Novel serratia marcescens for producing natural pigment and application of serratia marcescens

Abstract

The invention discloses novel serratia marcescens for producing natural pigments and application thereof, and belongs to the field of microorganisms. The invention provides a novel serratia marcescens GXMZU-S. The preservation number of the serratia marcescens GXMZU-S is GDMCC.No.65230, the preservation date is September 29, 2024, the preservation unit is Guangdong Microbiological Culture Collection Center, and the preservation address is Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Middle Road, Guangzhou City, China. The serratia marcescens GXMZU-S screened by the method disclosed by the invention does not generate pigments after being subjected to shake culture, needs to be subjected to standing culture, has no strict requirements on growth temperature, and can reduce the cost required by shake and heat preservation measures when being applied to industrial production; the GXMZU-S pigment solution is used for dyeing, the steps are simple, the color fastness is high, safety is achieved, and use is convenient and fast.

Description

Technical Field

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

[0002] Serratia marcescens is a bacterium widely found in nature that can produce pigments through secondary metabolism. At present, pigments are divided into two types: natural pigments and artificially synthesized pigments. Natural pigments are mainly extracted from plants and some insects themselves, such as cochineal, while artificially synthesized pigments are synthesized through industrial chemistry. The former is difficult to obtain, and the yield is not high, requiring a large number of breeding to obtain, and the pigment acquisition cycle is long. The latter is mainly synthesized through chemical industry, which is not only consuming a lot and easy to cause environmental pollution, but some synthetic pigments entering human daily life can easily cause life and health problems. 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] At present, many strains of Serratia marcescens have been isolated and identified, but the development and utilization of Serratia marcescens is still relatively small. Some patents have been used to study the insect and disease resistance of the secondary metabolites of Serratia marcescens, but there are few applications in dyeing and textiles, and the Serratia marcescens strains reported in the prior art all require shaking culture to produce pigments, which consumes industrial costs. The present invention has discovered a new Serratia marcescens strain GXMZU-S, which can produce pigments without shaking culture and has a good dyeing effect. Summary of the invention

[0004] The purpose of the present invention is to provide a new natural pigment-producing Serratia marcescens and an application thereof 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 applied to industrial production, the cost required for shaking and heat preservation measures can be reduced.

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

[0009] The fermentation liquid was centrifuged, and 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 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] Using the method for preparing a pigment solution, a pigment solution is prepared;

[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: starting from 30°C, after dyeing for one minute, the temperature is increased by 3°C per minute until the temperature reaches 60°C, and the temperature is continued for 30 minutes, and then the temperature is increased to 80°C and dyeing is continued for 20 minutes.

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

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

[0019] The currently disclosed Serratia marcescens all require shaking culture to produce pigments. The Serratia marcescens GXMZU-S screened by the present invention does not produce pigments after shaking culture, must be cultured statically, and has no strict requirements on the growth temperature. When applied to industrial production, it can reduce the costs required for shaking and heat preservation measures.

[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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0023] Figure 2 This 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 fermentation liquid of GXMZU-S 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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: Guangxi Darongshan Forest Nature Reserve.

[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, 121°C high pressure steam sterilization for 20 min.

[0035] 1. Isolation of strains

[0036] Take the soil sample and grind it thoroughly, add it to sterile water, add appropriate amount of glass beads, shake and culture it at 30℃, 200rpm for 3h, shake the soil sample thoroughly; take the above turbid liquid and inoculate it into LB at 10% inoculum, 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 liquid of each concentration was evenly spread on the LB solid plate, cultured at 30°C for 1-7 days, and 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°C 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 three segments were sequenced and spliced ​​into the full-length 16srRNA sequence. After comparison with the NCBI database, it was found 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 16SrRNA base sequences are shown in SEQ ID No.1-3 respectively.

[0041] SEQ ID No.1:

[0042]

[0043] SEQ ID No.2:

[0044] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[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 Microbiological Culture Collection Center. The preservation date is September 29, 2024. The preservation address is the Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Middle Road, Guangzhou, China, and the preservation 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 whether 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 solution and cultured into two bottles of sterile liquid LB medium at a 1% inoculum. 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, and 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 pigments. Only under static culture conditions can GXMZU-S produce pigments.

[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 cultured in a sterile liquid LB medium at 30°C for 7 days (see Figure 4 ), the fermentation liquid was centrifuged, the supernatant was filtered to obtain the GXMZU-S pigment aqueous solution, methanol was added to the precipitate, and the mixture was crushed using a flash extractor, and the mixture was centrifuged, and the supernatant 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 Color 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 solutions with a volume fraction of 0.1% and 0.2% using 95% ethanol. The solutions were placed under natural light for 0 h, 12 h, 24 h, 48 h, 72 h, and 96 h, 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 light stability.

[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 microplate reader to measure the OD 535 The maximum absorption peak A value was measured, and the experiment was repeated three times to obtain the average value. The results are shown in Table 3, which shows that the pigment methanol solution has good thermal stability.

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

[0068]

[0069] 6. Optimization of dyeing ratio

[0070] The cotton fabric was cut into 1×1 cm cloth, and the dye methanol solution (original solution) and the experimental groups with different ratios of the volume ratio of the dye methanol solution to the aqueous solution of 1:5, 1:10, 1:15, 1:20, 1:25, and 1:30 were set to soak the cloth, and it was dyed by hot dip method (starting from 30°C, dyeing for one minute, increasing the temperature by 3°C per minute until the temperature reached 60°C, dyeing for 30 minutes, and then increasing the temperature to 80°C and continuing dyeing for 20 minutes), cooled and washed, and the washed cloth was placed in a 60°C oven to dry, and the ratio with the best dyeing effect and the expected color that best meets 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] The pigment methanol solution and the aqueous solution were mixed in a volume ratio of 1:10, and a 30×30cm cotton fabric was immersed in the pigment aqueous solution and dyed by hot dip method, starting from 30℃. After dyeing for one minute, the temperature was increased by 3℃ per minute until the temperature reached 60℃, and the temperature was continued for 30min. Then the temperature was increased to 80℃ and dyeing was continued for 20min. After being removed from the fabric and cooled, it was washed with water and dried in an oven at 60℃.

[0074] Color fastness test: The dyed cotton fabrics were sent to China Textile Standard (Shenzhen) Testing Co., Ltd. for testing of washing fastness, acid perspiration color fastness, alkali perspiration color fastness, rubbing color fastness, washing color fastness, light color fastness and non-chlorine bleaching color fastness. 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 detection, and the effect is very good. Only the color fastness of light fastness is level 2, and the effect is poor. It may be necessary to add additional light fastness and color fixation for practical application. In general, the overall color fastness of the pigment aqueous solution provided by this application is strong and suitable for application in the textile industry.

[0079] In addition, by using the pigment aqueous solution to dye cotton fabric, it was found that the 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 static culture without shaking culture, has no strict requirements on growth temperature, and can reduce the cost of shaking and heat preservation measures when applied to industrial production. The pigment can be extracted by water or chemical reagents, and the dyeing steps of the pigment aqueous solution and the pigment methanol solution are simple, the color fastness is high, and it is safe and easy to use.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A new strain of Serratia marcescens GXMZU-S, characterized in that: 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: The colony of Serratia marcescens GXMZU-S was inoculated into a liquid LB medium and statically cultured to obtain a fermentation liquid; The fermentation liquid was centrifuged, and 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, after dyeing for one minute, the temperature is increased by 3°C per minute until the temperature reaches 60°C, which is continued for 30 minutes, and then the temperature is increased to 80°C and dyeing is continued for 20 minutes.

Images