradability.

[0003] Microbial pigments, possessing unique advantages distinct from animal and plant pigments, are poised to become an inevitable trend in the industrial production of natural pigments. A wide variety of microorganisms, such as actinomycetes, filamentous fungi, yeasts, and algae, are available in nature for fermentation production of natural pigments. The production process of microbial pigments offers advantages such as a wide range of raw material sources, rapid cell growth, short production cycles, no seasonal or geographical limitations, economic efficiency, energy conservation, and ease of large-scale industrial production. These advantages completely overcome the shortcomings of other types of pigments in terms of production processes and applicability, demonstrating enormous potential for development and utilization. Of particular note is actinomycetes, an important microbial resource, which possess unique advantages among fermentation industrial strains due to the production of various antibiotics and non-antibiotic active substances, including vitamins, enzyme inhibitors, immunomodulators, antiparasitic agents, and natural pigments, during their fermentation process.

[0004] The main challenges in the large-scale application of microbial pigments in textile dyeing include: a shortage of high-pigment-producing microbial strains; immature and environmentally unfriendly extraction and purification conditions for microbial pigments; and low color fastness and unstable dyeing performance of microbial pigments on fabrics. CN202011344426.0 discloses a method for preparing and dyeing with *Streptomyces spectinomyces* red pigment, which is a natural pigment with characteristics of safety, non-toxicity, non-carcinogenicity, and biodegradability. Dissolving *Streptomyces spectinomyces* red pigment in an appropriately proportioned solution, combined with a suitable dyeing process, results in a high dyeing rate. After dyeing, the color fastness to washing and rubbing of silk fabrics are both grade 4, meeting the dyeing standards for silk fabrics. Furthermore, it exhibits certain resistance to Gram-positive bacteria such as *Staphylococcus aureus*, Gram-negative bacteria such as *Klebsiella pneumoniae*, and fungi such as *Candida albicans*. However, the water-soluble extracellular pigments in the microbial pigments disclosed in this patent are mainly produced directly through liquid fermentation. In contrast, the lipid-soluble intracellular pigments are largely present inside the cell, usually bound to proteins and lipids, and rarely released into the fermentation broth, resulting in low solubility in water. Furthermore, due to negative feedback inhibition, the accumulation of pigments within the cell inhibits further production, significantly limiting the final pigment yield. Summary of the Invention

[0005] The technical problem this invention aims to solve is the shortcomings of existing methods that use microbial strains as intracellular pigments, resulting in high extraction difficulty and low yield. This invention provides a red pigment-producing *Streptomyces spectabilis* strain (…). Streptomyces spectabilis Pigment extraction method of S-13.

[0006] Another technical problem of the present invention is to provide a *Streptomyces spectabilis* strain that produces erythropoietin (…). Streptomyces spectabilis Application of pigments extracted from S-13.

[0007] The objective of this invention is achieved through the following technical solution: A spectacular Streptomyces that produces red pigment ( Streptomyces spectabilis The method for extracting pigments from S-13, wherein the Streptomyces speciosa S-13 is deposited at the China General Microbiological Culture Collection Center, China, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on July 8, 2021, with accession number CGMCC No. 22845.

[0008] The above-mentioned method for extracting pigments from *Streptomyces speciosa* S-13, which produces red pigments, includes the following steps: S1. Inoculate the spore suspension of Streptomyces spectinomyces S-13 into a sterile liquid culture medium, and culture it in the dark at room temperature with shaking to obtain a seed liquid. Then, inoculate the seed liquid into a liquid fermentation medium, add a surfactant, and then culture the inoculated fermentation medium with shaking in the dark to obtain a fermentation broth containing the pigment of Streptomyces spectinomyces S-13. S2. After centrifuging the fermentation broth, the fermentation supernatant and precipitated mycelium are obtained. The fermentation supernatant is concentrated and dried to obtain red pigment powder.

[0009] Furthermore, the sterile liquid culture medium and the liquid fermentation medium were Gao's No. 1 liquid culture medium, the components of which included: 20 g / L soluble starch, 1 g / L KNO3, 0.5 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O and 0.3 g / L yeast extract.

[0010] Further, the surfactant is one or more of polyethylene glycol octylphenyl ether, polyethylene glycol, and polyoxyethylene sorbitan monooleate. Preferably, the surfactant is polyethylene glycol octylphenyl ether.

