A method for screening coenzyme q10 high-yield strain based on colony morphology

Abstract

The application discloses a method for screening coenzyme Q10 high-yield strains based on colony morphology, and belongs to the technical field of microbial fermentation and strain breeding, and comprises the following steps: diluting and coating a starting bacterial liquid on a flat plate culture medium, and directly performing preliminary screening according to the morphological characteristics of single colonies after static culture, wherein the single colony with the following characteristics is selected as a target strain: the color is dark green, the morphology is circular, the edge is complete, and the diameter is 8-12 mm; then, the target strain is sequentially subjected to slant expansion culture, seed liquid culture and fermentation culture; finally, the coenzyme Q10 high-yield strain is verified and obtained by measuring the bacterial concentration of the fermentation liquid and the coenzyme Q10 yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and strain selection technology, and in particular relates to a method for screening high-yielding coenzyme Q10 strains based on colony morphology. Background Technology

[0002] Coenzyme Q10, also known as ubiquinone 10, is a lipid-soluble quinone compound widely found in the mitochondria of eukaryotic cells. It acts as a proton carrier and electron transporter in the cellular respiratory chain, and is a key cofactor in oxidative phosphorylation and adenosine triphosphate (ATP) synthesis. Furthermore, coenzyme Q10 possesses significant antioxidant activity, scavenging free radicals, protecting the integrity of biological membrane structures, and playing important roles in maintaining cardiovascular function, regulating immunity, and anti-aging. With its expanding applications in pharmaceuticals, health foods, and cosmetics, the market demand for high-purity, highly bioactive coenzyme Q10 is increasing daily.

[0003] Currently, the main production methods for Coenzyme Q10 include chemical synthesis, extraction from animal and plant tissues, and microbial fermentation. Chemical synthesis involves complex steps, demanding reaction conditions, and is prone to producing byproducts with mixed stereostructures, resulting in low bioavailability of the final product and potentially introducing harmful residues. Animal and plant extraction methods are limited by the seasonal and regional fluctuations of raw material sources, involve complex extraction processes, and have low yields, making them unsuitable for large-scale, standardized industrial production and economically unviable.

[0004] In contrast, microbial fermentation offers a range of advantages, including a short production cycle, controllable culture conditions, environmental friendliness, ease of scalability, and consistent natural product conformation, making it the mainstream technology for the industrial production of coenzyme Q10. However, one of the core bottlenecks of this technology lies in the selection and preservation of high-yield strains. During long-term subculturing, preservation, or fermentation, production strains are prone to physiological degeneration, genetic variation, or metabolic pathway dysregulation, leading to a decline in the target product's synthetic capacity and severely affecting the stability of the fermentation process and the yield efficiency of the final product.

[0005] To obtain and maintain high-yielding strains, traditional screening methods typically rely on extensive shake-flask fermentation for secondary screening, which is labor-intensive, time-consuming, and costly in terms of manpower and materials. Although some rational screening strategies based on genomics and metabolomics have been developed, they are complex to operate and highly dependent on equipment, making them unsuitable for large-scale primary screening in production settings. Therefore, there is an urgent need in this field to establish a rapid, intuitive, low-cost primary screening method that can stably correlate strain phenotype with production performance, aiming to efficiently identify strains with high-yield potential at an early stage, providing reliable and high-quality germplasm resources for subsequent fermentation processes, thereby ensuring the efficiency and effectiveness of industrial-scale coenzyme Q10 production. Summary of the Invention

[0006] This invention provides a method for screening high-yielding coenzyme Q10 strains based on colony morphology. This method does not require complex instruments or genetic modification, and can achieve rapid initial screening of a large number of strains through only visual observation and simple measurement, which greatly improves screening efficiency and provides a stable and efficient seed source for subsequent fermentation processes.

