Aspergillus nidulans strain with high yield of theabrownin and application thereof

Abstract

The invention relates to an aspergillus nidulans strain with high yield of theabrownin and application thereof. The aspergillus nidulans strain is preserved in China General Microbiological Culture Collection Center on December 1, 2025, the preservation name of the aspergillus nidulans strain is Aspergillus nidulans A-17, and the preservation number of the aspergillus nidulans strain is CGMCC (China General Microbiological Culture Collection Center) No. 42334. The aspergillus nidulans provided by the invention has efficient theabrownin production capacity, does not produce mycotoxin, and can be applied to theabrownin industrial production; meanwhile, the strain can also be inoculated with tea leaves and a tea extracting solution for fermentation, and is used for industrially producing high-theabrownin dark tea and high-theabrownin instant tea. In addition, the strain can significantly reduce the delicate flavor and astringent taste of tea leaves, endows the tea leaves with stale flavor and mellow taste, and can be used for producing high-quality dark tea. The aspergillus nidulans provided by the invention has strong capability of producing theabrownin, has no safety risk, provides high-quality microbial germplasm resources for preparing theabrownin, high-theabrownin tea products and high-quality dark tea, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology application, and in particular relates to a high-yielding Aspergillus nidulans strain and its application. Background Technology

[0002] Theabrownins are the core active ingredients of dark tea (such as Pu-erh tea, green brick tea, and Liubao tea). They are high-molecular-weight, water-soluble brown substances formed by the oxidation and polymerization of tea polyphenols under the action of microorganisms. Theabrownins not only give dark tea its bright reddish-brown liquor color and mellow, smooth taste, but are also key functional components that enable dark tea to exert its antioxidant, blood sugar-lowering, gut microbiota-regulating, and anti-cancer activities.

[0003] Currently, the production of theabrownins relies on the pile fermentation process of black tea, which introduces environmental microorganisms to promote the gradual oxidation and polymerization of tea polyphenols into theabrownins. However, this process has significant drawbacks: a long production cycle and low theabrownin content. Traditional pile fermentation takes 45-60 days, during which parameters such as temperature, humidity, and oxygen concentration are easily affected by the environment, resulting in low theabrownin content. The open fermentation environment easily introduces other microorganisms, potentially producing harmful substances such as aflatoxin, threatening consumer health. Furthermore, the traditional process has low conversion efficiency of the tea's internal components, resulting in low theabrownin content, poor tea taste, and limited health benefits.

[0004] To overcome the bottlenecks of traditional processes, researchers achieved efficient synthesis of theaflavins by screening superior strains and optimizing conditions.

[0005] Chinese patent CN106635820B discloses a strain of Aspergillus niger (CGMCC No. 11276) for use in the production of theabrownins or high-theabrownin instant black tea from liquid fermented green tea concentrate. However, Aspergillus niger ( Aspergillus niger The activity of extracellular enzymes such as peroxidase and polyphenol oxidase secreted by the cells is easily affected by fermentation conditions.

[0006] Chinese patent CN108835328A discloses a method for producing fermented tea, using multiple microbial strains (such as Clostridium butyricum and Aspergillus nidulans) to ferment tea powder, thereby improving the quality and nutritional value of the tea infusion. Although this method involves the use of Aspergillus nidulans (scientific name: *Aspergillus nidulans*), *Aspergillus nidulans*... Aspergillus nidulans var. cristatus ) is a fungus belonging to the order Chyromycetes, family Hypomycetes, and is related to Aspergillus nidus ( Aspergillus nidulans It is not a single strain. Furthermore, the application involves processes such as co-frying tea leaves (e.g., Gynostemma pentaphyllum) with wheat bran and liquid fermentation, but does not focus on the high theabrownin production of a single strain.

