Streptomyces thermophilus with high melanin yield and application thereof

Abstract

The invention discloses streptomyces thermophilus B1633 with high melanin yield, which is preserved in the China Center for Type Culture Collection on June 16, 2025, and has the preservation number of CCTCC M 20251381. According to the streptomyces thermophilus with high melanin yield, the highest liquid fermentation yield can reach 11.97 g / L. The fermentation liquor has the effect of removing DPPH free radicals, and when the fermentation liquor is added to the liquor fermentation process, the effects that the ethyl acetate content of raw liquor is increased by 50.57%, the total ester content is increased by 40.25%, and the fusel oil content is reduced by 7.55% can be achieved, and the active influence on the liquor flavor is achieved. The streptomyces thermophilus is separated from Luzhou-flavor distiller's yeast, can be used for producing white spirit, and provides a new functional strain for improving the quality of the white spirit.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a high-melanophora-producing thermophilic streptomyces and its applications. Background Technology

[0002] As a typical representative of traditional fermented foods, the flavor and quality of Chinese Baijiu are closely related to its brewing microbial community. The complex microbial system in the starter culture drives the fermentation of raw materials and produces flavor substances such as esters and acids. In recent years, with consumers' increasing demands for the health attributes and flavor profiles of Baijiu, strengthening the starter culture with functional microorganisms to improve the quality of the liquor has become an important research direction in the industry.

[0003] Melanin, as a natural high-molecular-weight pigment, possesses significant antioxidant, anti-radiation, and metal ion chelating abilities. Microbial melanin has garnered significant attention in the food and pharmaceutical fields due to its safety and physiological activity. Studies have shown that melanin can scavenge free radicals generated during fermentation, protecting the activity of functional microorganisms. Furthermore, its degradation products may participate in the formation of flavor compounds, indirectly affecting the flavor of the liquor. However, high-melanin-producing strains are currently scarce in baijiu (Chinese liquor) brewing environments, and their application potential in baijiu brewing has not yet been fully explored.

[0004] Actinomycetes are an important component of the microbial community in brewing koji (fermentation starter), renowned for their diverse metabolites. Some strains can synthesize melanin, but traditional isolation methods struggle to efficiently screen actinomycete strains from the complex koji system. Current technologies primarily utilize melanin-producing bacteria from soil or extreme environments, requiring thorough verification of their suitability and safety for food fermentation. Directly isolating functional strains from koji, however, offers both environmental adaptability and production application potential, representing a more promising industrialization approach. Therefore, targeted screening of high-melanin-producing actinomycetes from traditional koji and their application in baijiu (Chinese liquor) brewing holds promise for providing a new strategy to improve baijiu quality and expanding the practical value of microbial melanin in food fermentation. Summary of the Invention

[0005] The purpose of this invention is to provide a thermophilic Streptomyces thermomalaceus B1633 that produces high levels of melanin. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251381, deposited on June 16, 2025, at Wuhan University, Wuhan, China.

[0006] The thermophilic Streptomyces B1633 strain disclosed in this invention was isolated from a strong-aroma type of brewing yeast. The colony morphology of this strain after 5 days of cultivation on modified Gao's plates is as follows: nearly circular colonies with a rough, pale yellow surface, a raised center, regular edges, and pale yellow hyphae within the substrate.

[0007] The thermophilic Streptomyces thermoviolaceus B1633 involved in this invention is obtained by the following method:

[0008] Strong-aroma koji was used as the source for strain screening. After pulverizing the koji sample, purified actinomycete strains were obtained by separation using a modified Gao's No. 1 medium supplemented with naphthylpyridinium acid and potassium dichromate. Strains capable of staining plates black were selected and inoculated into liquid fermentation medium. After fermentation, the strain with the highest melanin production was selected by comparing the absorbance values ​​of the fermentation broth supernatant at 420 nm and then molecularly identified. The fermentation medium formulation for this strain was then optimized, and the DPPH free radical scavenging ability of the purified melanin solution was measured.

[0009] This invention also discloses the application of brewing starter containing *Streptomyces thermophilus* B1633 in the fermentation of baijiu (Chinese liquor). The high-melanin-producing *Streptomyces thermophilus* is fermented to obtain a melanin-rich inoculum; this inoculum is then mixed with the brewing starter to prepare a microbial compound starter for fermentation and brewing.

[0010] Compared with existing technologies, the present invention has the following advantages: the high-yield melanin-producing thermophilic Streptomyces of the present invention can achieve a maximum yield of 11.97 g / L, and also improves the flavor of baijiu to a certain extent. This thermophilic Streptomyces is isolated from strong-aroma koji and can be used in baijiu production, providing a new functional strain for improving the quality of baijiu. Attached Figure Description

[0011] Figure 1 This is a diagram showing the colony morphology of strain B1633.

