A type of Fu brick tea with a floral aroma and its processing method

Abstract

This invention relates to the field of dark tea processing technology, specifically to a fungal-aroma Fu brick tea and its processing method. The invention utilizes high-throughput screening to obtain three key bacterial strains with complementary metabolic characteristics from Fu brick tea: *Aspergillus cristatus* 430682HH-13B, *Aspergillus chevalieri* 320282HH-22, and *Eurotium herbariorum* 533100HH-56. Combining these three strains in Fu brick tea processing allows for targeted regulation of the tea's aroma and shortens the processing cycle to 15 days.

Description

Technical Field

[0001] This invention relates to the field of dark tea processing technology, specifically to a fungus-scented Fu brick tea and its processing method. Background Technology

[0002] Fu brick tea, as a core category of dark tea, relies heavily on the metabolic activity of the "golden flower fungus" community during the "fermentation" process to determine its quality. However, traditional processes and existing technologies face systemic technical bottlenecks that urgently need to be overcome. In traditional processes, the uncontrollable environmental microbial community in natural inoculation leads to a low proportion of key strains (Aspergillus cristatus). This results in the following limitations in Fu brick tea processing: First, the imbalance in the internal microbial community structure causes significant fluctuations in the activity of extracellular hydrolytic enzymes, leading to inconsistent conversion of tea polyphenols in the tea matrix. Second, the relatively single-strain-dominated metabolic system results in fewer volatile compounds, with a high proportion of 1-octen-3-one as the key aroma active substance, while lacking aroma types represented by other characteristic aroma components such as linalool oxide and α-terpineol. Furthermore, the natural fermentation cycle of traditional processing is as long as 28±2 days, with a total processing cycle exceeding 40 days, and the sluggish response to temperature and humidity results in mycelial biomass below 0.85g / 100g (20×10⁻⁶). 4 The critical failure rate of (cfu / g) is 20%.

[0003] Although artificial inoculation techniques have shortened the flowering cycle to 15-20 days in recent years, the problem of strain antagonism is prominent in mixed-strain fermentation, often leading to flowering failure. Furthermore, existing processes still have the following problems: ① lack of multi-strain metabolic kinetic models, leading to intensified nutrient competition; ② mismatch between culture parameters and strain growth curves (e.g., delayed spore germination of *Cymbidium cladosporum* at a constant temperature of 30℃ by 6-8 hours); ③ lack of quantitative standards for aroma component regulation (aroma correlation model R...). 2 <0.35). Summary of the Invention

[0004] This invention obtained three key bacterial strains with complementary metabolic characteristics from Fu brick tea and dark tea through high-throughput screening: *Aspergillus cristatus* 430682HH-13B, *Aspergillus chevalieri* 320282HH-22, and *Eurotium herbariorum* 533100HH-56. Using these three strains in combination for Fu brick tea processing enables targeted regulation of the tea's aroma and shortens the processing cycle to 15 days.

[0005] Aspergillus cristatus 430682HH-13B was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China) with accession number CCTCC NO: M2025900.

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

[0007] Aspergillus chevalieri 320282HH-22 was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China) with accession number CCTCC NO: M2025901.

[0008] When used in the processing of Fu brick tea, Aspergillus chevalieri 320282HH-22 efficiently expresses linalool oxide (≥101.95±22.97μg / L, which is 40.06% higher than that of traditional processes) through the terpene synthase gene cluster.

[0009] Eurotium herbariorum 533100HH-56 was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China) with accession number CCTCC NO: M2025902.

[0010] When used in the processing of Fu brick tea, Eurotium herbariorum 533100HH-56 activates the key enzyme for α-terpineol synthesis (≥91.67±33.94μg / L, which is 109% higher than the traditional process).

[0011] This invention also provides the application of Aspergillus cristatus 430682HH-13B in the processing of Fu brick tea.

[0012] This invention also provides the application of Aspergillus chevalieri 320282HH-22 in the processing of Fu brick tea.

[0013] This invention also provides the application of Eurotium herbariorum 533100HH-56 in the processing of Fu brick tea.

[0014] The present invention also provides a mixed microorganism, including 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 consist 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 aforementioned mixed bacteria can achieve a balanced increase of 38%-65% in the activity of extracellular enzyme systems (cellulase, pectinase, protease, etc.).

