Fungus flower fragrance type Fuzhuan tea and processing method thereof

Abstract

The invention relates to the technical field of dark tea processing, in particular to fungus flower fragrance type Fuzhuan tea and a processing method thereof. According to the invention, three key strains with metabolic complementation characteristics, namely, Aspergillus cristatus 430682HH-13B (Aspergillus cristatus 430682HH-13B), Aspergillus scherzeri 320282HH-22 (Aspergillus cereus 320282HH-22) and Eurotium cereus 533100HH-56 (Eurotium cereus 533100HH-56), are obtained from the Fu tea through high throughput screening, and the three key strains are used for preparing the Fu tea with the metabolic complementation characteristics, namely the Aspergillus cristatus, the Aspergillus cereus, the Aspergillus cereus, the Aspergillus cereus, the Aspergillus cereus, the Aspergillus cereus and the Eurotium cereus. The three strains are jointly used for processing the Fuzhuan tea, so that directional regulation and control of the aroma of the Fuzhuan tea can be realized, and the process period is shortened to 15 days.

Description

Technical Field

[0001] The invention relates to the technical field of dark tea processing, and in particular to a fungus-flower-aroma type Fu'er tea and a processing method thereof. Background Art

[0002] As a core category of dark tea, the quality of Fucha is highly dependent on the metabolic activity of the "golden flower fungus" group in the "flowering" process. However, there are systemic technical bottlenecks in the traditional process and existing technologies that need to be overcome. In the traditional process, the natural inoculation method results in a low proportion of the key strain (Aspergillus cristatus) due to the uncontrollable environmental microbial community. This phenomenon will lead to the following limitations in Fucha processing: First, the imbalance of the internal bacterial community structure of Fucha causes large fluctuations in its extracellular hydrolase activity, resulting in different degrees of conversion of tea polyphenols in the tea matrix; second, the metabolic system dominated by a relatively single strain leads to a small number of volatile compounds, and the category with 1-octen-3-one as the key aroma active substance accounts for a high proportion, while the aroma types represented by other characteristic aroma components such as linalool oxide and α-terpineol are lacking. In addition, the natural flowering cycle of the traditional processing technology is as long as 28±2 days, and the total process cycle exceeds 40 days. The mycelial biomass is less than 0.85g / 100g (20×10 4 cfu / g) with a critical failure rate of 20%.

[0003] In recent years, although artificial inoculation technology has shortened the flowering period to 15-20 days, the problem of strain antagonism in mixed strain fermentation is prominent, which often leads to flowering failure. In addition, the existing process still has the following problems: ① The lack of a multi-strain metabolic kinetic model leads to intensified nutrient competition; ② The culture parameters do not match the strain growth curve (for example, the germination of spores of Eurotium wax leaves is delayed by 6-8 hours at a constant temperature of 30°C); ③ There is a lack of quantitative standards for the regulation of aroma components (the aroma correlation model R 2 <0.35). Summary of the Invention

[0004] The present invention uses high-throughput screening to identify three key strains with metabolic complementarity from Fu'er tea and dark tea: Aspergillus cristatus (430682HH-13B), Aspergillus chevalieri (320282HH-22), and Eurotium herbariorum (533100HH-56). Combining these three strains in Fu'er tea processing allows for targeted regulation of tea aroma and shortens the process cycle to 15 days.

[0005] Aspergillus cristatus 430682HH-13B was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025900.

[0006] When used in Fu tea processing, Aspergillus cristatus (430682HH-13B) exhibits unique advantages in the (E,E)-2,4-heptadienal synthesis pathway (content ≥6.13±0.43μg / L, an increase of 81.9% compared with traditional processes).

[0007] Aspergillus chevalieri 320282HH-22 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025901.

[0008] When used in Fu tea processing, Aspergillus chevalieri (320282HH-22) efficiently expressed linalool oxide (≥101.95±22.97μg / L, a 40.06% increase compared to traditional processes) through the terpene synthase gene cluster.

[0009] Eurotium herbariorum 533100HH-56 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025902.

[0010] When used in Fu tea processing, Eurotium herbariorum 533100HH-56 activated the key enzyme for α-terpineol synthesis (≥91.67±33.94μg / L, an increase of 109% compared with the traditional process).

[0011] The invention also provides application of Aspergillus cristatus (430682HH-13B) in processing Fu'er tea.

[0012] The invention also provides application of Aspergillus chevalieri (320282HH-22) in processing Fu'er tea.

[0013] The invention also provides application of Eurotium herbariorum 533100HH-56 in processing Fu'er tea.

[0014] The invention also provides a mixed fungus, comprising Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56.

[0015] In a specific embodiment of the present invention, the mixed bacteria consists of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56.