[0012] Figure 2 The melanin production of strain B1633 on a solid plate;

[0013] Figure 3 Line graph of DPPH free radical scavenging experiment. Detailed Implementation

[0014] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0015] Example 1

[0016] This embodiment provides a method for obtaining a strain of thermophilic Streptomyces, including the following steps:

[0017] 1. Screening of melanin-producing actinomycetes

[0018] 1.1 Preparation of Culture Medium

[0019] Gao's No. 1 medium: potassium nitrate 1.0 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, soluble starch 20.0 g / L, solid medium with added agar 15.0 g / L, sterilized at 121℃ for 20 min;

[0020] Fermentation medium: glucose 0.1%, tyrosine 0.2%, ammonium sulfate 0.5%, calcium chloride 0.01%, sodium chloride 0.5%, magnesium sulfate 0.2%, beef extract 0.2%, peptone 0.5%.

[0021] 1.2 Isolation and purification of melanin-producing actinomycetes

[0022] Potassium dichromate and nalidixic acid were added to Gao's No. 1 culture medium as inhibitors to inhibit fungi and non-target bacteria, respectively. The yeast samples were heat-treated: the samples were placed in a 100℃ oven for 1 hour, then added to an Erlenmeyer flask containing sterile water and shaken at 37℃ for 30 minutes. After treatment, the samples were diluted to 10⁻¹⁰ on plates. -1 10 -2 10 -3 10 -4 Gradient coating was performed, and the mixture was incubated at 37°C for one week before observation.

[0023] Colonies exhibiting actinomycete characteristics are selected from screening plates: the colony surface is rough and dry, tightly bound to the culture medium and difficult to pick; hyphae are visible under a microscope, and the hyphae are significantly finer compared to those of molds. These strains are then streaked onto modified Gao's No. 1 medium until single colonies are obtained. Strains that stain the medium black are identified as melanin-producing strains (e.g., [missing information]). Figure 2 ).

[0024] 1.3 Liquid fermentation of strains

[0025] Single colonies of the actinomycetes obtained in the initial screening stage were inoculated into liquid Gao's No. 1 medium and cultured at 37℃ and 180 rpm for 48 hours to obtain the seed culture. 1 mL of seed culture was added to every 100 mL of liquid fermentation medium and cultured at 37℃ and 180 rpm for 7 days. After fermentation, the supernatant was diluted to a suitable concentration, and its absorbance at 420 nm was measured using a spectrophotometer. The formula for calculating melanin content is: In the formula, C represents the melanin content (mg / mL), and A represents the absorbance of the sample solution (A0). 420nm ), 0.269 represents the absorbance of 1.0 mL of aqueous solution containing 0.1 mg of melanin.

[0026] Plate culture results showed that two actinomycete strains, B1627 and B1633, could stain the culture medium black. The absorbance values ​​of the diluted fermentation supernatant at 420 nm were 0.270 and 0.424, respectively. Therefore, B1633 was used for subsequent experiments.

[0027] 1.4 Optimization of Fermentation Medium

[0028] To investigate the optimal culture medium formulation for melanin production by B1633, an orthogonal experiment was designed. Seven factors were included: glucose, ammonium sulfate, calcium chloride, sodium chloride, magnesium sulfate, beef extract, and peptone, with three levels for each factor. The culture medium was prepared according to the software-designed scheme. After one week of cultivation, the supernatant of the fermentation broth was diluted to an appropriate concentration, and its absorbance at 400 nm was measured using a spectrophotometer. Based on the software analysis results, the optimal formulation was determined to be: 0.1% glucose, 0.5% ammonium sulfate, 0.03% calcium chloride, 0.5% sodium chloride, 0.2% magnesium sulfate, 0.2% beef extract, and 1.0% peptone. Calculations based on the formula showed that the optimized melanin yield reached 11.97 g / L.

[0029] 1.5 Determination of DPPH free radical scavenging capacity of pure melanin

[0030] First, the melanin from B1633 fermentation was purified: the fermentation broth was centrifuged to obtain the supernatant, the supernatant was concentrated and the pH was adjusted to 1-2, and after standing for one day, it was centrifuged again and the precipitate was taken as crude melanin. The crude melanin was soaked in 5 mol / L-7 mol / L hydrochloric acid solution for 4-5 hours, then washed with anhydrous ethanol and acetone in sequence, and dried at room temperature to constant weight to obtain pure melanin.