[0007] However, existing strains still have the following problems: ① Their ability to produce theaflavins is low, making it difficult to meet the needs of industrial-scale production. For example, Aspergillus niger (… Aspergillus nigerAs a food-grade safe microbial strain, it is widely used in the production of theabrownins. However, the activity of its secreted extracellular enzymes, such as peroxidase and polyphenol oxidase, is easily affected by fermentation conditions. For example, fermentation at 37℃ and 150 rpm for 12 days can produce theabrownins content of 25.4%, but the strain is prone to degeneration during continuous subculturing or large-scale production, leading to reduced enzyme activity and a theabrownin conversion rate of less than 20%. ② Risk of mycotoxin contamination, threatening human health. Traditional solid-state fermentation processes are prone to introducing toxin-producing strains such as Aspergillus flavus and Aspergillus parasiticus due to the open environment, resulting in mycotoxin contamination such as aflatoxin B1. Although liquid fermentation can reduce the risk through pure culture, some strains (such as Aspergillus fumigatus) may still produce mycotoxins, threatening food safety. For example, Aspergillus fumigatus... Aspergillus fumigatus Although it has a strong ability to produce theabrownins, it may produce fungal toxins, which limits its application in the food industry. Summary of the Invention

[0008] To address the problems of low efficiency and poor safety in the production of theabrownins by strains in existing technologies, this invention provides a high-yield Aspergillus nidulans strain and its applications.

[0009] Meanwhile, the present invention further optimizes its fermentation process, and has good industrial application prospects in the production of theabrownins, high theabrownin tea products, and high-quality black tea.

[0010] The objective of this invention can be achieved through the following technical solutions: The first aspect of the present invention is: providing a strain of Aspergillus nidulans, isolated from tea leaves undergoing pile fermentation, and named Aspergillus nidulans (… Aspergillus nidulans A-17, the Aspergillus nidulans was deposited on December 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42334; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0011] The Aspergillus nidus described herein has the ability to efficiently produce theabrownin.

[0012] Aspergillus nidus ( Aspergillus nidulans The ITS gene of A-17 is shown in SEQ ID NO.1, as detailed below: .

[0013] The second aspect of this invention is: providing a method for isolating *Aspergillus nidulans*: using a gradient dilution plating method, a black tea sample is dissolved in sterile water and shaken for 20 min, then successively diluted to different gradient bacterial suspensions, plated on Martin agar plates, and streaked twice to obtain typical single colonies. The fungal ITS gene is used for PCR amplification, and the obtained sequence is annotated by BLASTN comparison with sequences in the National Center for Biotechnology Information (NCBI) database to clarify the taxonomic position of the strain.

[0014] This invention uses the National Center for Biotechnology Information (NCBI) database for retrieval, and combines the colony morphology and microscopic characteristics of the strain to prove that the strain is *Aspergillus nidulans* (…). Aspergillus nidulans ).

[0015] A third aspect of the present invention is to provide the application of the Aspergillus nidulans as a strain for the industrial production of theabrownins.

[0016] More specifically, selected from one of the following applications: 1) Inoculate tea leaves for pile fermentation to produce high-theaflavin black tea; 2) It can be inoculated with tea extract for liquid fermentation to produce high-theabrownin instant tea.

[0017] The Aspergillus nidus described in this invention is used to reduce the umami and astringency of tea, while imparting a mellow and aged flavor. When Aspergillus nidus is inoculated into black tea for pile fermentation, the tea polyphenol content decreases by more than 50% after the 9th day of fermentation.

[0018] In some embodiments of the present invention, the tea leaves are selected from one or more of fresh tea leaves, black tea, green tea, white tea, yellow tea, oolong tea, red tea, or dark tea.

[0019] In some embodiments of the present invention, the high-theabrownin black tea includes high-theabrownin Pu-erh tea, high-theabrownin Fu brick tea, high-theabrownin Qing brick tea, high-theabrownin Kang brick tea, and high-theabrownin Liubao tea.

[0020] In some embodiments of the present invention, the tea extract is selected from one or more of the following: fresh tea leaves, black tea, green tea, white tea, yellow tea, oolong tea, red tea, and dark tea extracts.

