A *Metacarpa* strain for improving tea quality and its application

Abstract

This invention belongs to the field of tea production fermentation technology, specifically disclosing a *Fungi* strain for improving tea quality and its application, namely *Fungi* strain Fungi37, classified and named... Diaspora masirevicii The strain, with accession number CGMCC No. 42364, is a rare endophytic fungus found in tea plants and is the first reported strain in the field of tea. Experiments have shown that this strain can significantly enhance the brightness and vividness of tea infusion and play a certain regulatory role in the flavor presentation of tea. Metabolomics analysis shows that this bacterium can promote the large-scale conversion and synthesis of active ingredients such as methyl caffeate, methyl gallate, ethyl gallate, hesperidin, and isosulfanol during tea fermentation. Furthermore, in an exogenous sugar-added environment, it can promote the conversion and synthesis of beneficial components such as dihydromyricetin, yarrow aglycone, isochlorogenic acid A, plantainin, malic acid, sorbic acid, and gluconic acid.

Description

Technical Field

[0001] This invention belongs to the field of tea production fermentation technology, specifically relating to a *Metacarpa* strain that improves tea quality and its application. Background Technology

[0002] Tea is a globally significant plant-based beverage. The five core elements for evaluating its quality are liquor color, aroma, taste, infused leaves, and appearance. Among these, liquor color and taste account for 45% and 55% of the quality of black tea and powdered tea, respectively (refer to the national standard GB / T23776-2018). Therefore, the transformation of tea liquor color and taste during tea processing is extremely important for the formation of tea product quality.

[0003] Pu-erh ripe tea, as an important post-fermented tea, is favored by consumers for its unique and rich flavor and taste. In recent years, Pu-erh tea has gained widespread popularity due to its various health benefits, such as anti-oxidation, lowering blood lipids, lowering blood sugar, and regulating blood pressure. Pu-erh ripe tea is a tea product made from sun-dried green tea leaves processed from Yunnan large-leaf tea varieties, through a pile fermentation process. Fermentation is a crucial stage in the processing of Pu-erh ripe tea, in which microorganisms play an indispensable role. Endophytic bacteria are an important source of microbial composition during tea fermentation. However, current technologies have limited understanding of the functions of microorganisms during tea processing, relying heavily on manual experience for processing and quality assurance. This results in a lack of data support for tea quality. Therefore, isolating, identifying, and analyzing the functions of functional endophytic strains is of great significance for the targeted regulation of tea flavor and quality formation. Summary of the Invention

[0004] This invention aims to provide a type of inter-seat shell ( Diaporthe This invention describes the use of endophytic fungi from tea plants to improve tea fermentation quality. The invention isolates an endophytic fungus of the genus *Metacarpa* from fresh tea leaves, which has been sequence-identified as... Diaporthe masirevicii The function and application of this strain in tea fermentation are currently lacking in research. Specifically, this invention provides the following technical solution: The *Fungi* strain 37 provided in this invention is classified and named as follows: Diaporthe masirevicii The biological material (strain), with accession number CGMCC No. 42364 and accession date November 6, 2025, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The biological material (strain) was received by the collection center on November 6, 2025, and registered. The viability of the biological material (strain) was tested by the collection center on November 6, 2025, and the result was: viable.

[0005] Furthermore, the ITS-1F sequence of the strain Fungi37 is shown in SEQ ID NO.1, and the ITS4 sequence is shown in SEQ ID NO.2.

[0006] In some embodiments, the present invention provides the application of the strain Fungi37 in tea fermentation, the application including regulating the color transformation and / or flavor transformation of tea infusion.

[0007] Furthermore, the tea fermentation includes at least one of the following methods: a) Fermentation is carried out using black tea liquor as a base to reduce the acidity, bitterness, and astringency of the tea liquor; b) Fermentation is carried out using fresh tea leaves as a base, and the freshness and taste of the tea are enhanced by adding sucrose.

[0008] The Fungi strain 37 provided by this invention can significantly improve the brightness and vividness of tea soup. In the microbial fermentation process based on black tea soup, it can significantly reduce the acidity, bitterness and astringency of the tea soup. In the microbial fermentation process based on fresh tea leaves, the taste and freshness of the tea soup can be improved by adding sucrose.

