Eurotium cristatum and application thereof in white tea processing

Abstract

The invention provides eurotium cristatum and application thereof in white tea processing, and belongs to the technical field of microorganisms. The eurotium cristatum is Eurotium cristatum HNXY-61 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is Beijing, China, the preservation date is July 24, 2024, and the preservation number is CGMCC NO.41472. The eurotium cristatum HNXY-61 has the advantages that the eurotium cristatum HNXY-61 can be used for preparing the eurotium cristatum HNXY-61; the eurotium cristatum is applied to the eurotium cristatum growing process of the Xinyang white tea so as to improve the quality characteristics of the Xinyang white tea.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to golden flower fungus and application thereof in white tea processing. Background Art

[0002] Eurotium cristatum is a fungus of the genus Eurotium, and its asexual state is Aspergillus. It is commonly known as "golden flower". It is usually found in black tea and Fu tea. It is a beneficial fungus for the human body. Because of its round appearance and golden color, it is known as the "gold of tea". During the fermentation process, Eurotium cristatum promotes the metabolism of compounds in tea leaves by secreting extracellular enzymes, thereby affecting the quality of white tea. Eurotium cristatum plays an important role in the formation of biological and flavor compounds, and has shown many health benefits after consuming this tea.

[0003] Xinyang White Tea is a type of white tea. Its unique aroma and taste come from its simple production process, which only requires two steps: withering and drying. In particular, the long withering process gives white tea its unique quality. This helps the transformation and accumulation of substances in white tea. In the initial production process, white tea does not have the steps of rolling and frying, and the cell breakage rate is low. It is difficult for the contents to dissolve quickly during the brewing process, so the aroma of white tea is not significant, the taste is light, and it is difficult to be accepted by consumers in a short period of time.

[0004] The "flowering" process was studied relatively late in Xinyang white tea, but it is the key to the special quality of Xinyang white tea. Generally, the "flowering" process has very high requirements for the environment, requiring suitable temperature, humidity and ventilation conditions, and a long cycle; in this process, if it is not properly controlled, it may cause uneven flowering and affect the overall quality of the tea; for example, if the humidity is too high, it may cause the tea to mold; if the temperature is too low, it may inhibit the "flowering" process. Therefore, screening strains suitable for the "flowering" of Xinyang white tea is an important way to promote the continuous improvement of the quality of Xinyang white tea and the improvement of the process. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a golden flower fungus, which is applied to the flowering process of Xinyang white tea to improve the quality characteristics of Xinyang white tea.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a golden flower fungus, the golden flower fungus is Eurotium cristatum Eurotium cristatum HNXY-61 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: Beijing, China, deposit date: July 24, 2024, deposit number: CGMCC NO. 41472.

[0007] Optionally, the ITS base sequence of the golden flower fungus is the nucleotide sequence shown in SEQ ID No.1.

[0008] The invention also provides an application of the golden flower fungus in the production and fermentation of white tea.

[0009] Optionally, the use of the golden flower fungus in the production and fermentation of white tea includes: culturing the golden flower fungus to prepare a spore suspension; and inoculating the spore suspension into white tea for fermentation.

[0010] Optionally, the concentration of the spore suspension is 0.8×10 5 -0.8×10 8 Pieces / mL.

[0011] Optionally, the inoculation amount of the spore suspension is 1-4%.

[0012] Optionally, the fermentation temperature is 25-30°C.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The golden flower fungus HNXY-61 isolated from white tea by the present invention is Aspergillus Shevalnosum, and its morphological characteristics are greatly different from those of the existing Aspergillus Shevalnosum. Under a specific process, the golden flower fungus HNXY-61 can form golden flower particles on the surface of tea leaves, which significantly improves the quality characteristics of white tea. At the same time, the golden flower fungus HNXY-61 may have different functions from the existing Aspergillus Shevalnosum, and has great development and utilization value. DETAILED DESCRIPTION

[0014] In order to better understand the present invention, the content of the present invention is further clearly set forth in conjunction with the embodiments below, but the protection content of the present invention is not limited to the following embodiments. In the following description, a large number of specific details are provided in order to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details.