[0021] In some embodiments of the present invention, the high-theabrownin instant tea includes high-theabrownin instant Pu-erh tea, high-theabrownin instant Fu brick tea, high-theabrownin instant green brick tea, high-theabrownin instant Kang brick tea, or high-theabrownin instant Liubao tea.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The Aspergillus nidus A-17 provided by this invention has a highly efficient theabrownin production capacity. When inoculated into black tea and fermented for 9 days, the theabrownin content of the fermented tea produced reached as high as 167.93 g / kg. The theabrownin content of this Aspergillus nidus fermented tea is 5.23-5.90 times higher than that of traditional Pu-erh tea and Liubao tea, 7.26 times higher than that of green brick tea, and 9.90-12.00 times higher than that of Fu brick tea and Kang brick tea. When black tea is inoculated with functional bacteria for pure strain fermentation, the theabrownin production capacity of Aspergillus nidus (167.93 g / kg) is significantly higher than that of Aspergillus cristatus (30.49 g / kg), Aspergillus ryukyu (72.00 g / kg), and Penicillium mansoni (70.70 g / kg).

[0023] 2. The Aspergillus nidus A-17 provided by this invention can significantly improve the flavor of dark tea and can be used to produce high-quality dark tea. Inoculation of raw dark tea with Aspergillus nidus A-17 during fermentation can significantly reduce the umami and astringency of the tea, while imparting a mellow and rich flavor ("rich flavor" refers to the abundance of soluble solids in the brewed tea, and "mellow flavor" refers to the smooth and rounded taste of the tea soup). See details... Figure 4 .

[0024] 3. The Aspergillus nidus A-17 provided by this invention does not produce mycotoxins during fermentation and has high safety. The Aspergillus nidus of this invention has a strong ability to produce theabrownins, providing high-quality microbial germplasm resources for the preparation of theabrownins, high-theabrownin tea products, and high-quality dark tea, and has good prospects for industrial application. Attached Figure Description

[0025] Figure 1 Colony morphology and microscopic characteristics of Aspergillus nidulans in Martin's medium (A) and potato dextrose agar (PDA) medium (B) (C, D).

[0026] Figure 2 The gel electrophoresis results of the ITS gene show that the left band is the marker and the right band is the product of the ITS gene.

[0027] Figure 3 Aspergillus nidus inoculated with black tea for pile fermentation.

[0028] Figure 4 (A) Radar image of the flavor characteristics of Aspergillus nidulans fermented tea. (B) Dynamic changes of major compounds during Aspergillus nidulans fermentation. (C) Images of Aspergillus nidulans fermented tea used for sensory evaluation: tea leaves (bottom) and tea infusion (top).

[0029] Figure 5 Aspergillus nidus ( Aspergillus nidulans A photograph of A-17 placed in a storage tube. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific embodiments.

[0031] The culture medium used in the following examples is as follows: Martin's medium (g / L): 10g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.03g Bengal red, 0.03g streptomycin (added after sterilization and cooling to 50-60℃), 20g agar, dissolved in water and brought to a final volume of 1000ml, pH at rest, autoclaved (121℃, 20min).

[0032] PDA medium (g / L): 200g potato, 20g glucose, 20g agar, add 1L distilled water and cook the potato at 100℃ for 30min to obtain potato extract, add water to make up to 1000ml, pH at rest, autoclave (121℃, 20min).

[0033] Example 1: Obtaining Aspergillus nidulans A-17 1. Origin of Black Tea Dark tea produced by Hubei Zhao Liqiao Tea Factory Co., Ltd.

[0034] 2. Isolation of microorganisms A strain of Aspergillus nidulans was isolated from dark tea using a combination of plate dilution and streak plate isolation. Simply put, 5.0 g of dark tea was added to an Erlenmeyer flask containing 50 mL of sterile water and a sterile steel ball, and the mixture was rotated at 25°C and 180 rpm for 60 min. Different dilution factors (10...) were then used to isolate Aspergillus nidulans. -1 Up to 10 -7 The bacterial suspension was spread onto Martin broth and incubated at 30°C and 80% relative humidity for 3-7 days. Typical single colonies were obtained by streak isolation twice.