[0017] As a preferred embodiment of the present invention, the effective viable count ratio of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22, and Eurotium herbariorum 533100HH-56 in the mixed bacteria 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 the strain compatibility model established through co-culture experiments, when the three strains in the mixed culture are within the above-mentioned proportion 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 the application of the mixed bacteria in the processing of Fu brick tea.

[0020] This invention also provides a method for processing fungus-scented Fu brick tea, comprising:

[0021] 1) Adjust the moisture content of the raw materials for fermentation to ≤25% with purified water; sterilize with steam, and then cool to room temperature;

[0022] 2) The mixed bacteria are inoculated into the sterilized Fu brick tea raw material from step 1), and the following gradient fermentation is carried out:

[0023] In the initial stage, during fermentation from 0 to 120 hours: temperature 26-28℃, relative humidity 80-85%;

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

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

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

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

[0028] Preferably, when the raw material for fermenting Fu tea is first-grade black tea from Anhua, Hunan, the optimal ratio of viable bacteria (Aspergillus cristatus 430682HH-13B, Aspergillus schevalieri 320282HH-22, and Eurotium herbariorum 533100HH-56) in the mixed bacteria is 5:3:2, the prepared Fu tea with a green mushroom aroma will have the best effect.

[0029] Preferably, when the raw material for fermenting Fu brick tea is third-grade Fu brick tea from Jingyang, Shaanxi, the optimal ratio of viable bacteria (Aspergillus cristatus 430682HH-13B, Aspergillus schevalieri 320282HH-22, and Eurotium herbariorum 533100HH-56) in the mixed bacteria is 6:2:2, the resulting floral-scented Fu brick tea will have the best effect.

[0030] Preferably, when the raw material for fermenting Fu tea is Yunnan Pu'er ripe tea, the optimal ratio of viable bacteria of Aspergillus cristatus 430682HH-13B, Aspergillus chevalieri 320282HH-22, and Eurotium herbariorum 533100HH-56 in the mixed bacteria is 5:2.5:2.5, resulting in the best fermented aroma Fu tea.

[0031] In some specific embodiments, the steam sterilization conditions are 118-121°C for 15-20 minutes.

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

[0033] The three-stage gradient fermentation method employed in this invention promotes rapid mycelial growth, particularly the colonization of Aspergillus cristatus mycelium (biomass growth rate reaches 0.12 g / h) in the initial fermentation stage; in the middle stage of fermentation, it promotes the synthesis of secondary metabolites such as linalool oxide, (E,E)-2,4-heptadienol, and α-terpineol; among which, the peak rate of linalool synthesis by Aspergillus chevallaris secondary metabolite reaches 1.24 μg / (g·h); in the later stage of fermentation, it guides Aspergillus cereus to complete the aroma precursor conversion (α-terpineol accumulation increases by 109%), and can stabilize and lock in aroma components.

[0034] As a preferred embodiment of the present invention, the total inoculum amount of the mixed bacteria is (5-6)×10 based on the mass of the dried, unfermented Fu brick tea raw material. 6 CFU / g.

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

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

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

[0038] The method of this invention can process a variety of Fu tea products, including three types of Fu tea: Qingjunxiang (clear fungus aroma), Huajunxiang (floral fungus aroma), and Shujunxiang (ripe fungus aroma).

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

[0040] The method of this invention uses a gradient fermentation process to achieve targeted regulation of the aroma of Fu brick tea.

[0041] The present invention also includes the fungus-scented Fu brick tea prepared by the above method.

[0042] The processing method for Fu brick tea with a floral aroma of this invention shortens the processing cycle to 15 days (67% faster than traditional methods) and reduces energy consumption by at least 30% through real-time parameter control, while achieving a total content of characteristic aroma components exceeding 200 μg / L. Compared to traditional methods, the total amount of volatile aroma substances in the Fu brick tea processed by this invention is significantly increased. The colonization success rate of the microbial community in this invention is increased to 98.3% (compared to 80% in traditional methods), providing a complete engineering and technical solution for the upgrading of the Fu brick tea industry and yielding significant economic benefits. Attached Figure Description

[0043] Figure 1 Morphological diagram of Aspergillus cristatus (430682HH-13B).

[0044] Figure 2 Morphological diagram of Aspergillus chevalieri (320282HH-22).

[0045] Figure 3 Morphological diagram of the strain *Eurotium herbariorum* (533100HH-56).