[0009] In some embodiments, the present invention provides the application of the strain Fungi37 in the processing of novel tea beverages based on tea broth, the processing including regulating the color and flavor of tea broth by adding the strain Fungi37 exogenously, which can be used in tea fermentation aimed at transforming the color or flavor of tea broth or in the processing of novel tea beverages based on tea broth.

[0010] In some embodiments, the present invention provides the application of the strain Fungi37 in improving tea quality, wherein the strain Fungi37 promotes the synthesis of at least one of the following active ingredients during tea fermentation: methyl caffeate, methyl gallate, ethyl gallate, hesperidin, and isosulfanol.

[0011] In some embodiments, the present invention provides the application of the strain Fungi37 in improving tea quality. When sucrose is added to the fermentation system, the strain Fungi37 further promotes the synthesis of at least one of the following components: dihydromyricetin, cyanidin, isochlorogenic acid A, plantainin, malic acid, sorbic acid, and gluconic acid.

[0012] In one embodiment, the present invention also provides a tea fermentation method, comprising inoculating the aforementioned strain Fungi37 into a tea infusion base and culturing it with shaking at 30°C and 200 rpm for 72-96 hours.

[0013] Furthermore, the tea base is black tea or fresh leaf tea, and when the tea base is fresh leaf tea, 5 g / 100 mL of sucrose is added.

[0014] In one embodiment, the present invention also provides a tea product, which is tea or tea beverage obtained by fermentation after inoculation with the said strain Fungi37.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention isolates and purifies an endophytic fungal strain from tea leaves. After colony morphology and sequence comparison, the strain is identified as belonging to the genus *Metacarpa*. Diaporthe masirevicii The strain, named Fungi 37, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42364. This is the first report of this strain in the field of tea, and it belongs to a relatively rare endophytic fungus of tea plants. Experiments have shown that this strain can significantly improve the brightness and vividness of tea infusion and plays a certain role in regulating the flavor of tea. Metabolomics analysis shows that during the fermentation of tea infusion, this bacterium can promote the large-scale conversion and synthesis of active ingredients such as methyl caffeate, methyl gallate, ethyl gallate, hesperidin, and isosulfanol. In an exogenous sugar environment, it can also promote the conversion and synthesis of beneficial components such as dihydromyricetin, cyanidin, isochlorogenic acid A, plantainin, malic acid, sorbic acid, and gluconic acid. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of the *Fungi* 37 strain from the *Metacarpus* genus. Figure 2 Phylogenetic tree diagram of Fungi 37, an endophytic fungus in tea plants; Figure 3 The diagram shows the color transformation of tea liquor caused by the endophytic fungus Fungi 37 from the tea plant. A represents a color comparison diagram; B represents a statistical comparison of components. Figure 4 The color of the fermentation broth after adding sugar and without adding sugar is shown in the image. Figure 5 This diagram illustrates the flavor changes of strain Fungi 37 during fermentation using different tea infusions; Note: A: Fresh leaf infusion; B: Black tea infusion; Figure 6 A graph showing the flavor changes of tea strain Fungi 37 during fermentation with added sugar (tct) and without added sugar (ct); Figure 7 A statistical chart showing the content of functional components in the tea infusion fermentation broth (ct37); Figure 8A comparison of the content of functional components in the fermentation broth of tea infused with sugar-added culture medium (tct37) and tea infused without sugar-added culture medium (ct37). Detailed Implementation

[0017] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the testing methods, purchased goods, unless otherwise specified, shall be used under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in the practice and testing of this disclosure.

[0018] The *Fungi* strain 37 provided in this invention is classified and named as follows: Diaporthe masirevicii The biological material (strain), with accession number CGMCC No. 42364 and accession date November 6, 2025, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The biological material (strain) was received by the collection center on November 6, 2025, and registered. The viability of the biological material (strain) was tested by the collection center on November 6, 2025, and the result was: viable.

[0019] Example 1 1. Isolation and identification of endophytic fungi in tea plants Tea tree leaves were collected from a tea tree planting base in Menghai County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, and brought back to the laboratory for isolation and culture of endophytic fungi in the tea trees. The tea samples were then temporarily stored in a refrigerator at 4-10°C.

