Serratia marcescens strain HSU-5 capable of producing purpurin and application of serratia marcescens strain HSU-5

Abstract

The invention relates to a serratia marcescens strain HSU-5 for producing purpurin and application of the serratia marcescens strain HSU-5. The serratia marcescens strain HSU-5 capable of producing the purpurin is preserved in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, and the preservation number is CCTCC M 20241204. The serratia marcescens strain HSU-5 capable of producing purpurin has a smooth surface, has no obvious bulges, and can be separated as a single strain. Sequence comparison results show that the strain HSU-5 is a purpurine-producing strain belonging to serratia. The produced purpurin has good thermal stability, light stability, oxidation resistance and antibacterial activity, and the structure and the property of the pigment produced by the strain need to be deeply researched, so that the purpurin is expected to be popularized and applied in the industry.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a purple pigment-producing Serratia strain HSU-5 and applications thereof. Background Art

[0002] Pigments, as important additives, not only give products an attractive appearance and color, but also significantly enhance their market appeal and consumer purchasing power in industries such as food, pharmaceuticals, feed, and cosmetics. Traditional Chinese culinary culture places particular emphasis on the optimal combination of color, aroma, and flavor. Brightly colored foods often stimulate appetite and create a pleasant experience. However, as consumers become more health-conscious, safety requirements for pigments in food and related products are also increasing.

[0003] Pigments widely used in the current market are primarily categorized into two main categories: synthetic pigments and natural pigments. While synthetic pigments offer advantages such as strong coloring power, high stability, ease of use, and economical efficiency, they also carry potential health risks that should not be ignored. Some synthetic pigments and their metabolites may pose a risk to human health, and they are susceptible to contamination from pollutants such as heavy metals and organic solvents during production and processing, further exacerbating safety concerns. Therefore, the development of safe, reliable, and harmless alternative pigments has become a key focus of the industry.

[0004] Natural pigments are becoming a preferred choice in the pigment market due to their safety, non-toxicity, and nutritious properties. Natural pigments primarily originate from plants, animals, and microorganisms. While naturally pure, pigments extracted from plants and animals suffer from low content, difficulty in extraction, high cost, and susceptibility to oxidation and fading, limiting their large-scale application. In contrast, pigment extraction through microbial fermentation offers significant advantages, including low production costs, ease of industrial production, and the ability to effectively avoid these issues.

[0005] Microbial pigments, secondary metabolites of microorganisms, are abundant and diverse in color, including red, orange, yellow, green, blue, and purple. Purple pigments, in particular, have attracted considerable attention due to their broad application prospects in food, skincare, health supplements, and the textile industry. However, research on purple pigment-producing strains is relatively limited, particularly in China and abroad, and efficient and stable purple pigment-producing strains are particularly scarce.

[0006] Against this backdrop, we successfully isolated a Serratia strain named HSU-5 after extensive screening and cultivation. This strain possesses a stable purple pigment-producing capacity, capable of efficiently synthesizing a water-insoluble purple pigment under suitable conditions. The pigment is brightly colored and highly stable. Serratia, a genus of small, Gram-negative rods found widely in soil, water, plants, animals, and the human intestinal and respiratory tract, possesses stable biological properties and is easy to culture and control.

[0007] The HSU-5 strain has broad application prospects. In the food industry, the purple pigment it produces can be used in a variety of foods, including beverages, frozen desserts, candies, and seasonings. It not only enhances the product's color appeal but also serves as a pH indicator, enhancing the taste and texture of food. In the cosmetics field, purple pigment can be used in skincare products, adding a unique color and luster. Furthermore, the purple pigment produced by the HSU-5 strain has broad potential for application in the health supplement and textile industries. Summary of the Invention

[0008] In view of the above situation, the purpose of the present invention is to provide a purple pigment-producing Serratia marcescens strain HSU-5 with good thermal stability, light stability and antioxidant properties.

[0009] A first aspect of the present invention is to provide a purple pigment-producing Serratia sp. HSU-5 strain. The purple pigment-producing Serratia sp. HSU-5 strain was deposited with the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with a deposit number of CCTCC M 20241204. The deposit address is Wuhan University, Wuhan, China, and the depositor is the China Center for Type Culture Collection. The 16S rRNA gene sequence of the purple pigment-producing Serratia sp. HSU-5 strain is shown in SEQ ID NO. 1.

