Aspergillus awamori aspG1 and application thereof in pu'er tea fermentation

Abstract

This invention discloses a strain of Aspergillus oryzae AspG1 and its application in Pu-erh tea fermentation, belonging to the field of microbial strain resource development and deep processing technology of tea. The method provided by this invention includes: mixing sun-dried Pu-erh tea raw materials with water at a 1:1 ratio, sterilizing to obtain a solid tea base, inoculating with AspG1 strain, and then carrying out solid-state fermentation under staged temperature control conditions. Experiments showed that the fermentation product exhibited sweet and fruity aroma characteristics on day 14, and purified to floral aroma on day 21; the protease activity of AspG1 strain was increased by 229.42% compared with the control, and the β-glucosidase activity was increased by 58.21%; gas chromatography-mass spectrometry analysis showed that the cis-linalool OAV value in the AspG1 fermentation product reached 275.41 (approximately 31.8 times higher than the control), and 1-octen-3-one was specifically detected (OAV reached 228967.43). This invention provides microbial resources and process solutions for the targeted regulation of aroma in the solid-state fermentation of Pu-erh tea.

Description

Technical Field

[0001] This invention relates to the field of microbial strain resource development and deep processing technology of tea, and in particular to a strain of Aspergillus oryzae AspG1 and its application in the fermentation of Pu-erh tea. Background Technology

[0002] Pu-erh ripe tea is a post-fermented tea made from Yunnan large-leaf sun-dried green tea leaves through a solid-state pile fermentation process within a specific geographical indication protection area (GB / T 22111-2008). During solid-state fermentation, under the synergistic effect of the microbial community and its secreted extracellular enzyme system with a humid and hot environment, the internal components of the tea undergo profound oxidation, polymerization, degradation, and transformation, gradually forming the unique color, aroma, and flavor quality system of Pu-erh ripe tea.

[0003] Aroma is the primary sensory dimension for evaluating tea quality. From a flavor chemistry perspective, the material basis of tea aroma lies in the compositional spectrum, abundance distribution, and human olfactory threshold of volatile organic compounds. Existing research indicates that in traditionally naturally fermented Pu-erh tea, methoxybenzene derivatives, represented by 1,2,3-trimethoxybenzene and 1,2-dimethoxybenzene, are the main chemical entities constituting undesirable flavors such as "aged flavor" and "earthy flavor." These substances have a prominent relative proportion under natural fermentation conditions, and due to their low olfactory threshold, they easily mask the pleasant floral and fruity aromas imparted by terpenoids. Simultaneously, key terpenoids that contribute to the superior floral and fruity aromas of tea, such as linalool, cis-linalool oxide, and geraniol, are either insufficient in content or have low aroma activity values ​​in naturally fermented products, making it difficult to form a perceptible dominant aroma profile.

[0004] This chemical pattern, where "aged flavor masks floral aroma," constitutes a sensory quality bottleneck for the Pu'er tea industry in expanding its consumer market. Intervening at the microbial source, and using superior aroma-producing strains selected independently from the fermentation system for enhanced inoculation, is a scientifically feasible path to achieve targeted regulation of aroma quality.

[0005] Aspergillus awamori is one of the dominant fungal groups in the pile fermentation process of Pu-erh tea, and its metabolic activities have a significant impact on the transformation of tea components and the formation of tea quality. However, current research on Aspergillus awamori in Pu-erh tea fermentation mainly focuses on its enzyme production characteristics (such as proteases and saccharifying enzymes), and a systematic technical solution has not yet been developed for its aroma-producing function, especially its ability to synthesize characteristic floral and fruity aroma substances under solid-state fermentation, aroma evolution kinetics, and enzyme-aroma correlation mechanisms. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Aspergillus awamori AspG1 and its application in Pu-erh tea fermentation, in order to solve the problems existing in the prior art. The core of this invention lies in providing a Aspergillus awamori AspG1 strain that has been systematically screened and verified, and a method for directional regulation of aroma during solid-state fermentation of Pu-erh tea based on this strain.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a strain of Aspergillus awamori AspG1, which was deposited on June 12, 2024 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41367.

[0008] The second technical solution of the present invention is a microbial inoculant, comprising Aspergillus pumilus AspG1.

[0009] The third technical solution of the present invention is the application of Aspergillus oryzae AspG1 or the microbial agent in the fermentation preparation of Pu-erh ripe tea.