[0016] Experiments have shown that, based on the constructed multi-species metabolic network model, the use of the mixed bacteria can achieve a balanced increase of 38%-65% in the activity of the extracellular enzyme system (cellulase, pectinase, protease, etc.).

[0017] As a preferred embodiment of the present invention, in the mixed bacteria, the ratio of the effective viable counts of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56 is (5-6):(2-3):(2-2.5), for example, 5:3:2, 6:2:2 or 5:2.5:2.5.

[0018] Experiments have shown that, based on a strain compatibility model established through co-culture experiments, when the three bacteria in the mixed bacteria are within the above-mentioned ratio range, the colony growth inhibition rate can be reduced from 42.3% in traditional mixed culture to 6.8%.

[0019] The present invention also provides application of the mixed bacteria in processing Fu'er tea.

[0020] The present invention also provides a method for processing mushroom-flower-scented Fu'er tea, comprising:

[0021] 1) adjusting the moisture content of the to-be-fermented Fu'er tea raw material to ≤25% with purified water; steam sterilizing, and then cooling to room temperature;

[0022] 2) inoculating the mixed bacteria into the sterilized Fu'er tea raw material in step 1) and performing the following gradient fermentation:

[0023] Initial stage, fermentation 0-120h: temperature 26-28℃, relative humidity 80-85%;

[0024] Mid-stage, fermentation 121-288h: temperature 30-32°C, relative humidity 70-78%;

[0025] Late stage, fermentation 289-360h: temperature 25-28℃, relative humidity 60-65%.

[0026] In some specific embodiments, the moisture content of the to-be-fermented Fu'er tea raw material is adjusted to 18-25% with purified water.

[0027] In some specific embodiments, the raw materials for the Fu tea to be fermented include Hunan Anhua first-grade dark tea, Shaanxi Jingyang third-grade Fu brick tea, and Yunnan Pu'er cooked tea.

[0028] Preferably, when the raw material of Fu'er tea to be fermented is Hunan Anhua first-grade dark tea, when the ratio of the effective live bacteria count of Aspergillus cristatus 430682HH-13B, Aspergillus schevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56 in the mixed bacteria is 5:3:2, the prepared green fungus-flavored Fu'er tea has the best effect.

[0029] Preferably, when the raw material of Fu tea to be fermented is Shaanxi Jingyang third-grade Fu brick tea, when the ratio of the effective live bacteria count of Aspergillus cristatus (430682HH-13B), Aspergillus schevalieri (320282HH-22) and Eurotium herbariorum (533100HH-56) in the mixed bacteria is 6:2:2, the prepared flower fungus aroma Fu tea has the best effect.

[0030] Preferably, when the raw material of Fu'er tea to be fermented is Yunnan Pu'er cooked tea, when the ratio of the effective live bacteria count of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56 in the mixed bacteria is 5:2.5:2.5, the prepared cooked fungus-flavored Fu'er tea has the best effect.

[0031] In some specific embodiments, the steam sterilization condition is 118-121° C., 15-20 min.

[0032] In some specific embodiments, the inoculation method is uniform spraying.

[0033] The three-stage gradient fermentation adopted by the present invention can enable rapid growth of hyphae in the initial fermentation stage, especially promoting the colonization of Aspergillus cristatus hyphae (biomass growth rate reaches 0.12g / h); in the middle fermentation stage, it can promote the synthesis of secondary metabolites such as linalool oxide, (E,E)-2,4-heptadienal, and α-terpineol; among them, the peak synthesis rate of linalool, the secondary metabolite of Aspergillus shevarium, reaches 1.24μg / (g·h); in the late fermentation stage, it can guide the conversion of aroma precursors in Eurotium corylifolia (the cumulative amount of α-terpineol increases by 109%), and can stabilize and lock in aroma components.

[0034] As a preferred embodiment of the present invention, based on the mass of the dry raw material of Fu'er tea to be fermented, the total inoculation amount of the mixed bacteria is (5-6)×10 6 CFU / g.

[0035] As a preferred embodiment of the present invention, a two-stage drying method is adopted after the fermentation is completed: drying at 75-80°C for 15-20 minutes, and then drying at 55-60°C for 2-3 hours; drying to a moisture content of ≤10%.

[0036] In some specific embodiments, after fermentation, two-stage drying is adopted: drying at 80° C. for 15-20 minutes, and then drying at 55° C. for 2-3 hours, until the moisture content is ≤10%.

[0037] Experiments have shown that the two-stage drying method of the present invention can lock in the aroma and retain volatile aroma substances after fermentation.

[0038] The method of the present invention can process various Fu tea products, and can process three types of Fu tea with clear mushroom aroma, flower mushroom aroma and cooked mushroom aroma.

