HSU-17 strain and application thereof in producing haematochrome

Abstract

The invention relates to an HSU-17 strain and application of the HSU-17 strain in producing haematochrome. The HSU-17 strain is preserved in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, and the preservation number of the HSU-17 strain is CCTCC M 20241205. The invention aims to provide an HSU-17 strain which has good thermal stability, light stability and oxidation resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an HSU-17 strain and application thereof in producing red pigment. Background Art

[0002] Natural pigments, with their significant advantages such as safety, non-toxicity, natural color, and environmental friendliness, have demonstrated broad application potential in a variety of fields, including food, cosmetics, medicine, and textiles. In the textile industry in particular, natural pigments not only impart natural, healthy colors to textiles, but also meet consumers' pursuit of environmental protection and sustainability. In addition to the well-known successful applications of curcumin and lac pigment in the dyeing of cellulose, protein fibers, and synthetic fibers, respectively, with the advancement of science and technology, more and more natural pigments from plant sources have been discovered and applied to the dyeing and printing of textiles, such as madder red and indigo. These pigments are not only rich in color but also harmless to the human body, in line with the development trend of green textiles.

[0003] However, as mentioned above, the supply of plant pigments is indeed limited by natural factors such as long growth cycles, strong geographical dependence, and significant seasonal variations, which to some extent hinders the pace of large-scale industrial production. To overcome these challenges, researchers are actively exploring other sources of natural pigments, among which animal pigments and microbial pigments have become important research directions.

[0004] While animal pigments like cochineal are favored in certain markets for their unique color and high stability, their limited sources and complex extraction process make them a difficult choice for mainstream use. In contrast, microbial pigments have attracted considerable attention due to their unique advantages: a wide variety of microorganisms, rapid growth rates, and the ability to efficiently produce a variety of colors under controlled conditions without geographical or seasonal restrictions. This provides a new path to a stable supply of natural pigments.

[0005] Red pigment, in particular, is one of the most striking and widely used colors in nature and has enormous market potential in areas such as food coloring, pharmaceutical labeling, and textile dyeing. However, research on microbial red pigment production is still in its infancy, and many key scientific issues, such as optimizing optimal production conditions, improving pigment stability, and in-depth exploration of its specific functional properties (such as antioxidant and antibacterial properties), require more research data to support this. Based on this, the present invention provides an HSU-17 strain and its application in red pigment production. Summary of the Invention

[0006] In view of the above situation, the purpose of the present invention is to provide a HSU-17 strain with good thermal stability, light stability and antioxidant properties.

[0007] A first aspect of the present invention is to provide an HSU-17 strain, which was deposited with the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with the deposit number CCTCC M20241205. The 16S rRNA gene sequence of the HSU-17 strain is shown in SEQ ID NO. 1.

[0008] Furthermore, the culture medium comprises: 5.05 g of peptone, 2.45 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, 10 g of agar, and has a pH value of 7.2.

[0009] The second aspect of the present invention is:

[0010] The HSU-17 strain or its fermentation liquid is used in producing red pigment.

[0011] The application of the HSU-17 strain or its fermentation liquid in anti-oxidation.

[0012] The HSU-17 strain or its fermentation liquid is used in the preparation of anticancer, antitumor and antiviral drugs.

[0013] The advantages of the present invention are:

[0014] The present invention inoculates the obtained red pigment-producing strain into liquid LB culture medium, activates and cultures it for several days, then extracts it. The extracted red pigment is then subjected to property tests. The results show that the microbial-derived red pigment is readily soluble in anhydrous ethanol, soluble in methanol and acetone, and insoluble in ethyl acetate, making it an alcohol-soluble pigment. Natural light has a certain effect on the photostability of the red pigment, and the red pigment must be stored away from light. The red pigment has better thermal stability at 30°C or 60°C, and storage under these conditions is conducive to its stability. The red pigment has a good free radical scavenging effect and exhibits a certain antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Figure 1 The results of isolating red pigment-producing strains from diluted soil solution in Example;

[0017] Figure 2 The results of purification of the red pigment-producing strain HSU-17 in Example;

[0018] Figure 3 The 16S rRNA sequence comparison results of the red pigment-producing strain HSU-17 in Example;

[0019] Figure 4The results of two-day fermentation culture of the red pigment-producing strain HSU-17 in Example;

[0020] Figure 5 The results of four-day fermentation culture of the red pigment-producing strain HSU-17 in Example;

[0021] Figure 6 The crude red pigment solution extracted by ethanol extraction in the embodiment;

[0022] Figure 7 The results after the red pigment is concentrated are shown in the example;

[0023] Figure 8 The heat resistance test results of the red pigment in the embodiment are shown below:

[0024] Figure 9 The light stability results of the red pigment in the example are as follows;

[0025] Figure 10 The figure shows the scavenging effect of red pigment at different dilution ratios on ABTS free radicals. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] This embodiment provides an HSU-17 strain, which has been deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with the deposit number CCTCC M 20241205.