[0035] 3. Observation and microscopic characterization of colony morphology The purified colonies were inoculated onto Martin medium and potato dextrose agar (PDA) medium, respectively, for morphological characterization. Figure 1 As shown in Figure A, after culturing for 5 days on Martin medium at 30°C and 80% relative humidity, the *Aspergillus nidulans* strain formed sub-circular colonies with a diameter of 55 mm. These colonies exhibited a bluish-yellow central region surrounded by a distinct white outer ring, with a velvety surface texture and a dense, slightly elevated center. Figure 1 As shown in Figure B, the strain grew on PDA medium at 30°C and 80% relative humidity for 3 days, with a colony diameter of 37 mm. The morphological characteristics of *Aspergillus nidulans* on PDA medium were similar to those on Martin's medium, but the central pigmentation was darker, and the periphery hyphae were longer. Notably, *Aspergillus nidulans* spores dispersed very easily on PDA medium, forming small colonies.

[0036] Microscopic features of Aspergillus nidulans were observed using an Olympus IX73 inverted microscope (Tokyo, Japan). For example... Figure 1 As shown in CD, under a microscope, the hyphae of *Aspergillus nidulans* consist of isolated hyphae, with swollen terminal cells forming vesicles at the hyphal tips. Multicellular conidia are produced in the apical region of the hyphae, and the stem branches 2-5 times before vesicle formation. The terminal cells of each branch undergo mitosis, producing conidial chains arranged in a radial, broom-like structure. The conidia are predominantly spherical to ellipsoidal in shape.

[0037] Example 2 Identification of the ITS gene in Aspergillus nidulans Genomic DNA was extracted from strain A-17. Using primers ITS1-F (SEQ ID NO.2, 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4-R (SEQ ID NO.3, 5'-TCCTCCGCTTATTGATATGC-3'), the extracted DNA was used as a template for PCR amplification to obtain the target fragment.

[0038] The PCR reaction system is shown in Table 1: Table 1 PCR reaction system The basic PCR reaction conditions were: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 72℃ pre-extension for 10 min, and storage at 4℃, for a total of 30 cycles. After the reaction, the PCR products were examined by 1.5% agarose gel electrophoresis, and the results were detected and recorded using a gel imaging system. The gel electrophoresis results of the ITS gene are shown below. Figure 2 As shown, the left band is the marker, and the right band is the product of the ITS gene. The sequencing results were compared using BLAST on the NCBI website to determine the species of the strain.

[0039] The ITS gene of strain A-17 is shown in SEQ ID NO.1.

[0040] Sequence comparison with the NCBI GenBank database showed that strain A-17 isolated from dark tea is related to Aspergillus nidulans (…). Aspergillus nidulans The strain exhibited 100% sequence identity (Per. Ident.). Combined with an e-value of 0 for this match, the strain was identified as *Aspergillus nidulans*. Aspergillus nidulans ).

[0041] Example 3: Aspergillus nidulans inoculation and pile fermentation of black tea Before inoculation, *Aspergillus nidulans* was cultured on Martin's medium at 30°C for 72 h, and the spores were washed with 0.75% (w / v) NaCl solution. The spore suspension was then diluted with sterile water to a final concentration of 2 × 10⁻⁶. 6 CFU / mL. After sterilizing 1000 g of black tea at 121℃ for 20 min, it was inoculated with 500 mL of diluted spore suspension and fermented for 9 days at 30℃ and 80% relative humidity. Figure 3 Tea samples were collected on days 0, 3, 5, 7, and 9 of fermentation and named AN0, AN3, AN5, AN7, and AN9, respectively. They were stored at −80℃ for further analysis.