[0015] Unless otherwise specified, all raw materials are from commercially available products and do not contain other unspecified components except inevitable impurities unless otherwise specified.

[0016] Example 1 A golden flower fungus, Eurotium cristatum Eurotium cristatum HNXY-61 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: Beijing, China, deposit date: July 24, 2024, deposit number: CGMCC NO. 41472.

[0017] The golden flower fungus of the present invention is separated from white tea of ​​the Huangmiao Ecological Tea Factory in Shihe Port, Shihe District, Xinyang City, Henan Province. The morphological characteristics of the golden flower fungus of the present invention are: on the 4th day, a colony is formed, the colony diameter is 13 mm, and the entire colony is golden yellow; on the 6th day, the colony diameter is 19 mm, and the colony is white, light yellow to nearly olive yellow from the outside to the inside; on the 10th day, the colony diameter gradually increases to 29 mm, and the color of the middle part continues to deepen to brown; on the 12th day, the colony diameter is 32 mm, the center of the colony gradually turns dark brown, and the edge is light yellow hyphae.

[0018] Molecular biological identification of the golden flower fungus of the present invention: the ITS base sequence is the nucleotide sequence shown in SEQ ID No.1, and the ITS base sequence is: AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGCCCTCTGGGTCCAACCTCCCATCCGTGTCTATCTGTACCCTGTGGCTACGGCCCGCCGAAGACTAACATTTGAACACTGTCTGAAGTTTGCAGTCTGAGTTtttAGTTAAACAATAATTAAAACTTTCAACAACGGATCTCTTGGTTCCG.

[0019] Through BLAST comparison analysis of NCBI, the similarity of the golden flower fungus of the present invention to Aspergillus chevalieri is the highest, with a similarity of 100.0%, so it is presumed to be Aspergillus chevalieri.

[0020] Embodiment 2: A kind of application of golden flower fungus in Xinyang white tea production and fermentation, comprising the following steps: The golden flower fungus of Example 1 was prepared into a spore suspension, comprising: culturing the golden flower fungus at 28° C. for 12 days until the colony produces a large number of spores; first dripping a small amount of sterile water onto a PDA plate, gently scraping off the hyphae in the PDA plate with a sterile inoculation needle, then washing the PDA plate with a small amount of sterile water for 3 times, collecting the washing liquid and adding sterile water to a total volume of 4 mL; fully shaking at room temperature for 10 minutes to disperse the spores, filtering through 3 layers of sterile gauze, determining the spore concentration with a hemocytometer, and adjusting the concentration to a use concentration of about 0.8×10 7 Pieces / mL; The spore suspension was inoculated into white tea for fermentation, including: in an ultra-clean environment, a concentration of 0.8×10 715 mL of spore suspension with a concentration of spores / mL is sprayed on 1500 g of white tea, and after air drying, it is cultured in a 28°C incubator for 7-10 days, and the flowering is observed; the loose tea with good flowering is crushed into tea powder, inoculated into the finished white tea at a weight ratio of 1%, and mixed evenly; the inoculated white tea is divided into containers made of food-grade polypropylene, covered with a lid, and placed in a fermentation room. The temperature in the fermentation room is controlled at 25-30°C, and the flowering is observed at any time. The flowering can be completed in about 7 days, and the container lid is opened, and it is dried at 35-40°C for 3 days. After passing the inspection, it is packed and stored.

[0021] Example 3: The difference from Example 2 is that the finished white tea is inoculated at a weight ratio of 2%.

[0022] Example 4: The difference from Example 2 is that the finished white tea is inoculated at a weight ratio of 3%.

[0023] Example 5: The difference from Example 2 is that the finished white tea is inoculated at a weight ratio of 4%.