[0039] The method of the present invention is applicable to the processing of Fu tea in the main dark tea producing areas in the country (including core producing areas such as Anhua in Hunan and Jingyang in Shaanxi).

[0040] The method of the invention adopts a gradient fermentation process to achieve directional control of the aroma of Fu tea.

[0041] The present invention also includes the mushroom-flower-scented Fu'er tea prepared by the method.

[0042] The present invention's method for processing fungus-flower-scented Fu'er tea shortens the process cycle to 15 days (67% faster than traditional processes) by regulating parameters in real time, reduces energy consumption by at least 30%, and achieves a total content of characteristic aroma components exceeding 200 μg / L. Compared with traditional processes, the total amount of volatile aroma substances in the Fu'er tea processed by the present invention is significantly increased. The method's bacterial colonization success rate is increased to 98.3% (compared to 80% for traditional processes), providing a complete engineering and technical solution for the upgrading of the Fu'er tea industry and achieving significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a morphological diagram of the strain Aspergillus cristatus (430682HH-13B).

[0044] Figure 2 This is a morphological diagram of the strain Aspergillus chevalieri (320282HH-22).

[0045] Figure 3 This is the strain morphology of Eurotium herbariorum (533100HH-56).

[0046] Figure 4 This is a photo of the mushroom-flower-scented Fu'er tea product prepared in Example 1.

[0047] Figure 5 This is the GC-MS spectrum of the characteristic aroma compounds of the fungus-flower-flavored Fu'er tea prepared in Example 1.

[0048] Figure 6 This is a photo of the mushroom-flower-scented Fu'er tea product prepared in Example 4.

[0049] Figure 7 This is the GC-MS spectrum of the characteristic aroma compounds of the fungus-flower-flavored Fu'er tea prepared in Example 4.

[0050] Figure 8 This is a photo of the fungus-flower-scented Fu'er tea product prepared in Example 7.

[0051] Figure 9 This is the GC-MS spectrum of the characteristic aroma compounds of the fungus-flower-flavored Fu'er tea prepared in Example 7.

[0052] Figure 10 This is the process flow chart of Fu tea processing using the traditional processing method of comparative example 1.

[0053] Figure 11 This is a diagram of the three strains' collaborative metabolic network according to an embodiment of the present invention. DETAILED DESCRIPTION

[0054] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0055] PDA solid culture medium: 200 g potatoes (peeled), 20 g glucose, 14 g agar, add 1 L of distilled water or deionized water, stir and heat to boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, and sterilize by high pressure at 121°C for 15 min.

[0056] Czapek medium: 3.0 g sodium nitrate, 1.0 g dimethyl phosphate, 0.5 g magnesium sulfate, 0.5 g potassium chloride, 0.01 g ferrous sulfate, 30.0 g sucrose, 15.0 g agar, add 1 L distilled water or deionized water, stir and heat to boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, and sterilize by autoclave at 121°C for 15 min.

[0057] 20% sucrose Czapek medium: sodium nitrate 3.0g, dimethyl phosphate 1.0g, magnesium sulfate 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, sucrose 230.0g, agar 15.0g, add distilled water or deionized water 1L, stir and heat to boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, and sterilize by autoclave at 121℃ for 15min.

[0058] The contents of (E,E)-2,4-heptadienal, linalool oxide, and α-terpineol in the fungus-flower-scented Fu tea were determined by the following method.

[0059] HS-SPME extraction of volatile components: Accurately weigh 5 g of crushed tea sample and place it in a 100 mL headspace bottle, add 50 ml of boiling water, add a certain amount of internal standard (ethyl decanoate), and then insert the SPME handheld device equipped with a 50 / 30 μm DVB / CAR / PDM extraction head into the headspace of the extraction bottle through the rubber pad of the bottle cap. After equilibration in a 60°C water bath for 10 minutes, push out the fiber head and adsorb for 50 minutes. After the extraction is completed, retract the fiber head and immediately insert it into the inlet of the gas chromatograph for desorption for 3 minutes.

[0060] GC-MS analysis

[0061] GC conditions: A DB-5MS (30 m × 0.25 mm × 0.25 μm) flexible quartz capillary column was used. The inlet temperature was 240°C, and the carrier gas was high-purity helium (>99.999%) at a flow rate of 1.0 mL / min. Manual pulsed splitless injection was used. The column temperature program was as follows: initial temperature at 50°C (hold for 5 min), then increased at 3°C / min to 180°C (hold for 2 min), then increased at 10°C / min to 250°C (hold for 3 min).

