A new natural pigment-producing Serratia marcescens strain and its application

Abstract

The present invention discloses a new Serratia marcescens producing natural pigment and its application, belong to the field of microorganisms. The present invention provides a new Serratia marcescens (Serratia marcescens) GXMZU-S, the deposit number of the Serratia marcescens GXMZU-S is GDMCC.No.65230, and the deposit date is September 29, 2024, and the depository unit is Guangdong Province Microbial Culture Collection Center, and the deposit address is Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Middle Road, Guangzhou, China. Currently disclosed Serratia marcescens all require shaking culture to produce pigment, and the Serratia marcescens GXMZU-S screened by the present invention does not produce pigment after shaking culture, must be statically cultured, and is not strictly required to growth temperature, and can reduce the cost required for shaking and heat preservation measures when applied to industrial production; using GXMZU-S pigment solution dyeing, step is simple, and color fastness is high, safe, easy to use.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to a new natural pigment-producing Serratia marcescens strain and application thereof. Background Art

[0002] Serratia marcescens is a bacterium widely found in nature that can produce pigments through secondary metabolism. Currently, pigments are divided into two types: natural pigments and synthetic pigments. Natural pigments are mainly extracted from plants and some insects themselves, such as cochineal, while synthetic pigments are synthesized through industrial chemicals. The former are difficult to obtain, and the yield is not high, requiring large-scale breeding to obtain, and the pigment acquisition cycle is long. The latter are mainly synthesized through chemical synthesis, which is not only consuming a lot and easily causes environmental pollution, but some synthetic pigments can easily cause life and health problems when they enter human daily life. At present, extracting pigments synthesized by microorganisms' own metabolism is a newer form, with convenient culture conditions, high pigment yield, fast strain propagation speed, and only consuming water and culture medium, which is a greener and healthier way.

[0003] Currently, many strains of Serratia marcescens have been isolated and identified, but their development and utilization remain limited. Some patents have investigated the insect and disease resistance of Serratia marcescens' secondary metabolites, but their application in dyeing and textile applications is limited. Furthermore, all Serratia marcescens strains reported in the prior art require shaking culture to produce pigment, which increases industrial costs. The present invention has discovered a new Serratia marcescens strain, GXMZU-S, that can produce pigment without shaking culture and achieves excellent dyeing results. Summary of the Invention

[0004] The present invention aims to provide a new natural pigment-producing Serratia marcescens strain and its application to solve the problems existing in the above-mentioned prior art. The present invention has discovered a new Serratia marcescens strain GXMZU-S, which can produce pigment without shaking culture and has good dyeing effect. When used in industrial production, it can reduce the costs required for shaking and heat preservation measures.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a new strain of Serratia marcescens GXMZU-S. The deposit number of the Serratia marcescens GXMZU-S is GDMCC.No.65230, the deposit date is September 29, 2024, the deposit unit is Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Guangzhou, China.

[0007] The present invention also provides a method for preparing a pigment solution using the Serratia marcescens GXMZU-S, comprising the following steps:

[0008] A colony of Serratia marcescens GXMZU-S was inoculated into a liquid LB medium and statically cultured to obtain a fermentation broth;

[0009] The fermentation liquid was centrifuged, the precipitate was added with methanol, crushed, and centrifuged, and the supernatant was taken to obtain the pigment solution.

[0010] Furthermore, the static culture condition is 30° C. for 7 days.

[0011] The present invention also provides application of Serratia marcescens GXMZU-S in cotton fabric dyeing. The deposit number of the Serratia marcescens GXMZU-S is GDMCC.No.65230.

[0012] The present invention also provides a method for dyeing cotton fabric using Serratia marcescens GXMZU-S, comprising the following steps:

[0013] preparing a pigment solution using the method for preparing a pigment solution;

[0014] The pigment solution and the aqueous solution are mixed at a volume ratio of 1:(10-15) to obtain a pigment aqueous solution, cotton fabric is immersed in the pigment aqueous solution, dyed by a hot dip method, and then washed and dried.

