Pseudomonas citronellolis and application thereof in production of melanin

Abstract

The application discloses a strain of Pseudomonas citronellolis and application of the strain in production of melanin, and belongs to the technical field of microbial engineering. Pseudomonas citronellolis ZY-01 GDMCC No:65662 and application thereof in preparation of melanin. The yield of the strain ZY-01 is increased by 20.69 times through design and optimization of a fermentation medium, the yield of melanin produced by shake flask fermentation can reach 54.315 g / L, and the melanin has good reproducibility and scalability. The extracted melanin is identified as natural melanin by ultraviolet-visible spectroscopy, infrared spectroscopy and solubility analysis, and does not contain protein and nucleic acid impurities. The Pseudomonas citronellolis of the application is a high-yield and high-quality strain meeting industrial production, and can be used for preparation of many products containing melanin, such as drugs, drug carriers, foods, cosmetics, melanin composite materials and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial engineering, and particularly relates to a strain of pseudomonas citronellolis and application thereof in production of melanin. BACKGROUND

[0002] Melanin, as the most abundant natural pigment in nature, is a kind of polymeric phenolic or indolic substances with extremely complex chemical structure and non-homogeneity, and widely exists in various animals, plants and microorganisms. As a secondary metabolite, melanin is not essential for the growth and development of organisms, but can effectively improve the survival and competitiveness of organisms. Its universal existence is considered to be the result of long-term mutual adaptation between life and environment in the process of evolution. According to the different synthesis pathways and intermediate metabolites, melanin can be mainly divided into three categories: eumelanin, pheomelanin and allomelanin. Among them, allomelanin includes pyomelanin, 1,8-dihydroxynaphthalene (DHN) melanin and the like.

[0003] Melanin has various biological functions. It can absorb ultraviolet rays, reduce the damage of ultraviolet rays to the DNA of skin cells, reduce the risk of skin cancer, and exhibit good anti-radiation ability. Melanin also has advantages such as biological antioxidant and immune promotion. A large number of free radicals in the body can cause cell damage, accelerate aging and trigger various diseases. As an antioxidant, melanin can neutralize free radicals, and its ability to scavenge free radicals is 5 times that of vitamin E, which can effectively protect cells from oxidative damage. At the same time, melanin can also regulate the function of the immune system, enhance the immunity of the body, participate in visual protection, maintain the normal physiological function of the eye, and the like. These characteristics make melanin have great application potential in many fields such as medicine, industry, agriculture, food, cosmetics and the like. In the field of medicine, melanin can be used for developing new drug carriers, sunscreens and anti-tumor drugs, and the like; in the food industry, it can be used as a natural food additive to improve the color and stability of food; in the field of cosmetics, the antioxidant and ultraviolet absorption properties of melanin make it an important ingredient for developing high-end skin care products. In addition, melanin also has strong application potential in melanin composite materials, integration of heavy metals using melanin, photo-thermal conversion and the like, and has a very broad application prospect.

[0004] Natural melanin is widely distributed in animals, plants and microorganisms: animal melanin is in various tissues, including skin, hair, eyes, inner ear and mucosa; plant melanin mainly exists in the epidermis of seeds and fruits; many molds, bacteria and actinomycetes can produce melanin through fermentation. Due to the long growth cycle of animals and plants, low extraction rate of melanin, and many random variables, etc., the world is more inclined to obtain natural melanin through microbial fermentation. Microbial fermentation method is not limited by region and season, has short production cycle, small environmental pollution, high product safety, is easy for industrialized production and has low cost, and has better competitive advantage in the melanin production market. Breeding of high-yield strains is crucial for fermentation production, and is an important factor to determine the industrial value of fermentation products. Since melanin synthesis involves the synergistic action of many genes and enzymes, the regulation mechanism is complex and has not been fully elucidated; and the melanin synthesis pathway differs in different microorganisms, which increases the complexity of breeding high-yield strains suitable for industrial production. At present, the resources of high-yield strains suitable for industrial production are still insufficient, which limits the development of melanin industry to some extent.

[0005] With the continuous expansion of the application field of melanin and the rapid growth of market demand, mining of high-yield melanin strains can not only improve production efficiency, but also effectively control production cost, improve product quality, enhance market competitiveness of melanin products, and is conducive to promoting the sustainable development of melanin industry. SUMMARY

[0006] The purpose of the present application is to provide a high-yield melanin strain and a method for producing melanin using the strain. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above-mentioned purpose, the present application first provides a strain of Pseudomonas citronellolis, which is Pseudomonas citronellolis ZY-01, the preservation number of which in Guangdong Microbial Culture Collection Center is GDMCC No: 65662.

[0008] The present application also provides a composition for preparing melanin, which contains the Pseudomonas citronellolis ZY-01.

[0009] The composition can be a culture or a microbial agent.

[0010] The culture can refer to the general term of the liquid or solid product (all the substances in the culture container) which grows with the microbial population after artificial inoculation and culture. That is, the product obtained by growing and amplifying the microorganism, which can be a biologically pure culture of microorganism, or can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The culture also includes the subculture obtained by subculturing the microorganism, which can be a culture of a certain generation, or a mixture of several generations.

[0011] Further, the culture can be a fermentation culture (such as a fermentation broth). The fermentation culture can be obtained by fermenting the Pseudomonas citronellolis ZY-01 in a fermentation medium, which contains bacterial cells, bacterial metabolites and fermentation medium.