[0062] MS conditions: ion source EI, electron energy 70 eV, ion source temperature 230 °C; scanning mass range m / z 50-550; quadrupole temperature 150 °C.

[0063] Qualitative and quantitative analysis of tea aroma components

[0064] Qualitative analysis: Identification of volatile compounds was based on retention indices (RIs), mass spectral matching with the NIST17 standard library on the instrument workstation, combined with relevant literature, and retention times. A standard mixture of C7-C40 n-alkanes was analyzed under the same chromatographic conditions, and RIs were calculated as follows: RI = 100n + 100 × [(RTx - RTn) / (RTn+1 - RTn)], where: RI is the retention index of the analyte; RTx is the retention time of the analyte; RTn is the retention time of an n-alkane with n carbon atoms; and RT(n+1) is the retention time of an n-alkane with (n+1) carbon atoms.

[0065] Quantitative Analysis: Quantitative analysis primarily utilizes a combination of internal and external standards. For all volatile aroma components identified by HS-SPME / GC-MS, 10 μL of ethyl decanoate was used as the internal standard, and quantification was performed by comparing the peak area of each compound to the peak area of the internal standard. For compounds for which commercially available standards were available, a standard curve was constructed using the standard concentration as the horizontal axis and the ratio of the standard peak area to the internal standard peak area as the vertical axis, and quantification was performed using the external standard curve. For compounds for which standards were not available, estimates were made using standards with the same functional groups, similar chemical structures, and similar carbon number.

[0066] Screening, identification and preservation of Aspergillus cristatus

[0067] In October 2024, two strains of "Golden Flower" fungi were isolated from Fu tea produced in Linxiang, Hunan, and were numbered 430682HH-13A and 430682HH-13B.

[0068] Strain Isolation Method: Take Linxiang Fucha tea and place it in sterile water with glass beads (1:10). Shake thoroughly to disperse the colonies. Then, spread the solution onto Czapek medium plates using a 10-fold dilution method and incubate at 28°C until single colonies emerge. Observe the growth of individual colonies, number the best-performing "Golden Flower" colonies, and record their growth every other day. After incubating at 28°C for 8 days, select a plate containing the appropriate dilution concentration and, using an inoculating needle, pick the best-performing colonies. Streak them onto Czapek medium and, after incubation at 28°C, transfer them to a slant plate once they have grown into single colonies. Incubate for 4 days before storing.

[0069] The number of "golden flower" bacteria per unit weight was used as an indicator for screening, and the results are shown in Table 1. The results showed that strain 430682HH-13B was the dominant "golden flower" bacteria.

[0070] Table 1 Number of “golden flower” fungi in Fu tea produced in Linxiang

[0071]

[0072] After strain 430682HH-13B was revived at room temperature for 24 hours, it was transferred to Czapek medium and 20% sucrose Czapek medium respectively, and cultured in a constant temperature incubator at 28°C. The growth of the colonies was observed every day and records were kept. The direct slide observation and morphological observation methods were used. The sterilized coverslip was inserted obliquely into the solid culture medium, and the strain was observed every other day starting from the 3rd day. When the hyphae expanded to the coverslip, it was removed, and a few drops of cotton blue stain were applied to the slide. The coverslip was carefully placed to avoid the formation of bubbles, and observed under a double-hole microscope. The morphology of strain 430682HH-13B can be seen Figure 1 .

[0073] Multi-gene phylogenetic analysis of strain 430682HH-13B was conducted based on the sequences of three genes: β-tubulin, calmodulin, and RNA polymeraseⅡ.

[0074] Comparisons within the NCBI GenBank database and multiple sequence alignment using ClusterX software revealed that strain 430682HH-13B naturally clustered with Aspergillus cristatus in the β-tubulin, calmodulin, and RNA polymerase II gene sequences, with a similarity of 99%. Therefore, strain 430682HH-13B was identified as Aspergillus cristatus.

[0075] Based on the above morphological observations, physiological and biochemical identifications and the results of multi-gene phylogenetic analysis of three gene sequences, it can be determined that this strain is Aspergillus cristatus and is named Aspergillus cristatus (Aspergillus cristatus430682HH-13B).

[0076] Aspergillus cristatus 430682HH-13B was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025900.

[0077] Screening, identification and preservation of Aspergillus shevarium

[0078] In October 2024, a strain of "Golden Flower" was isolated from Fu tea produced in Yixing, Jiangsu, and was numbered 320282HH-22.

[0079] The strain isolation method is the same as in Example 1.

[0080] Screening was conducted using the number of "golden flower" bacteria per unit weight as an indicator, and the results are shown in Table 2 below.