[0029] The isolation and purification steps of the HSU-17 strain are as follows:

[0030] (1) Sample collection: Soil was collected from the shore of Tingsong Lake at Huangshan University using the five-point sampling method. Before sampling, the topsoil of about 5 cm was removed, and only soil samples from 5 to 15 cm were selected. The collected soil samples were placed in sterilized ziplock bags and sealed for storage.

[0031] (2) Sample pretreatment: Weigh 15 g of the above soil sample on an electronic balance, place it in a sterilized 250 mL conical flask, add 100 mL of sterile double-distilled water, seal it with sealing film and cotton thread, place it on a shaker, and treat it for about 24 h.

[0032] (3) Preparation of culture medium: Prepare 500 mL of culture medium; first, weigh 5.05 g of peptone, 2.45 g of yeast powder, 5.10 g of sodium chloride, 10 g of agar, and 500 mL of distilled water in a 1 L conical flask prepared in advance on an electronic balance, and check whether the pH of the culture medium is around 7.0. If it is, 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 out the LB culture medium plate and set it aside.

[0033] (4) Screening of red pigment-producing strains:

[0034] Take out the soil dilution from the incubator, take 1mL of the supernatant with a pipette, add it to a pre-prepared test tube containing 9mL of sterile double-distilled water, mix well, and mark it one; then take 1mL of the solution in test tube one with a pipette and add it to another test tube containing 9mL of sterile double-distilled water, mix well, and mark it two; take 1mL of the mixed solution from test tube two with a pipette and add it to test tube three containing 9mL of sterile double-distilled water, mix well; then take 1mL from test tube three and add it to test tube four containing 9mL of sterile double-distilled water, stir well; repeat the previous steps to obtain test tubes four and five. Finally, a gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was applied to LB medium plates and placed in a 37°C constant temperature incubator. During the incubation period, the cells were observed every 12 h, 24 h, and 48 h, and corresponding experimental records were kept.

[0035] The first strain isolated from the diluted soil solution was Figure 1 As shown. Figure 1 The results showed that a red pigment-producing strain was isolated from the soil dilution sample and numbered HSU-17.

[0036] (5) Purification and preservation of red pigment-producing strains: On a clean bench, use a sterile inoculation loop to pick the above strain HSU-17 and streak it onto a freshly prepared LB medium plate. Place it in a 37°7 constant temperature incubator and incubate it for 24 hours to observe the purity of the strain. Repeat this step until a single colony is obtained. Figure 2 shown.

[0037] In a sterile clean bench, use a sterile inoculation loop to pick the purified strain HSU-7 and inoculate it into 3 mL of LB liquid medium. Incubate it in a shaker at 37°C until the logarithmic growth phase. Then, take 1 mL of fermentation broth and 1 mL of 40% glycerol, mix them, label them, and store them in a -80 8 refrigerator until use.

[0038] (6) Identification of strain HSU-17: The strain HSU-17 preserved above was taken out from the -80℃ refrigerator, inoculated into a freshly prepared 3mL LB liquid culture medium, and cultured at 37℃ for 14h. The genomic DNA was extracted using a bacterial genomic DNA extraction kit. The 16S rRNA was amplified using universal primers 27F and 1492R and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The obtained sequencing sequence was BLAST-aligned in the NCBI database, and the 16S rRNA sequences of other strains with higher sequence characteristics than this strain were selected from the alignment results; then, the 16S rRNA sequences of similar strains were compared using clustalX2 software to obtain the corresponding results. Finally, the gene sequence alignment results were processed and analyzed using MEGA software to obtain the evolutionary tree of the HSU-17 strain and a preliminary identification of its composition was performed. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that the 16S rRNA sequence of the red pigment-producing strain HSU-17 is highly similar to that of Serratia marcescers strain NBRC (Serratia species), so it is preliminarily determined that this red pigment-producing strain HSU-17 belongs to Serratia.

[0039] (7) Fermentation and extraction of red pigment-producing strains: Prepare 100 mL of LB 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 ℃ for 30 min.

[0040] The LB liquid culture medium comprises: 5.05 g of peptone, 2.45 g of yeast powder, 5.10 g of sodium chloride, and 500 mL of distilled water, and has a pH value of 7.2.

[0041] 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 red pigment-producing strain HSU-17. Figure 5 The results of the four-day fermentation of the red pigment-producing strain HSU-17 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 deep red.

[0042] Extraction of red pigment: Use an inoculating loop to dip a small amount of the strain obtained from the laboratory and inoculate it into a 10mL test tube containing liquid LB medium. Place it in a shaker at 28℃ and culture it for 7 hours until the liquid becomes turbid. Then take it out and inoculate the bacterial liquid in the test tube into a 1000mL conical flask containing liquid LB medium.