[0011] Further, the concentration of the surfactant is 15-45 g / L. Preferably, the concentration of the surfactant is 25 g / L.

[0012] Furthermore, the inoculation amount of the fermentation culture seed liquid is 0.5-9%. Preferably, the inoculation amount of the fermentation culture seed liquid is 7%.

[0013] Furthermore, the fermentation culture time is 3-12 days. Preferably, the fermentation culture time is 5 days.

[0014] Furthermore, the fermentation culture temperature is 25-37℃. Preferably, the fermentation culture temperature is 35℃.

[0015] Furthermore, the initial pH of the fermentation culture is controlled at 5-10. Preferably, the initial pH of the fermentation culture is controlled at 7.

[0016] Application of red pigment obtained from the above-mentioned method of pigment extraction from Streptomyces spectabilis S-13, which produces red pigment, in fabric dyeing.

[0017] Furthermore, the dyeing steps for silk fabrics include: at room temperature, immersing the silk fabric in anhydrous ethanol containing Streptomyces speciosa S-13 red pigment at a certain dyeing bath ratio, heating to the dyeing temperature for constant temperature dyeing, and after dyeing, soaping and washing to obtain the dyed silk fabric.

[0018] Further, the staining bath ratio is 25-100:1; the anhydrous ethanol concentration is 25-100%; the pigment concentration is 5-20 g / L; the staining temperature is 50-100℃; the staining time is 15-120 min; and the staining pH value is 4-9.

[0019] Compared with existing technologies, the beneficial effects are: This invention utilizes *Streptomyces spectinomyces* S-13 isolated from soil. By employing a fermentation culture system with added surfactants, *Streptomyces spectinomyces* S-13 releases pigments that are normally difficult to access intracellularly directly into the fermentation broth during its growth and reproduction. This reduces the inhibitory effect of high-concentration intracellular pigments, significantly increasing pigment yield. Furthermore, this invention allows for the direct extraction of intracellular red pigments produced during *Streptomyces spectinomyces* S-13 fermentation into the extracellular fermentation broth, avoiding the complex steps of collecting and breaking mycelia after fermentation and extracting intracellular red pigments using large amounts of organic solvents. This greatly simplifies the pigment extraction and purification process.

[0020] The pigment extraction method described in this invention has the advantages of short production cycle, economic and energy saving, safety and environmental protection. The red pigment produced has good stability, pH sensitivity, antioxidant activity and antibacterial activity, and can be used as an excellent functional pigment in the printing and dyeing field. Attached Figure Description

[0021] Figure 1 This is a diagram showing the culture characteristics of Streptomyces speciosa S-13 strain in Example 1 and the distribution of pigments produced under different fermentation conditions; Among them, the morphological characteristics of a-Streptomyces spectinomyces S-13 strain on Gao's No. 1 solid plate; the fermentation products of b-Streptomyces spectinomyces S-13 strain in ordinary Gao's No. 1 liquid medium (producing only intracellular pigments); and the fermentation products of c-Streptomyces spectinomyces S-13 strain in a surfactant liquid fermentation system (producing a large amount of extracellular pigments).

[0022] Figure 2 This is a phylogenetic tree of Streptomyces spectabilis S-13 strain constructed based on 16S rDNA sequence homology.

[0023] Figure 3 This is a comparison chart of fermentation products under liquid fermentation systems with different types of surfactants; Where a represents no surfactant added; b represents PEG 400 added; c represents Triton X-100 added; d represents Tween 20 added; e represents SDS added; and f represents Tween 80 added.

[0024] Figure 4Examples 1 show the UV-Vis spectra and color value statistics of pigments in liquid fermentation systems with different types of surfactants. Where 'a' represents the UV-Vis spectra of intracellular pigments in liquid fermentation systems with different types of surfactants. b shows the UV-Vis spectra of extracellular pigments in liquid fermentation systems with different types of surfactants, and c shows the statistical graphs of intracellular pigment color value, extracellular pigment color value, and total color value.

[0025] Figure 5 This is a graph showing the color value changes of extracellular pigment solutions in the liquid fermentation system of different concentrations of Triton X-100 in Example 2.