[0010] The fourth technical solution of this invention is a method for preparing ripe Pu-erh tea, comprising the following steps: (1) After pulverizing the sun-dried green tea, mix it with water and sterilize it to obtain a solid tea base; (2) The Aspergillus oryzae AspG1 or the microbial agent is inoculated into the solid tea base and solid fermentation is carried out under staged temperature control to obtain Pu'er ripe tea.

[0011] The fifth technical solution of the present invention is the Pu-erh ripe tea prepared by the preparation method.

[0012] The sixth technical solution of the present invention is the application of Aspergillus oryzae AspG1 or the microbial agent in the fermentation preparation of protease and β-glucosidase.

[0013] Based on the above technical solution, the present invention has the following technical effects: This invention isolated a strain of *Aspergillus oryzae*, AspG1, from the dominant microbial community during the fermentation process of Pu-erh tea. Enzyme activity analysis showed that this strain had significantly higher levels of protease and β-glucosidase secretion than other tested strains. Aroma component analysis further confirmed that this strain could synthesize large amounts of key floral active substances such as linalool and cis-oxidized linalool during fermentation, and could specifically produce 1-octen-3-one, which other strains could not synthesize. Sensory evaluation results showed that under solid-state fermentation conditions, this strain could drive an orderly evolution of tea aroma from "tea aroma" to "sweet aroma → fruity aroma → floral aroma". Attached Figure Description

[0014] Figure 1 The fermentation process of tea by AspG1 strain. Detailed Implementation

[0015] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0016] This invention provides a strain of Aspergillus awamori AspG1, which was deposited on June 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41367.

[0017] This invention also provides a microbial inoculant, including the Aspergillus pumilus AspG1.

[0018] This invention also provides the application of Aspergillus oryzae AspG1 or the microbial agent in the fermentation preparation of Pu-erh ripe tea.

[0019] This invention also provides a method for preparing ripe Pu-erh tea, comprising the following steps: (1) After pulverizing the sun-dried green tea, mix it with water and sterilize it to obtain a solid tea base; (2) The Aspergillus oryzae AspG1 or the microbial agent is inoculated into the solid tea base and solid fermentation is carried out under staged temperature control to obtain Pu'er ripe tea.

[0020] In some specific implementation schemes, the sun-dried green tea is Yunnan large-leaf variety sun-dried green tea, which is crushed and passed through a 40-mesh sieve; the mass ratio of the sun-dried green tea to water is 1:1; the sterilization is carried out at 121℃ and 0.1 MPa for 30 minutes.

[0021] In some specific implementations, the Aspergillus pumilus AspG1 or the microbial agent is activated and prepared as a seed solution for inoculation; The seed culture was prepared by inoculating Aspergillus pumilus AspG1 into potato dextrose liquid medium and incubating at 37°C with shaking at 180 r / min until OD500 was reached. 600 The value is 0.8; The inoculation amount of the seed liquid is 5% (mL / g) of the solid tea base mass.

[0022] In some specific implementation schemes, the total solid-state fermentation time is 21 to 21 days. The phased temperature control conditions are as follows: 0-2 days, fermentation temperature is 37℃; 3-21 days, fermentation temperature is 50℃.

[0023] This invention also provides ripe Pu-erh tea prepared by the aforementioned method.

[0024] This invention also provides the application of the Aspergillus oryzae AspG1 or the microbial agent in the fermentation preparation of proteases and β-glucosidase.

[0025] This invention discloses a novel Aspergillus awamori strain, AspG1, obtained through targeted screening from the traditional pile fermentation system of Yunnan Pu'er tea, which has the ability to synthesize terpene alcohol-like floral compounds, and a method for targeted regulation of aroma quality using this strain in the solid-state fermentation process of Pu'er tea.

[0026] Example 1 The strain AspG1 was isolated from tea mash samples during the mid-stage of traditional pile fermentation of Yunnan Pu'er tea in Pu'er City, Yunnan Province, using a combination of serial dilution and streak plating. A single pure culture of strain AspG1 was obtained after purification and cultivation on potato dextrose agar (PDA) plates.

[0027] Morphological characteristics: When incubated at 37°C for 72 hours on PDA plates, the colonies are initially white and fluffy, later turning yellowish-brown to dark brown. The hyphae are well-developed, and the conidial heads are radial or loosely columnar.

[0028] Molecular biological identification: Genomic DNA was extracted from the strain, the ITS rDNA sequence was amplified and sequenced, and the obtained sequence was compared with the NCBI GenBank database using BLAST homology. The results showed that the ITS sequence of this strain had the highest homology with the type strain of Aspergillus awamori.