[0012] The microbial agent can refer to a microbial preparation made of one or several microorganisms. The microbial agent can include solid microbial agents (such as slant strains, freeze-dried strains, etc.) and liquid microbial agents (such as bacterial liquid, glycerol bacteria, etc.). The conventional preparation method of microbial agents is well known to those skilled in the art, for example, the slant strain can be obtained by directly preserving the bacterial cells after inoculating and culturing on a solid slant medium; the freeze-dried strain can be obtained by dehydrating the bacterial cells using freeze-drying technology to form a dry powder; the bacterial liquid can be obtained by directly dispensing after culturing in a liquid medium; the glycerol bacteria can be prepared by mixing the bacterial cells with a cryopreservation solution containing glycerol, which can play an antifreeze protection role.

[0013] The application also provides the use of the Pseudomonas citronellolis ZY-01 or the composition in the preparation of melanin.

[0014] Further, the use includes preparing melanin by fermentation method.

[0015] The application also provides a method for preparing (producing) melanin, which comprises culturing the Pseudomonas citronellolis ZY-01 in a culture medium to obtain a culture, and collecting melanin from the culture.

[0016] The culture medium can refer to a mixture of various nutrients prepared in a certain proportion, which provides the growth and reproduction of microorganisms and the biosynthesis of various metabolites.

[0017] In the above method, the culture medium can contain a carbon source, a nitrogen source, inorganic salts and a substrate, the carbon source can be selected from at least one of glucose, sucrose, maltose, glycerol and xylose; the nitrogen source can be selected from at least one of tryptone and peptone; the inorganic salt can be selected from at least one of CuSO4, CaCl2, FeCl3 and MgCl2; the substrate can be selected from at least one of L-tyrosine and phenylalanine.

[0018] In the method, the culture medium can contain glucose, tryptone, L-tyrosine and CuSO4.

[0019] Further, the CuSO4 can be CuSO4·5H2O.

[0020] In the method, the concentration of the glucose can be 1-30 g / L, the concentration of the tryptone can be 5-30 g / L, the concentration of the L-tyrosine can be 1-20 g / L, and the concentration of the CuSO4 can be 0.01-0.9 g / L.

[0021] Further, the concentration of the glucose can be 10-15 g / L, the concentration of the tryptone can be 17-21 g / L, the concentration of the L-tyrosine can be 13-17 g / L, and the concentration of the CuSO4 can be 0.02-0.08 g / L.

[0022] Further, the concentration of the glucose can be 13 g / L, the concentration of the tryptone can be 19 g / L, the concentration of the L-tyrosine can be 15 g / L, and the CuSO4 can be CuSO4·5H2O with a concentration of 0.05 g / L.

[0023] Further, the culture medium can be composed of glucose 13 g / L, tryptone 19 g / L, L-tyrosine 15 g / L, CuSO4·5H2O 0.05 g / L, NaCl 5 g / L, and the rest is water.

[0024] In the method, the pH value of the culture medium can be 5-9. Further, the pH value of the culture medium can be 8.

[0025] The culture can refer to the growth and reproduction of microorganisms and the biosynthesis of various metabolites under suitable culture medium and culture conditions. The culture can be carried out according to the conventional methods in the art, including but not limited to hole plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and the conditions of various cultures such as temperature and rotation speed can be appropriately adjusted according to the actual situation.

[0026] The culture according to the present application includes culturing the P. cymbium ZY-01 under conditions suitable for culturing P. cymbium to produce melanin.

[0027] Further, the temperature of the culture can be 35-39℃. Further, the temperature of the culture can be 37℃.

[0028] In the method, the culture medium can have the following composition: 13 g / L of glucose, 19 g / L of tryptone, 15 g / L of L-tyrosine, 0.05 g / L of CuSO4·5H2O, 5 g / L of NaCl, and the rest is water; the pH value of the culture medium can be 8, and the culture temperature can be 37°C.

[0029] The culture conditions can further include a rotation speed of 200 r / min.

[0030] The method for preparing melanin can be a fermentation method for preparing melanin, which can include the following steps:

[0031] (1) inoculating the Pseudomonas citronellolis ZY-01 into a seed culture medium for seed culture to obtain a seed liquid;

[0032] (2) inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid;

[0033] (3) separating melanin from the fermentation liquid.

[0034] The seed culture medium can refer to any culture medium used for culturing the Pseudomonas citronellolis ZY-01 to obtain a seed liquid for inoculating a bioreactor (such as a shake flask, a fermentation tank, etc.). In some embodiments, the composition of the seed culture medium is 3 g / L of beef extract, 10 g / L of protein peptone, and 5 g / L of NaCl, and the rest is water; the culture conditions of the seed culture include 37°C and 200 rpm.

[0035] Further, the inoculation amount of the seed liquid inoculated into the fermentation culture medium for fermentation culture can be 1%-20% (volume percentage). In some embodiments, the inoculation amount is 1%.

[0036] Further, the liquid loading amount (i.e., the volume percentage of the fermentation culture medium in the bioreactor) of the fermentation culture can be 10%-85%. In some embodiments, the liquid loading amount is 20%.

[0037] In some embodiments of the present application, the separation of melanin from the fermentation liquid adopts an alkali dissolution and acid precipitation method, and the specific steps include: adjusting the pH value of the centrifuged fermentation liquid to 2 to precipitate melanin, suspending the melanin in HCl, dissolving the melanin in NaOH, and then acid precipitating again, and drying to obtain purified melanin.

[0038] The extracted melanin has a maximum absorption wavelength of 290 nm in the ultraviolet-visible spectrum, and the infrared spectrum shows 3344.85 cm -1 (O-H), 1653.9 cm -1The extracted melanin has no solubility in common polar organic solvents such as ethanol, methanol, acetonitrile, acetone and tetrahydrofuran, has low solubility in deionized water and under acidic conditions, and is completely dissolved in high-polarity aprotic solvents such as DMF (N,N-dimethylformamide) and DMSO (dimethyl sulfoxide); and is completely dissolved in 1 mol / L NaOH solution.