[0043] After culturing in a 28°C incubator for 10 days, 10 mL of the bacterial solution was pipetted into multiple centrifuge tubes, balanced, and centrifuged at 12,000 rpm for 20 minutes. The supernatant was removed, and 10 mL of anhydrous ethanol was added to the precipitate. After mixing, the precipitate was allowed to stand in a cool place for 60 minutes, and then centrifuged at 12,000 rpm for 20 minutes to obtain the extracted red pigment. Figure 6 .

[0044] According to this step, 800mL of bacterial solution in the 1000mL conical flask was gradually extracted, and the crude extract was placed in a rotary evaporator at 70℃ for 1-2h, and the concentrated red pigment was stored in the refrigerator. Figure 7 .

[0045] Research and analysis on the properties of red pigment produced by the red pigment-producing strain HSU-17:

[0046] 1. Analysis of heat resistance of red pigment:

[0047] Pipette 1mL of pure red pigment into a 2mL EP tube, mark and seal it, and place it in a preheated water bath. The temperature setting intervals are 30℃, 60℃, and 90℃. Set up 3 experimental objects in each interval. Measure the absorbance of the red pigment after heating at different temperatures for 2h, 4h, 6h, 8h, 10h, and 12h. Each group has 3 sets of values. After judging their correctness, take the average value. The experiment is repeated 3 times to finally determine the thermal stability of the red pigment. The results are shown in the figure. Figure 8 The experimental results show that the absorbance and color of the red pigment do not change significantly compared with those of the control group, indicating that the red pigment has good thermal stability at 30°C and 60°C.

[0048] 2. Analysis of photostability of red pigment:

[0049] 1mL of pure red pigment was added to a 2mL EP tube, marked and sealed, and divided into three groups: CK group, light group, and dark group, with three experimental subjects in each group. They were placed together in a dark place and treated under continuous light conditions and normal conditions. During this period, the color changes of each group were observed every 24 hours, and the OD value was measured at 537nm to determine the light stability of the red pigment. The results are shown in the figure. Figure 9 The experimental results show that when the red pigment is stored in the dark, its absorbance and color do not change significantly compared with the absorbance and color of the control group as the illumination time increases, indicating that the red pigment has good light stability under dark conditions.

[0050] 3. Solubility analysis of red pigment:

[0051] Take 500 μL of pure red pigment and place it in 500 μL of anhydrous ethanol, acetone, ethyl acetate, and methanol respectively to make red pigment ethanol solutions. Use a pipette to mix them evenly. Observe the color change and dissolution to determine its solubility. The results are shown in Table 1.

[0052] From the experimental results, we can see that red pigment is easily soluble in ethanol, soluble in methanol and acetone, and insoluble in ethyl acetate. It is an alcohol-soluble pigment.

[0053] Table 1 Dissolution of pigments in different solvents

[0054]

[0055] 4. Analysis method of antioxidant activity of red pigment:

[0056] Use a pipette to draw up pure red pigment as a stock solution. Dilute it with anhydrous ethanol to 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the stock solution concentration, then pipette 400 μL into each test tube. Pipette 2.45 mmol / L K₂S₂O₂ solution and 7 mmol / L ABTS solution in a 1:1 ratio by volume, mix thoroughly, and react in the dark for 12 hours. Dilute the reacted ABTS solution with anhydrous ethanol until the absorbance at 734 nm is 0.68-0.72.

[0057] In test tube 1, mix 400 μL of red pigment aqueous solution and 4 mL of ABTS solution evenly, then protect from light and react. Measure the absorbance after 6 minutes of reaction. Repeat the same steps to react the remaining diluted red pigment aqueous solution with ABTS solution according to the above ratio for 6 minutes, then measure the absorbance and record it. At the same time, take another test tube and add 400 μL of anhydrous ethanol and 4 mL of ABTS solution to it, mix well and protect from light as a blank control group. Measure its absorbance after 6 minutes of reaction and record the relevant data. The results are shown in the table. Figure 10 .

[0058] The experimental results show that the relationship between the dilution multiple of the red pigment and the clearance rate conforms to the linear regression equation. When the dilution multiple is 1, the clearance rate of the red pigment for ABTS free radicals can reach 97%. It is speculated that when the red pigment reaches a certain concentration, its clearance rate will be higher.

[0059] 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. An HSU-17 strain, characterized in that The HSU-17 strain was deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with the deposit number CCTCC M20241205.

2. The HSU-17 strain according to claim 1, characterized in that The 16S rRNA gene sequence of the HSU-17 strain is shown in SEQ ID NO.

1.

3. Use of a culture medium in culturing the HSU-17 strain according to claim 1, characterized in that: The culture medium comprises: 5.05 g of peptone, 2.45 g of yeast powder, 5.10 g of sodium chloride, 500 mL of distilled water, and 10 g of agar, and has a pH value of 7.

2.

4. Use of the HSU-17 strain according to claim 1 or its fermentation liquid in producing red pigment.

5. Use of the HSU-17 strain or its fermentation broth according to claim 1 in anti-oxidation.

6. Use of the HSU-17 strain or its fermentation liquid according to claim 1 in the preparation of anticancer, antitumor and antiviral drugs.

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