[0015] Furthermore, the pigment solution and the aqueous solution are mixed in a volume ratio of 1:10.

[0016] Furthermore, the hot dip method is as follows: starting from 30°C, dyeing for one minute, raising the temperature by 3°C per minute until the temperature reaches 60°C, continuing for 30 minutes, and then raising the temperature to 80°C and continuing dyeing for 20 minutes.

[0017] The present invention discloses the following technical effects:

[0018] The present invention discovered a new strain of Serratia marcescens GXMZU-S, which has strong reproductive ability, strong adaptability, fast growth cycle and produces a large amount of purple pigment.

[0019] Currently available Serratia marcescens strains all require shaking culture to produce pigments. However, the Serratia marcescens strain GXMZU-S screened by the present invention does not produce pigments after shaking culture and must be cultured statically. It also has no strict requirements on the growth temperature and can reduce the costs required for shaking and heat preservation measures when applied to industrial production.

[0020] The pigment produced by Serratia marcescens GXMZU-S can be extracted using water or chemical reagents. The dyeing process is simple, and it has high color fastness, is safe, and is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The colony of GXMZU-S cultured for 7 days;

[0023] Figure 2 is the similarity comparison result of GXMZU-S;

[0024] Figure 3 The fermentation broth of GXMZU-S under different culture conditions; the left bottle is the result of static culture, and the right bottle is the result of shaking culture;

[0025] Figure 4 The GXMZU-S fermentation broth was cultured for 7 days;

[0026] Figure 5 Shows the dyeing results under different ratios; original: represents the original pigment methanol solution, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30: represent the volume ratio of pigment methanol solution to aqueous solution is 1:5, 1:10, 1:15, 1:20, 1:25, 1:30 respectively. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1 Isolation and identification of strains

[0033] Soil sample: Dayongshan Forest Nature Reserve, Guangxi.

[0034] Isolation medium: LB medium: tryptone (10 g / L), yeast extract powder (5 g / L), sodium chloride (10 g / L), agar (15 g / L), pH = 6.9-7.1, high-pressure steam sterilization at 121°C for 20 min.

[0035] 1. Isolation of strains

[0036] After fully grinding the soil sample, add it to sterile water, add appropriate amount of glass beads and shake it at 30℃ and 200rpm for 3h to fully shake the soil sample; take the above turbid liquid and inoculate it into LB at 10% inoculum, and culture it at 200rpm for 7d. Then take 1mL of fermentation liquid and dilute it with sterile water to the following concentrations: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Then, 100 μL of the diluted fermentation broth of each concentration was evenly spread on the LB solid plate and cultured at 30°C for 1-7 days. Single colonies that obviously produced purple pigment were selected for purification and streaking, and then stored at low temperature in 25% glycerol tubes.

[0037] 2. Morphological observation

[0038] The isolated and purified strain was inoculated into the culture medium. The colony was normal and good, the strain was purple-red, and the surface was smooth. After culturing at 28℃ for 7 days, obvious purple-red pigment could be observed in the culture medium (see Figure 1 ).

[0039] 3. Identification of bacterial species

[0040] The isolated strain was sent to Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. for identification. The bacterial 16Sr RNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used. After PCR sequencing, the full-length 16SrRNA sequence was assembled into three segments. Comparison with the NCBI database revealed that the strain was a new strain. Finally, the most similar strain was identified as Serratia marcescens (see Figure 2 ), named Serratia marcescens GXMZU-S. The three 16S rRNA base sequences are shown in SEQ ID No. 1-3 respectively.

[0041] SEQ ID No. 1:

[0042]

[0043] SEQ ID No.2:

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[0045] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046]

[0047] The above-mentioned Serratia marcescens GXMZU-S has been deposited in the Guangdong Provincial Microbial Culture Collection Center. The deposit date is September 29, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Guangzhou, China, and the deposit number is GDMCC.No.65230.