[0039] The melanin yield obtained by the method for preparing melanin provided by the application can be greater than or equal to 40 g / L.

[0040] In view of the problems of low yield (generally <10 g / L), low substrate conversion efficiency and high production cost in the production of melanin by the existing microbial fermentation method, the application provides a new strain of Pseudomonas citronellolis with high melanin yield, and an optimized fermentation medium and culture condition thereof, thereby laying a foundation for realizing efficient and low-cost industrial production of melanin.

[0041] The key to the development of fermentation engineering products is to screen new excellent strains, and the inventors of the application screen a Pseudomonas citronellolis strain with high melanin yield through extensive and in-depth mining. Pseudomonas citronellolis ZY-01, which is identified by 16S rRNA and has a similarity of 99.59% with Pseudomonas citronellolis NBRC 103043 T The Pseudomonas citronellolis ZY-01 is gram-negative, and the colony is milky white and round with a diameter of 1-1.5 mm. The nucleotide sequence of the 16S rRNA gene of the Pseudomonas citronellolis ZY-01 is shown as SEQ ID NO: 1.

[0042] In order to fully exert the production potential of the strain ZY-01 and meet the industrial requirements of yield increase and cost saving, the fermentation medium of the strain ZY-01 is further designed and optimized. The efficient fermentation medium condition is obtained by combining the single-factor method, the Plackett-Burman design and the response surface method (Box-Behnken design), and the optimal medium composition is determined as follows: glucose 13 g / L, tryptone 19 g / L, L-tyrosine 15 g / L, CuSO4·5H2O 0.05 g / L and NaCl 5 g / L, and the rest is water. The initial pH value is 8.0, and the strain is cultured under the fermentation conditions of a temperature of 37℃, a rotation speed of 200 rpm, an inoculation amount of 1% and a liquid loading amount of 20%, so that the melanin yield obtained by the shake flask fermentation can reach 54.315 g / L, reaching a leading level.

[0043] The melanin in the fermentation liquor is extracted and purified by the alkali dissolution and acid precipitation method, and the product characteristics are identified. The ultraviolet-visible light spectrum has a maximum absorption peak of 290 nm, the logarithmic absorbance and the wavelength have a linear relationship in the range of 400-600 nm, the infrared spectrum characteristic peak and the solubility are consistent with the characteristics of the natural melanin reported in the literature.

[0044] Technical effects: The citronellol Pseudomonas ZY-01 screened by the application can efficiently synthesize and accumulate melanin in the fermentation process, and the yield of melanin prepared by shake flask fermentation can reach 54.315 g / L, far exceeding the prior art level, laying a foundation for industrial scale production. The yield of citronellol Pseudomonas ZY-01 is increased by 20.69 times by designing and optimizing the fermentation medium, and the preferred low-cost carbon source glucose and high-cost-performance nitrogen source tryptone significantly reduce the production cost. In addition, the extracted melanin is verified by ultraviolet spectrum to be free of protein and nucleic acid impurities, which meets the characteristics of natural melanin. The optimized medium and fermentation parameters are verified by PB experiment and BBD experiment, and have good reproducibility and scalability. The citronellol Pseudomonas of the application is a high-yield and high-quality strain for industrial production, which can reduce cost, increase profit and enhance the market competitiveness of melanin products. The citronellol Pseudomonas of the application can be used for preparing many products containing melanin, such as drugs, drug carriers, foods, cosmetics, melanin composite materials and the like, and has wide application prospect.

[0045] Deposit description

[0046] Strain name: Citronellol Pseudomonas

[0047] Latin name: Pseudomonas citronellolis

[0048] Classification name: Pseudomonas citronellolis

[0049] Strain number: ZY-01

[0050] Deposit unit: Guangdong Microbial Culture Collection Center

[0051] Abbreviation of deposit unit: GDMCC

[0052] Address: 5th Floor, Building 59, 100, Martyrs' Road, Guangzhou

[0053] Deposit date: December 23, 2024

[0054] Center registration number: GDMCC No: 65662. Brief description of drawings

[0055] Figure 1 It is a colony morphology diagram of strain ZY-01.

[0056] Figure 2 It is a gram staining result diagram of strain ZY-01 observed under an optical microscope.

[0057] Figure 3 It is a 16S rRNA adjacent phylogenetic tree of strain ZY-01.

[0058] Figure 4 The purified melanin was extracted from the fermentation broth.

[0059] Figure 5 The UV-Vis spectrum of the purified melanin.

[0060] Figure 6 The linear relationship between the logarithmic absorbance and the wavelength of the purified melanin.

[0061] Figure 7 The infrared spectrum of the purified melanin.

[0062] Figure 8 The standard curve of the melanin extracted from the fermentation broth of the strain ZY-01.

[0063] Figure 9 The single-factor experiment results (OD 400 is the value after 10-fold dilution).

[0064] Figure 10 The single-factor experiment results (OD 400 is the value after 10-fold dilution).

[0065] Figure 11 The contour plot and 3D surface plot of the interaction between the glucose concentration and the trypsin concentration on the melanin production.

[0066] Figure 12 The contour plot and 3D surface plot of the interaction between the glucose concentration and the L-tyrosine concentration on the melanin production.

[0067] Figure 13 The contour plot and 3D surface plot of the interaction between the trypsin concentration and the L-tyrosine concentration on the melanin production. DETAILED DESCRIPTION

[0068] The present application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0069] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources, unless otherwise specified.