[0010] After isolation and purification, the purple pigment-producing Serratia strain HSU-5 had a smooth surface and could be isolated. Purple secretions surrounded the strain. Over four days of fermentation, the color of the fermentation liquid gradually deepened, eventually turning a deep purple.

[0011] Furthermore, the fermentation culture step of the Serratia strain HSU-5 includes: preparing 100 mL of liquid culture medium in a 250 mL conical flask, sterilizing it in a sterilizer at 115°C for 20 minutes, dipping a single strain with a pipette tip, and fermenting it in the liquid culture medium; the liquid culture medium is: 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, 20 g of agar, and the pH value is 7.2.

[0012] The second aspect of the present invention is:

[0013] The culture medium is used in culturing the purple pigment-producing Serratia strain HSU-5. The culture medium comprises: 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, 20 g of agar, and has a pH value of 7.2.

[0014] The third aspect of the present invention is:

[0015] The purple pigment-producing Serratia strain HSU-5 or its fermentation liquid is used in anti-oxidation.

[0016] The purple pigment-producing Serratia strain HSU-5 or its fermentation liquid is used in the preparation of anticancer, antitumor and antiviral drugs.

[0017] The advantages of the present invention are:

[0018] The surface of the strain HSU-5 produced in the present invention is smooth without obvious protrusions and can be isolated as a single strain. Sequence comparison results show that the strain HSU-5 belongs to a purpurogenous strain of the genus Serratia.

[0019] This invention utilizes an anhydrous ethanol extraction method to extract purple pigment and investigates its stability and selected physicochemical properties. Thermal stability experiments demonstrate that the purple pigment exhibits good thermal stability within the temperature range of 30°C to 60°C. Light stability experiments reveal that after 6 hours of illumination under white light, the OD value of the purple pigment solution decreases, but the color change is not noticeable, demonstrating the pigment's good light stability. Solubility experiments reveal that the crude purple pigment extract is slightly soluble in n-hexane and soluble in n-butanol, xylene, and ethyl acetate. In antioxidant tests, the ABTS clearance rate reached a maximum of approximately 54% after a two-fold dilution of the crude pigment solution. It is speculated that this clearance rate increases when the purple pigment solution reaches a certain concentration. Antibacterial experiments reveal that a distinct zone of inhibition forms around a sterilized paper disc containing purple pigment added to a Bacillus subtilis culture medium, demonstrating that the purple pigment exhibits some antibacterial activity against Bacillus subtilis, but the effect is relatively poor. In summary, the purple pigment has good thermal stability, light stability, antioxidant and antibacterial properties. The structure and properties of the pigment produced by this strain need further research in order to promote its application in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 The results of isolating purple pigment-producing strains from diluted soil solution in Example;

[0022] Figure 2 The results of purification of the purple pigment-producing strain HSU-5 in Example;

[0023] Figure 3 The 16S rRNA sequence comparison results of the purple pigment-producing strain HSU-5 in Example;

[0024] Figure 4 The results of two-day fermentation culture of the purple pigment-producing strain HSU-5 in Example;

[0025] Figure 5 The results of the four-day fermentation culture of the purple pigment-producing strain HSU-5 in Example;

[0026] Figure 6 The crude extract solution of purple pigment extracted by ethanol extraction in the embodiment;

[0027] Figure 7 This is a full spectrum scan of the extracted pigment using an ultra-micro spectrophotometer in the embodiment;

[0028] Figure 8 This is a diagram showing the results of the fermentation liquid after centrifugation of the crude purple pigment extracted in the embodiment;

[0029] Figure 9 This is a crude extraction result diagram of the purple pigment obtained in the embodiment;

[0030] Figure 10 This is a test chart of the effect of temperature on the stability of purple pigment;

[0031] Figure 11 This is a test chart of the effect of white light on the absorbance of purple pigment;

[0032] Figure 12 This is a diagram showing the solubility of purple pigment in different organic solvents (from left to right the solvents are n-hexane, n-butanol, xylene, and ethyl acetate);

[0033] Figure 13 The effect of purple pigment at different dilution ratios on the scavenging rate of ABTS free radicals;

[0034] Figure 14 This is the antibacterial effect diagram of purple pigment on Bacillus subtilis;

[0035] Figure 15 This is a diagram showing the antibacterial effect of purple pigment on Escherichia coli. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments. The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0037] Example 1

[0038] This embodiment provides a purple pigment-producing Serratia strain HSU-5, which has been deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with a deposit number of CCTCC M20241204.