[0020] Culture medium preparation stage: PDA solid culture medium (potato extract powder 12 g / L, glucose 20 g / L, agar 15 g / L, the remainder is water, adjusted to 1L, final pH 5.6±0.2) was selected as the growth medium for endophytic fungi. No antibiotics were added to the culture medium. After weighing according to the formula, the medium was adjusted to 1L with deionized water, sterilized at 121℃ for 15 min, cooled to 50-60℃, and then unpacked in a clean bench for later use.

[0021] Material preparation stage: Before bacterial isolation, the tea samples were surface sterilized (washed with sterile water for 0.5 min, then washed in 75% ethanol for 1 min, then washed with 2% NaClO for 3 min, then transferred to 75% sterile ethanol for 1 min, and finally washed with sterile water for 0.5 min). After surface sterilization, the tea leaves were prepared as follows: 1) Randomly select 3-5 leaves, cut them into small cubes of about 0.5 cm using a sterilized scalpel, and place them in a clean petri dish. 2) Take 10 g of leaves and place them in a sterilized small mixer with 50 mL of sterile water. Mix alternately for 1 min. Filter the resulting liquid through sterile medical gauze. Then, rinse the filtered liquid with sterile water for 10 minutes. 0 10 -1 10 -2 10 -3 10 -4 10 -5 The solution was diluted to six different levels, with each level of stock solution serving as the working solution for subsequent inoculation. The entire process of sterilizing the tea leaves and preparing the working solution was carried out in a clean bench.

[0022] Inoculation Stage: 1) Spread the cut leaf cubes evenly on PDA medium, distributing 4 leaf cubes evenly on each medium, and then place them in a biochemical incubator for cultivation. 2) Spread 100 μL of working solution onto PDA solid medium and place them in a biochemical incubator for cultivation. Set the biochemical incubator temperature to 24-28 degrees Celsius. The cultivation time depends on the growth, generally 3-5 days. After fungal colonies have grown, pick a single colony from the plate and transfer it to a new medium. Repeat this process until a pure culture is obtained.

[0023] Strain identification and preservation: The purified microorganisms were sequenced and identified using ITS1F / ITS4. Colony morphology was as follows: Figure 1 As shown in SEQ ID NO. 1 and SEQ ID NO. 2, the bacterial species is named Fungi 37, and its classification is as follows: Diaporthe masirevicii The accession number is CGMCC No.42364.

[0024] The laboratory strains were preserved in duplicate. 1) Using PDA medium (potato extract 12 g / L, glucose 20 g / L, agar 15 g / L, the remainder being water, adjusted to 1L, final pH 5.6±0.2) at 24-28℃, the strains were cultured until the mycelium covered 2 / 3 of the substrate. The culture was then transferred to a slant culture medium and cultured for another 2-3 days under the same conditions. After the fresh mycelium grew, the culture was stored at 4-8℃ and reactivated every six months. 2) After incubating in PDB medium (potato extract powder 6 g / L, glucose 20 g / L, the remainder is water, bring the volume to 1L, no pH adjustment is needed) for 2-3 days with constant temperature and shaking, a bacterial solution containing mycelial clusters is obtained. The bacterial solution containing mycelial clusters is mixed with 50% glycerol (1:1 v / v) and then frozen at -80℃. The 50% glycerol is prepared by mixing 0.9% NaCl with 100% glycerol (1:1 v / v).

[0025] 2. Phylogenetic tree construction of Fungi 37, an endophytic fungus in tea plants Genomic DNA was extracted from the purified endophytic fungus Fungi 37 from tea plants (using the Fungal Genomic DNA Extraction Kit - DP317 Tiangen Biotechnology). The ITS region was amplified using specific primers (ITS1 and ITS4). The successfully amplified products were sequenced to obtain the ITS sequence. The sequencing results were compared with known sequences in the NCBI database, and 21 homologous sequences with ≥98% similarity were selected for subsequent analysis. A phylogenetic tree was constructed using MEGA-X software. Figure 2 The maximum likelihood (ML) method was selected for calculation, with a bootstrap value of ≥1000. Phylogenetic tree construction results showed that this strain is genetically distant from other known strains of the same genus, classifying it as a newly discovered species.

[0026] Example 2 1. Preparation of tea liquor from fresh tea leaves and black tea base The functional analysis of endophytic bacteria used tea infusion as the culture condition. The fresh tea leaves and black tea products used in this invention are all derived from the representative Yunnan large-leaf tea variety "Yun Kang No. 10". All tea infusions were prepared on the spot.