[0046] Figure 4 Photograph of the fungus-scented Fu brick tea product prepared in Example 1.

[0047] Figure 5 The image shows the GC-MS spectrum of the characteristic aroma compounds of the fungus-scented Fu brick tea prepared in Example 1.

[0048] Figure 6 Photograph of the fungus-scented Fu brick tea product prepared in Example 4.

[0049] Figure 7 The image shows the GC-MS spectrum of the characteristic aroma compounds of the fungus-scented Fu brick tea prepared in Example 4.

[0050] Figure 8 Photograph of the fungus-scented Fu brick tea product prepared in Example 7.

[0051] Figure 9 The image shows the GC-MS spectrum of the characteristic aroma compounds of the fungus-scented Fu brick tea prepared in Example 7.

[0052] Figure 10 The flowchart of traditional processing method for Fu brick tea is shown in Comparative Example 1.

[0053] Figure 11 This is a synergistic metabolic network diagram of three strains in an embodiment of the present invention. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0055] PDA solid culture medium: 200g potato (peeled), 20g glucose, 14g agar, add 1L distilled water or deionized water, stir and heat to boiling until completely dissolved, dispense into test tubes or Erlenmeyer flasks, and autoclave at 121℃ for 15min.

[0056] Czapek's medium: Sodium nitrate 3.0g, dimethyl hydrogen phosphate 1.0g, magnesium sulfate 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, sucrose 30.0g, agar 15.0g, add 1L of distilled or deionized water, stir and heat to boiling until completely dissolved, dispense into test tubes or Erlenmeyer flasks, and autoclave at 121℃ for 15min.

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

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

[0059] HS-SPME extraction of volatile components: Accurately weigh 5g of crushed tea sample and place it in a 100mL headspace vial. Add 50ml of boiling water and a certain amount of internal standard (ethyl decanoate). Then, insert the SPME handheld device equipped with a 50 / 30μm DVB / CAR / PDM extraction head into the headspace of the extraction vial through the rubber gasket on the cap. After equilibration in a 60℃ water bath for 10min, push out the fiber head and allow adsorption for 50min. After extraction is complete, retract the fiber head and immediately insert it into the injection port of the gas chromatograph for desorption for 3min.

[0060] GC-MS analysis

[0061] GC conditions: DB-5MS (30m × 0.25mm × 0.25μm) flexible quartz capillary column. Injector temperature: 240℃; carrier gas: high-purity helium (purity >99.999%); flow rate: 1.0mL / min; manual pulse splitless injection. Column temperature program: initial temperature 50℃, hold for 5 min; increase to 180℃ at 3℃ / min, hold for 2 min; then increase to 250℃ at 10℃ / min, hold for 3 min.

[0062] MS conditions: EI ion source, electron energy 70 eV, ion source temperature 230 °C; scan quality 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 is based on retention indices (RIs), mass spectrometry matching with the NIST17 standard library on the instrument workstation, and integration with relevant literature, retention times, etc. A 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 n-carbon n-alkanes; and RT(n+1) is the retention time of (n+1)-carbon n-alkanes.

[0065] Quantitative Analysis: Quantitative analysis primarily employed 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 with that of the internal standard. For some compounds for which commercially available standards were available, a standard curve was constructed with the standard concentration on the x-axis and the ratio of the standard peak area to the internal standard peak area on the y-axis, and external standard curve quantification was performed. For compounds without standards, standards with the same functional groups, similar chemical structures, and similar carbon numbers were used for estimation.

[0066] Screening, identification and preservation of Aspergillus cristatus

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

[0068] Strain isolation method: Take Fu brick tea from Linxiang, place it in sterile water containing glass beads (1:10), shake thoroughly to disperse the colonies, and spread it onto Czapek's agar plates using a 10-fold dilution method. Incubate at 28℃ until single colonies grow. Observe the growth of individual colonies, number the "Golden Flower" colonies with better growth, and record the growth status every other day. After 8 days of incubation at 28℃, select plates with appropriate dilution concentrations, pick the best-performing colonies with an inoculation needle, streak them onto Czapek's agar plates, incubate at 28℃ until single colonies grow, then transfer to slant agar, incubate for 4 days, and then store.