[0007] The specific details of the invention are as follows: A method for screening high-coenzyme Q10-producing strains based on colony morphology includes the following steps: (1) Dilute the starting bacterial culture and spread it on a plate culture medium. Incubate at 28-34℃ for 10-12 days to obtain single colonies; (2) From the single colonies obtained in step (1), select single colonies that are dark green in color, round in shape with intact edges and a diameter of 8~12mm as the target strain; (3) Inoculate the target strains obtained in step (2) into slant culture medium and incubate them at 28~34℃ for 4~6 days to obtain slant seeds covered with mycelial moss; (4) Inoculate the slant seeds into liquid seed culture medium and culture them at 28-34℃ and 200-300rpm for 1-2 days to obtain seed liquid in the logarithmic growth phase; (5) Transfer the seed culture to the fermentation medium at an inoculation rate of 2-10%, and culture it at 28-34℃ and 200-300rpm for 3-5 days to obtain the fermentation broth; (6) Measure the bacterial concentration of the fermentation broth and screen strains with high bacterial concentration as high coenzyme Q10 producing strains.

[0008] Preferably, the formulation of the plate culture medium in step (1) is as follows: yeast powder 1.2%-1.8%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 0.15%-0.25%, ferrous sulfate 0.005%-0.015%, magnesium sulfate 0.03%-0.08%, agar powder 1.2%-2.0%, and pH 6.5-7.0.

[0009] Preferably, the slant culture medium in step (3) has the same composition as the plate culture medium in step (1).

[0010] Preferably, the liquid seed culture medium in step (4) is formulated as follows: yeast powder 1.2%-1.8%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 0.15%-0.25%, ferrous sulfate 0.005%-0.015%, magnesium sulfate 0.03%-0.08%, glucose 0.3%-0.6%, auxiliary solution 1ml / L, pH 6.5-7.0.

[0011] Preferably, the fermentation medium in step (5) has the following formulation: ammonium sulfate 1.5%-3.0%, anhydrous glucose 6%-10%, corn steep liquor 0.5%-2.0%, monosodium glutamate 0.3%-0.8%, magnesium sulfate 0.1%-0.2%, calcium carbonate 1.5%-3.0%, and pH 6.5-7.0. Preferably, the coenzyme Q10 fermentation units corresponding to the dark green colonies in step (2) are not less than 750 μg / ml.

[0012] Preferably, the starting strain is *Evodia rutaecarpa*, with the preservation number CCTCCNO: M2022181.

[0013] Preferably, the bacterial concentration in step (6) is determined by centrifugation or optical density method, and the coenzyme Q10 content is determined by high performance liquid chromatography with a detection wavelength of 275 nm.

[0014] A high-yield strain of coenzyme Q10 was obtained by screening using the method described above. Its colony morphology is dark green, round, with intact edges, and a diameter of 8-12 mm.

[0015] Application of a preferred high-yield strain of coenzyme Q10 in the preparation of coenzyme Q10.

[0016] This invention has achieved at least the following technological advancements: 1. Innovation in Screening Paradigm: This invention is the first to discover and establish a stable correlation between specific colony morphological characteristics and high coenzyme Q10 production. This allows the screening process to be moved to the plate culture stage, enabling the rapid identification of potential high-yielding strains at an early stage through visual observation and simple measurements, thus achieving a fundamental shift in the screening paradigm.

[0017] 2. Improved screening efficiency and throughput: This method shortens the initial screening process from a fermentation process that takes several days to immediate observation and judgment after 10-12 days of cultivation, eliminating the need to wait for fermentation and complex test results. It allows operators to simultaneously perform rapid evaluation and initial screening of dozens or even hundreds of single colonies on the same plate, resulting in an order-of-magnitude increase in screening throughput and significantly accelerating the strain selection process.

[0018] 3. Significantly reduced technical barriers and costs: This method does not rely on expensive analytical equipment such as high-performance liquid chromatography (HPLC) or mass spectrometry, nor does it involve complex biotechnologies such as gene editing or metabolomics analysis. It primarily relies on routine microbiological experimental skills, significantly reducing the technical barriers and the economic and time costs of screening, making this method easy to promote and apply in ordinary laboratories and even in production lines.