[0024] The finished white tea in the above-mentioned Examples 2-5 is Xinyang white tea obtained through conventional withering and drying processes.

[0025] The tea leaves used to make the finished white tea are small leaves or old leaves. Small leaves are the tender leaves that have just unfolded on the tea tree, one bud and one leaf, and old leaves are the mature leaves on the tea tree.

[0026] Effect evaluation case: The finished white tea fermented without inoculation of golden flower fungus was used as blank control. The following tests were performed on the white tea (small leaves, old leaves) prepared in Examples 2-5 and the finished white tea of ​​blank control.

[0027] 1. Sensory evaluation 1.0 g of blank control white tea sample (small leaf) and white tea sample (small leaf) prepared in Example 2 were weighed respectively and put into a standard evaluation cup, soaked in boiling water for 5 min, and the tea soup was filtered into the evaluation bowl according to the brewing steps. Referring to the white tea evaluation method in GB / T23776-2018 "Tea Sensory Evaluation Method", three qualified tea evaluation personnel conducted a sensory evaluation on the test tea.

[0028] 2. Active ingredient detection 2.1 Determination of tea polyphenols content The content of tea polyphenols in tea was determined by the Folin phenol method, and the concentration of gallic acid standard solution was 1 mg / mL.

[0029] (1) Preparation of gallic acid standard curve Accurately pipette 0 mL, 0.2 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of gallic acid standard solution into a 50 mL volumetric flask and dilute to the mark. Add 1.0 mL of gallic acid standard stock solution and 5.0 mL of folin phenol to a 10 mL graduated test tube. After standing for 6 min, add 4.0 mL of 7.5% sodium carbonate solution and dilute the solution to 10 mL with pure water. After 60 min of reaction, measure the absorbance at a wavelength of 765 nm.

[0030] (2) Extraction and content determination method of tea polyphenols in samples Weigh 0.2 g of the sample accurately and transfer it to a 10 mL centrifuge tube. Add 5 mL of 70% methanol aqueous solution to the centrifuge tube, place it in a 70°C water bath for 10 min, cool it down and place it in a centrifuge. Centrifuge it at 4,000 r / min for 10 min. Transfer the supernatant after centrifugation to a 10 mL volumetric flask. Extract the residue again with 5 mL of 70% methanol aqueous solution. Combine the two extracts and adjust the volume. Filter with a 0.45 um water membrane. After filtering, transfer it to a 100 mL volumetric flask and adjust the volume (to be tested). Add 2 mL of test solution, 1 mL of pure water, and 5 mL of folin phenol to a 25 mL volumetric flask, let it stand for 6 min, and determine the absorbance A in the same way as "Drawing the standard curve of gallic acid".

[0031] 2.2 Determination of free amino acid content in fermented white tea The ninhydrin colorimetric method was used to determine the content of free amino acids in tea. The concentration of glutamate standard solution was 10 mg / mL, which was diluted to standard solutions with concentrations of 0.0 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, and 0.6 mg / mL.

[0032] (1) Preparation of glutamate standard curve In a 25 mL volumetric flask, pipette 1 mL of glutamic acid standard solution, 0.5 mL of pH 8.0 phosphate buffer, and 0.5 mL of 2% ninhydrin solution, respectively. Heat in a boiling water bath for 15 min, cool to volume, and let stand for 10 min before measuring the absorbance at a wavelength of 570 nm.

[0033] (2) Extraction and content determination of free amino acids in samples Weigh 0.3 g of the sample accurately, add 45 mL of boiling distilled water to a 50 mL conical flask, immediately transfer to a boiling water bath, and extract for 45 min. After the extraction is completed, filter under reduced pressure while hot. Transfer the filtrate to a 50 mL volumetric flask, cool it down and make up the volume for testing. Add 2 mL of test solution, 1 mL of pH 8.0 phosphate buffer, and 1 mL of 2% ninhydrin solution to a 25 mL volumetric flask, heat it in a boiling water bath for 15 min, and make up the volume after cooling. Let it stand for 10 min and measure the absorbance at a wavelength of 570 nm.