[0026] Figure 6 This is a graph showing the trend of color value changes in the extracellular pigment solution under different fermentation times in the liquid fermentation system of Example 3.

[0027] Figure 7 This is a graph showing the color value changes of extracellular pigment solutions under different fermentation temperatures in the liquid fermentation system of Example 4.

[0028] Figure 8 This is a graph showing the color value changes of extracellular pigment solutions under different fermentation temperatures in the liquid fermentation system of Example 5.

[0029] Figure 9 This is a graph showing the trend of color value changes in extracellular pigment solutions under different inoculum amounts in the liquid fermentation system of Example 6.

[0030] Figure 10 This is a graph showing the trend of color value changes of extracellular pigment solutions under different liquid volumes in Example 7.

[0031] Figure 11 This is a graph showing the antibacterial activity of different pigment samples against the test bacteria in Example 14.

[0032] Among them, a-Staphylococcus aureus; b-Bacillus subtilis; c-Pseudomonas aeruginosa; d-Escherichia coli; e-Candida parapsilosis; 1-Glacial acetic acid; 2-Glacial acetic acid solution of pigment; 3-Anhydrous ethanol; 4-Ethanol solution of pigment; 5-Deionized water; 6-Penicillin.

[0033] Figure 12 This is a diagram showing the antioxidant activity of the pigment in Example 15; Where a represents the scavenging rate of DPPH· by different concentrations of pigment, and b represents the scavenging rate of ABTS by different concentrations of pigment. + • Clearance rate.

[0034] Figure 13 These are images of Streptomyces spectabilis S-13 pigment dye and its dyeing effect on silk fabrics. Wherein, a is the dye prepared from the red pigment of Streptomyces speciosa S-13; b is the dyeing effect of the dye on silk fabrics. Detailed Implementation

[0035] The following examples further explain and clarify the invention, but the specific examples do not limit the invention in any way. Unless otherwise specified, the methods and equipment used in the examples are conventional methods and equipment, and the raw materials used are all commercially available.

[0036] Example 1 This embodiment provides *Streptomyces spectabilis* (… Streptomyces spectabilis The method for extracting pigments from S-13 and the determination of pigment color value include the following steps: S1. Fermentation culture of the strain Spore suspensions of *Streptomyces spectinomyces* S-13 were inoculated at a 5% inoculum into Gao's No. 1 liquid culture medium and cultured at 28°C and 225 rpm in the dark for 30 h to obtain seed culture. 1.5 mL of each seed culture was inoculated into six 80 mL bottles of Gao's No. 1 liquid fermentation medium. One bottle was without surfactant, while the other five bottles were inoculated with the following surfactants at the following concentrations: 16 g / L Tween 80 (polyoxyethylene sorbitan monooleate), 22 g / L Triton X-100 (polyethylene glycol octylphenyl ether), 18 g / L Tween 20 (polyoxyethylene sorbitan monolaurate), 5 g / L SDS (sodium dodecyl sulfate), and 20 g / L PEG 400 (polyethylene glycol 400). The inoculated fermentation medium was then cultured at 30°C and 175 rpm in the dark for 7 days to obtain fermentation broth containing *Streptomyces spectinomyces* S-13 pigment.

[0037] S2. Extraction of extracellular pigments After fermentation, the fermentation broth was centrifuged at 4℃ and 9600 rpm. The supernatant was collected and transferred to a rotary evaporator. After being evaporated to a paste at 40℃, it was dried into pigment powder in a vacuum drying oven.

[0038] S3. Determination of extracellular pigment value Dissolve 0.05 g of pigment powder in 5 mL of glacial acetic acid, then dilute it three times. Perform a full wavelength scan within the range of 400–700 nm using a UV-Vis spectrophotometer, recording the wavelength at which the diluted pigment solution exhibits maximum absorbance. Then, apply the formula... ( A The absorbance of the test sample; α The extracellular pigment value was calculated based on the dilution factor. The pigment values ​​measured below are the sum of the values ​​at two wavelengths, 505 nm and 536 nm.

[0039] Example 2 This embodiment follows the method described in Example 1. Different concentrations of surfactant were used for fermentation culture in this embodiment. The specific steps are as follows: 1 mL of seed culture was transferred to 30 mL of fermentation medium (seed culture inoculum volume: 3%), and Triton X-100 concentrations were set at 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, and 45 g / L, respectively, with the pH adjusted to 7.5. The fermentation medium was incubated at 30℃ and 175 rpm in the dark for 7 days. The extracellular pigment value was measured to determine the optimal surfactant concentration required for pigment production by *Streptomyces spectinomyces* S-13 strain during fermentation.