[0070] The R2A solid culture medium in the following examples is composed of: 0.25 g / L of tryptone, 0.5 g / L of acid hydrolyzed casein, 0.5 g / L of yeast extract powder, 0.5 g / L of soluble starch, 0.3 g / L of potassium phosphate, 0.1 g / L of magnesium sulfate, 0.3 g / L of sodium pyruvate, 0.25 g / L of proteose peptone, 0.5 g / L of glucose, 20 g / L of agar, and the rest is water.

[0071] Example 1, screening and identification of Pseudomonas citronellolis ZY-01

[0072] 1. Screening of Pseudomonas citronellolis ZY-01

[0073] The strain described in the present application was isolated from a sewage sample from a sewage treatment plant in Shanghai, China by the inventors in May 2022. Considering that the strains in the screening range are derived from oligotrophic environments, the low-nutrient characteristics of R2A are more consistent with their original conditions, which helps to restore their physiological activity. Moreover, using R2A as the activation medium can maintain consistency in the culture conditions and reduce the risk of adaptive variation. Therefore, R2A medium was selected as the activation medium. Subsequently, the bacterial solution was streaked on the R2A plate using an inoculation loop, and incubated at 37°C for 24-72 h in an incubator. Then, single colonies on the plate were picked and preserved on a slant for subsequent screening experiments.

[0074] The metabolic pathway of microorganisms producing melanin mainly relies on L-tyrosine as a precursor, which is then continuously oxidized and polymerized to obtain natural melanin after being catalyzed by different key enzymes. Considering that melanin is an extracellular secreted pigment that can diffuse to the surrounding medium, it is convenient for visual screening. Therefore, L-Tyr melanin production medium was prepared: glucose 1 g, proteose peptone 5 g, NaCl 5 g, CaCl2 0.1 g, L-tyrosine 1 g, and water to 1 L, pH 7.2. The prepared screening medium was divided into 50 mL in 250 mL shake flasks and sterilized by high-pressure steam. Single colonies on the slant were picked and inoculated into the sterilized L-Tyr liquid medium, which was incubated at 37°C and 200 rpm in an incubator for 1-7 days. The color change of the medium in the shake flask was observed.

[0075] Screening results: Through shake flask fermentation screening, it was found that the strain ZY-01 described in the present application secreted melanin into the culture medium at 37°C for 48 h. During the fermentation of the strain to produce melanin, the color of the fermentation broth changed from light yellow to dark yellow, then to orange, then to pink, then to deep red, and finally to black.

[0076] 2. Identification of Pseudomonas citronellolis ZY-01

[0077] (1) Morphological identification

[0078] Morphological characteristics analysis: R2A solid medium was configured, the disposable culture dish was taken out to complete the plate inversion in the clean bench, and the plate was placed horizontally in the clean bench. The ultraviolet light was turned on, and after the plate was solidified, it was placed upside down in a 37°C incubator for 1 day. After 1 day, whether colonies grew on the plate was observed. If not, it indicated that the medium was not contaminated with bacteria, and the strain ZY-01 could be directly cultured by plate streaking method. After the single colony was cultured, the colony characteristics were observed and recorded. A clean glass slide was taken, a drop of 0.9% physiological saline was added to the middle position, a single colony was picked up with a inoculating loop and smeared on the drop of physiological saline, dried, fixed under the outer flame of an alcohol lamp, primary stained with crystal violet for 1 min, washed with water, mediately dyed with iodine solution for 1 min, washed with water, added with decolorizing solution, shook the slide, decolorized for about 20 s, washed with water, re-stained with safranine for 1 min, washed with water, and then the water on the slide was absorbed, and then oil immersion examination was performed.

[0079] The identification results are shown in Figure 1 and Figure 2 : the colony morphology of strain ZY-01 cultured at 37°C for 2 days on R2A solid medium plate Figure 1 Through observation, the colony was milk white, the colony surface was flat, the edge was smooth, and the morphology was a circular shape with a diameter of 1 mm-1.5 mm. The strain ZY-01 was subjected to gram staining, and after the staining was completed, the staining result was observed by optical microscope Figure 2 . Finally, it was stained red, indicating that the strain ZY-01 was a gram-negative bacterium.

[0080] (2) Molecular biology identification

[0081] 16S rRNA sequence PCR amplification: Since 16S rRNA gene includes conserved regions and variable regions, which are alternated with each other, reflecting the phylogenetic relationship between species, while the variable regions reflect the differences between species, therefore, the preliminary identification of bacteria usually detects the sequence information of 16S rRNA. The experimental strain ZY-01 was inoculated in R2A solid medium by plate streaking method, and single colonies were obtained after 37°C culture for 1 d, and colony PCR was performed. The single colony of strain ZY-01 was used as a template, and 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) and 1492R (5'-ACGGCTACCTTGTTACGACTT-3', SEQ ID NO:3) were used as upper and lower primers to amplify the 16S rRNA gene of strain ZY-01. The PCR amplification system was 50 μL: primer 27F (10 mM) 2 μL, primer 1492R (10 mM) 2 μL, Taq PCR Master Mix enzyme (2x blue dye) 25 μL, ddH2O 20 μL, and 1 μL of bacterial single colony. The PCR amplification program was 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 10 min.