[0048] Example 2 GXMZU-S pigment property determination

[0049] 1. Selection of culture conditions

[0050] Experimental verification has found that GXMZU-S can produce pigments regardless of whether it is placed in an incubator or at room temperature (winter or summer) of 20-30℃. No special insulation measures are required, and room temperature can be selected for application.

[0051] The purified GXMZU-S strain was inoculated and streaked onto an LB plate and cultured at 30°C for 2 days. After culture, a single colony was picked and cultured in sterilized liquid LB medium for 24 hours. This was used as the seed liquid and cultured into two bottles of sterile liquid LB medium at a 1% inoculum volume. One bottle was cultured at 30°C and 200 rpm for 2 days, and the other bottle was cultured at 30°C for 2 days. The culture conditions were observed.

[0052] After 2 days, the results are as follows Figure 3 As shown, under shaking culture, GXMZU-S only produces a yellow bacterial suspension (which will appear in normal culture strains) and does not produce pigment. Only under static culture conditions can GXMZU-S produce pigment.

[0053] 2. Fermentation culture and pigment solution preparation

[0054] The purified strain was inoculated and streaked onto an LB plate and cultured at 30°C for 2 days. A single colony was taken and placed into a sterile liquid LB medium and cultured at 30°C for 7 days (see Figure 4 ), the fermentation broth was centrifuged, and the supernatant was filtered to obtain a GXMZU-S pigment aqueous solution. Methanol was added to the precipitate and the mixture was crushed using a flash extractor. The mixture was then centrifuged to obtain the supernatant, which was the GXMZU-S pigment methanol solution.

[0055] 3. The color of pigments at different pH values

[0056] Take a certain amount of pigment methanol solution and adjust the pH value using 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide respectively.

[0057] Table 1 Colors of pigments at different pH values

[0058] pH 2 3 4 5 6 7 8 9 10 11 color Rose Red Rose Red Rose Red Rose Red pink pink pink light pink light pink light pink

[0059] From the color of the pigment at different pH values ​​in Table 1, it can be seen that the pink color is maintained in the pH range of 2-8, and the pink color becomes lighter when the pH is greater than 9, so the pigment is relatively stable under acidic conditions.

[0060] 4. Photostability of pigments

[0061] The pigment methanol solution was prepared into 0.1% and 0.2% solutions with 95% ethanol by volume, and placed under natural light for 0h, 12h, 24h, 48h, 72h, and 96h, and the OD was measured. 535 The maximum absorbance A was obtained by repeating the experiment three times and taking the average value. The results are shown in Table 2. It can be seen that the pigment solutions with volume fractions of 0.1% and 0.2% have good photostability.

[0062] Table 2 OD values ​​of pigment methanol solutions with different concentrations at different times

[0063]

[0064]

[0065] 5. Thermal stability of pigments

[0066] Take the pigment methanol solution and heat it at 20℃, 40℃ and 70℃ for 20min, 35min, 50min and 65min respectively, then use the enzyme marker to measure the OD 535 The maximum absorption peak A value was obtained by repeating the experiment three times and taking the average value. The results are shown in Table 3. It can be seen that the pigment methanol solution has good thermal stability.

[0067] Table 3 OD values ​​of pigment methanol solutions heated at different temperatures for different times

[0068]

[0069] 6. Optimization of dyeing ratio

[0070] Cotton fabric was cut into 1×1 cm cloth, and the experimental groups with different ratios of pigment methanol solution (original solution) and pigment methanol solution to water solution at a volume ratio of 1:5, 1:10, 1:15, 1:20, 1:25, and 1:30 were set to soak the cloth, and hot dip dyeing was performed (starting from 30℃, dyeing for one minute, increasing the temperature by 3℃ per minute until the temperature reached 60℃, dyeing for 30 minutes, and then increasing the temperature to 80℃ and continuing dyeing for 20 minutes) and then cooling and washing. The washed cloth was placed in a 60℃ oven to dry, and the ratio with the best dyeing effect and the expected color that best met the requirements was selected as the optimal dyeing ratio.