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

[0070] Table 1. Quantity of "Golden Flower" Fungus in Fu Tea Produced in Linxiang

[0071]

[0072] After reviving strain 430682HH-13B at room temperature for 24 hours, it was transferred to Czapek's medium and 20% sucrose Czapek's medium, respectively, and incubated in a 28℃ incubator. Colony growth was observed and recorded daily. Direct slide observation and morphological observation methods were used. Sterile coverslips were obliquely inserted into the solid culture medium, and the strain was observed every other day starting from day 3. When the hyphae extended to the coverslip, it was removed, a few drops of cotton blue stain were applied to the slide, and the coverslip was carefully placed back on, avoiding air bubbles. Observation was performed under a two-hole microscope. The morphology of strain 430682HH-13B is shown in the figure. Figure 1 .

[0073] Multigene phylogenetic analysis of strain 430682HH-13B based on the gene sequences of three genes: β-tubulin, calmodulin, and RNA polymerase II.

[0074] Comparisons were performed in the NCBI GenBank database using ClusterX software for multiple sequence alignment. The results showed that strain 430682HH-13B exhibited natural clustering with the β-tubulin, calmodulin, and RNA polymerase II gene sequences of *Aspergillus cristatus*, 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 polygenic phylogenetic analysis of three gene sequences, this strain can be identified as Aspergillus cristatus and named Aspergillus cristatus (430682HH-13B).

[0076] Aspergillus cristatus 430682HH-13B was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China), with accession number CCTCC NO: M2025900.

[0077] Screening, identification and preservation of Aspergillus chevaleri

[0078] In October 2024, a strain of "golden flower" fungus was isolated from Fu tea produced in Yixing, Jiangsu Province, with the identification number 320282HH-22.

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

[0080] The number of "golden flower" bacteria per unit weight was used as an indicator for screening, and the results are shown in Table 2 below.

[0081] Table 2. Quantity of "Golden Flower" Fungus in Fu Tea Produced in Yixing, Jiangsu Province

[0082]

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

[0084] Phylogenetic analysis of strain 320282HH-22 based on the gene sequences of β-tubulin, calmodulin, and RNA polymerase II was performed. Comparisons were made in the NCBI GenBank database using ClusterX software for multiple sequence alignment. The results showed that strain 320282HH-22 exhibited natural clustering with Aspergillus chevalieri in the β-tubulin, calmodulin, and RNA polymerase II 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 polygenic phylogenetic analysis of the three gene sequences, this strain can be identified as Aspergillus chevalieri, and named Aspergillus chevalieri 320282HH-22.

[0086] Aspergillus chevalieri 320282HH-22 was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China) with accession number CCTCC NO: M2025901.

[0087] Screening, identification and preservation of *Cymbidium clavatum*

[0088] In October 2024, a strain of "golden flower" fungus was isolated from dark tea produced in Dehong, Yunnan Province, with the identification number 533100HH-56.

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

[0090] The number of "golden flower" bacteria per unit weight was used as an indicator for screening, and the results are shown in Table 3 below.

[0091] Table 3. Quantity of "Golden Flower" bacteria in dark tea produced in Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province

[0092]

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

[0094] Phylogenetic analysis of strain 533100HH-56 based on the sequences of three genes: β-tubulin, calmodulin, and RNA polymerase II. Comparisons were performed in the NCBI GenBank database using ClusterX software for multiple sequence alignment. The results showed that strain 533100HH-56 exhibited a natural clustering with *Eurotium herbariorum* in terms of the β-tubulin, calmodulin, and RNA polymerase II 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 multigene phylogenetic analysis of three gene sequences, this strain can be identified as *Aspergillus cristatus*, and named *Eurotium herbariorum* 533100HH-56.

[0096] The strain *Eurotium herbariorum* 533100HH-56 was deposited on April 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China) with accession number CCTCC NO: M2025902.

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

[0098] Example 1: Green Mushroom-Flavored Fu Brick Tea and its Preparation

[0099] This embodiment provides a fungus-scented Fu brick tea, the processing method of which is as follows:

[0100] 1) Using first-grade black tea from Anhua, Hunan as raw material, the moisture content is adjusted to 22% with purified water, and then sterilized by steam (121℃, 20min); then cooled to room temperature;

[0101] 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 5:3:2 to form a mixed culture, and inoculate (spray evenly) this mixture into the sterilized black tea from step 1) for gradient fermentation; the total inoculation amount of the mixed culture is 5 × 10⁻⁶ based on the mass of the dried black tea. 6 CFU / g.