[0082] 16S rRNA sequence alignment: After the amplified gene was detected by 1% agarose gel electrophoresis, the clear PCR amplification product of the running gel strip was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for 16S rRNA sequencing, and the sequencing results were processed after sequencing. First, use Chromas software to cut a large number of unqualified base sequences with double peaks, then use MEGAversion 6.0 software to splice the cut gene sequences, the spliced gene sequences are the 16S rRNA gene sequences of the strain ZY-01 to be identified, and the EzTaxon-eserver (http: / / eztaxon-e.ezbiocloud.net / ) database is used to compare with the 16S rRNA gene sequences of other published strains in the same genus. If the similarity is greater than 99%, the strain information of the strain can be preliminarily determined. The obtained 16S rRNA sequence with 100% integrity is compared with the known bacterial 16S rRNA gene sequence, and the top ten bacterial 16S rRNA gene sequences with the closest similarity to the compared sequence are downloaded. Use ClustalW tool in MEGAversion 6.0 software to align the sequences in the fasta file. After alignment, save it in mas file format. Use the neighbor-joining method to build a tree, with a bootstrap value of 1000, and use Kimura's two-parameter model to calculate the evolutionary distance.

[0083] The results of the identification are as follows Figure 3 As shown: The 16S rRNA gene sequence of strain ZY-01 was amplified and sequenced, revealing a 1551 bp length (nucleotide sequence as shown in SEQ ID NO:1), with 100% sequence integrity. The 16S rRNA clone of strain ZY-01 was compared with the EZbiocloud database, and it was found to be similar to that of strain ZY-01. Pseudomonas citronellolis NBRC 103043 T The similarity was close to 99.59%. Based on the alignment results, the 16S rRNA gene sequences of the ten bacteria with the closest sequence similarity to strain ZY-01 were downloaded, and a phylogenetic tree was constructed using the nearest neighbor method. Figure 3 Phylogenetic analysis based on 16S rRNA sequences showed that strain ZY-01 always resided in the same clump as other species in the genus *Pseudomonas*, indicating a relatively distant phylogenetic relationship with species from neighboring genera. Strain ZY-01 and... Pseudomonas citronellolis NBRC 103043 T The strain exhibits the highest proximity, belonging to the same lineage. Therefore, based on the 16S rRNA sequence, this high-melanin-producing strain ZY-01 is identified as *Citronellol Pseudomonas*. Pseudomonas citronellolis .

[0084] Based on morphological and molecular biological identification, strain ZY-01 was finally identified as *Citronellol Pseudomonas*. Pseudomonas citronellolis The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on December 23, 2024, with accession number GDMCC No:65662.

[0085] Example 2: Identification of melanin in fermentation broth

[0086] 1. Production of melanin by fermentation using strain ZY-01

[0087] (1) Citronellol Pseudomonas ZY-01 was inoculated into seed culture medium for seed culture to obtain seed liquid.

[0088] (2) The seed liquid is inoculated into the fermentation medium for fermentation culture to obtain the fermentation broth.

[0089] The seed culture medium was NB medium: beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L.

[0090] Fermentation medium: glucose 1 g, peptone 5 g, NaCl 5 g, CaCl20.1 g, L-tyrosine 1 g, make up to 1 L, pH 7.2.

[0091] Strain ZY-01 was inoculated from R2A solid medium into a 50 mL sterile centrifuge tube containing 5 mL NB liquid medium, and incubated at 37°C, 200 rpm for 4 h, OD 600 The OD was measured to be between 0.6-0.8, and then inoculated into a 250 mL flask containing 50 mL of fermentation medium at an inoculation amount of 1%, and incubated at 37°C, 200 rpm for 7 d to obtain a fermentation broth.

[0092] 2. Identification of melanin in the fermentation broth

[0093] Purification of melanin in the fermentation broth: The alkaline solution and acid precipitation method was used to extract melanin from the fermentation broth. Most natural melanins are not soluble in water, but are soluble in strong alkaline solution at pH≈11, and precipitate in solution at pH<3. The steps of extraction and purification are as follows: First, centrifuge the fermentation broth at 10000 rpm for 10 min, and retain the supernatant, in order to remove the bacterial cells and other impurities; adjust the pH of the supernatant to 2 with 6 mol / L HCl solution, and stand for 12 h, in order to allow the melanin to precipitate fully; then centrifuge at 10000 rpm for 10 min to retain the precipitate, in order to obtain the crude extract of melanin; suspend the crude extract with 6 mol / L HCl solution and stand for 12 h, in order to degrade the proteins and lipids connected to the melanin; centrifuge at 10000 rpm for 10 min, and retain the precipitate, which is dissolved with 1 mol / L NaOH solution, and then centrifuged at 10000 rpm for 10 min, in order to remove the proteins and lipids connected to the melanin; then adjust the pH to 2 with 6 mol / L HCl solution, and stand for 12 h, in order to precipitate the melanin; finally, centrifuge at 10000 rpm for 10 min to obtain the flocculent precipitate, which is freeze-dried in a freeze-dryer for 8 h, in order to dry it fully; the obtained melanin is the purified melanin, which is stored at 4°C for later use.

[0094] Solubility analysis: several portions of 50 mg of the extracted and purified melanin were weighed, and dissolved in 1 mL of ethanol, methanol, acetonitrile, acetone, tetrahydrofuran, DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), deionized water, 6 mol / L HCl solution, and 1 mol / L NaOH solution, respectively. After vortexing for 1 min, the solubility of the melanin was observed.

[0095] UV-Vis UV-visible spectrum analysis: The natural melanin after extraction and purification was weighed, dissolved in 0.1 mol / L NaOH solution, and diluted to 0.1 g / L of melanin solution. The absorption spectrum of melanin in the range of 200-800 nm was determined by UV-visible spectrometer, and the blank reference solution was 1 mol / L NaOH solution, so as to determine the maximum absorption wavelength of the melanin extracted from the fermentation broth of strain ZY-01.