[0039] The steps for isolating and purifying the purple pigment-producing Serratia strain HSU-5 are as follows:

[0040] (1) Sample collection: The soil in the blueberry garden was collected using the five-point sampling method. A soil sample of about 15 cm in length was selected from the soil layer about 10 cm from the surface. The processed soil sample was then placed in a sterilized ziplock bag and sealed for storage.

[0041] (2) Sample pretreatment: The collected soil samples were screened to remove visible impurities and gravel, and then dried. The soil samples were then ground into fine particles. 15 g of the ground sample was weighed using an electronic balance and placed in a clean and sterilized 250 mL conical flask. 100 mL of distilled water was added, and the flask was sealed with sealing film and cotton thread and placed on a shaker for about 24 h. The remaining soil samples were sealed and stored in sterilized polyethylene plastic bags for future use.

[0042] (3) Sterilization and aseptic operation: High temperature has a lethal effect on many microorganisms. Therefore, during the microbial transfer experiment, it is generally necessary to carry out the experiment next to the flame of an alcohol lamp. The inoculation loop is first disinfected with 75% ethanol and then directly burned with a flame to achieve the sterilization effect. The inoculation loop can only be used after it has cooled to prevent the high temperature from inactivating the microorganisms to be inoculated.

[0043] (4) Preparation of culture medium: Prepare 500 mL of culture medium; first, weigh 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 20 g of agar, and 500 mL of distilled water in a 1 L conical flask prepared in advance on an electronic balance, test the pH of the culture medium, wrap it with sealing film, mark it, and put it into a sterilizer (121°C) together with the culture dish for sterilization for about 30 minutes, then take it out for use.

[0044] While the culture medium is being sterilized, open the clean bench for sterilization (about 30 minutes) in preparation for the next experimental operation. After the sterilization is completed, take out the sterilizer, wait for the solution to cool to about 50-60℃, take it out together with the culture dish and put it on the workbench for packaging. Before the experiment, use 75% ethanol to disinfect your hands. You need to pour the plate next to the alcohol lamp, pour the culture solution into the culture dish, and shake it slightly. The culture solution needs to cover the bottom of the culture dish a little. Then put it on the clean bench to cool, turn it upside down after solidification, and prepare for the next experiment.

[0045] (5) Screening of purple pigment-producing strains:

[0046] The experiment was performed on a clean bench. Remove the soil dilution from the incubator. Use a pipette to remove 1 mL of the supernatant and add it to a pre-prepared test tube containing 9 mL of purified water. Mix thoroughly and mark it one. Then, use a pipette to remove 1 mL of the solution from test tube one and add it to another test tube containing 9 mL of purified water. Mix thoroughly and mark it two. Use a pipette to remove 1 mL of the mixed solution from test tube two and add it to test tube three containing 9 mL of purified water. Mix thoroughly. Then, use 1 mL from test tube three and add it to test tube four containing 9 mL of purified water. Repeat the above steps for test tubes four and five. Finally, a gradient of dilutions of 10⁻¹, 10⁻², 10⁻³, 10⁻4, and 10⁻5 was obtained. Note that the procedure must not be performed outside the clean bench. The pipette tip must be replaced after each use. The operation should be performed near an alcohol lamp. The prepared culture medium was divided into four equal parts, with two culture media in each part. Each part was used for inoculation and marked on the culture dishes.

[0047] Divide the prepared culture medium into five equal portions, two in each portion, for inoculation. Mark the portions on the culture dishes. Use a pipette to draw 10 μL of the solution and transfer it to the marked medium in sequence, making sure to change the pipette tip after each use.

[0048] Use a pre-sterilized coating rod to spread the culture medium in a regular pattern. Let it rest for a few minutes, then invert it. Repeat the previous steps to inoculate each culture medium. Sterilize the coating rod after each use to prevent contamination and experimental errors.