[0040] Example 3 This embodiment follows the method described in Example 2. Different fermentation times were used in this embodiment, and the specific steps are as follows: 1 mL of seed culture was transferred to 30 mL of fermentation medium (seed culture inoculation amount was 3%), the Triton X-100 concentration was set to 45 g / L, the pH was adjusted to 7.5, and the fermentation medium was placed in a shaker at 30℃ and 175 rpm in the dark for shaking culture. Starting from the third day of culture, the extracellular pigment value was measured every 24 h to determine the optimal production time of pigment by fermentation of Streptomyces spectinomyces S-13 strain.

[0041] Example 4 This embodiment follows the method described in Example 2. Different fermentation temperatures were used for fermentation in this embodiment. The specific steps are as follows: 1 mL of seed culture was transferred to 30 mL of fermentation medium (seed culture inoculum volume: 3%). The Triton X-100 concentration was set to 45 g / L, and the pH was adjusted to 7.5. The shaker temperatures were set to 25℃, 28℃, 30℃, 35℃, and 37℃, respectively, and cultured at 175 rpm in the dark for 7 days. After the culture was completed, the extracellular pigment value was measured to determine the optimal fermentation temperature for pigment production by *Streptomyces spectinomyces* S-13 strain.

[0042] Example 5 This embodiment follows the method described in Example 2. Different initial pH values ​​were used for fermentation culture in this embodiment. The specific steps are as follows: One mL of seed culture was transferred to 30 mL of fermentation medium at pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively (seed culture inoculum size 3%). The Triton X-100 concentration was 45 g / L. The fermentation medium was incubated at 30℃ and 175 rpm in the dark for 7 days. After incubation, the extracellular pigment value was measured to determine the optimal initial pH for pigment production by *Streptomyces spectinomyces* S-13 strain.

[0043] Example 6 This embodiment follows the method described in Example 2. Different inoculum sizes were used for fermentation culture in this embodiment. The specific steps are as follows: The inoculum sizes were set to 0.5%, 1%, 3%, 5%, 7%, and 9%, respectively. The pH was adjusted to 7.5, and the Triton X-100 concentration was 45 g / L. The fermentation medium was incubated at 30℃ with shaking at 175 rpm in the dark for 7 days. After the culture was completed, the extracellular pigment value was measured to determine the optimal inoculum size for pigment production by *Streptomyces spectinomyces* S-13 strain.

[0044] Example 7 This embodiment follows the method described in Example 2. Fermentation was carried out in 250 mL Erlenmeyer flasks using the same liquid volume. The specific steps are as follows: Seed culture was transferred to fermentation medium with volumes of 10 mL, 20 mL, 30 mL, 40 mL, and 50 mL, respectively, with a seed culture inoculum size of 3%. The pH was adjusted to 7.5, and the Triton X-100 concentration was 45 g / L. The fermentation medium was incubated at 30℃ with shaking at 175 rpm in the dark for 7 days. After the incubation period, the extracellular pigment value was measured to determine the optimal liquid volume for pigment production by *Streptomyces spectinomyces* S-13 strain.

[0045] Analysis of Experimental Results Depend on Figure 1 It is known that the mycelium of Streptomyces S-13 in this invention is bright orange-red and contains a large amount of red pigment. Moreover, the pigment produced by Streptomyces S-13 is basically present in the mycelium and is an intracellular pigment.

[0046] Depend on Figure 2 It was found that the 16S rDNA sequence of Streptomyces S-13 showed 100% homology with Streptomyces spectabilis (KY604743.1) in the GenBank database. Based on the colony morphology and physiological and biochemical indicators, Streptomyces S-13 was preliminarily identified as Streptomyces spectabilis (KY604743.1). Streptomyces spectabilis ).