[0031] Prepare 0.1 mM DPPH solution. Dilute melanin solution to 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 mg / L. Prepare vitamin E solution of the same concentration. Take 2 ml of DPPH solution and 2 ml of sample solutions of different concentrations into test tubes, mix well, and react in the dark for half an hour. Measure the absorbance of the solution at 520 nm. Calculate the DPPH free radical scavenging rate of melanin solution and vitamin E solution respectively. The calculation formula is: In the formula, A0 represents the absorbance of the blank solution containing only DPPH, A1 represents the absorbance of the sample solution containing DPPH, and A2 represents the absorbance of the sample solution without DPPH.

[0032] The results show that... Figure 3 As shown, melanin solution has a good scavenging effect on DPPH free radicals, and the free radical scavenging rate increases with the increase of melanin concentration, indicating that melanin solution has good antioxidant capacity. Its scavenging capacity for DPPH free radicals is weaker than that of vitamin E, about 50% of that of vitamin E.

[0033] 1.6 Fermentation of Light Aroma Baijiu

[0034] Rinse the sorghum three times, add water to cover the grain, and place it at 65℃ for 24 hours. Drain the soaked grain, steam it in an autoclave at 115℃ for 10 minutes, remove it, soak it in hot water for 5 minutes, drain it again, put it back into the autoclave, steam it again at 111℃ for 10 minutes, and then let it cool naturally.

[0035] Weigh 1 kg of steamed grain into a clean tray, spread it out and let it cool. The control group was given 1% of homemade yeast (Rhizopus + Saccharomyces cerevisiae), while the experimental group received 1% of the above-mentioned melanin fermentation liquid in addition to the control. After thorough mixing, the mixture was placed in a sealed bag and left open in a 30℃ incubator for saccharification and incubation for 24 hours. Then, the fermented mash after distillation in the workshop was sterilized at 121℃ for 20 minutes. After cooling, 500 g each of the saccharified mash and the sterilized mash were weighed and mixed thoroughly (three replicates per sample), and placed in a gas collection bag for anaerobic fermentation for 7 days.

[0036] 1.7 Distillation of Light Aroma Type Raw Spirit

[0037] Add approximately 500 mL of water to a conical flask used for distillation, then pour in the fermented mash. Insert the flask containing the sample into the stopper connected to the condenser tube, and heat it on an electric stove. When distillation is observed in the delivery tube, collect 100 mL of the distillate using a graduated cylinder. Analyze the flavor components in the distillate using gas chromatography.

[0038] The test results are shown in the table below: In the light-aroma base wine after the addition of fermentation broth, the contents of ethyl acetate and total esters were significantly increased, reaching 893.54 mg / L and 1022.73 mg / L, respectively. The contents of n-propanol and fusel oil were significantly reduced compared with the blank, indicating that the melanin fermentation broth has a certain effect on improving the flavor of the light-aroma base wine.

[0039] Table 1. Content of major flavor components in wine samples

[0040]

[0041] 2. Identification of strain B1633

[0042] 2.1 Morphological observation of strain B1633

[0043] Strain B1624 was inoculated onto modified Gao's No. 1 solid medium and cultured at 37℃ for 5 days. Afterward, the colonies were observed to be nearly round, with a rough, pale yellow surface, a raised center, and regular edges. The mycelia within the substrate were pale yellow. Figure 1 As shown.

[0044] 2.2 Molecular identification of strain B1633

[0045] Genomic DNA was extracted from the strain using a soil genomic DNA extraction kit. Using this as a template, the ribosomal RNA subunit encoding gene (16S rDNA) was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system consisted of 2 μL template, 0.5 μL 27F, 0.5 μL 1492R, 15 μL PremixTaq, and 12 μL dd H2O. The PCR amplification program was as follows: 95℃-3 min; 93℃-60 s, 50℃-60 s, 72℃-70 s; 30 cycles; 72℃-10 min. The amplified product was sent to BGI Genomics for sequencing. The sequenced data was compared with the National Center for Biotechnology Information (NCBI) website, and the results showed that strain B1633 is *Streptomyces thermophilus*.

[0046] The above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all aspects of the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention is defined by the claims.

Claims

1. A thermophilic Streptomyces thermoviolaceus B1633, characterized in that, This strain is deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC M 20251381.

2. The application of the thermophilic Streptomyces of claim 1 in the fermentation production of melanin.

3. The application of the thermophilic Streptomyces according to claim 1 in the brewing of light-aroma baijiu, characterized in that, The microbial agent containing thermophilic Streptomyces is mixed with yeast and then fermented.

4. A microbial inoculant, characterized in that, It includes the thermophilic Streptomyces thermoviolaceus B1633 with accession number CCTCC M20251381 as described in claim 1.

5. A microbial inoculant according to claim 4, characterized in that, The microbial agent can be solid or liquid.

6. A light-aroma type of baijiu, characterized in that, It is brewed from a microbial inoculum containing Streptomyces thermoviolaceus B1633, which has the accession number CCTCC M20251381 as described in claim 1.

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