[0034] 2.3 Determination of polysaccharide content in fermented white tea The polysaccharide content in the samples was determined by the phenol-sulfuric acid method, and the concentration of the glucose standard solution was 20 mg / mL.

[0035] (1) Preparation of glucose standard curve In a 10 mL volumetric flask, add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution, respectively, and add water to make up to the volume for testing. In each 20 mL test tube, take 1 mL of the test solution, use 1 mL of distilled water as a control, add 2.0 mL of phenol solution to the test solution, shake well and let it stand, then add 7.0 mL of concentrated sulfuric acid, mix well with a vortex mixer, heat in a boiling water bath for 10 min, immediately move to an ice water bath, cool to room temperature, let it stand for 30 min, and measure the absorbance at a wavelength of 490 nm.

[0036] (2) Extraction and content determination of polysaccharides in samples Weigh 0.3 g of the sample accurately on an electronic balance, transfer it to a 50 mL volumetric flask, add 5 mL of distilled water for pre-soaking for 1 h, use an ultrasonic extractor to perform ultrasonic-assisted extraction at 80 °C for 3 times, 60 min each time, and add 5 mL of distilled water, and combine the extracts. Transfer the extract to a rotary evaporator and concentrate it to 2.5 mL under reduced pressure, add 95% ethanol precipitated at 4 times the volume of the concentrate, let it stand for 12 h, pour the solution and precipitate into a centrifuge tube, centrifuge at 4000 r / min for 15 min. Take 1 mL of the sample extract for detection, and the detection operation is the same as the standard curve.

[0037] 2.4 Determination of flavonoid content in fermented white tea The flavonoid content was determined by aluminum nitrate colorimetry, and the concentration of rutin standard solution was 0.3 mg / mL.

[0038] (1) Preparation of rutin standard curve Accurately pipette 0.0 mL, 5.0 mL, 10.0 mL, 15.0 mL, and 20.0 mL of the reference solution into a 50 mL volumetric flask, add 50% ethanol solution to each volume to 25 mL, add 1.5 mL of 5% sodium nitrite solution, react for 6 min, add 1.5 mL of 10% aluminum nitrate solution, react for another 6 min, add 20 mL of 1% sodium hydroxide solution, dilute to 50 mL with 50% ethanol, and let stand for 15 min. Use the solution in the first volumetric flask as a blank control and measure its absorbance at 510 nm.

[0039] (2) Extraction and content determination method of flavonoids in samples Weigh 1.5 g of sample, record the sample volume, use 40 mL of 95% ethanol for ultrasonic extraction for 45 min, transfer the extract to a 50 mL volumetric flask, and add 95% ethanol to make up to 50 mL. Accurately transfer 5 mL of the above extract, and perform the detection operation the same as the standard curve.

[0040] 3. Experimental results 3.1 Sensory evaluation analysis of white tea 3.2 Content of tea polyphenols The results of tea polyphenol content determination are shown in the following table: Note: The values ​​in the table are mean ± standard error. * "and" # ” indicates significant difference compared with the corresponding blank control (P<0.05).

[0041] The results in the table show that the tea polyphenols content of white tea leaflets fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions has significant differences compared with the blank control; the tea polyphenols content of white tea old leaves fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions has significant differences compared with the blank control; this indicates that the introduced Chrysopsis pilosula can metabolize and transform tea polyphenols, reduce the bitterness in the tea soup, and improve the nutritional value of tea.

[0042] 3.2 Free amino acid content The results of free amino acid content determination are shown in the following table: Note: The values ​​in the table are mean ± standard error. * "and" # ” indicates significant difference compared with the corresponding blank control (P<0.05).