[0102] Gradient fermentation is as follows:

[0103] Stage 1 (Initial stage, 0-120h): Temperature 28℃, relative humidity 85%, to promote rapid mycelial growth;

[0104] Phase 2 (mid-term, 121-288h): Temperature 32℃, relative humidity 75%, promotes the synthesis of (E,E)-2,4-heptadienalol;

[0105] Stage 3 (late stage, 289-360h): Temperature 25℃, relative humidity 65%, locking in green aromatic components.

[0106] After fermentation, a two-stage drying process is adopted (i.e., drying at 80℃ for 15-20 minutes first, and then drying at 55℃ for 2-3 hours) until the moisture content is ≤10%, locking in the aroma and retaining volatile aroma substances to obtain the fungal-scented Fu tea.

[0107] The mushroom-aroma type Fu brick tea prepared in this embodiment has a sensory evaluation of "fresh mushroom aroma and long-lasting sweet aftertaste". See product photos below. Figure 4 The GC-MS spectra of characteristic aroma compounds are shown in [reference needed]. Figure 5 .

[0108] Example 2: Green Mushroom-Flavored Fu Brick Tea and its Preparation

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

[0110] Example 3: Green Fungus-Flavored Fu Brick Tea and its Preparation

[0111] This embodiment provides a fungal-scented Fu brick tea, the processing method of which differs from that of Embodiment 1 only in that the effective live bacteria ratio of Aspergillus cristatus, Aspergillus chevalieri and Eurotium herbariorum in the mixed fungus is 1:5:4.

[0112] Example 4: Floral and Fungal Fragrance-Type Fu Brick Tea and its Preparation

[0113] This embodiment provides a fungus-scented Fu brick tea, the processing method of which is as follows:

[0114] 1) Using Shaanxi Jingyang Grade 3 Fu brick tea as raw material, the moisture content is adjusted to 18% with purified water, and then steam sterilized (121℃, 20min); then cooled to room temperature;

[0115] 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 6:2:2 to form a mixed culture, and inoculate (spray evenly) this mixture into the sterilized Grade 3 Fu brick tea from step 1) for gradient fermentation; the total inoculation amount of the mixed culture is 5 × 10⁻⁶ based on the mass of the dried Grade 3 Fu brick tea. 6 CFU / g.

[0116] Gradient fermentation is as follows:

[0117] Stage 1 (Initial stage, 0-120h): Temperature 28℃, relative humidity 85%, to promote rapid mycelial growth;

[0118] Stage 2 (intermediate stage, 121-288h): Temperature 32℃, relative humidity 78%, maximizes the synthesis of linalool oxide;

[0119] Phase 3 (late stage, 289-360h): Temperature 25℃, relative humidity 65%, locking in floral aroma components.

[0120] After fermentation, a two-stage drying process is adopted (i.e., drying at 80℃ for 15-20 minutes first, and then drying at 55℃ for 2-3 hours) until the moisture content is ≤10%, locking in the aroma and retaining volatile aroma substances to obtain the fungal-scented Fu tea.

[0121] The fungus-aroma type Fu brick tea prepared in this embodiment has a rich floral fragrance and a bright orange-yellow liquor. See product photos below. Figure 6 The GC-MS spectra of characteristic aroma compounds are shown in [reference needed]. Figure 7 .

[0122] Example 5: Floral and Fungal Fragrance-Type Fu Brick Tea and its Preparation

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

[0124] Example 6: Floral and Fungus-Flavored Fu Brick Tea and its Preparation

[0125] This embodiment provides a fungal-scented Fu brick tea, the only difference between its processing method and that of Embodiment 4 is that the effective viable count ratio of Aspergillus cristatus, Aspergillus chevalieri and Eurotium herbariorum in the mixed fungus is 1:5:4.

[0126] Example 7: Preparation of Fermented Fu Brick Tea

[0127] This embodiment provides a fungus-scented Fu brick tea, the processing method of which is as follows:

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

[0129] 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 5:2.5:2.5 to form a mixed culture, and inoculate (spray evenly) this mixture into the sterilized third-grade Fu brick tea from step 1) for gradient fermentation; based on the mass of dried Yunnan Pu'er ripe tea, the total inoculation amount of the mixed culture is 5 × 10⁻⁶. 6 CFU / g.

