Leavening agent and application thereof in coffee preparation

Abstract

The invention relates to a leavening agent and application of the leavening agent in coffee preparation. The method comprises the following steps: creating a flora with functional properties, and compounding to obtain a leavening agent; the sensory quality is evaluated through an SCA cup test method, fermentation parameters (including an initial pH value, an inoculum size, temperature and time) are improved through a single factor experiment and a response surface method, and the adding proportion of a leavening agent and the proportion of each yeast strain are determined; the leavening agent is added into coffee for fermentation, so that coffee with better sensory quality is obtained.

Description

Technical Field

[0001] This application relates to the field of coffee processing technology, and in particular to a method for producing specialty coffee by fermentation using yeast preparations. Background Technology

[0002] Coffee is widely popular due to its complex and diverse sensory characteristics, and it has also received considerable attention in health research for its functional components, making it one of the world's three major beverages. The sensory quality of coffee is determined by chemical components such as caffeine and chlorogenic acid. Coffee also has biological functions such as antioxidation, anti-inflammation, and neuroprotection.

[0003] However, traditional natural fermentation relies on environmental microorganisms (such as wild yeast and bacteria), and its microbial community structure is random and unstable. 酒香酵母属 Overgrowth of wild yeasts such as *Brucea javanica* produces excessive volatile phenols (e.g., 4-ethylguaiacol), resulting in off-flavors like "stable" or "medicinal." Metabolism by other microorganisms (e.g., acetic acid bacteria, molds) produces short-chain acids such as acetic acid and propionic acid, leading to a sharp, pungent acidity and disrupting the acid-bitter balance. This results in significant fluctuations in coffee sensory quality, low aroma complexity, and poor acid-bitter balance (SCA score ≤ 70). The enzyme systems of these microorganisms (esterases / phenolic enzymes) degrade chlorogenic acid (loss rate > 27%) and deplete flavor amino acid precursors (decreased by 20%), leading to reduced antioxidant activity and insufficient Maillard reaction substrates. Furthermore, the lack of highly efficient demethylating strains during natural fermentation results in caffeine degradation relying on slow chemical hydrolysis, significant bitter residue (caffeine ≥ 12.98 mg / g; SCA bitterness score ≥ 6), and low ester synthesis enzyme activity in wild yeast, with ester content < 8%, failing to meet the market demand for decaffeinated coffee.

[0004] Therefore, in order to solve the above-mentioned defects, there is an urgent need for a new fermentation agent for coffee fermentation that can improve the sensory quality of coffee and optimize its functional properties and flavor profile. Summary of the Invention

[0005] To address or partially address the problems existing in the related technologies, this application provides a fermentation agent and its application in coffee preparation. Using this fermentation agent to ferment coffee can significantly improve the quality of coffee and greatly increase the content of volatile substances such as chlorogenic acid and amino acids.

[0006] This application provides a fermentation agent, which is composed of brewing yeast (Saccharomyces cerevisiae). 酿酒酵母属 酿酒酵母 ), Brett's yeast ( 布鲁塞尔酒香酵母 Bayer conjugated yeast ( 拜耳接合酵母 ), Candida astrosa ( 星状假丝酵母 ), fermented Pichia pastoris ( 毕赤酵母属 发酵毕赤酵母 ), fission yeast (粟酒裂殖酵母 ) and Kluyveromyces clarithii ( 克勒克酵母属 尖端克勒克酵母 The mixture is obtained by compounding; its mass ratio is 1:2:1:2:1:2:1 or 1:1:1:1:1:1:1.

[0007] Furthermore, the preservation number of the brewing yeast is: NR_111984.1; The accession number of the *Brethia brevicornu* is: NR_125501.1; The accession number of the Bayer conjugating yeast is: NR_442885.1; The accession number of the *Candida astrosa* is: NR_111808.1; The preservation number of the fermented Pichia pastoris is: NR_119085.1; The accession number of the fission yeast is: NR_044978.1; The accession number of the Kluyveromyces clarithii is: NR_119622.1.

[0008] On the other hand, this application also provides a method for preparing coffee, the method comprising the following steps: 1) Add the above-mentioned starter culture agent to the processed fresh coffee cherries for fermentation; after fermentation, fermented green coffee beans are obtained; The fermentation conditions were as follows: initial pH of 7, fermentation agent addition of 24.9%, temperature of 35.2℃, and time of 86.5h. 2) The fermented green beans in 1) are medium-roasted to make roasted coffee beans, which are then ground and brewed to obtain coffee.

[0009] Furthermore, the mass ratio in 1) is 1:2:1:2:1:2:1.

[0010] Furthermore, the processing method of the fresh coffee cherries in 2) is to remove their outer skin to obtain the processed fresh coffee cherries.

[0011] Furthermore, the temperature for medium baking in step 2) is 200°C.

[0012] On the other hand, this application also provides the application of the above-described fermenting agent in the preparation of coffee.

[0013] Beneficial effects 1. This application provides a fermentation agent, which is made from brewer's yeast ( 酿酒酵母属 酿酒酵母 ), Brett's yeast ( 布鲁塞尔酒香酵母 Bayer conjugated yeast ( 拜耳接合酵母 ), Candida astrosa ( 星状假丝酵母 Pichia pastoris fermentation ( 毕赤酵母属发酵毕赤酵母 ), fission yeast ( 粟酒裂殖酵母 ) and Kluyveromyces clarithii ( 克勒克酵母属 尖端克勒克酵母 The mixture was prepared by blending the seven yeast strains at a mass ratio of 1:2:1:2:1:2:1. This was based on the functional verification of isolated kombucha (Table 1) and the blend optimization experiment (Tables 2 / 7), which showed that the seven yeast strains had clearly defined roles. (1) Saccharomyces / Wickerhamomyces is a "binary ester synthesis" (accounting for 28.6%), driving the generation of fruit aroma substances such as ethyl acetate; (2) Zygosaccharomyces / Candida (28.6%) ensures the supply of flavor precursors and a rich taste; (3) Schizosaccharomyces / Kloeckera (28.6%) constructs a closed loop of "chlorogenic acid protection-flavor release"; (4) Pichia (14.3%) targets and degrades caffeine; (5) Brettanomyces (14.3%): The proportion is strictly controlled to balance complexity and odor risk.