[0096] FTIR Fourier infrared spectrum analysis: The extracted and purified melanin and potassium bromide (KBr) were dried to constant weight in an oven at 70°C, 2 mg of sample and 40 mg of KBr were weighed respectively, ground uniformly, and pressed into a tablet by a tablet press. The scanning range was 4000-500 cm -1 , and the scanning was determined by Fourier infrared spectrometer.

[0097] Experimental results: The melanin solid powder obtained by alkali dissolution and acid precipitation method (Fig. 1) Figure 4 ) presents uniform and deep black, which is a fine, dry, slightly soft agglomerate with typical matte appearance, no obvious visible impurity particles or color spots, and no odor.

[0098] The extracted melanin is not soluble in common polar organic solvents such as ethanol, methanol, acetonitrile, acetone, tetrahydrofuran; the solubility is low in deionized water and acidic conditions; but it is completely dissolved in high-polarity aprotic solvents such as DMF (N,N-dimethylformamide) and DMSO (dimethyl sulfoxide), and it is found that the extracted melanin is completely dissolved in 1 mol / L NaOH solution.

[0099] From the UV-Vis ultraviolet-visible spectrum (Fig. 2) Figure 5 ), the maximum absorption wavelength of the melanin extracted from the fermentation broth of strain ZY-01 is 290 nm (Fig. 3) Figure 5 ), and no obvious absorbance peak is observed at 230 nm, 260 nm and 280 nm, indicating that the extracted and purified melanin almost does not contain impurities such as carbohydrates, nucleic acids and proteins; the logarithmic absorbance of the extracted melanin has a linear relationship with the wavelength between 400-600 nm (Fig. 4) Figure 6 . Due to the characteristics of the complex conjugated structure of melanin, the linear relationship between the logarithmic absorbance and the wavelength between 400-600 nm is one of the most important standards for identifying melanin, and the slope of the linear curve is often used to identify melanin. FTIR Fourier infrared spectrum analysis is shown in Fig. 5 Figure 7 .

[0100] Example 3, fermentation medium optimization

[0101] (1) Quantitative analysis of strain ZY-01:

[0102] The melanin in the fermentation broth of strain ZY-01 (obtained in Example 2) was extracted by alkali dissolution and acid precipitation, and the extracted and purified melanin was dissolved in 1 mol / L NaOH solution to prepare gradient standard solutions of 6 g / L, 5 g / L, 4 g / L, 3 g / L, 2 g / L and 1 g / L. Then, the gradient standard solutions were diluted 50 times and the OD was measured. 400 A standard curve was drawn and the concentration was calculated with 1 mol / L NaOH solution as a blank control.

[0103] (2) Selection of carbon source, nitrogen source, inorganic salt and substrate:

[0104] The basic fermentation medium (basic L-Tyr liquid medium) was prepared by dissolving 1 g of glucose, 5 g of protein peptone, 5 g of NaCl, 0.1 g of CaCl2 and 1 g of L-tyrosine in 1 L of water and adjusting the pH to 7.2. In order to save fermentation cost and shorten production cycle, the four key medium components, i.e. carbon source, nitrogen source, inorganic salt and substrate, which may affect the strain, were optimized. The basic L-Tyr liquid medium was used as a control, and single variable method was used to design experiments. Different carbon sources, nitrogen sources, inorganic salts and substrates were used, and each experiment was set in triplicate (Table 1). The fermentation parameters were uniformly controlled as follows: 20% liquid loading, 1% inoculum, 200 rpm shaking speed, 37°C constant temperature, 100 mL shake flask was used for 7 days of culture, and then OD 400 value was determined and compared to select the optimal medium formula.

[0105]

[0106] (3) Determination of the optimal concentration of carbon source, nitrogen source, inorganic salt and substrate

[0107] With initial pH value of 7.2, fermentation temperature of 37°C, rotation speed of 200 r / min and inoculum amount of 1% as the initial culture conditions, single factor experiment was used to study the effects of carbon source concentration, nitrogen source concentration, metal ion concentration, substrate concentration and initial pH value in the medium components on the melanin yield (Table 2). Each experiment was set in triplicate to determine the control range of subsequent fermentation parameters.

[0108]

[0109] (4) Plackett-Burman (PB) experiment

[0110] According to the single factor experiment results, the Design Expert 13.0 software was used to design the PB experimental scheme of glucose concentration, tryptone concentration, CuSO4·5H2O concentration, L-tyrosine concentration and initial pH value, 1 low level (-1) and 1 high level (1) were set for each variable, 3 parallel experiments were set in each group, and the melanin yield was used as the response value to screen the significant factors affecting the strain ZY-01 fermentation synthesis of melanin. The experimental factors and levels are shown in Table 3.

[0111]

[0112] (5) Box-Behnken (BBD) experiment

[0113] Box-Behnken design is a commonly used response surface design method, Design Expert 13.0 software was used in this experiment, and BBD test design was carried out on the basis of PB experiment, 1 low level (-1), 1 middle level (0) and 1 high level (1) were set for each variable, 3 parallel experiments were set in each group, and the parameters with significant influence were screened. Other factors were obtained from the single factor experiment with the highest melanin yield, the melanin yield was used as the response value to optimize the fermentation medium conditions of strain ZY-01, and the predicted melanin yield by response surface method was verified. The experimental factors and levels are shown in Table 4.