[0130] Gradient fermentation is as follows:

[0131] Stage 1 (Initial stage, 0-120h): Temperature 28℃, relative humidity 85%, to promote rapid mycelial growth;

[0132] Phase 2 (mid-term, 121-288h): Temperature 33℃ and relative humidity 70% can accelerate the accumulation of α-terpineol;

[0133] Phase 3 (late stage, 289-360h): Temperature 25℃, relative humidity 65%, locking in the aroma components of the cooked mushroom.

[0134] After fermentation, a two-stage drying process is adopted (i.e., drying at 80℃ for 15-20 minutes first, and then drying at 55℃ for 2-3 hours) until the moisture content is ≤10%, locking in the aroma and retaining volatile aroma substances to obtain the fungal-scented Fu tea.

[0135] The mushroom-flavored Fu brick tea prepared in this embodiment has a mellow and rich aroma and a smooth taste. See product photos below. Figure 8 The GC-MS spectra of characteristic aroma compounds are shown in [reference needed]. Figure 9 .

[0136] Example 8: Cooked Fermented Fu Brick Tea and its Preparation

[0137] This embodiment provides a fungal-scented Fu brick tea, the only difference between its processing method and that of Embodiment 7 is that the effective live bacteria ratio of Aspergillus cristatus, Aspergillus chevalieri and Eurotium herbariorum in the mixed fungus is 3:2:5.

[0138] Example 9: Cooked Fermented Fu Brick Tea and its Preparation

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

[0140] Comparative Example 1

[0141] This comparative example provides a Fu brick tea, using the same first-grade Hunan Anhua black tea as in Example 1, and employing traditional processing methods (traditional brick-shaped Fu brick tea produced by Yiyang Tea Factory), without adding any microbial agents, relying solely on natural fermentation. For detailed processing procedures, please refer to... Figure 10 .

[0142] Comparative Example 2

[0143] This embodiment provides a fungal-scented Fu brick tea, the only difference between which processing method is used and that of Embodiment 1 is that only Aspergillus cristatus is inoculated.

[0144] Comparative Example 3

[0145] This embodiment provides a fungal-scented Fu brick tea, the only difference between which processing method is used and that of Embodiment 1 is that only Aspergillus chevalieri is inoculated.

[0146] Comparative Example 4

[0147] This embodiment provides a fungal-scented Fu brick tea, the only difference between its processing method and that of Embodiment 1 is that it is inoculated only with Eurotium herbariorum.

[0148] Comparative Example 5

[0149] This embodiment provides a fungus-scented Fu brick tea, the only difference between its processing method and that of Embodiment 1 is that it does not use gradient fermentation, but only ferments for 360 hours at a constant temperature of 28°C and a relative humidity of 85%.

[0150] Comparative Example 6

[0151] This embodiment provides a fungus-scented Fu brick tea, the only difference between which processing method is used and that of Embodiment 1 is: after fermentation, it is dried at a constant temperature of 80℃ for 2-3 hours until the moisture content is ≤10%.

[0152] Experiment 1

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

[0154] Based on the constructed multi-species metabolic network model, the use of the aforementioned mixed bacteria can achieve a balanced increase of 38%-65% in the activity of extracellular enzyme systems (cellulase, pectinase, protease, etc.).

[0155] Experiment 2 Sensory Evaluation

[0156] Following the national standard methods for sensory evaluation, GB / T 23776-2018 "Sensory Evaluation Methods for Tea" and GB / T14487-2017 "Terminology for Sensory Evaluation of Tea," three tea experts with extensive experience in tea evaluation conducted sensory evaluations on the Fu brick tea samples from the above examples and comparative examples. Scores were assigned based on the tea's appearance, taste, aroma, liquor color, and infused leaf appearance, and the scores were weighted and calculated. The results are shown in Table 4.