[0042] Example 4 Sensory evaluation of Aspergillus nidulans fermented tea Sensory evaluation of Aspergillus nidulans fermented teas (AN0, AN3, AN5, AN7, and AN9) was conducted according to the national standard method for tea evaluation, GB / T 23776-2018. Simply put, 3.00 g of tea samples were steeped in 150 mL of boiling water for 8 minutes. The tea infusion was then quickly transferred to a white porcelain bowl, and the sensory panel immediately evaluated the color and taste. The taste was characterized by attributes of umami, saturation, body, sweetness, bitterness, astringency, and mellowness. "Body" indicated the abundance of soluble solids in the brewed tea, and "mellowness" indicated the smooth and rounded mouthfeel of the tea. The evaluation panel scored the intensity of each taste attribute on a scale of 6, from "0" (imperceptible) to "5" (extremely strong). Specifically, different concentrations of theanine, sucrose, caffeine, and EGCG were used to correct the umami, sweetness, bitterness, and astringency of the tea infusion. The umami score of the theanine reference solution (0–1.0 mg / mL) ranged from 0 to 5, corresponding to concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. Similarly, control solutions of sucrose (0–1.0 mg / mL) and caffeine (0–1.0 mg / mL) were prepared to calibrate sweetness and bitterness intensity, respectively. The astringency score of the EGCG control solution (0–1.25 mg / mL) ranged from 0 to 5, corresponding to concentrations of 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mg / mL, respectively.

[0043] Sensory evaluation results as follows Figure 4 As shown in Figure C, the tea color ranges from dark green (AN0) to yellowish-brown (AN3), dark gray (AN5), and brown (AN7-AN9), reflecting the evolutionary nature of Aspergillus nidulans-driven fermentation. The color of the tea liquor is directly affected by the concentration of theaflavins, ranging from yellowish-green (AN0) to bright yellow (AN3-AN5), and finally to reddish-brown (AN7-AN9), reflecting the gradual accumulation of theaflavins during Aspergillus nidulans-driven fermentation. Simultaneously, the umami flavor of the tea significantly diminishes during fermentation, while the aged, full-bodied, and mellow flavors significantly increase. Figure 4 A). The sensory characteristics of the final fermented tea were aged flavor, full-bodied flavor, and mellow flavor, with average scores of 4.25, 4.00, and 4.50, respectively. Therefore, during the fermentation process of dark tea, Aspergillus nidulans reduces the umami and astringency of the raw dark tea, while imparting an aged flavor and mellow taste.

[0044] Example 5: Determination of tea polyphenol and theabrownin content in tea fermented with Aspergillus nidulans Gallic acid was used as a standard, and the total phenolic content in fermented tea samples was determined using the GB / T 8313-2018 Folin-Ciocalteu colorimetric method. Theabrownins were quantitatively analyzed using the method of Wang et al. (Reference: Effects of enzymatic action on the formation of theabrownin during solid statefermentation of Pu-erh tea). Figure 4 As shown in Figure B, the tea polyphenol content decreased significantly, from 168.25 g / kg in AN0 to 85.71 g / kg in AN3, 79.89-81.22 g / kg in AN5-AN7, and 72.22 g / kg in AN9; after fermentation with Aspergillus nidus, the tea polyphenol content decreased by 57.08%. The theabrownin content increased from 97.52 g / kg in AN0 to 110.51-111.83 g / kg in AN3-AN5, 144.78 g / kg in AN7, and 167.93 g / kg in AN9; after fermentation with Aspergillus nidus, the theabrownin content increased by 72.20%. The theabrownin content of the tea fermented with *Aspergillus nidulans* (167.93 g / kg) is 5.23–5.90 times higher than that of traditional Pu-erh and Liubao teas, 7.26 times higher than that of green brick tea, and 9.90–12.00 times higher than that of Fu brick tea and Kang brick tea (see reference: Integration of non-targeted metabolomics and E-tongue evaluation reveals the chemical variation and taste characteristics of five typical dark teas). When black tea is inoculated with functional bacteria for pure culture fermentation, the theabrownin production capacity of *Aspergillus nidulans* (167.93 g / kg) is significantly higher than that of *Aspergillus cristatus* (30.49 g / kg), *Aspergillus ryukiensis* (72.00 g / kg), and *Penicillium mansoni* (70.70 g / kg) (see reference: Metabolic function and quality contribution of tea-derived microbes, and their safety risk in dark tea manufacture).