[0027] The preparation method for the tea infusion based on fresh tea leaves was carried out according to the method provided in the national standard GB-T 8305-2013 "Determination of Tea Extractives": Fresh leaf samples obtained from tea trees were freeze-dried and then ground into tea powder. 3g of tea powder was weighed into a 500mL Erlenmeyer flask, 300mL of boiling distilled water was added, and the flask was immediately transferred to a boiling water bath for extraction for 45min (shaking once every 10min). After extraction, the flask was filtered under reduced pressure while hot. The tea residue was washed several times with about 150mL of boiling distilled water and then transferred to a 500mL volumetric flask. After cooling to room temperature, the flask was diluted to volume with deionized water. The diluted tea infusion was transferred to a reagent bottle, and the tea infusion was prepared in the same proportion as required for the experiment. The solution was sterilized at 120℃ for 20min and then placed in a clean bench for later use.

[0028] The preparation method of the black tea base tea soup is based on the sensory evaluation method of black tea provided in the national standard GB / T23776-2018 "Sensory Evaluation Method of Tea": Take 5.0g of sun-dried black tea product, place it in the corresponding tea evaluation cup at a tea-to-water ratio (mass-volume ratio) of 1:50, fill it with boiling water, cover it, start the timer, brew for 5 minutes, filter out the tea soup, prepare the tea soup in the same proportion according to the experimental requirements, sterilize it at 120℃ for 20 minutes, and then place it on an ultra-clean workbench for later use.

[0029] 2. The endophytic fungus Fungi 37 in tea trees has the function of changing the color of tea liquor. Functional analysis of tea liquor color transformation by the endophytic fungus Fungi 37 in tea trees was conducted. Fresh tea leaf infusion was used as the nutrient environment, with a blank infusion as the control. The tea was cultured in a shaker at 30℃ and 200rpm for 72 hours. The control group showed no significant change in tea liquor color, but its clarity slightly decreased. In contrast, the tea liquor from the microbial fermentation group exhibited a bright, clear, and vibrant red color. Differences in the content of the three tea components among the groups were analyzed using ultraviolet spectrophotometry. The results showed… Figure 3 ), Diaporthe masirevicii The content of theabrownin and thearubigin in the culture environment of (Fungi 37) was significantly higher than that in the blank control, and the content of theabrownin was 3.76 times that of the blank control (CK) and the content of thearubigin was 5.84 times that of the blank control.

[0030] A fermentation experiment was conducted by adding sucrose to the tea infusion base, simulating the production method of kombucha. Two control groups were established: a blank tea infusion and a blank tea infusion with added sucrose (5g sucrose per 100ml of tea infusion). The tea infusion base was treated with and without added sugar. The bacterial strains were cultured on a shaker at 30℃ and 200rpm for 96 hours. No significant changes in tea color were observed in either control group. The tea infusion from the unsuccessful fermentation exhibited a deep red color, clear and bright, while the tea infusion from the sugar-treated group showed a similar orange-yellow color to the control tea infusion, clear and bright. Figure 4This suggests that the color of the soup can be stabilized by adding sugar from external sources.

[0031] 3. Analysis of changes in tea flavor The flavor transformation function of the endophytic fungus Fungi 37 in tea was analyzed. Fresh tea leaves and black tea base infusions were used, with two blank infusions as controls. The mixtures were cultured in a shaker at 30℃ and 200rpm, and samples were taken every 24 hours. An electronic tongue device was used to collect data on six flavor profiles (saltiness, bitterness, astringency, umami, saltiness, and richness) in the tea infusions from different treatments. The results showed that during fermentation using black tea base, the strain exhibited a decrease in acidity, bitterness, and astringency, and an increase in umami and saltiness. Figure 5 This indicates that the strain plays a role in improving the flavor of tea products or fermented tea beverages based on black tea infusion.

[0032] During the fermentation process using fresh tea leaves as a base, the strain exhibited a change in taste, with increased acidity, bitterness, and astringency, and decreased umami and saltiness. This indicates that the strain can promote the transformation of flavor compounds during the development of tea products based on fresh leaves or raw tea infusion. A sugar-treated group was set up to simulate the fermentation formula of kombucha. Electronic tongue measurement results showed that the sugar-treated group exhibited a decrease in acidity, bitterness, and astringency, and an increase in umami and saltiness compared to the untreated group. Figure 6 This indicates that the strain has potential applications in processing novel tea drinks using tea broth as a base.