[0049] Place the coated LB medium in an incubator for cultivation. Observe the culture medium every 12 hours, 24 hours, and 48 hours, and keep corresponding experimental records.

[0050] The first strain isolated from the diluted soil solution was Figure 1 As shown. Figure 1 The results show that a purple pigment-producing strain was isolated from the soil dilution sample and numbered HSU-5. The next step is to isolate and purify the desired purple pigment-producing strain.

[0051] (6) Purification of purple pigment-producing strains: After the desired strains are screened out, use new culture dishes for separation and purification. Place the inoculation loops and new culture dishes required for the experiment into a biosafety cabinet, and then sterilize the biosafety cabinet with ultraviolet light. Before the experiment begins, light an alcohol lamp with a lighter and use the flatbed line drawing method to separate and purify the strains. After each separation and purification, accurately write the marking information with a marker and then place it in a constant temperature incubator at an appropriate temperature for cultivation. Repeat the previous experimental steps until a single colony appears.

[0052] In a sterilized biosafety cabinet, use a sterile inoculating loop to pick up 3 mL of purified liquid culture medium and place it in a 28°C shaker to culture until the logarithmic growth phase. Then, mix 1 mL of fermentation broth and 1 mL of 40% glycerol, label it, and store it in a -80°C culture storage cabinet.

[0053] The purpurogenous strain isolated from the soil dilution medium was separated and purified several times to finally obtain a single colony, e.g. Figure 2 As shown. Figure 2 The experimental results show that the surface of the HSU-5 strain that produces purple pigment is smooth and can be isolated separately. There is purple secretion around the strain.

[0054] Visual observation revealed a convex, smooth surface and oval-shaped individual colonies, indicating that the strain was initially identified as a bacterium. A 16S rRNA sequence comparison was then performed. A sample of the purple-pigmented strain produced by HSU-5 was sent to a genetic testing center for identification, resulting in the acquisition of the 16S rRNA sequence of HSU-5. This sequence was compared with BLAST analysis results, revealing 16S rRNA sequences of other strains with similar sequence characteristics. This sequence was then compared with the 16S rRNA sequences of similar strains using clustalX2 software, yielding corresponding results. Finally, the resulting gene sequence alignment was analyzed using MEGA software to construct a phylogenetic tree of the HSU-5 strain and provide a preliminary analysis of its composition.

[0055] The determination and comparison of the total length of the 16S rRNA gene nucleotide sequence is more conducive to various designs and studies of bacteria. The obtained strain HSU-5 was subjected to 16S rRNA sequence comparison and phylogenetic tree analysis using BLAST, clustalX2, MEGA and other software. The results are as follows Figure 3 shown.

[0056] from Figure 3 It can be seen that the 16S rRNA sequence of the purple pigment-producing strain HSU-5 is highly similar to that of the bacteria Serratia marcescers strain NBRC (Serratia species), so it is preliminarily judged that this purple pigment-producing strain HSU-5 belongs to Serratia.

[0057] (7) Fermentation culture of purple pigment-producing strains: Prepare 100 mL of liquid culture medium in a 250 mL conical flask, wrap it with sealing film and cotton thread, and sterilize it in a sterilizer at 115°C for 20 minutes. After ultraviolet disinfection on the clean bench, open the sterilized pipette tip, take out the pipette tip with tweezers, dip the front end of the pipette tip into a single colony, and then drop the pipette tip into the sterilized liquid culture medium. Then mark it with a marker and place it in a constant temperature incubator for culture. Observe the fermentation of the strain regularly and keep good experimental records.

[0058] The liquid culture medium comprises: 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, and 20 g of agar, and has a pH value of 7.2.

[0059] Dip the tip of the pipette into a single strain and then ferment it in liquid culture medium. After four days of cultivation, the results are as follows: Figure 4 and Figure 5 shown. Figure 4 This is the result of two days of fermentation of the purple pigment-producing strain HSU-5. Figure 5 The results of the four-day fermentation of the purple pigment-producing strain HSU-5 are shown in Figure 2. Based on the observations of the above experiment, we can see that after about four days of fermentation, the color of the fermentation liquid gradually deepens and finally turns into dark purple.