[0081] Table 2 Number of “golden flower” fungi in Fu tea produced in Yixing, Jiangsu

[0082]

[0083] The morphology of strain 320282HH-22 is shown in Figure 2 The observation method is the same as in Example 1.

[0084] Multigene phylogenetic analysis of strain 320282HH-22 was conducted based on the sequences of three genes: β-tubulin, calmodulin, and RNA polymerase II. Multiple sequence alignment was performed using ClusterX software, comparing strain 320282HH-22 to Aspergillus chevalieri in the β-tubulin, calmodulin, and RNA polymerase II gene sequences within the NCBI GenBank database. The results showed that strain 320282HH-22 naturally clustered with Aspergillus chevalieri in these three gene sequences, with a similarity of 99%. Therefore, strain 320282HH-22 was identified as Aspergillus chevalieri.

[0085] Based on the above morphological observations, physiological and biochemical identifications and the results of multi-gene phylogenetic analysis of three gene sequences, it can be determined that this strain is Aspergillus chevalieri and named Aspergillus chevalieri 320282HH-22.

[0086] Aspergillus chevalieri 320282HH-22 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025901.

[0087] Screening, identification and preservation of Eurotium cereus

[0088] In October 2024, a strain of "Golden Flower" was isolated from dark tea produced in Dehong, Yunnan, and was numbered 533100HH-56.

[0089] The strain isolation method is the same as in Example 1.

[0090] Screening was conducted using the number of "golden flower" bacteria per unit weight as an indicator, and the results are shown in Table 3 below.

[0091] Table 3 Number of “golden flower” fungi in dark tea produced in Dehong Dai and Jingpo Autonomous Prefecture, Yunnan

[0092]

[0093] The morphology of strain 533100HH-56 is shown in Figure 3 The observation method is the same as in Example 1.

[0094] Multigene phylogenetic analysis of strain 533100HH-56 was conducted based on the sequences of three genes: β-tubulin, calmodulin, and RNA polymerase II. Multiple sequence alignment was performed using ClusterX software, comparing strain 533100HH-56 to those of Eurotium herbariorum in the β-tubulin, calmodulin, and RNA polymerase II gene sequences within the NCBI GenBank database. The results showed that strain 533100HH-56 naturally clustered with those of Eurotium herbariorum in these three gene sequences, with a similarity of 99%. Therefore, strain 533100HH-56 was identified as Eurotium herbariorum.

[0095] Based on the above morphological observations, physiological and biochemical identifications and the results of multi-gene phylogenetic analysis of three gene sequences, the strain was identified as Aspergillus cristatus and named Eurotium herbariorum 533100HH-56.

[0096] The strain Eurotium herbariorum 533100HH-56 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on April 27, 2025, and its deposit number is: CCTCC NO: M2025902.

[0097] The following strains were used: Aspergillus cristatus, accession number: M2025900; Aspergillus chevalieri, accession number: M2025901; Eurotium herbariorum, accession number: M2025902.

[0098] Example 1: Green fungus-flavored Fu'er tea and its preparation

[0099] This embodiment provides a fungus-flower-scented Fu'er tea, and its processing method is as follows:

[0100] 1) Using Hunan Anhua first-grade dark tea as raw material, adjust its moisture content to 22% with purified water, steam sterilize (121°C, 20 min), and then cool to room temperature;

[0101] 2) Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum were mixed in a ratio of 5:3:2 in terms of effective viable bacteria count, and the mixture was inoculated (evenly sprayed) into the sterilized dark tea in step 1) to perform gradient fermentation; the total inoculum amount of the mixture was 5×10 6 CFU / g.

[0102] Gradient fermentation is as follows:

[0103] Stage 1 (initial stage, 0-120h): temperature 28°C, relative humidity 85%, to allow mycelium to grow rapidly;

[0104] Stage 2 (mid-term, 121-288h): temperature 32°C, relative humidity 75%, promoting the synthesis of (E,E)-2,4-heptadienaldol;

[0105] Stage 3 (late stage, 289-360h): temperature 25°C, relative humidity 65%, locking the green fragrance components.

[0106] After fermentation, two-stage drying is adopted (i.e., drying at 80°C for 15-20 minutes and then drying at 55°C for 2-3 hours) to reduce the moisture content to ≤10%, thereby locking the aroma and retaining the volatile aroma substances to obtain the fungus-flower-scented Fu'er tea.

[0107] The fungus-flavored Fu'an tea prepared in this embodiment has a sensory evaluation of "fresh fungus fragrance and lasting sweetness". Figure 4 , the GC-MS spectrum of characteristic aroma compounds is shown in Figure 5 .

[0108] Example 2: Green fungus-flavored Fu'er tea and its preparation

[0109] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 1 only in that the ratio of the effective viable bacteria count of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 3:2:5.