[0157] Table 4

[0158] sample Appearance, aroma, color, and taste score Example 1 The fruit is covered with golden flecks, light yellow in color, plump, with a fresh and elegant mushroom aroma and a long-lasting sweet aftertaste. 94.00 Example 2 The mushrooms are covered with golden flecks, slightly dark yellow in color, with plump grains, a pure mushroom aroma, and a mellow flavor. 92.90 Example 3 The fruit is covered with golden flecks, yellow in color, with small grains, a pure mushroom aroma, and a mellow taste. 91.98 Example 4 The mushroom flowers are abundant, bright yellow in color, plump, and have a rich aroma; the soup is a bright orange-red. 94.33 Example 5 The mushrooms are covered with golden flecks, yellow in color, and the grains are still plump. They have a pure mushroom aroma and a mellow taste. 92.85 Example 6 The mushrooms are covered with golden flecks, yellow in color, and the grains are relatively plump. They have a pure mushroom aroma and a mellow taste. 91.85 Example 7 The flowers are full of golden flecks, yellow in color, plump, with a rich and mellow aroma and a smooth taste. 94.15 Example 8 The mushrooms are covered with golden flecks, slightly dark yellow in color, with plump grains, a pure mushroom aroma, and a mellow flavor. 92.90 Example 9 The mushrooms are covered with golden flecks, yellow in color, and the grains are relatively plump. They have a pure mushroom aroma and a mellow taste. 91.85 Comparative Example 1 It has abundant blooms, a pure and mellow flavor, and a floral aroma. 90.30 Comparative Example 2 The flowers are abundant, light yellow in color, and the grains are still plump. They have a pronounced mushroom aroma and a mellow, sweet aftertaste. 90.93 Comparative Example 3 The flowers are abundant, yellow in color, and the grains are relatively plump, with a pronounced mushroom aroma and a pure taste. 91.05 Comparative Example 4 The mushroom flowers are abundant, slightly dark yellow in color, with plump grains, a pure mushroom aroma, and a mellow flavor. 90.85 Comparative Example 5 The flowers are abundant, yellow in color, and the grains are relatively plump with a distinct mushroom aroma and a pure taste. 90.75 Comparative Example 6 The fruit is covered with golden flecks, its color is dark yellow, the grains are fairly plump, the aroma of mushrooms is slightly fishy, ​​and the taste is fairly mellow. 86.75

[0159] As shown in Table 4, the co-fermentation of *Aspergillus cristatus* (430682HH-13B), *Aspergillus chevalieri* (320282HH-22), and *Eurotium herbariorum* (533100HH-56) significantly improves the quality and quantity of "golden flower" fungi in Fu brick tea, resulting in a tea with abundant golden flowers and plump granules. Furthermore, by selecting the optimal ratio of fungal strains based on the different tea leaves, it imparts aroma characteristics such as a fresh fungal aroma, a rich floral aroma, and a mellow, ripe fragrance. In addition, co-fermentation enhances the fermentation process, improving the brightness of the tea liquor while also improving the mellowness and sweetness of the 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. This indicates that the proportion of limited viable counts of the three strains in Examples 1, 4, and 7 are most suitable for the preparation of Fu brick tea with green, floral, and cooked aromas, respectively.

[0160] Experiment 3

[0161] The content of key aroma active substances in the above embodiments and comparative Fu brick tea samples is 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 Aspergillus cristatus (430682HH-13B), Aspergillus chevalieri (320282HH-22), and Eurotium herbariorum (533100HH-56) are co-fermented, the optimal ratio of microorganisms, depending on the tea raw material, can impart aroma and quality characteristics to Fu brick tea, such as a fresh fungal aroma, a rich floral aroma, and a mellow, ripe aroma. This is consistent with the results of the detection of key aroma active substances. Compared with Comparative Example 1, the concentrations of (E,E)-2,4-heptadienal, linalool oxide, and α-terpineol in Example 1 were increased by 81.90%, 22.01%, and 48.52%, respectively; while in Example 4, the concentrations of (E,E)-2,4-heptadienal, linalool oxide, and α-terpineol were increased by 22.55%, 151%, and 82.37%, respectively.

[0166] Experiment 4

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

[0168] Table 6 shows the number of "Golden Flower Fungus" at the end of fermentation.

[0169] sample Golden flower fungus count (cfu / g) Example 1 <![CDATA[4.6×10 6 ]]> Comparative Example 5 <![CDATA[3.5×10 5 ]]>

[0170] Note: In Table 6, "Golden Flower Fungus" refers to the total number of the three fungi: Aspergillus cristatus, Aspergillus chevalieri, and Eurotium herbariorum.