[0029] Based on the combined morphological and molecular identification results, the strain was identified as Aspergillus awamori and named strain AspG1.

[0030] Aspergillus awamori AspG1 was deposited on June 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41367.

[0031] Example 2 Step 1: Preparation of solid-state fermentation substrate Using fresh leaves of the Yunnan large-leaf variety (Camellia sinensis var. assamica) "Yun Kang 10" (one bud and three leaves) as raw material, the process involves withering (2 hours), fixing (280℃, 8 minutes), rolling (30 minutes, following a light-heavy-light cycle), breaking up clumps, and sun-drying until the moisture content is below 10%, resulting in sun-dried green tea. The sun-dried green tea is then pulverized and passed through a 40-mesh standard sieve to obtain a solid-state fermented tea base. The tea base is weighed and thoroughly mixed with distilled water at a 1:1 mass ratio, stirred evenly, and then divided into petri dishes and sealed with sealing film.

[0032] Step 2: Sterilization The packaged solid tea base was placed in a vertical pressure steam sterilizer and sterilized at 121℃ and 0.1 MPa for 30 minutes. After sterilization, it was allowed to cool naturally to room temperature, then transferred to a clean bench and sterilized by ultraviolet irradiation for 30 minutes. Finally, it was resealed with sterile plastic wrap for later use.

[0033] Step 3: Seed culture preparation and inoculation Aspergillus buergerian strain AspG1 was inoculated into potato dextrose liquid medium (formulation: 200 g / L potato, 20 g / L glucose, pH 7.2-7.6, sterilized at 121℃ for 30 minutes) and cultured at 37℃ with shaking at 180 r / min until OD reached. 600 The seed solution was obtained with a value of 0.8. In a clean bench, the seed solution was aseptically inoculated into the sterilized solid tea base prepared in step two, with an inoculation amount of 5% (mL / g) of the tea base mass. Following a ratio of 5 mL of seed solution per 100 g of solid tea base (wet weight), i.e., an inoculation amount of 5% of the wet weight of the solid tea base (volume-to-weight ratio, v / w, mL / g), the seed solution was evenly dropped onto the surface of the obtained sterilized solid tea base.

[0034] Meanwhile, a blank control group (CK group) without inoculation was set up, and an equal amount of sterile water was used to replace the seed liquid inoculated into the tea base.

[0035] Step 4: Staged temperature-controlled solid-state fermentation The inoculated and control group solid tea bases were placed in a constant temperature incubator and solid-state fermentation was carried out according to the following staged temperature control procedure: Table 1

[0036] Step 5: Sampling and Quality Inspection Samples were taken on day 0 (before inoculation), day 7, day 14, and day 21 of fermentation. Each sampling was performed under aseptic conditions. The samples were used for sensory evaluation and subsequent physicochemical and aroma component analysis.

[0037] The sensory evaluation team, composed of professionals, conducted the evaluation at room temperature (25°C). Quantitative descriptive analysis (QDA) was used, and after multiple rounds of discussion and screening, the main aroma attributes were determined to be: sweet, floral, fruity, tea-like, mushroom-like, sour, and musty. A 6-point linear scale (0-5 points) was used, with 0 indicating no perception of the attribute and 5 indicating the highest perceived intensity. Each sample was evaluated independently three times by each person, and the arithmetic mean was taken as the final score for that aroma attribute.

[0038] Table 2

[0039] Experimental results show that the method of this invention achieves targeted and orderly regulation of tea aroma under solid-state fermentation conditions: the 14th day of fermentation is the first application window for the target aroma, with the product characterized by sweet and fruity aromas, and the aroma intensity reaching the highest value (4 points) in this experiment; the 21st day is the second application window, with the aroma purified into floral aroma, which can be used for the targeted development of higher-end products. The control group showed no aroma evolution throughout the entire process.

[0040] Quantitative data from sensory evaluation show that the method of this invention has a significant targeted regulatory effect on the evolution of tea aroma during solid-state fermentation: The control group (CK) maintained an aroma type of "tea aroma and sweet aroma" throughout the 21-day fermentation cycle, with a sensory intensity score of 2 points, and failed to exhibit floral or fruity aroma characteristics.

[0041] The AspG1 inoculation group showed a clear phased aroma evolution trajectory: Day 7 of fermentation: The aroma is mainly "sour and musty", scoring 2 points. This is a typical metabolic byproduct of the rapid microbial proliferation stage in the early stage of fermentation.