[0060] (8) Crude extraction of purple pigment: Use a pipette to draw the fermentation liquid of the purple pigment-producing strain into 6 centrifuge tubes, with 20 mL in each centrifuge tube. After balancing, centrifuge with a speed of 12000 r / min and a centrifugation time of 20 min. After the centrifugation is completed, pour out the supernatant in the centrifuge tube and retain the precipitate (purple pigment-producing strain), add 20 mL of distilled water and shake until the precipitate is completely dissolved in the distilled water. Place a centrifuge tube in the middle of a plastic cup filled with crushed ice. The crushed ice is used to absorb the heat generated during the ultrasonic crushing process. Rub the amplitude of the ultrasonic cell crusher into the liquid surface to a depth of about 10 mL, ensuring that the amplitude does not touch the wall of the centrifuge tube. Set the crusher power to 150 W, the interval time to 1 s, the ultrasonic time to 1 s, and the full time to 15 min for crushing. After the five centrifuge tubes are crushed, balance them and centrifuge with a speed of 10000 r / min and a centrifugation time of 20 min. After the centrifugation is completed, collect the supernatant (purple pigment aqueous solution) in the centrifuge tube and remove the precipitate. The collected purple pigment aqueous solution and the control group strain aqueous solution were measured with a full spectrum analyzer and the data were recorded.

[0061] (9) Pigment extraction

[0062] Prepare 3 bottles of 100 mL liquid culture medium and sterilize them in an autoclave at 115°C for 20 min. In an ultra-clean workbench, use a pipette to inoculate 100 μL of the seed culture medium from step (6) at a ratio of 1:1000 into the sterilized liquid culture medium. Pipette thoroughly to mix, seal, and incubate in a constant temperature incubator at 26°C for three days to obtain a fermentation broth with good color.

[0063] Pipette 25 mL of bacterial solution into a 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, add anhydrous ethanol, soak for 30 min, centrifuge again at 10000 rpm for 10 min, collect the supernatant and pour it into a rotary evaporator for concentration, and rotary evaporate at 45 ° C for a period of time to obtain a concentrated pigment crude extract solution.

[0064] Full spectrum scan of the pigment: First, clean the sample stage. Place 2 μL of sterile double-distilled water on the sample stage for 10 seconds, then wipe with a dust-free tissue. Repeat the procedure with sample buffer. Prepare a purple pigment sample of appropriate concentration and place 2 μL on the sample stage for measurement. Using anhydrous ethanol solution as a blank, perform a full spectrum scan, measure the absorbance of the pigment solution, and determine the wavelength of maximum absorption.

[0065] The strain was fermented and cultured in liquid culture medium, and the pigment was extracted by ethanol extraction. Then, the sample concentrate was obtained by evaporation and concentration. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that the pigment concentrated by the rotary evaporator is purple in color, clear and free of impurities.

[0066] Using an ultra-micro spectrophotometer, the extracted pigment was scanned in full spectrum with anhydrous ethanol solution as blank control. The results are as follows: Figure 7 From the experimental results, it can be clearly observed that the maximum absorption wavelength of the pigment produced by the strain is around 537nm, which is the wavelength of purple-red.

[0067] The bacterial liquid in the fermentation broth was first centrifuged at high speed, the supernatant was discarded, and then ultrapure water was added. The supernatant was crushed using an ultrasonic crusher and then centrifuged a second time. The supernatant after centrifugation was the crude extracted purple pigment solution. The experimental results are as follows Figure 8 and Figure 9 shown.

[0068] according to Figure 8 The results showed that after the first centrifugation of the fermentation broth, the strain precipitated at the bottom of the centrifuge tube and appeared purple. From this, it can be inferred that this strain produces pigments intracellularly and needs to be crushed to extract the pigments. Figure 9 It is the crude pigment extract obtained after crushing and centrifugation. It can be clearly seen that the pigment solution is purple, which is the result required by the experiment.

