A brown pigment-producing Achromobacterium pulmonaryis PHS-001 and its applications

Abstract

This invention relates to a brown pigment-producing *Achromobacterium pulmonaryum* strain PHS-001 and its applications. The *Achromobacterium pulmonaryum* strain PHS-001 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37838. The *Achromobacterium pulmonaryum* strain PHS-001 obtained by this invention can produce brown pigment, and the fermentation cost is low, the brown pigment extraction process is simple, and it can be used not only for dyeing protein fibers such as silk and wool, but also for dyeing synthetic fiber materials such as nylon. Furthermore, it can achieve good coloring results on fabrics containing cationic groups.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a brown pigment-producing bacterium pulmonaryis PHS-001 and its applications. Background Technology

[0002] Most commercially available brown dyes used in textiles are petroleum-based raw materials, synthesized through chemical methods. The dye production process is highly polluting, and the products pose a risk of skin allergies. Some brown dyes are metal complexes, carrying the risk of heavy metal contamination. Plant-derived brown pigments, typically from plants like coffee beans and walnut shells, are costly to extract. However, bio-fermentation technology, with its advantages of short production cycles (usually 7-14 days), precisely controllable cultivation conditions, and independence from natural environmental constraints, is becoming a key breakthrough in the development of natural pigments and can be used to produce affordable brown dye products.

[0003] However, research on microbial brown pigments is still in its early stages. Large-scale production faces bottlenecks such as low strain yields, poor dye stability (susceptible to pH and temperature variations), and immature dyeing processes (insufficient colorfastness), resulting in very limited practical textile applications. Therefore, isolating and screening a new, high-yielding, stable, easily extractable, and suitable brown pigment-producing strain for textile dyeing is of great strategic significance for promoting the development of green dyeing technology and achieving a low-carbon transformation of the textile industry. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the prior art and provide a brown pigment-producing bacterium pulmonaryis PHS-001 and its application, which can produce brown pigment in large quantities, with low fermentation cost, simple brown pigment extraction process and good staining effect.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A brown pigment-producing bacillus pulmonaryis, PHS-001, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37838.

[0007] The application of the aforementioned Achromobacterium pulmonaryis PHS-001 in the preparation of brown pigment.

[0008] A fermentation medium for producing brown pigment from *Achromobacterium pulmonaryis* PHS-001 comprises the following raw materials at the following concentrations: yeast extract: 1-5 g / L, peptone: 1-10 g / L, glucose: 50-70 g / L, (NH4)2SO4: 1-5 g / L, MgSO4: 0.5-1.5 g / L, NaCl: 10-20 g / L, K2HPO4: 1.0-2.0 g / L, FeSO4: 0.05-0.15 g / L, CuSO4: 0.01-0.08 g / L, with a pH of 6.0-6.5.

[0009] A fermentation culture method for the aforementioned *Achromobacterium pulmonale* PHS-001 includes the following steps:

[0010] Step 1, Seed culture preparation: The activated Achromobacterium pulmonale PHS-001 was inoculated into LB liquid medium and cultured with shaking at 30 ℃ and 200 r / min until the OD600 nm reached 1.0-1.2 to obtain the seed culture;

[0011] Step 2: Inoculate the seed culture into the brown pigment-producing fermentation medium. The seed culture inoculation amount is 2%. Shake culture at 30℃ and 200 r / min for 72-90 hours to obtain the strain fermentation broth.

[0012] A method for extracting brown pigment, comprising the following steps:

[0013] The fermentation broth of the strain was heated to 70-80 ℃ and kept at that temperature for 0.5-1.5 h. The supernatant was collected by centrifugation. Then, an alkaline solution was added to the supernatant until the pH was ≥10. The residual protein was precipitated by alkaline treatment. The supernatant was collected by centrifugation again. Then, the supernatant was concentrated to 1 / 2 of its original volume at 60-70 ℃ to obtain a crude extract of brown pigment.