[0042] Day 14 of fermentation: The aroma undergoes a fundamental transformation, with "sweetness" and "fruitiness" becoming the dominant aroma types, and the score jumps to 4 points (out of 5), the highest aroma intensity value throughout the entire experiment. This stage indicates that as fermentation enters the middle and late stages, the floral and fruity aroma substances synthesized by the AspG1 strain accumulate in large quantities, and their aroma intensity has significantly surpassed the aroma of the initial fermentation by-products.

[0043] Day 21 of fermentation: The aroma is further purified, exhibiting a distinct "floral" characteristic, scoring 2 points. The initial sour and musty smells have dissipated, and the aroma has shifted from rich to a delicate floral scent.

[0044] The above data confirms that the AspG1 strain can drive the tea aroma to complete the directional and orderly evolution of "tea aroma → sweet aroma / fruit aroma → floral aroma" in solid-state fermentation, and the aroma evolution pattern is predictable in time and controllable in operation.

[0045] Example 3 Enzymatic mechanism verification of aroma-producing characteristics of AspG1 strain (1) Experimental design Using "Yun Kang No. 10" sun-dried green tea as raw material, tea infusion was prepared at a tea-to-water ratio of 1:10 (w / v, 10 mL distilled water per 1 g of tea leaves). After dispensing, the infusion was sterilized at 121℃ for 30 minutes. Activated AspG1 seed culture was inoculated, and a control group (CK) was also included. Fermentation was carried out at a constant temperature of 37℃. Samples were taken on days 0, 5, 10, 15, and 20. Protease activity was determined using the Folin-Ciocalteu method, and β-glucosidase activity was determined according to relevant literature methods.

[0046] (2) Experimental results Table 3

[0047] Note: Aspergillus buergerianus AspG1 can secrete proteases under culture conditions, and the proteases are at least one of acidic proteases and neutral proteases.

[0048] The significantly high activity of proteases efficiently hydrolyzes stored proteins in the tea matrix into free amino acids. Amino acids are not only major contributors to the fresh and delicious flavor of tea, but also important precursor substrates for the subsequent Maillard reaction, which generates roasted aroma compounds such as pyrazines and pyrroles, as well as floral and fruity aroma compounds. The high activity of β-glucosidase directly acts on terpene alcohol aroma precursors (such as linalool glucoside) stored in tea cells in glycosidic form, releasing free linalool and other floral active substances after hydrolysis of the glycosidic bonds. These two enzymatic pathways work synergistically to drive the presentation of floral and fruity aroma characteristics in the AspG1 group solid-state fermentation products.

[0049] Example 4 Chemical identification and quantitative analysis of characteristic aroma compounds of AspG1 strain (1) Sample pretreatment and detection conditions One mL of the liquid fermentation broth from AspG1 fermented for 21 days was placed in a 20 mL headspace vial, and 10 μL of deuterated n-hexanol-d13 internal standard solution was added. After headspace adsorption at 50 °C for 20 min using an SPME extraction head, desorption was performed at 250 °C for 5 min using a GC injector. Chromatography was performed using a DB-Heavy Wax capillary column (30 m × 250 μm × 0.5 μm), with the following temperature program: 40 °C for 3 min → ramp to 200 °C at 6 °C / min → ramp to 250 °C at 10 °C / min and hold for 5 min. Mass spectrometry was performed using a TOF high-resolution mass spectrometer (electron impact source 70 eV, scan range m / z 35-550). Compound identification was based on NIST database matching, and quantification was performed using the internal standard peak area ratio method.

[0050] Two comparison groups were set up: tea extract (Group S, sun-dried green tea, which is made from fresh leaves of Yunnan large-leaf variety through withering, fixation, rolling and sun drying, and is the raw material of Pu'er raw tea (raw tea), which has not yet undergone pile fermentation. Therefore, the extract prepared from it is an extract of unfermented tea (raw tea)) and a natural fermentation control (Group CK).

[0051] (2) Results of OAV analysis of key floral fragrance compounds Table 4

[0052] Cis-O-Linol was completely absent in group S and present only in trace amounts in group CK, but its OAV value reached 275.41 in group AspG1, an increase of approximately 31.8 times, demonstrating that this strain has the ability to synthesize or efficiently convert this floral aroma compound de novo. 1-Octen-3-one was not detected in any of the control treatments, but it accumulated specifically in group AspG1 with an extremely high OAV value, constituting a unique aroma marker. Linol showed high OAV in all three groups, providing a floral aroma base. The synergistic effect of these three key compounds constitutes the unique three-layered complex aroma of "floral-woody-mushroom" in the AspG1 fermentation product.