[0019] 4. Improved stability and reproducibility of high-yield strain screening: By establishing clear and quantifiable morphological standards, the screening process is freed from excessive reliance on personal experience, achieving standardization and objectivity. This correlation, validated by extensive experimental data, ensures the stability and high success rate of screening results, providing a reliable guarantee for obtaining high-quality production strains with consistent performance. Attached Figure Description

[0020] Figure 1 Single colonies selected for isolation plates. Detailed Implementation

[0021] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0022] In the following description of the embodiments, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that this application may also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of this application with unnecessary detail.

[0023] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0024] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0025] As used in this application specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0026] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0027] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0028] Example 1: Description of the screening method for high-yield Coenzyme Q10 strains A method for screening high-yielding coenzyme Q10 strains includes the following steps: Step 1: Plate separation and initial screening: After melting the coenzyme Q10 liquid starting bacterial solution, use a 1ml sterile pipette to draw 1ml of the bacterial solution and add it to a test tube containing 9ml of physiological saline. Shake well to obtain the desired result. Diluent; perform serial dilutions of 10-fold to... Select , , Three dilutions were prepared, with 0.05 ml to 0.3 ml of bacterial suspension spread evenly onto agar plates using a triangular rake. The plates were then inverted in a biochemical incubator and incubated statically at (28±2)℃ for 10–12 days. After incubation, single colonies that were dark green, round with intact edges and a diameter of 8–12 mm were selected as the target strain.

[0029] Step 2: Slant seed expansion culture: Use an inoculation spatula to inoculate the target colonies selected in Step 1 one-to-one onto the slant culture medium, and incubate at 32±2℃ for 4-6 days until the slant is covered with dark green mycelial moss free of contamination, thus obtaining slant seeds.

[0030] Step 3: Seed culture: Use an inoculation loop to pick up a slant of bacterial growth about 1 cm long and inoculate it into an Erlenmeyer flask containing liquid seed culture medium. Incubate at 32±2℃ and 200-300 rpm for 1-2 days to obtain a seed culture in the logarithmic growth phase. The OD value of the seed culture at 700 nm should be between 0.2 and 0.8.

[0031] Step 4: Fermentation Culture and High-Yield Strain Identification: Inoculate the seed culture into the fermentation medium at an inoculum rate of 2-10% and culture for 3-5 days at 32±2℃ and 200-300 rpm. After fermentation, measure the bacterial concentration of the fermentation broth and detect the coenzyme Q10 content using high-performance liquid chromatography. Select strains with high bacterial concentration and high coenzyme Q10 yield as high-yield strains.

[0032] Example 2: Specific Screening Process The screening process was specifically demonstrated using Luteovulumsphaeroides (CCTCC NO: M2022181) as the starting strain.

[0033] 1. Plate isolation and colony selection: Take cryopreserved tubes of *Oomyces cerevisiae*, dilute and plate them according to the method in Example 1, and incubate at 32±2℃ for 10 days. After incubation, select 20 single colonies that meet the criteria of "dark green, round, with intact edges, and a diameter of 8-12 mm", number them 1-20, and use them as target strains for subsequent experiments.

[0034] 2. Slant culture: The above 20 single colonies were inoculated into slant culture medium and incubated at 32±2℃ for 5 days. All of them yielded slant seeds with good growth, dark green color and no contamination.