[0069] Research and analysis on the properties of purple pigment produced by the purple pigment-producing strain HSU-5:

[0070] 1. Analysis method of heat resistance of purple pigment:

[0071] Use a pipette to draw 200mL of the extracted purple pigment crude extract as the mother liquor, then use a spectrophotometer to measure its absorbance and record it. Prepare 4 test tubes numbered 1, 2, 3, and 4 respectively, use a pipette to draw 5mL of purple pigment aqueous solution from the mother liquor in turn and add it to the 4 test tubes, and finally plug the test tube mouth of each test tube with a rubber stopper. Debug the water bath and set the temperature to room temperature (25℃), 30℃, 60℃, and 90℃ respectively, and wait for each water bath to reach the specified temperature. Put the test tubes numbered 1, 2, 3, and 4 into the water bath at room temperature (25℃), 30℃, 60℃, and 90℃ at the same time and heat them in a water bath for 1.5h. After the water bath heating is completed, observe the changes in each test tube, measure the absorbance of the purple pigment aqueous solution in the 6 test tubes and record them.

[0072] 2. Heat resistance of purple pigment:

[0073] The purple pigment extracted with ethanol was divided into four groups and stored in a water bath at room temperature (25°C), 30°C, 60°C, and 90°C for 6 hours, and the absorbance was measured every hour. The changes in the absorbance of the purple pigment over time at different temperatures are as follows Figure 10 As shown. Figure 10 It can be seen that the temperature has little effect on the stability of purple pigment at 30℃ to 60℃, but the absorbance value of purple pigment fluctuates greatly under 90℃ treatment, showing an overall downward trend.

[0074] 3. Photostability analysis method of purple pigment:

[0075] Equal amounts of the crude purple pigment extract after ethanol extraction were set up into two groups. One group was placed under white light, and the other group was placed in the dark. They were left to stand for 6 hours. The OD value of the purple pigment aqueous solution in the EP tube was measured and recorded every 1 hour.

[0076] 4. Photostability of purple pigment:

[0077] Place two groups of purple pigment crude extracts, one under white light and the other in dark conditions, and let them stand for 6 hours. Measure the OD537 nm value of the purple pigment aqueous solution in the EP tube every hour and record the results. Figure 11 As shown in the figure, after 6 hours of light treatment, the OD537 nm value of the purple pigment showed a downward trend under both dark and white light conditions. The change was more pronounced under dark conditions, while white light had some effect on the purple pigment, but the impact was minor. This suggests that this purple pigment is more suitable for storage in locations with milder light exposure and is not suitable for storage in the dark.

[0078] 5. Solubility analysis method of purple pigment:

[0079] Use a pipette to pipette 500 μL of n-hexane, n-butanol, xylene, and ethyl acetate into four EP tubes, numbering them 1, 2, 3, and 4. Then, use a pipette to pipette 500 μL of the purple pigment aqueous solution into each of the four EP tubes. Thoroughly shake the purple pigment aqueous solution and the reagents in the four EP tubes, then let them stand for 45 minutes and observe the changes in the purple pigment in the EP tubes.

[0080] 6. Solubility of purple pigment:

[0081] The purple pigment aqueous solution was thoroughly shaken with the organic solvent in the four EP tubes and then allowed to stand for 45 minutes. The changes in the purple pigment in the EP tubes were observed. The results were as follows: Figure 12 As shown. Figure 12 It can be observed that purple pigment dissolves differently in different solvents. From left to right, the solvents are n-hexane, n-butanol, xylene, and ethyl acetate. The crude extract of purple pigment is slightly soluble in n-hexane and soluble in n-butanol, xylene, and ethyl acetate. However, the pigment becomes lighter in n-hexane and xylene, and fades in ethyl acetate.

[0082] 7. Analysis method of antioxidant activity of purple pigment:

[0083] 0.0384g ABTS was diluted to 10mL, and 0.0134g potassium persulfate was diluted to 10mL. The two reagents were mixed in a 1:1 ratio and protected from light for 12 hours to obtain ABTS. ABTS was diluted with double-distilled water to a pH of 0.68-0.72 at 734nm for later use. The extracted pigment was diluted 2-, 4-, 8-, 16-, and 32-fold as experimental groups and a blank control group was set up. The reaction was allowed to proceed in the dark for a period of time, and the data was measured. The ABTS free radical scavenging rate was calculated to investigate the antioxidant activity of the pigment.