[0110] Example 3: Green fungus-flavored Fu'er tea and its preparation

[0111] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 1 only in that the ratio of the effective viable bacteria counts of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 1:5:4.

[0112] Example 4 Flower Fungus Fragrance Fu Tea and its Preparation

[0113] This embodiment provides a fungus-flower-scented Fu'er tea, and its processing method is as follows:

[0114] 1) Using Shaanxi Jingyang third-grade Fuzhuan tea as raw material, the water content was adjusted to 18% with purified water, and steam sterilized (121°C, 20 min); then cooled to room temperature;

[0115] 2) Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum were mixed in an effective viable bacterial count ratio of 6:2:2, and the mixture was inoculated (evenly sprayed) into the third-grade Fuzhuan tea sterilized in step 1) for gradient fermentation; the total inoculation amount of the mixed bacteria was 5×10 6 CFU / g.

[0116] Gradient fermentation is as follows:

[0117] Stage 1 (initial stage, 0-120h): temperature 28°C, relative humidity 85%, to allow mycelium to grow rapidly;

[0118] Phase 2 (mid-term, 121-288 h): temperature 32 °C, relative humidity 78%, to maximize the synthesis of linalool oxide;

[0119] Stage 3 (late stage, 289-360h): temperature 25°C, relative humidity 65%, locking the fungus fragrance components.

[0120] After fermentation, two-stage drying is adopted (i.e., drying at 80°C for 15-20 minutes and then drying at 55°C for 2-3 hours) to reduce the moisture content to ≤10%, thereby locking the aroma and retaining the volatile aroma substances to obtain the fungus-flower-scented Fu'er tea.

[0121] The fungus-flavored Fu'an tea prepared in this embodiment has a strong floral aroma and a bright orange soup color. Figure 6 , the GC-MS spectrum of characteristic aroma compounds is shown in Figure 7 .

[0122] Example 5: Flower Fungus Fragrance Fu Tea and Its Preparation

[0123] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 4 only in that the ratio of the effective viable bacteria count of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 3:2:5.

[0124] Example 6: Flower Fungus Fragrance Fu Tea and Its Preparation

[0125] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 4 only in that the ratio of the effective viable bacteria counts of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 1:5:4.

[0126] Example 7 Preparation of Ripe Fungus Fragrance Fu Tea

[0127] This embodiment provides a fungus-flower-scented Fu'er tea, and its processing method is as follows:

[0128] 1) Using Yunnan Pu'er cooked tea as raw material, adjust its moisture content to 20% with purified water, steam sterilize (121°C, 20 min), and then cool to room temperature;

[0129] 2) Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum were mixed at an effective viable count ratio of 5:2.5:2.5 as a mixed bacteria, and inoculated (evenly sprayed) into the third-grade Fuzhuan tea sterilized in step 1) for gradient fermentation; based on the mass of dry Yunnan Pu'er cooked tea, the total inoculum amount of the mixed bacteria was 5×10 6 CFU / g.

[0130] Gradient fermentation is as follows:

[0131] Stage 1 (initial stage, 0-120h): temperature 28°C, relative humidity 85%, to allow mycelium to grow rapidly;

[0132] Stage 2 (mid-stage, 121-288h): Temperature 33°C, relative humidity 70%, which can accelerate the accumulation of α-terpineol;

[0133] Stage 3 (late stage, 289-360h): temperature 25°C, relative humidity 65%, locking in the aroma of cooked mushrooms.

[0134] After fermentation, two-stage drying is adopted (i.e., drying at 80°C for 15-20 minutes and then drying at 55°C for 2-3 hours) to reduce the moisture content to ≤10%, thereby locking the aroma and retaining the volatile aroma substances to obtain the fungus-flower-scented Fu'er tea.

[0135] The fungus-flavored Fu'an tea prepared in this embodiment has a mellow aroma and a smooth taste. Figure 8 , the GC-MS spectrum of characteristic aroma compounds is shown in Figure 9 .

[0136] Example 8 Ripe Fungus Fragrance Fu Tea and Its Preparation

[0137] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 7 only in that the ratio of the effective viable bacteria count of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 3:2:5.

[0138] Example 9: Cooked Mushroom Fragrance Fu Tea and Its Preparation

[0139] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 7 only in that the ratio of the effective viable bacteria count of Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum in the mixed bacteria is 1:5:4.

[0140] Comparative Example 1

[0141] This comparative example provides a kind of Fu tea, which uses the same Hunan Anhua first-grade dark tea as Example 1 as raw material, adopts the traditional processing method (Yiyang Tea Factory produces traditional brick-shaped Fu brick tea), does not add bacteria, and only ferments naturally. Figure 10 .