[0035] 3. Seed culture and detection: Seeds from each slant were inoculated into seed culture medium and cultured at 32±2℃ and 250rpm for 1 day. The pH and OD value (700nm) of the seed culture were measured, and the results are as follows: serial number PH OD serial number PH OD 1 6.98 0.248 11 6.87 0.268 2 7.03 0.249 12 6.89 0.261 3 6.89 0.269 13 6.84 0.277 4 6.87 0.271 14 6.81 0.266 5 6.90 0.266 15 6.80 0.269 6 6.87 0.263 16 6.80 0.272 7 6.88 0.258 17 6.82 0.274 8 6.85 0.274 18 6.83 0.265 9 6.86 0.268 19 6.77 0.256 10 6.95 0.273 20 6.86 0.258 All seed solutions had OD values ​​in the range of 0.2-0.8, which meets the requirements for the logarithmic growth phase.

[0036] Simultaneously, a large number of single colonies in different colony states were selected and inoculated into subsequent seed bottles and fermentation bottles, and the bacterial concentration and fermentation units of the fermentation broth were statistically analyzed. Table 1 shows the bacterial concentration and fermentation units of the fermentation broth for single colonies in different states.

[0037] Table 1

[0038] As can be seen from the table, the high-yielding strains are those that are dark green, round in shape without gaps, and have a colony diameter of 8-12 mm.

[0039] 4. Fermentation Culture and Product Detection: The above seed culture was inoculated into the fermentation medium at a 4% inoculum and cultured at 32±2℃ and 240rpm for 5 days. After fermentation, pH, bacterial concentration, and coenzyme Q10 yield were measured, and the results are as follows: serial number PH Bacterial concentration % Coenzyme Q10 units (μg / ml) serial number PH Bacterial concentration % Coenzyme Q10 units (μg / ml) 1 6.80 13 782.2 11 6.69 12 756.5 2 6.77 14 817.7 12 6.72 13 767.3 3 6.76 13 793.4 13 6.84 13 771.7 4 6.72 13 779.5 14 6.73 13 774.6 5 6.72 13 775.5 15 6.73 13 783.3 6 6.75 14 802.9 16 6.69 13 796.1 7 6.71 13 760.3 17 6.68 13 787.9 8 6.69 13 761.8 18 6.67 13 770.5 9 6.68 13 782.0 19 6.70 13 786.7 10 6.71 13 790.2 20 6.68 12 759.0 5. Screening Conclusion The coenzyme Q10 yields of the 20 strains were all above 750 μg / ml, with a maximum of 817.7 μg / ml and an average yield of approximately 780 μg / ml. The bacterial concentrations were all above 12%. These results demonstrate that the strains screened using the morphological criteria described in this invention exhibit stable fermentation performance and high yields, validating the reliability and effectiveness of this screening method.

[0040] 6. Culture medium formulation (prepared by weight / volume percentage) Plate culture medium (%): yeast extract 1.5, dipotassium hydrogen phosphate 0.2, sodium chloride 0.2, ferrous sulfate 0.01, magnesium sulfate 0.05, agar powder 1.5, auxiliary solution 1 ml / L, pH 6.80, sterilized at 121℃ for 30 minutes.

[0041] Seed culture medium (%): yeast powder 1.5, dipotassium hydrogen phosphate 0.2, sodium chloride 0.2, ferrous sulfate 0.01, magnesium sulfate 0.05, glucose 0.4, auxiliary solution 1ml / L, pH 6.80, sterilized at 121℃ for 30 minutes, dispensed into 250ml Erlenmeyer flasks, 50ml per flask.

[0042] Fermentation medium (%): ammonium sulfate 2, anhydrous glucose 8, corn steep liquor 1, monosodium glutamate 0.5, magnesium sulfate 0.14, auxiliary solution 1ml / L, auxiliary solution 2ml / L, calcium carbonate 2, pH 6.80, sterilized at 121℃ for 30 minutes, dispensed into 250ml Erlenmeyer flasks, 50ml per flask.

[0043] 7. Coenzyme Q10 Detection Method Sample preparation: Accurately pipette 10 ml of fermentation broth into a 100 ml brown volumetric flask, add 1-2 drops of 0.6 mol / L hydrochloric acid and 1 ml of hydrogen peroxide, gently shake, add 20 ml of acetone, dilute to volume with anhydrous ethanol, extract by ultrasonication at 35 °C for 40 minutes, filter through a 0.45 μm filter membrane, and then inject the sample.