[0084] Clearance calculation formula:

[0085] Clearance rate = (A0-A) / A0×100%

[0086] Where A0 is the A734 nm value when the sample concentration is 0; A is the A734 nm value of the sample.

[0087] 8. Antioxidant properties of purple pigment

[0088] The measured data were used to calculate the clearance rate of ABTS by pigments with different dilution ratios. Figure 13 It can be seen that the higher the concentration of purple pigment, the higher the free radical scavenging rate, and the longer the reaction time, the better the scavenging effect. Among them, when the reaction time is 10 minutes, the purple pigment solution with a dilution factor of 2 (the highest concentration) has the highest ABTS scavenging rate, about 54%. From this, it can be inferred that when the purple pigment solution reaches a certain concentration, the ABTS scavenging rate will be even higher. Therefore, it can be concluded that purple pigment has good antioxidant properties.

[0089] 9. Analysis method of antibacterial activity of purple pigment

[0090] Prepare 500mL of LB solid culture medium and sterilize it in an autoclave at 115℃ for 20min, then pour it into the plate. Use a spreader to spread Escherichia coli and Bacillus subtilis on the LB solid culture medium in the clean bench, and spread 4 LB solid culture media for each of the two bacteria. After spreading, divide the LB solid culture medium into four parts and label them 1, 2, 3, and 4. Use the paper diffusion method to conduct the experiment. Use tweezers to place a sterilized paper in the center of the four equally divided areas. Use a pipette to draw 5μL of sterile water and inject it into the sterilized paper in areas 1 and 2 of the culture medium in turn (blank control), and then draw 5μL of the extracted purple pigment aqueous solution and inject it into the sterilized paper in areas 3 and 4 of the culture medium. Place all LB solid culture media in a constant temperature incubator at 28℃ and culture for 48h to observe whether there is an antibacterial effect.

[0091] 10 Antibacterial properties of purple pigment:

[0092] The LB solid culture medium for the antibacterial experiment was placed in a constant temperature incubator at 28°C for 48 hours to observe whether there was an antibacterial effect. The results are as follows: Figure 14 and Figure 15 As shown. Figure 14 It can be seen that in the antibacterial experiment of paper discs on LB solid medium coated with Bacillus subtilis, an obvious antibacterial zone appeared around the sterilized paper discs with purple pigment added, but the inhibition zone was small, so purple pigment had a certain antibacterial effect on Bacillus subtilis, but the effect was poor. Figure 15 It can be seen that in the paper antibacterial experiment conducted on LB solid culture medium coated with Escherichia coli, there was no obvious inhibition zone around the sterilized paper with purple pigment added, and there was no difference compared with the blank control with sterile water added, so purple pigment had no antibacterial effect on Escherichia coli.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A purple pigment-producing Serratia strain HSU-5, characterized in that The purple pigment-producing Serratia marcescens strain HSU-5 was deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with the deposit number CCTCC M20241204.

2. The purple pigment-producing Serratia strain HSU-5 according to claim 1, characterized in that The 16S rRNA gene sequence of the purple pigment-producing Serratia marcescens strain HSU-5 is shown in SEQ ID NO.

1.

3. The purple pigment-producing Serratia strain HSU-5 according to claim 1, characterized in that The fermentation and culturing steps of the Serratia marcescens strain HSU-5 include: preparing 100 mL of a liquid culture medium in a 250 mL conical flask, sterilizing the culture medium in an autoclave at 115° C. for 20 minutes, dipping a single strain with a pipette tip, and fermenting the culture medium in the liquid culture medium; the liquid culture medium comprises: 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, and 20 g of agar, and has a pH value of 7.

2.

4. Use of the culture medium in culturing the purple pigment-producing Serratia strain HSU-5 according to claim 1, characterized in that: The culture medium comprises: 4.95 g of peptone, 2.40 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, and 20 g of agar, and has a pH value of 7.

2.

5. Use of the purple pigment-producing Serratia strain HSU-5 or its fermentation liquid according to claim 1 in anti-oxidation.

6. Use of the purple pigment-producing Serratia strain HSU-5 or its fermentation liquid according to claim 1 in the preparation of anticancer, antitumor and antiviral drugs.

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