[0114]

[0115] Experimental results: Quantitative analysis results of strain ZY-01 producing melanin: After acid precipitation and alkali dissolution extraction and quantitative analysis, the melanin yield was 2.5045 g / L, and the linear equation obtained from the standard curve (Y=0.1796X+0.0134) was as follows: Figure 8

[0116] The results of the selection of carbon source, nitrogen source, inorganic salt and substrate Figure 9 ​): The optimal carbon source, nitrogen source, inorganic salt and substrate of strain ZY-01 were selected by single factor significant experiment results. It was found that glucose with a predicted yield of 2.551 g / L became the optimal carbon source, although the melanin yield with glycerol and xylose as carbon source was similar, but the advantage of using glucose as carbon source for fermentation to produce melanin was not only in the fermentation efficiency, but also in the cost benefit, its unit price was lower, which met the cost control needs of industrial production. The yield of tryptone was 2.986 g / L, which was better than other nitrogen sources, although the yield of melanin of strain ZY-01 with tryptone as nitrogen source was close to that with peptone, but the price was lower, which highlighted the strategy of optimizing resource allocation under the premise of ensuring yield, so tryptone was selected as the best nitrogen source. When selecting the best inorganic salt, it was found that metal ions played a key role in the fermentation of strain ZY-01 to synthesize melanin, the addition of CuSO4 made the melanin yield jump to 4.811 g / L, which was more than 65% higher than the basic group, the significant promotion of copper ions to the yield suggested that there might be a metal-dependent enzymatic mechanism in the synthesis of melanin. L-tyrosine as a precursor of melanin, its yield was significantly higher than that of salicylic acid, phenylalanine and other substitutes, which confirmed the dependence of melanin biosynthesis pathway on specific substrate. Therefore, through multidimensional screening, the optimal carbon source was glucose, the optimal nitrogen source was tryptone, the optimal inorganic salt was CuSO4, and the optimal substrate was L-tyrosine, which not only took into account the high yield of melanin and controlled the low cost, but also laid the foundation for industrial application.

[0117] Determination of the concentration of the optimal carbon source, nitrogen source, inorganic salt, substrate and initial pH Figure 10 : The effects of glucose, tryptone, CuSO4·5H2O, L-tyrosine and initial pH on the yield of melanin produced by strain ZY-01 were investigated by single factor experiment. The concentration of glucose significantly affected the synthesis of melanin by strain ZY-01, when the concentration was 15 g / L, OD 400 reached 1.759, and the yield of melanin was the highest at 4.823 g / L, which was higher than or lower than this concentration, indicating that 15 g / L was the optimal carbon source concentration, and excessive glucose might cause carbon metabolic inhibition. As an organic nitrogen source, with increasing concentration, the yield of melanin produced by strain ZY-01 showed a trend of first increasing and then decreasing, at 15 g / L, it synergistically promoted cell growth and product accumulation, at this time, the yield of melanin was the highest, at 5.153 g / L, excessive nitrogen source might destroy the carbon-nitrogen balance and inhibit secondary metabolism, so 15 g / L was the optimal tryptone concentration. With increasing concentration of CuSO4·5H2O, the yield of melanin produced by strain ZY-01 showed a decreasing trend, indicating that high concentration of Cu 2+Cytotoxicity. When the concentration of CuSO4 was 0.05 g / L, the highest melanin yield was 4.604 g / L, so the optimal fermentation concentration of CuSO4·5H2O was 0.05 g / L. L-tyrosine as a precursor of melanin synthesis, its concentration had the most significant impact on the yield, with the increasing concentration of L-tyrosine, the yield of melanin synthesized by strain ZY-01 fermentation showed a trend of first increasing and then decreasing, excessive substrate may trigger feedback inhibition or osmotic stress. When the concentration of L-tyrosine was 10 g / L, the highest yield of melanin was expected to be 13.723 g / L, so the optimal fermentation concentration of L-tyrosine was determined to be 10 g / L. With the increasing initial pH, the yield of melanin synthesized by strain ZY-01 showed a trend of first increasing and then decreasing, the initial pH affected the enzyme activity and transmembrane transport to regulate metabolism, alkaline environment would promote the activity of key enzymes, while strong alkaline (pH>8.0) would damage the integrity of the cell membrane. When the initial pH was 8.0, the highest yield of melanin was 5.136 g / L, so the optimal fermentation initial pH value was determined to be 8.0.

[0118] Plackett-Burman (PB) test screening results: according to the single factor optimization results, taking the yield of melanin as the response value, PB experiment was used to screen the significant factors of glucose concentration, tryptone concentration, CuSO4·5H2O concentration, L-tyrosine concentration and initial pH value of fermentation medium. The experimental results are shown in Table 5. As can be seen from the analysis of variance in Table 6, the P value of the model is 0.0168<0.05, which indicates that it is significant, so the model is reliable, which shows that the model fits well in the whole regression region studied. By comparing the P values of each parameter, it can be seen that the concentration of L-tyrosine and the concentration of tryptone have a significant impact on the yield of melanin, among which the P value of L-tyrosine concentration is 0.0072, which is less than 0.01, which indicates that the concentration of L-tyrosine is the most significant factor affecting the synthesis of melanin by strain ZY-01 during fermentation. The coefficient estimate value of each factor in Table 6 indicates that the concentration of glucose and CuSO4·5H2O should be controlled during the synthesis of melanin by strain ZY-01, because excessive concentration will inhibit the synthesis of melanin. Based on the above analysis, the concentration of tryptone and L-Tyr is the significant factor affecting the synthesis of melanin by strain ZY-01 in the medium conditions. At the same time, considering that subsequent amplification culture will be carried out, the factor of glucose concentration is additionally selected for the subsequent response surface experiment.

[0119]

[0120]

[0121] Note: In the table, "*" means significant (P<0.05), "**" means extremely significant (P<0.01).