[0171] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A processing method for a fungus-scented Fu brick tea, characterized in that, include: 1) Adjust the moisture content of the raw materials for fermentation to ≤25% with purified water; sterilize with steam, and then cool to room temperature; 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 5:3:2 to form a mixed culture; the total inoculum amount of the mixed culture is (5-6) × 10⁻⁶ based on the mass of the dried, unfermented Fu brick tea raw material. 6 CFU / g; The mixed bacteria are inoculated into the sterilized Fu brick tea raw material in step 1), and the following gradient fermentation is carried out: In the initial stage, fermentation from 0 to 120 hours: temperature 28℃, relative humidity 85%; Mid-stage fermentation, 121-288 hours: temperature 32℃, relative humidity 75%; Later stage of fermentation, 289-360 hours: temperature 25℃, relative humidity 65%; After fermentation, a two-stage drying process is adopted: first, drying at 80℃ for 15-20 minutes, and then drying at 55℃ for 2-3 hours; drying until the moisture content is ≤10% to obtain fungal-scented Fu brick tea; The preservation number of the *Aspergillus cristatus* is: CCTCCNO: M2025900; The accession number of the Aspergillus chevalieri is: CCTCCNO: M2025901; The accession number of the *Eurotium herbariorum* is: CCTCCNO: M2025902.

2. The processing method of the fungus-scented Fu brick tea according to claim 1, characterized in that, The raw material for the Fu brick tea to be fermented is first-grade black tea from Anhua, Hunan.

3. A processing method for a fungus-scented Fu brick tea, characterized in that, include: 1) Adjust the moisture content of the raw materials for fermentation to ≤25% with purified water; sterilize with steam, and then cool to room temperature; 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 6:2:2 to form a mixed culture; the total inoculum amount of the mixed culture is (5-6) × 10⁻⁶ based on the mass of the dried, unfermented Fu brick tea raw material. 6 CFU / g; The mixed bacteria are inoculated into the sterilized Fu brick tea raw material in step 1), and the following gradient fermentation is carried out: In the initial stage, fermentation from 0 to 120 hours: temperature 28℃, relative humidity 85%; Mid-stage fermentation, 121-288 hours: temperature 32℃, relative humidity 78%; Later stage of fermentation, 289-360 hours: temperature 25℃, relative humidity 65%; After fermentation, a two-stage drying process is adopted: first, drying at 80℃ for 15-20 minutes, and then drying at 55℃ for 2-3 hours; drying until the moisture content is ≤10% to obtain fungal-scented Fu brick tea; The preservation number of the *Aspergillus cristatus* is: CCTCCNO: M2025900; The accession number of the Aspergillus chevalieri is: CCTCCNO: M2025901; The accession number of the *Eurotium herbariorum* is: CCTCCNO: M2025902.

4. The processing method of the fungus-scented Fu brick tea according to claim 3, characterized in that, The raw material for the Fu brick tea to be fermented is third-grade Fu brick tea from Jingyang, Shaanxi.

5. A processing method for a fungus-scented Fu brick tea, characterized in that, include: 1) Adjust the moisture content of the raw materials for fermentation to ≤25% with purified water; sterilize with steam, and then cool to room temperature; 2) Mix *Aspergillus cristatus*, *Aspergillus chevalieri*, and *Eurotium herbariorum* at a viable count ratio of 5:2.5:2.5 to form a mixed culture; the total inoculum amount of the mixed culture is (5-6)×10⁻⁶ based on the mass of the dried, unfermented Fu brick tea raw material. 6 CFU / g; The mixed bacteria are inoculated into the sterilized Fu brick tea raw material in step 1), and the following gradient fermentation is carried out: In the initial stage, fermentation from 0 to 120 hours: temperature 28℃, relative humidity 85%; Mid-stage fermentation, 121-288 hours: temperature 33℃, relative humidity 70%; Later stage of fermentation, 289-360 hours: temperature 25℃, relative humidity 65%; After fermentation, a two-stage drying process is adopted: first, drying at 80℃ for 15-20 minutes, and then drying at 55℃ for 2-3 hours; drying until the moisture content is ≤10% to obtain fungal-scented Fu brick tea; The preservation number of the *Aspergillus cristatus* is: CCTCCNO: M2025900; The accession number of the Aspergillus chevalieri is: CCTCCNO: M2025901; The accession number of the *Eurotium herbariorum* is: CCTCCNO: M2025902.

6. The processing method of the fungus-scented Fu brick tea according to claim 5, characterized in that, The raw material for the Fu tea to be fermented is Yunnan Pu'er ripe tea.

7. A type of Fu brick tea with a floral aroma, characterized in that, It is prepared by the processing method described in any one of claims 1-6.

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