[0045] Example 6 Safety evaluation of tea fermented with Aspergillus nidulans Fermented tea samples (AN0, AN3, AN5, AN7, AN9) were ground into a fine powder (100 mesh). 30 mg of each sample was mixed with 0.7 mL of 70% (v / v) methanol and extracted with ultrasound for 15 min. The extract was centrifuged at 10000×g for 15 min, and the tea residue was extracted again under the same conditions. The mixed supernatant was centrifuged at 10000×g for 15 min at 4℃. Ultra-high performance liquid chromatography-time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS) was used to detect the levels of mycotoxins, including aflatoxin B1 / B2 / G1 / G2, citrinin, fumonisin B1 / B2, ochratoxin A, deoxynivalenol, and zearenone, in the samples (see reference: Distinct changes of metabolic profile and sensory quality during Qingzhuan tea processing revealed by LC-MS-based metabolomics). Standard curves were plotted using mycotoxin standard solutions of different concentrations to perform qualitative and quantitative analysis of mycotoxins in Aspergillus nidulans fermented tea.

[0046] The safety of Aspergillus nidulans fermented tea was evaluated using UPLC-QTOF-MS / MS. The mycotoxins detected in the fermented tea by UPLC-QTOF-MS / MS are shown in Table 2. It can be seen that no mycotoxins were detected in the Aspergillus nidulans fermented tea samples.

[0047] Table 2. Detection of mycotoxins in Aspergillus nidulans fermented tea by UPLC-QTOF-MS / MS method The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A strain of Aspergillus nidulans, characterized in that, Named Aspergillus nidus ( Aspergillus nidulans A-17, the Aspergillus nidulans described therein was deposited on December 1, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42334.

2. The ITS gene of Aspergillus nidulans as described in claim 1, characterized in that, The ITS gene sequence of Aspergillus nidulans is shown in SEQ ID NO.

1.

3. The application of the Aspergillus nidulans strain as described in claim 1, characterized in that, The application of Aspergillus nidulans as a strain for the industrial production of theabrownins.

4. The application according to claim 3, characterized in that, Choose from one of the following applications: 1) Inoculate tea leaves for pile fermentation to produce high-theaflavin black tea; 2) Inoculate with tea extract for liquid fermentation to produce high-theabrownin instant tea.

5. The application according to claim 4, characterized in that, Aspergillus nidus is used to reduce the umami and astringency of tea, while giving it a mellow and aged flavor. When Aspergillus nidus is inoculated into black tea for pile fermentation, the content of tea polyphenols decreases by more than 50% after the 9th day of fermentation, and the content of theabrownins reaches more than 150 g / kg.

6. The application according to claim 4, characterized in that, The tea leaves are selected from one or more of the following: fresh tea leaves, black tea, green tea, white tea, yellow tea, oolong tea, red tea, or dark tea.

7. The application according to claim 4, characterized in that, The high-theabrownin black tea includes high-theabrownin Pu-erh tea, high-theabrownin Fu brick tea, high-theabrownin Qing brick tea, high-theabrownin Kang brick tea, and high-theabrownin Liubao tea.

8. The application according to claim 4, characterized in that, The tea extract is selected from one or more of the following: fresh tea leaves, black tea, green tea, white tea, yellow tea, oolong tea, red tea, and dark tea extracts.

9. The application according to claim 4, characterized in that, The high-theabrownin instant tea includes high-theabrownin instant Pu-erh tea, high-theabrownin instant Fu brick tea, high-theabrownin instant Qing brick tea, high-theabrownin instant Kang brick tea, and high-theabrownin instant Liubao tea.

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