[0053] 1-Octen-3-one (OT = 0.000003 mg / kg) is a volatile ketone compound with a strong mushroom aroma. Its olfactory threshold is extremely low, and even trace amounts can make a significant contribution to the overall aroma.

[0054] This "present / absent" specific detection characteristic constitutes a unique identifier for the aroma-producing function of the AspG1 strain. The combination of 1-octen-3-one with cis-oxidized linalool and linalool endows AspG1 fermented tea products with a triple complex aroma of "floral + woody + mushroom", which has a differentiated olfactory recognition that cannot be reproduced by any other tested strains.

[0055] GC-MS full-spectrum quantitative data showed that the total amount of volatile aroma compounds in the AspG1 fermentation broth was 177.79 mg / L, ranking first among all treatment groups, 2.2 times that of the CK group (80.99 mg / L) and 1.65 times that of the tea extract S group (107.97 mg / L). In terms of compound distribution, 42 alcohols were detected in the AspG1 group, with a total content of 69.52 mg / L, accounting for 39.1% of its total aroma, leading in both diversity and absolute abundance of alcohols. Alcohols are the main carriers of floral, fruity, and woody aromas in tea, and their high abundance is completely consistent with the outstanding floral and fruity aroma scores of the AspG1 group in the aforementioned sensory evaluation.

[0056] In summary, the method provided by this invention includes: mixing sun-dried Pu-erh tea raw materials with water at a 1:1 ratio, sterilizing to obtain a solid tea base, inoculating with AspG1 strain, and then carrying out solid-state fermentation for 21 days under staged temperature control conditions (37℃ for 0-2 days, 50℃ for 3-21 days). Experiments confirmed that the fermentation product exhibited sweet and fruity aroma characteristics on day 14 (sensory score 4 / 5), and purified to a floral aroma on day 21; the protease activity of AspG1 strain was increased by 229.42% compared to the control, and the β-glucosidase activity was increased by 58.21%; gas chromatography-mass spectrometry analysis showed that the cis-linalool OAV value in the AspG1 fermentation product reached 275.41 (approximately 31.8 times higher than the control), and 1-octen-3-one was specifically detected (OAV reaching 228967.43). This invention provides strain resources and process solutions for the targeted regulation of aroma in the solid-state fermentation of Pu-erh tea.

[0057] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Aspergillus awamori AspGl, characterized in that, This strain was deposited on June 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41367.

2. A microbial inoculant, characterized in that, Includes Aspergillus bufotae as described in claim 1.

3. The application of Aspergillus oryzae AspG1 as described in claim 1 or the microbial agent as described in claim 2 in the fermentation preparation of Pu-erh ripe tea.

4. A method for preparing ripe Pu-erh tea, characterized in that, Includes the following steps: (1) After pulverizing the sun-dried green tea, mix it with water and sterilize it to obtain a solid tea base; (2) The Aspergillus oryzae AspG1 of claim 1 or the microbial agent of claim 2 is inoculated into the solid tea base and solid fermentation is carried out under staged temperature control to obtain Pu'er ripe tea.

5. The preparation method according to claim 4, characterized in that, The sun-dried green tea is Yunnan large-leaf variety sun-dried green tea, which is crushed and passed through a 40-mesh sieve; the mass ratio of the sun-dried green tea to water is 1:1; the sterilization is carried out at 121℃ and 0.1 MPa for 30 minutes.

6. The preparation method according to claim 4, characterized in that, The Aspergillus pumilum AspG1 of claim 1 or the microbial agent of claim 2 is activated and prepared as a seed liquid for inoculation. The preparation method of the seed liquid is as follows: the A. shirousamensis AspG1 is inoculated into a potato glucose liquid culture medium, and cultured at 37℃ and 180 r / min oscillation until the OD 600 value is 0.

8. The inoculation amount of the seed liquid is 5% (mL / g) of the solid tea base mass.

7. The preparation method according to claim 4, characterized in that, The total time for solid-state fermentation is 21 days to 21 days. The phased temperature control conditions are as follows: 0-2 days, fermentation temperature is 37℃; 3-21 days, fermentation temperature is 50℃.

8. The ripe Pu-erh tea prepared by the preparation method according to any one of claims 4-7.

9. The use of Aspergillus oryzae AspG1 as described in claim 1 or the microbial agent as described in claim 2 in the fermentation preparation of proteases and β-glucosidases.

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