[0142] Comparative Example 2

[0143] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which is different from that of Example 1 only in that only Aspergillus cristatus is inoculated.

[0144] Comparative Example 3

[0145] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which is different from that of Example 1 only in that only Aspergillus chevalieri is inoculated.

[0146] Comparative Example 4

[0147] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which is different from that of Example 1 only in that only Eurotium herbariorum is inoculated.

[0148] Comparative Example 5

[0149] This embodiment provides a fungus-flower-scented Fu'er tea, the processing method of which differs from that of Example 1 only in that gradient fermentation is not used, and fermentation is performed for 360 hours under a constant temperature of 28° C. and a relative humidity of 85%.

[0150] Comparative Example 6

[0151] This embodiment provides a fungus-flower-scented Fu'er tea, and its processing method is different from that of Example 1 only in that: after fermentation, the tea is dried at a constant temperature of 80° C. for 2-3 hours to a moisture content of ≤10%.

[0152] Experiment 1

[0153] In the above embodiment, Aspergillus cristatus, Aspergillus schevalieri and Eurotium herbariorum were used for fermentation. The synergistic metabolic network diagram of the three strains is shown in FIG. Figure 10 .

[0154] According to the constructed multi-species metabolic network model, the use of the mixed bacteria can achieve a balanced increase of the activity of the extracellular enzyme system (cellulase, pectinase, protease, etc.) by 38%-65%.

[0155] Experiment 2 Sensory Evaluation

[0156] Three tea experts with extensive tea evaluation experience conducted a sensory evaluation of the above examples and comparative Fu'er tea samples in accordance with the national sensory evaluation standards GB / T 23776-2018, "Tea Sensory Evaluation Method," and GB / T 14487-2017, "Tea Sensory Evaluation Terminology." They assigned scores based on the tea's appearance, flavor, aroma, tea soup color, and leaf base, and used a weighted calculation to generate a score. The results are shown in Table 4.

[0157] Table 4

[0158] sample Appearance, aroma, soup color, taste score Example 1 Full of golden flowers, light yellow in color, plump grains, fresh and elegant mushroom fragrance, long-lasting sweetness 94.00 Example 2 The golden flowers are full, the color is slightly dark yellow, the grains are still full, the mushroom fragrance is pure, and the taste is mellow. 92.90 Example 3 The golden flowers are full, yellow in color, small in size, with a pure mushroom aroma and a mellow taste. 91.98 Example 4 The golden flower is full, the color is bright yellow, the grains are full, the mushroom flower fragrance is rich, and the soup is bright orange-red. 94.33 Example 5 The golden flowers are full, yellow in color, the grains are still full, the mushroom fragrance is pure, and the taste is mellow. 92.85 Example 6 The golden flowers are full, the color is yellow, the grains are still full, the mushroom aroma is relatively pure, and the taste is mellow. 91.85 Example 7 The golden flowers are full, yellow in color, full in grain, mellow in aroma and smooth in taste. 94.15 Example 8 The golden flowers are full, the color is slightly dark yellow, the grains are still full, the mushroom fragrance is pure, and the taste is mellow. 92.90 Example 9 The golden flowers are full, the color is yellow, the grains are still full, the mushroom aroma is relatively pure, and the taste is mellow. 91.85 Comparative Example 1 The flowers are lush, the taste is mellow, and there is a fungus-flower fragrance 90.30 Comparative Example 2 The golden flowers are lush, light yellow in color, the grains are still full, the mushroom fragrance is obvious, and the mellow and sweet taste 90.93 Comparative Example 3 The golden flowers are lush, yellow in color, the grains are still full, the mushroom fragrance is obvious, and the taste is mellow. 91.05 Comparative Example 4 The golden flowers are lush, the color is slightly dark yellow, the grains are still full, the mushroom fragrance is pure, and the taste is mellow. 90.85 Comparative Example 5 The golden flowers are lush, yellow in color, the grains are plump, the mushroom aroma is obvious, and the taste is mellow. 90.75 Comparative Example 6 The golden flowers are full, the color is dark yellow, the grains are still full, the mushroom fragrance is slightly fishy, and the taste is mellow. 86.75

[0159] As shown in Table 4, co-fermentation with Aspergillus cristatus (430682HH-13B), Aspergillus chevalieri (320282HH-22), and Eurotium herbariorum (533100HH-56) significantly improves the quality and quantity of the "golden flower" fungi in Fu'er tea, resulting in a tea with abundant golden flowers and plump tea granules. Furthermore, by optimizing the ratio of bacterial strains based on the specific tea base, the tea imparts aroma characteristics such as a refreshing fungal aroma, a rich fungal-flower aroma, and a mellow, mature aroma. Furthermore, co-fermentation enhances the fermentation process, improving the tea's brightness while also enhancing the body and sweetness of the tea's flavor. The sensory scores of Examples 1-9 were significantly better than those of the comparative samples, and Example 1 was significantly better than Examples 2 and 3, Example 4 was significantly better than Examples 5 and 6, and Example 7 was significantly better than Examples 8 and 9, indicating that the limited viable cell count ratios of the three strains of bacteria in Examples 1, 4, and 7 were most suitable for the preparation of green fungus aroma, flower fungus aroma, and cooked fungus aroma Fu'er tea, respectively.