[0044] Chromatographic conditions: C18 column (250 mm); mobile phase: ethanol:methanol = 7:3 (v / v); detection wavelength: 275 nm; column temperature: 35 ℃; flow rate: 2.0 ml / min; injection volume: 20 μl.

[0045] It should be noted that the method of this invention does not involve specific microorganisms. Theoretically, as long as there is a correlation between the content of fermentation products and the colony morphology, color, and size, this method is applicable. The strain selected in this embodiment was deposited at the China Center for Type Culture Collection on March 9, 2022, and its name is *uteovulumsphaeroides* (accession number: CCTCCNO: M2022181).

[0046] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for screening high-yielding coenzyme Q10 strains based on colony morphology, characterized in that, Includes the following steps: (1) Dilute the starting bacterial culture and spread it on a plate culture medium. Incubate at 28-34℃ for 10-12 days to obtain single colonies; (2) From the single colonies obtained in step (1), select single colonies that are dark green in color, round in shape with intact edges and a diameter of 8~12mm as the target strain; (3) Inoculate the target strains screened in step (2) into slant culture medium and incubate them at 28~34℃ for 4~6 days to obtain slant seeds covered with mycelial moss; (4) Inoculate the slant seeds into liquid seed culture medium and culture them at 28-34℃ and 200-300rpm for 1-2 days to obtain seed liquid in the logarithmic growth phase; (5) Transfer the seed culture to the fermentation medium at an inoculation rate of 2-10%, and culture it at 28-34℃ and 200-300rpm for 3-5 days to obtain the fermentation broth; (6) Measure the bacterial concentration of the fermentation broth and screen strains with high bacterial concentration as high coenzyme Q10 producing strains.

2. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The formulation of the plate culture medium in step (1) is as follows: yeast powder 1.2%-1.8%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 0.15%-0.25%, ferrous sulfate 0.005%-0.015%, magnesium sulfate 0.03%-0.08%, agar powder 1.2%-2.0%, and pH 6.5-7.

0.

3. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The slant culture medium in step (3) has the same composition as the plate culture medium in step (1).

4. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The formula of the liquid seed culture medium in step (4) is as follows: yeast powder 1.2%-1.8%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 0.15%-0.25%, ferrous sulfate 0.005%-0.015%, magnesium sulfate 0.03%-0.08%, glucose 0.3%-0.6%, auxiliary solution 1ml / L, pH 6.5-7.

0.

5. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The fermentation medium formula in step (5) is: ammonium sulfate 1.5%-3.0%, anhydrous glucose 6%-10%, corn steep liquor 0.5%-2.0%, monosodium glutamate 0.3%-0.8%, magnesium sulfate 0.1%-0.2%, calcium carbonate 1.5%-3.0%, and pH 6.5-7.

0.

6. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The coenzyme Q10 fermentation units corresponding to the dark green colonies in step (2) are not less than 750 μg / ml.

7. The method for screening high-yielding coenzyme Q10 strains based on colony morphology according to claim 1, characterized in that, The starting strain was *Evodia rutaecarpa*, with accession number CCTCCNO: M2022181.

8. A method for screening high-yielding coenzyme Q10 strains based on colony morphology according to any one of claims 1-7, characterized in that, The bacterial concentration in step (6) is determined by centrifugation or optical density method, and the coenzyme Q10 content is determined by high performance liquid chromatography with a detection wavelength of 275 nm.

9. A high-yield strain of coenzyme Q10, characterized in that, The colonies obtained by screening using the method described in any one of claims 1-8 are dark green, round, with intact edges, and have a diameter of 8-12 mm.

10. The application of the high-yield coenzyme Q10 strain according to claim 9 in the preparation of coenzyme Q10.

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