[0043] From the results in the table, it can be seen that the content of free amino acids in white tea leaflets fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions is significantly different from that in the blank control group; the content of free amino acids in old white tea leaves fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions is significantly different from that in the blank control group; this shows that the inoculation of Chrysopsis pilosula into white tea for "flowering" reduces the content of free amino acids, which may be related to the occurrence of decarboxylation and deamination, coupled oxidation reactions and Maillard reactions; in addition, amino acids are also believed to provide nutrition for the large-scale reproduction of microorganisms such as Aspergillus.

[0044] 3.3 Polysaccharide content The results of polysaccharide content determination are shown in the following table: Note: The values ​​in the table are mean ± standard error. * "and" # ” indicates significant difference compared with the corresponding blank control (P<0.05).

[0045] From the results in the table, it can be seen that the polysaccharide content of white tea leaflets fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions is significantly different from that of the blank control group; the polysaccharide content of white tea old leaves fermented with different inoculation amounts of Chrysopsis pilosula spore suspensions is significantly different from that of the blank control group; this shows that the inoculation of Chrysopsis pilosula into white tea for fermentation can effectively promote the synthesis and conversion of polysaccharides, and significantly increase its content.

[0046] 3.4 Flavonoid content The results of flavonoid content determination are shown in the following table: Note: The values ​​in the table are mean ± standard error. * "and" # ” indicates significant difference compared with the corresponding blank control (P<0.05).

[0047] From the results in the table, it can be seen that the flavonoids content of white tea leaflets fermented with different inoculation amounts of golden flower fungus spore suspension is significantly different from that of the blank control group; the flavonoids content of white tea old leaves fermented with different inoculation amounts of golden flower fungus spore suspension is significantly different from that of the blank control group; this shows that after the white tea is inoculated with golden flower fungus for fermentation, the flavonoids content decreases, which may be caused by the hydrolysis of flavonoids and flavonoid glycosides by golden flower fungus during the fermentation process. The release of flavonoids in water during deglycosylation helps to reduce the bitterness and sourness of tea, thereby improving the bitterness and astringency of tea.

[0048] 3.4 Catechins content The results of catechin content determination are shown in the following table: Note: The values ​​in the table are mean ± standard error. * "and" # ” indicates significant difference compared with the corresponding blank control (P<0.05).

[0049] From the results in the table, it can be seen that the catechin content of white tea leaflets fermented with different inoculation amounts of Chrysopsis chrysanthemi spore suspension is significantly different from that of the blank control group; the catechin content of white tea old leaves fermented with different inoculation amounts of Chrysopsis chrysanthemi spore suspension is significantly different from that of the blank control group; this shows that after Chrysopsis chrysanthemi fermented in white tea, it plays a certain role in the degradation or transformation of catechins, and the reduction of catechins can improve the bitter taste of white tea.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A golden flower fungus, characterized in that: Eurotium cristatum Eurotium cristatum HNXY-61 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: Beijing, China, deposit date: July 24, 2024, deposit number: CGMCC NO. 41472.

2. The golden flower fungus according to claim 1, characterized in that: The ITS base sequence is the nucleotide sequence shown in SEQ ID No.

1.

3. An application of the golden flower fungus as claimed in claim 1 or 2 in the production and fermentation of white tea.

4. The use of a golden flower fungus in white tea production and fermentation as claimed in claim 3, characterized in that: include: Cultivating the golden flower fungus to prepare a spore suspension; The spore suspension is inoculated into white tea for fermentation.

5. The use of golden flower fungus in white tea production and fermentation as claimed in claim 4, characterized in that: The concentration of the spore suspension is 0.8×10 5 -0.8×10 8 Pieces / mL.

6. The use of golden flower fungus in white tea production and fermentation as claimed in claim 5, characterized in that: The inoculum amount of the spore suspension is 1-4%.

7. The use of golden flower fungus in white tea production and fermentation as claimed in claim 6, characterized in that: The fermentation temperature is 25-30°C.

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