[0071] from Figure 5It can be seen that after washing and drying, the dyeing ratios of 1:10 and 1:15 are most in line with expectations and do not fade. The subsequent use of the 1:10 ratio is the optimal dyeing ratio.

[0072] Example 3 Fabric Dyeing

[0073] After the pigment methanol solution and the aqueous solution are mixed in a volume ratio of 1:10, a 30×30 cm cotton fabric is immersed in the pigment aqueous solution and dyed by hot dip method. Starting from 30℃, after dyeing for one minute, the temperature is increased by 3℃ per minute until the temperature reaches 60℃, and the temperature is continued for 30 minutes. Then the temperature is increased to 80℃ and dyeing is continued for 20 minutes. After being removed from the fabric and cooled, it is washed with water and dried in an oven at 60℃.

[0074] Color fastness testing: The dyed cotton fabrics were sent to China Textile Standard (Shenzhen) Testing Co., Ltd. for testing of wash fastness, acid perspiration, alkali perspiration, rubbing, washing, light, and non-chlorine bleaching. The test results are shown in Table 4.

[0075] Table 4 Color fastness test results

[0076]

[0077]

[0078] In Table 4, color fastness ≥ 3 can be judged as qualified. Furthermore, 4-5 is the highest level of the test, and the effect is very excellent. Only the color fastness of light fastness is level 2, which is poor. It may be necessary to add additional light fastness and color fixation for practical application. Overall, the pigment aqueous solution provided by this application has strong overall color fastness and is suitable for application in the textile industry.

[0079] In addition, by utilizing pigment aqueous solution to dye cotton fabric, it was found that pigment aqueous solution can also dye cotton fabric with good effect and high color fastness.

[0080] In summary, the present invention provides a new strain of Serratia marcescens GXMZU-S, which can produce pigment by simply culturing it in static culture, without the need for shaking. It has a relaxed growth temperature requirement, which reduces the cost of shaking and heat preservation measures when used in industrial production. Furthermore, the pigment can be extracted using either water or chemical reagents, and both aqueous and methanolic pigment solutions offer simple dyeing procedures, high color fastness, and are safe and easy to use.

[0081] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A new strain of Serratia marcescens GXMZU-S, characterized by: The deposit number of the Serratia marcescens GXMZU-S is GDMCC.No.65230.

2. A method for preparing a pigment solution using the Serratia marcescens GXMZU-S according to claim 1, characterized in that: The following steps are involved: A colony of Serratia marcescens GXMZU-S was inoculated into a liquid LB medium and statically cultured to obtain a fermentation broth; The fermentation liquid was centrifuged, the precipitate was added with methanol, crushed, and centrifuged, and the supernatant was taken to obtain the pigment solution.

3. The method for preparing a pigment solution according to claim 2, wherein The static culture condition is 30° C. for 7 days.

4. The application of Serratia marcescens GXMZU-S in cotton fabric dyeing is characterized in that: The deposit number of the Serratia marcescens GXMZU-S is GDMCC.No.65230.

5. A method for dyeing cotton fabric using Serratia marcescens GXMZU-S, characterized in that: The following steps are involved: The pigment solution is prepared by the method for preparing a pigment solution according to any one of claims 2 to 3; The pigment solution and the aqueous solution are mixed at a volume ratio of 1:(10-15) to obtain a pigment aqueous solution, cotton fabric is immersed in the pigment aqueous solution, dyed by a hot dip method, and then washed and dried.

6. The method according to claim 5, characterized in that The pigment solution and the aqueous solution are mixed in a volume ratio of 1:

10.

7. The method according to claim 5, characterized in that The hot dip method is as follows: starting from 30°C, dyeing for one minute, raising the temperature by 3°C per minute until the temperature reaches 60°C, continuing for 30 minutes, and then raising the temperature to 80°C and continuing dyeing for 20 minutes.

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