[0047] Depend on Figure 3-4It was found that without the addition of surfactant, *Streptomyces speciosa* S-13 exhibited high intracellular pigment values ​​and extremely low extracellular pigment values. When Triton X-100 was added, the fermentation broth showed a distinctly dark red color, with a significant decrease in intracellular pigment values ​​and the highest extracellular pigment values. When PEG 400 and Tween 20 were added, more intracellular pigments were retained within the mycelia, and the extracellular pigment values ​​measured in the fermentation broth were lower than those obtained with Triton X-100. When SDS and Tween 80 were added, there was virtually no cell growth in the culture medium, and the fermentation broth obtained from the liquid fermentation system was turbid and showed no cell growth, making it impossible to determine the intracellular and extracellular pigment values. Therefore, in this liquid fermentation system, Triton X-100 is the optimal surfactant for pigment production. Since the extracellular pigments dissolved from Triton X-100 in the fermentation broth showed two relatively obvious absorption peaks at 505 nm and 536 nm, it indicates that the extracellular pigments produced by Streptomyces speciosa S-13 contain at least two major components.

[0048] Since the extracellular pigments dissolved from Triton X-100 in the fermentation broth showed two relatively obvious absorption peaks at 505 nm and 536 nm, it indicates that the extracellular pigments produced by *Streptomyces speciosa* S-13 contain at least two major components. Therefore, the sum of the color values ​​of the extracellular pigments at these two wavelengths was used as the evaluation index for the subsequent single-factor experiments. Figures 5-10 As shown, within the range of each single-factor optimization experiment, the optimal Triton X-100 concentration for the production of extracellular pigments by *Streptomyces speciosa* S-13 is 25 g / L; the optimal initial pH is 7; the optimal liquid volume is 30 mL in a 250 mL culture space; the optimal inoculum size is 7%; the optimal fermentation temperature is 35℃; and the optimal fermentation time is 5 days.

[0049] Example 8 This embodiment demonstrates the effect of temperature on pigment stability, with the following steps: Weigh 0.015 g of pigment powder and dissolve it completely in 10 mL of glacial acetic acid. Place the pigment solution in a constant temperature water bath at room temperature (25℃), 30℃, 50℃, 70℃, and 90℃ for 30 min. After removing it and letting it stand for 10 min, observe the color change. Mix 1 mL of pigment solution with 2 mL of glacial acetic acid and perform a full wavelength spectral scan in the range of 400~700 nm.

[0050] When the pigment solution was treated at 25~90℃ for 30 min, the color, characteristic absorption peak and maximum absorbance of the pigment solution did not change significantly, indicating that the pigment has high stability under different temperature treatments.

[0051] Example 9 This embodiment demonstrates the effect of light on pigment stability, and the steps are as follows: The pigment solution was placed under ultraviolet light, sunlight, and red light for irradiation. Every 30 minutes, it was taken out and the full wavelength spectrum was scanned in the range of 400~700 nm. After the scan was completed, the absorbance value at 536 nm was recorded and compared.

[0052] After the pigment solution was treated with red light, ultraviolet light and sunlight for 30 to 120 minutes, there were no significant changes in the color, characteristic absorption peak and maximum absorbance of the pigment solution, indicating that the pigment has high stability under different light treatments.

[0053] Example 10 This embodiment demonstrates the effect of pH on pigment stability, and the steps are as follows: The pigment solution was adjusted to pH values ​​of 4, 5, 6, 7, 8, 9, and 10 using hydrochloric acid and sodium hydroxide solutions, respectively. The pigment solution at each pH value was then subjected to a full-wavelength spectral scan in the wavelength range of 400-700 nm. The absorbance value at 536 nm was recorded and compared after the scan.

[0054] When the pH value increases from 4.0 to 10.0, the color and characteristic absorption peak of the pigment solution change significantly, and the color changes from rose red to bright red and then to yellow, indicating that the pigment is a pH-sensitive pigment.

[0055] Example 11 This embodiment demonstrates the effect of metal ions on pigment stability, and the steps are as follows: Add Ca to the pigment solution respectively 2+ Mg 2+ Zn 2+ Ag + K + Each pigment solution was subjected to a full-wavelength spectral scan in the wavelength range of 400-700 nm. After the scan, the absorbance value at 536 nm was recorded and compared.

[0056] When Ca 2+ Mg 2+ Zn 2+ When these metal ions are present, the maximum absorbance of the pigment solution increases significantly, indicating that these metal ions have a certain color-enhancing effect on the pigment.