[0122] Box-Behnken experimental results: Based on the previous experimental results and the subsequent process amplification requirements, three parameters, glucose concentration, L-tyrosine concentration and tryptone concentration, were selected to design the parameter optimization experiment with melanin yield as the response value. The response surface experiment design and results are shown in Table 7. According to the variance analysis in Table 8, the model has extremely high significance (P<0.01), and the lack of fit item does not reach the significant level (P>0.05), indicating that the model is reliable. In addition, the model has high goodness of fit (R²=0.9564) and can better predict the yield of strain ZY-01 in the fermentation production of melanin. From the experimental results, the effects of factors B (tryptone concentration) and C (L-tyrosine concentration) on the yield of melanin are significant, while only BC (interaction between tryptone and L-tyrosine) is significant in the interaction term, and AC and BC are not significant. In addition, the effects of quadratic terms A², B² and C² on the response value are also significant, indicating that the relationship between each factor and the yield of melanin is not a simple linear relationship. In summary, the order of the effects of each factor is: tryptone concentration (B) > L-tyrosine concentration (C) > glucose concentration (A).

[0123]

[0124]

[0125] Note: In the table, "*" means significant (P<0.05), "**" means extremely significant (P<0.01).

[0126] The experimental data were statistically analyzed by Design Expert13 to obtain a quadratic multinomial regression equation for the yield of melanin (Y): Y=127.19063+8.49799xA+7.00276xB+5.81817xC+0.022370xAB-0.093735xAC+0.405494xBC-0.281023xAxA-0.328533xBxB-0.415666xCxC (R 2 =0.9564).

[0127] According to the regression equation, the response surface analysis diagram was drawn by Design Expert13, including contour plot and 3D surface plot, which can reflect the effects of two-factor interaction on the yield of melanin. Through Figure 11 It can be seen that the contour lines of glucose concentration and tryptone concentration are relatively sparse and approximately circular, indicating that the interaction between glucose concentration and tryptone concentration is not significant (P>0.05); throughFigure 12 It can be seen that the contour lines of glucose concentration and L-tyrosine concentration are sparse and approximately circular, indicating that the interaction of glucose concentration and L-tyrosine concentration is not significant (P>0.05); through Figure 13 It can be seen that the change surface corresponding to L-tyrosine concentration is flatter than the change surface corresponding to tryptone concentration, which indicates that the effect of tryptone concentration on the melanin absorbance is greater than that of L-tyrosine concentration, which is consistent with the result of variance analysis. At the same time, it can be seen from the contour map that the contour lines are elliptical, indicating that the interaction of tryptone concentration and L-tyrosine concentration is extremely significant (P<0.01).

[0128] Optimization result verification: The fermentation medium conditions for strain ZY-01 to synthesize melanin were optimized by using Design Expert 13 software. Through model prediction, the optimal concentrations of glucose, tryptone and L-tyrosine were 13.4105 g / L, 19.3481 g / L and 14.6394 g / L, respectively. Under the optimized conditions, the predicted average of melanin was 47.4143 g / L. At a confidence level of 95%, the actual melanin yield was between 36.556 g / L and 58.2725 g / L, which was within the predicted range of the model. In order to verify the predicted results of the established model, the fermentation conditions were set as follows: glucose concentration 13 g / L, tryptone concentration 19 g / L, L-tyrosine concentration 15 g / L, CuSO4·5H2O concentration 0.05 g / L, NaCl concentration 5 g / L, initial pH value 8.0, temperature 37℃, and shaking speed 200 r / min. The seed medium and culture conditions were the same as those in Example 2. Three parallel experiments were set up, and the actual average melanin yield of the three groups was 54.315 g / L. Compared with the melanin yield before optimization of the fermentation medium conditions (2.5045 g / L), the melanin yield was 21.69 times higher than before optimization, which was increased by 20.69 times, and could be used in subsequent large-scale fermentation.

[0129] The above describes the present application in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. A strain of Pseudomonas citronellolis, characterized in that, The citronellol Pseudomonas is Pseudomonas citronellolis ZY-01, having a preservation number of GDMCC No: 65662 at Guangdong Microbial Culture Collection Center.

2. A composition for preparing melanin, characterized by, The composition contains the citronellolis Pseudomonas of claim 1.

3. Use of the citronellolis Pseudomonas of claim 1 or the composition of claim 2 in the preparation of melanin.

4. A method of preparing melanin, characterized by, The method comprises culturing the citronellolis Pseudomonas of claim 1 in a culture medium to obtain a culture, collecting melanin from the culture, the culture medium containing a carbon source selected from at least one of glucose, sucrose, maltose, glycerol and xylose; a nitrogen source selected from tryptone; an inorganic salt selected from at least one of CuSO4, CaCl2, FeCl3 and MgCl2; and a substrate selected from at least one of L-tyrosine and phenylalanine.

5. The method of claim 4, wherein, The culture medium contains glucose, tryptone, L-tyrosine and CuSO4.

6. The method of claim 5, wherein, The concentration of the glucose is 1-30 g / L, the concentration of the tryptone is 5-30 g / L, the concentration of the L-tyrosine is 1-20 g / L, and the concentration of the CuSO4 is 0.01-0.9 g / L.

7. The method of claim 6, wherein, The concentration of the glucose is 13 g / L, the concentration of the tryptone is 19 g / L, the concentration of the L-tyrosine is 15 g / L, and the CuSO4 is CuSO4·5H2O with a concentration of 0.05 g / L.

8. The method according to any one of claims 4-7, characterized by, The pH value of the culture medium is 5-9.

9. The method according to any one of claims 4-7, characterized by, The composition of the culture medium is: glucose 13 g / L, tryptone 19 g / L, L-tyrosine 15 g / L, CuSO4·5H2O 0.05 g / L, NaCl 5 g / L, and the rest is water; the pH value of the culture medium is 8, and the culture temperature is 37℃.

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