[0160] Experiment 3

[0161] The contents of key aroma active substances in the above examples and comparative examples of Fu'er tea samples are shown in Table 5 below.

[0162] Table 5 Content of key aroma active substances (μg / L)

[0163]

[0164]

[0165] As shown in Table 5, when co-fermented with Aspergillus cristatus (430682HH-13B), Aspergillus chevalieri (320282HH-22), and Eurotium herbariorum (533100HH-56), the optimal strain ratio, tailored to the specific tea base, imparts Fucha with aroma characteristics such as a refreshing fungus aroma, a rich fungus-flower aroma, and a mellow, mature aroma. This is consistent with the results of key aroma-active substance testing. Compared with Comparative Example 1, the contents of (E,E)-2,4-heptadienal, linalool oxide, and α-terpineol in Example 1 increased by 81.90%, 22.01%, and 48.52%, respectively; and the contents of (E,E)-2,4-heptadienal, linalool oxide, and α-terpineol in Example 4 increased by 22.55%, 151%, and 82.37%, respectively.

[0166] Experiment 4

[0167] The number of golden flower fungi at the end of fermentation in Example 1 and Comparative Example 5 is shown in Table 6 below.

[0168] Table 6 Number of “Golden Flower Fungi” at the end of fermentation

[0169] sample Number of golden flower fungi (cfu / g) Example 1 <![CDATA[4.6×10 6 ]]> Comparative Example 5 <![CDATA[3.5×10 5 ]]>

[0170] Note: The “golden flower fungi” in Table 6 refers to the total number of Aspergillus cristatus, Aspergillus chevalieri and Eurotium herbariorum.

[0171] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Eurotium herbariorum 533100HH-56, its deposit number is: CCTCCNO: M2025902.

2. Aspergillus cristatus 430682HH-13B, its deposit number is: CCTCCNO: M2025900.

3. Aspergillus chevalieri 320282HH-22, its deposit number is: CCTCCNO: M2025901.

4. A mixed fungus comprising the Eurotium herbariorum 533100HH-56 of claim 1, the Aspergillus cristatus 430682HH-13B of claim 2, and the Aspergillus chevalieri 320282HH-22 of claim 3.

5. The mixed bacteria according to claim 4, characterized in that The ratio of the effective viable counts of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22 and Eurotium herbariorum 533100HH-56 is (5-6):(2-3):(2-2.5), preferably 5:3:2, 6:2:2 or 5:2.5:2.

5.

6. Use of the Eurotium herbariorum 533100HH-56 according to claim 1, the Aspergillus cristatus 430682HH-13B according to claim 2, the Aspergillus chevalieri 320282HH-22 according to claim 3, or the mixed fungus according to claim 4 or 5 in processing Fu'er tea.

7. A method for processing fungus-flower-scented Fu'er tea, characterized in that: include: 1) adjusting the moisture content of the to-be-fermented Fu'er tea raw material to ≤25% with purified water; steam sterilizing, and then cooling to room temperature; 2) inoculating the mixed bacteria into the sterilized Fu'er tea raw material in step 1) and performing the following gradient fermentation: Initial stage, fermentation 0-120h: temperature 26-28℃, relative humidity 80-85%; Mid-stage, fermentation 121-288h: temperature 30-32°C, relative humidity 70-78%; Late stage, fermentation 289-360h: temperature 25-28℃, relative humidity 60-65%.

8. The processing method of the mushroom-flower-scented Fu'er tea according to claim 7, characterized in that: Based on the mass of the dry raw material of Fucha to be fermented, the total inoculation amount of the mixed bacteria is (5-6)×10 6 CFU / g.

9. The processing method of the mushroom-flower-scented Fu'er tea according to claim 7 or 8, characterized in that: After fermentation, two-stage drying is adopted: first drying at 75-80℃ for 15-20 minutes, then drying at 55-60℃ for 2-3 hours; drying to a moisture content of ≤10%.

10. A fungus-flower-scented Fu'er tea, characterized in that: The invention is prepared by the processing method according to any one of claims 7 to 9.

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