[0014] The application of the aforementioned Achromobacterium pulmonaryis PHS-001 in the dyeing of textile materials.

[0015] As a further technical solution, the textile material includes any one of silk, wool, nylon, cationic modified cotton, and cationic modified fiber.

[0016] A method for dyeing textile materials, wherein the textile materials are dyed using a crude extract of brown pigment obtained by the extraction method.

[0017] As a further technical solution, the dyeing bath ratio is 1:20-50, and the dyeing temperature is 60-100 ℃.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention provides the first screening of a novel achromobacter pulmonis strain, PHS-001, from wetland soil, which produces brown pigments and has the following advantages:

[0020] 1) It can produce a high yield of brown pigments, and the produced brown pigments have good thermal and light stability.

[0021] 2) Fermentation can be carried out using conventional culture media, yeast powder, glucose and other ingredients to produce brown pigment. The fermentation cost is low and the components are clearly defined, making it suitable for large-scale fermentation.

[0022] 3) The brown pigment produced is easy to extract from the fermentation broth. The extraction process is simple, requires no organic solvents, and is environmentally friendly.

[0023] 4) It can be used for dyeing various textile materials, and can be used not only with brown pigments but also directly with crude extracts from fermentation broth. It offers good dyeing results, a clean and sustainable overall process, and aligns with green manufacturing trends. When the produced brown pigment is used to dye textile materials, the wet rubbing color fastness can reach grade 3-4 or higher, and the dry rubbing color fastness can reach grade 4-5, demonstrating excellent color fastness performance and overcoming the technical bottleneck of insufficient color fastness in existing microbial brown pigment dyeing. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of Pulmonary Achromobacterium pulmonaryis PHS-001 on LB agar plates;

[0025] Figure 2 This is the phylogenetic tree of Pulmonary Achromobacterium pulmonaryis PHS-001;

[0026] Figure 3 This is a sample image of the fermentation broth of Achromobacterium pulmonale PHS-001;

[0027] Figure 4 These are images showing the dyeing effects of a crude extract of brown pigment from P. 001 pulmonary achromobacterium on different fabrics.

[0028] exist Figure 4 In the text, a: silk; b: wool; c: nylon; d: cationic modified cotton. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0030] 1. Strain PHS-001, classified as Achromobacter pulmonis; depositary institution: China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO. 37838, deposit date: March 4, 2026; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0031] 2. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0032] Example 1: Isolation, Screening, Identification and Preservation of Strains

[0033] Soil samples (5-10 cm) were collected from the Jiulongshan National Forest Park in Pinghu City, Zhejiang Province using a five-point sampling method. After sterile water shaking treatment, the samples were serially diluted (10⁻⁶ m³ / h). -2 10 -3 10 -4 10 -5 10 -6 The culture was plated on LB medium and incubated at 30 °C. Strains producing brown pigment were observed on the plates. Colonies were picked and purified multiple times using the streak plate method to obtain single colonies. These colonies were then inoculated into liquid fermentation medium for fermentation. Strains that stably grew in liquid medium and produced brown pigment were selected and named strain PHS-001. After reaching the logarithmic growth phase, the strain was mixed with an equal volume of 50% glycerol and stored at -80 °C. Genomic DNA was extracted from the revived strain, and 16S rDNA was amplified using 27F / 1492R universal primers and sequenced. The sequences were then compared using BLAST in the NCBI database to construct a strain phylogenetic map. Figure 2 )

[0034] The nucleotide sequence of the 16S rDNA of strain PHS-001 is shown in SEQ ID NO.1, as follows:

[0035] CTGGCTGGGCGGAGCTTACACATGCAGTCGAACGGCAGCACGGACTTCGGTCTGGTGGCGAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGCGGGGGATAACTACGCGAAAGCGTAGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGATCTTCGGACCTTGCACTATTGGAGCGGCCGATATCGGATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCCGTAGCTGGTTTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCCATCCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGGAAAGAAACGTCGCGGGTTAATACCCCGCGAAACTGACGGTACCTGCAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAGAAAGATGTGAAATCCCAGAGCTTAACTTTGGAACTGCATTTTTAACTACCGGGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCCTAAACGATGTCAACTAGCTGTTGGGGTCTTCGGGACCTTGGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAATTGACAGGGACCCGCACAAGCGGTGGGATGATGTGGATTTAATTTCGATGCAACGCCGAAAAACCTTACCCTACTCTTTGACATGTCTGGGAAATGCCCGAAGA

[0036] Upon identification, strain PHS-001 belongs to *Achromobacter pulmonis*. Therefore, this strain is named *Achromobacter pulmonis* PHS-001 and deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 37838, deposit date March 4, 2026; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] Example 2: Shake-flask fermentation of Achromobacter pulmonis PHS-001 and determination of brown pigment

[0038] 1. Culture medium

[0039] Seed culture medium: yeast extract 5.0 g / L, peptone 10.0 g / L, sodium chloride 10.0 g / L, agar 15.0 g / L, pH 6.8-7.0;

[0040] Fermentation medium: yeast extract 2.5 g / L, peptone 5 g / L, glucose 60 g / L, (NH4)2SO4 3 g / L, MgSO4 1 g / L, NaCl 15 g / L, K2HPO4 1.5 g / L, FeSO4 0.1 g / L, CuSO4 0.05 g / L, pH 6.0-6.5.

[0041] 2. Shake-flask fermentation

[0042] Single colonies were picked from agar plates and transferred to seed culture medium. The culture was maintained at 30 °C and 200 r / min until the OD600 reached 1.0–1.2. Then, a 2% inoculum was transferred to fermentation medium and cultured at 30 °C and 200 r / min for 72 h to obtain the fermentation broth of *Achromobacter pulmonis* PHS-001 (e.g., ...). Figure 3 (As shown).

[0043] Example 3: Extraction of brown pigment from fermentation broth

[0044] The fermentation broth of *Achromobacter pulmonis* PHS-001 was heated to 75 °C and incubated for 1 h. After centrifugation at 9000 r / min for 10 min, the supernatant was collected, and the bacterial precipitate was removed to obtain a supernatant containing brown pigment. A 1 mol / L NaOH solution was added for alkali treatment to adjust the pH to alkaline (pH 10), causing residual protein to precipitate. The mixture was centrifuged at 9000 r / min for 10 min, and the supernatant was collected. The supernatant was then dried at 70 °C to reduce the water content by half, increasing the brown pigment concentration, thus obtaining a crude extract of brown pigment from *Achromobacter pulmonis* PHS-001.

[0045] Example 4: Application in textile dyeing

[0046] 1. Silk dyeing

[0047] (1) Silk pretreatment: Soak the silk in hot water, take it out and squeeze out the excess water, and use absorbent paper to absorb the surface moisture.

[0048] (2) Dyeing process: The pH value of the crude brown pigment extract obtained in Example 3 was adjusted to 4 as the dyeing solution, the bath ratio was 1:50, the dyeing temperature was 90 ℃, and the solution was kept warm for 30 min before being taken out.

[0049] (3) Washing: Prepare a 5 g / L soap solution with a bath ratio of 1:20 and wash at 60 ℃ for 15 min. Rinse with clean water and hang to dry.

[0050] The rubbing resistance and color fastness of silk fabrics were determined according to GB / T3920-2008. The results showed that the dry rubbing fastness of silk could reach grade 4-5, and the wet rubbing fastness could reach grade 3-4, indicating high color fastness.

[0051] 2. Wool dyeing

[0052] (1) Wool pretreatment: Soak the wool in hot water, take it out and squeeze out the excess water, and use absorbent paper to absorb the surface moisture.

[0053] (2) Dyeing process: The pH value of the crude brown pigment extract obtained in Example 3 was adjusted to 4 as the dyeing solution, the bath ratio was 1:30, the dyeing temperature was 100 ℃, and the solution was kept warm for 30 min before being taken out.