[0057] Example 12 This embodiment provides the effects of oxidants, reducing agents, and food additives on pigment stability, and the steps are as follows: H2O2 solutions with concentrations of 4%, 8%, 12%, 16%, and 20% were prepared respectively. 500 μL of pigment solution was added to 500 μL of the above H2O2 solutions of different concentrations and mixed thoroughly. After standing for 30 min, the full wavelength spectrum was scanned in the wavelength range of 400-700 nm. The absorbance value at 536 nm was recorded and compared after the scan.

[0058] Na₂O₄S₂ solutions with concentrations of 4%, 8%, 12%, 16%, and 20% were prepared respectively. 500 μL of pigment solution was added to 500 μL of each of the above Na₂O₄S₂ solutions and mixed thoroughly. After standing for 30 min, a full-wavelength spectral scan was performed in the wavelength range of 400-700 nm. The absorbance value at 536 nm was recorded and compared after the scan.

[0059] Sodium chloride, sodium citrate, and sucrose solutions with concentrations of 4%, 8%, 12%, 16%, and 20% were prepared respectively. A 1.5 mL centrifuge tube containing 500 μL of pigment solution was taken, and 500 μL of the above-mentioned sucrose, sodium citrate, or sodium chloride solutions of different concentrations were added and thoroughly mixed. After standing for 30 min, a full-wavelength spectral scan was performed in the wavelength range of 400-700 nm. The absorbance value at 536 nm was recorded and compared after the scan.

[0060] When pigment solutions are treated with different concentrations of H₂O₂, Na₂O₄S₂, or food additives, H₂O₂ has a certain color-enhancing effect on the pigments, while Na₂O₄S₂, which has a reducing effect, negatively impacts the stability of the pigments. After treatment with different concentrations of food additives such as sodium chloride, sodium citrate, and sucrose, the characteristic absorption peaks of the pigments remain almost unchanged, indicating good stability.

[0061] Example 13 This embodiment demonstrates the effect of prolonged darkness on pigment stability, and the steps are as follows: Take 15 mL of pigment solution and store it at room temperature in the dark. Take 1 mL of pigment solution every 1 day, centrifuge at 4000 rpm for 5 min, and then perform a full wavelength spectrum scan in the wavelength range of 400-700 nm. After the scan, record the absorbance value at 536 nm and compare it.

[0062] After standing in the dark for 10 consecutive days, the color of the pigment solution remained stable without significant fading. During the standing process, the pigment showed a slight increase in solubility during the first 1-4 days, gradually stabilizing during the latter 5-10 days. This is likely due to the slow increase in solubility of the pigment in glacial acetic acid solution initially, reaching equilibrium later. The minimal change in absorbance at 536 nm over the 10 days indicates good stability of the pigment when stored in the dark.

[0063] Example 14 This embodiment provides the antibacterial performance testing of pigments, and the specific steps are as follows: Weigh 0.015 g of pigment powder and dissolve it thoroughly in 10 mL of glacial acetic acid and anhydrous ethanol to prepare two pigment solutions. These solutions were then analyzed with *Escherichia coli* (E. coli). Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Bacillus subtilis ( Bacillus subtilis Staphylococcus aureus ( Staphylococcus aureus ), Candida parapsilosis ( Candida parapsilosis Using penicillin as an indicator bacterium, 0.05 mg / mL penicillin was set as a positive control, and glacial acetic acid and anhydrous ethanol were set as negative controls. After placing the culture dishes at 4℃ for 1-2 h, they were then incubated upside down at 37℃ (bacteria) or 30℃ (yeast) for 24 h (bacteria, yeast). Each experiment was performed in triplicate. After all the test bacteria had grown, the diameter of the inhibition zone was measured and recorded.

[0064] Analysis of Experimental Results Depend on Figure 11 It can be seen that, compared with the positive and negative controls, the glacial acetic acid solution of the pigment showed excellent antibacterial effects against Escherichia coli, Staphylococcus aureus, and Candida glabrata, with inhibition zone diameters of 20.63±0.54 mm, 37.43±0.56 mm, and 10.13±0.17 mm, respectively. In addition, the ethanol solution of the pigment did not show significant antibacterial effects against the above-mentioned test bacteria, indicating that the antibacterial active component of the pigment produced by the surfactant liquid fermentation system is easily soluble in glacial acetic acid but insoluble in anhydrous ethanol.