[0033] 4. Functional metabolite analysis Tea infusion was extracted from fresh tea leaves. A blank tea infusion (ctck) was used as a control. Treatment groups included tea infusion shaken with fungus (ct37), tea infusion with added sugar (tctck), and tea infusion shaken with added sugar (tct37). 5g of sucrose was added to 100ml of tea infusion. Holes were punched in densely packed mycelia using a 5mm diameter punch, with 5 identical mycelial blocks added to each treatment to ensure consistent inoculum levels. After pretreatment in a clean bench, the mixture was incubated at 30℃ on a shaker for 72h. Broad-target metabolomics analysis was performed using a Vanquish (ThermoFisher Scientific) ultra-high performance liquid chromatography (UHPLC). The results showed that the content of active ingredients in the tea infusion, including methyl caffeate, methyl gallate, ethyl gallate, hesperidin, and isosulfanol, which have been reported to possess antioxidant, anti-aging, anti-tumor, and anti-inflammatory activities, increased by 13.24–1724 times after fermentation with the endophytic fungus Fungi37. Figure 7 (Table 1) confirmed the function of this endophytic strain in the transformation of functional components.

[0034]

[0035] After fermentation with added sugar, the levels of active ingredients in the fermentation broth, such as dihydromyricetin, yarrow aglycone, isochlorogenic acid A, and plantainin, increased by 6.13 to 114 times compared to the fermentation broth without added sugar (Tables 2 and 3). Beneficial components such as malic acid, sorbic acid, and gluconic acid, which aid digestion, blood sugar control, and beverage preservation, also increased by more than 5 times. Figure 8 This indicates that the strain can further utilize sucrose and convert it into beneficial functional components, thus avoiding the adverse effects of sugar intake on human health.

[0036]

[0037] .

Claims

1. A Fungi 37 strain of the genus *Metacarpus*, characterized in that, Category Naming Diaporthe masirevicii Its accession number is CGMCC No.42364, and its accession date is November 6, 2025.

2. The strain Fungi37 as described in claim 1, characterized in that, Its ITS-1F sequence is shown in SEQ ID NO.1, and its ITS4 sequence is shown in SEQ ID NO.

2.

3. The application of strain Fungi37 as described in claim 1 or 2 in tea fermentation, characterized in that, The applications include regulating the color and / or flavor transformation of tea infusions.

4. The application as described in claim 3, characterized in that, The tea fermentation includes at least one of the following methods: a) Fermentation is carried out using black tea liquor as a base to reduce the acidity, bitterness, and astringency of the tea liquor; b) Fermentation is carried out using fresh tea leaves as a base, and the freshness and taste of the tea are enhanced by adding sucrose.

5. The application of strain Fungi37 as described in claim 1 or 2 in the processing of novel tea beverages based on tea broth, characterized in that, The processing includes regulating the color and flavor of the tea soup by adding exogenous strain Fungi37.

6. The application of strain Fungi37 as described in claim 1 or 2 in improving tea quality, characterized in that, The strain Fungi37 promotes the synthesis of at least one of the following active ingredients during tea fermentation: methyl caffeate, methyl gallate, ethyl gallate, hesperidin, and isosulfanol.

7. The application of strain Fungi37 as described in claim 1 or 2 in improving tea quality, characterized in that, When sucrose is added to the fermentation system, strain Fungi37 further promotes the synthesis of at least one of the following components: dihydromyricetin, cyanidin, isochlorogenic acid A, plantagoside, malic acid, sorbic acid, and gluconic acid.

8. A tea fermentation method, characterized in that, This includes inoculating the strain Fungi37 described in claim 1 or 2 into a tea infusion base and culturing it with shaking at 30°C and 200 rpm for 72-96 hours.

9. The method as described in claim 8, characterized in that, The tea soup base is either black tea soup or fresh leaf tea soup, and when the tea soup base is fresh leaf tea soup, 5 g / 100 mL of sucrose is added.

10. A tea product, characterized in that, Tea or tea beverage obtained by inoculating with the strain Fungi37 of claim 1 or 2 for fermentation.

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