[0054] (3) Washing: Prepare a 5 g / L soap solution with a bath ratio of 1:20 and wash at 60℃ for 15 minutes. Rinse with clean water and hang to dry.

[0055] 3. Nylon dyeing

[0056] (1) Dyeing process: Take nylon cloth, adjust the pH value of the crude brown pigment extract obtained in Example 3 to 5 as the dyeing solution, the bath ratio is 1:30, the dyeing temperature is 90 ℃, keep warm for 30 min, and then take it out.

[0057] (2) Washing: Prepare a 5 g / L soap solution with a bath ratio of 1:20 and wash at 60℃ for 15 min. After rinsing with clean water, dry in an oven.

[0058] 4. Cationic modified cotton dyeing

[0059] (1) Cotton modification treatment: 5 g / L cationic modifier, bath ratio (g / ml) 1:20, 60℃ modification for 20 min.

[0060] (2) Dyeing process: The pH value of the crude brown pigment extract obtained in Example 3 was adjusted to 10 as the dyeing solution, the bath ratio was 1:20, the dyeing temperature was 60 ℃, and the solution was kept warm for 45 min before being taken out.

[0061] (3) Washing: Prepare a 5 g / L soap solution with a bath ratio of 1:20 and wash at 60℃ for 15 min. After rinsing with clean water, dry in an oven.

[0062] The staining results of the above materials are shown in the figure. Figure 4 ,from Figure 4 It is known that the crude extract of brown pigment from Achromobacter pulmonis PHS-001 of the present invention can be used not only for dyeing protein fibers such as silk and wool, but also for dyeing chemical fiber materials such as nylon, and it can also achieve good coloring effect on fabrics containing cationic groups.

[0063] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A brown pigment-producing Achromobacter iowii PHS-001, characterized by, It is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 37838.

2. The use of A. lwoffii PHS-001 in the preparation of brown pigment according to claim 1.

3. A brown pigment producing fermentation medium for use in the production of P. hurellii PHS-001 according to claim 1, characterized in that, Each raw material includes the following concentrations: yeast powder: 1-5 g / L, peptone: 1-10 g / L, glucose: 50-70 g / L, (NH4)2SO4 1-5 g / L, MgSO4 0.5-1.5 g / L, NaCl 10-20 g / L, K2HPO4 1.0-2.0 g / L, FeSO4 0.05-0.15 g / L, CuSO4 0.01-0.08 g / L, and pH is 6.0-6.

5.

4. The fermentation culture method of the Klebsiella pneumoniae PHS-001 according to claim 1, characterized in that, The method comprises the following steps: Step 1, seed liquid preparation: inoculate activated A. lwoffii PHS-001 into LB liquid medium, and cultivate at 30°C and 200 r / min until OD600 nm reaches 1.0-1.2 to obtain a seed liquid; Step 2, inoculate the seed liquid into the brown pigment-producing fermentation medium according to claim 2, and cultivate for 72-90 hours to obtain a strain fermentation liquid.

5. A method for extracting brown pigments, characterized by, The method comprises the following steps: Heat the strain fermentation liquid according to claim 4 to 70-80°C, and keep the temperature for 0.5-1.5 hours, then centrifuge to collect the supernatant, then add alkali to the supernatant until pH≥10, and perform alkali treatment to precipitate residual proteins, then centrifuge to collect the supernatant, then concentrate the supernatant to 1 / 2 of the original volume at 60-70°C to obtain a brown pigment crude extract.

6. The use of A. lwoffii PHS-001 in the dyeing of textile materials according to claim 1.

7. Use according to claim 6, characterized in that, The textile material includes any one of silk, wool, nylon, cationic modified cotton, and cationic modified fiber.

8. A method of dyeing a textile material, characterized in that, The brown pigment crude extract obtained by the extraction method according to claim 5 is used for dyeing textile materials.

9. The method of claim 8, wherein, The bath ratio of dyeing is 1:20-50, and the dyeing temperature is 60-100°C.

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