[0065] Example 15 In this embodiment, the antioxidant activity of the pigments was determined using both the DPPH and ABTS methods. Figure 12 It is known that this pigment is effective against DPPH· and ABTS. + The clearance rate of · showed a clear concentration-dependent effect. As the pigment concentration increased, its effect on DPPH· and ABTS· decreased. + The removal rate of · also increased significantly, indicating a stronger removal effect. This result suggests that substances with antioxidant activity in the pigment gradually decompose or lose activity as the reaction time increases.

[0066] Example 16 This embodiment provides a method for dyeing silk fabrics using the pigment extracted in Example 2. The steps are as follows: At room temperature, the silk fabric is immersed in anhydrous ethanol containing Streptomyces speciosa S-13 red pigment at a bath ratio of 50:1. The pH is adjusted to 4-10, and the temperature is increased to 80°C at a heating rate of 3°C / min and then kept constant for 60 min. After dyeing, the excess dye is washed away with cold water, and then the fabric is boiled in a 5 g / L soap solution at 40°C for 30 min. After washing, the dyed silk fabric is obtained.

[0067] The color fastness to rubbing, washing, sunlight, and perspiration of dyed silk fabrics was determined in accordance with the national standards GB / T 3920-2008 "Textiles - Tests for Color Fastness to Rubbing", GB / T 3921-2008 "Textiles - Tests for Color Fastness to Rubbing", GB / T 8426-1998 "Textiles - Tests for Color Fastness to Rubbing", and GB / T 3922-2013 "Textiles - Tests for Color Fastness to Rubbing". The test results are shown in Table 1 below.

[0068] Table 1

[0069] Depend on Figure 13 It can be seen that the color of silk fabrics after being dyed with pigments extracted from Streptomyces speciosa S-13 remains consistent with the pigments. According to Table 1, the color fastness of silk fabrics dyed with Streptomyces speciosa S-13 pigments is around level 5 in terms of rubbing resistance, perspiration resistance, and soap washing resistance, which reaches the national superior grade level and has great application potential in the field of silk fabric dyeing.

[0070] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A spectacular Streptomyces species that produces red pigment ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The Streptomyces speciosa S-13 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 8, 2021, with accession number CGMCC No. 22845. The steps of the pigment extraction method include: S1. Inoculate the spore suspension of Streptomyces spectinomyces S-13 into a sterile liquid culture medium, and after shaking culture in the dark at room temperature, obtain the seed liquid. Then, inoculate the seed liquid into a liquid fermentation medium, add the surfactant polyethylene glycol octylphenyl ether, and then shake the fermentation medium after inoculation and ferment in the dark to obtain the fermentation broth containing the pigment of Streptomyces spectinomyces S-13. S2. After centrifuging the fermentation broth, the fermentation supernatant and mycelium are obtained. The fermentation supernatant is concentrated and dried to obtain red pigment powder.

2. The *Streptomyces spectabilis* species that produces red pigment according to claim 1 ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The sterile liquid culture medium and liquid fermentation medium were Gao's No. 1 liquid culture medium, which consisted of: 20 g / L soluble starch, 1 g / L KNO3, 0.5 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O and 0.3 g / L yeast extract, with the pH adjusted to 7.4~7.

6.

3. The *Streptomyces spectabilis* species producing red pigment according to claim 1 ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The concentration of the surfactant is 15-45 g / L.

4. The *Streptomyces spectabilis* species producing red pigment according to claim 1 ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The inoculum size for the fermentation culture seed liquid is 0.5-9%.

5. The *Streptomyces spectabilis* species producing red pigment according to claim 1 (… Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The fermentation time is 3-12 days.

6. The *Streptomyces spectabilis* species producing red pigment according to claim 1 ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The fermentation culture temperature is 25-37℃.

7. The *Streptomyces spectabilis* species producing red pigment according to claim 1 ( Streptomyces spectabilis The pigment extraction method of S-13 is characterized in that, The pH during cultivation is controlled at 5-10.

8. *Streptomyces spectabilis* producing red pigment according to any one of claims 1-7 ( Streptomyces spectabilis Application of red pigment obtained by the pigment extraction method of S-13 in fabric dyeing.

Images