[0014] Synergistic advantages: The 1:2:1:2:1:2:1 ratio was locked through response surface methodology (Table 3) and achieved at pH 7.0 / 35.2℃. (1) SCA score 86.5 (18.5% higher than natural fermentation); (2) Protects chlorogenic acid and enriches amino acids, with chlorogenic acid retention rate >92% and amino acid enrichment amount of 27.36 mg / g; (3) The inhibition rate of volatile phenols is >60%, and the diversity of esters is increased by 125%.

[0015] (4) Targeted degradation of caffeine (-18.3%); (5) The proportion of Brycetes yeast was reduced to 14.3% to eliminate phenolic odor.

[0016] 2. This application also provides a method for preparing coffee. By using the fermenting agent formulated in this application combined with optimized process conditions (optimized fermentation conditions are an initial pH of 7.0, an inoculum size of 24.9%, a temperature of 35.2°C, and a duration of 86.5 hours), the sensory quality of the coffee is significantly improved after fermentation. Volatile substance analysis shows that... 酿酒酵母属 The metabolic activity of the genus increases the amount of alcohols and esters (ethyl acetate, isoamyl alcohol), thereby enhancing the complexity of the aroma. 酒香酵母属As the proportion of genus decreases, the amount of volatile phenolic substances (4-ethylguaiacol) produced also decreases, thereby improving the harmony between flavors. Analysis of non-volatile substances shows that the content of chlorogenic acid and amino acids increases significantly, further optimizing the functional properties and flavor profile of coffee.

[0017] 3. This application utilizes a novel starter culture combined with an improved fermentation process to ferment coffee. Under the optimized fermentation conditions described in this application, the starter culture can achieve the following effects: (1) Neutral pH (7.0) is the optimal pH for β-glucosidase ( 尖端克勒克酵母 ) and protease ( 拜耳接合酵母 The optimal operating conditions for enzyme activity (>80%) are as follows: when pH < 6.0 (Table 4): 克勒克酵母属 Inactivation of β-glucosidase → 35% decrease in free phenol release ( Figure 6 (NFC group flavonoids 7.49 mg / L vs YC group 13.68 mg / L) 接合酵母属 Protease inhibition → amino acid synthesis decreased by 42% (NFC group 18.54 mg / g). pH 7.0 ensures efficient conversion of enzymatically hydrolyzed substrates (bound phenols / coffee proteins).

[0018] (2) Temperature 35.2℃ regulates the ecological niche of the microbial community. 酿酒酵母属 The optimal growth temperature is 32-36℃ (accounting for 35% of biomass), which dominates the initial fermentation stage; 裂殖酵母属 (Heat-resistant bacteria) initiate secondary metabolism at 35℃ (D2-D4 abundance increases by 15%). Figure 8 ), protecting chlorogenic acid. When the temperature is too high, it leads to 酿酒酵母属 Accelerated decay → insufficient ethanol substrate → ester synthesis decreased by 53% (response surface model F-value -1.77C) 2 ); 酒香酵母属 (Heat resistance) Outburst → Volatile phenols ↑120% ( Figure 7 NFC group phenol peak).

[0019] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0020] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0021] Figure 1 This is a flowchart of the overall scheme of this application.

[0022] Figure 2 This is a diagram showing the colony morphology of yeast isolated from kombucha in a culture medium.

[0023] Figure 3 It is an SCA cupping test.

[0024] Figure 4 The effect of fermentation conditions on SCA score; Note: A: Effect of starter culture addition on SCA score; B: Effect of initial pH on SCA score; C: Effect of fermentation temperature on SCA score; D: Effect of fermentation time on SCA score.

[0025] Figure 5 This involves cupping scores and sensory characteristic analysis of fermented samples; A: Cupping scores of different fermented sample groups; B: Sensory characteristic analysis of different fermented sample groups. Note: YC: Yeast-fermented coffee, NFC: Unfermented coffee, NC: Naturally fermented coffee.

[0026] Figure 6 This is a comparison of the physicochemical indicators between the NFC group and the YC group; Note: The difference is significant (P < 0.05). This indicates a highly significant difference (P < 0.01). P < 0.001 indicates extremely significant difference, ns indicates no significant difference (P ≥ 0.05), YC: yeast-fermented coffee, NFC: unfermented coffee.

[0027] Figure 7 This is a comparative analysis of the physicochemical indicators of the NC group and the YC group; Note: The difference is significant (P < 0.05). This indicates a highly significant difference (P < 0.01). ns indicates extremely significant difference (P < 0.001), ns indicates no significant difference (P ≥ 0.05), YC: yeast fermented coffee, NC: naturally fermented coffee.

[0028] Figure 8 It refers to the changes in the composition of the fungal community before and after fermentation.

[0029] Figure 9 This is the interaction between fermentation conditions and SCA score.

[0030] Figure 10 It is a classification of the main metabolites in green coffee beans.

[0031] Figure 11 A: Principal component analysis of yeast-fermented green coffee beans and unfermented green coffee beans; B: Differential metabolite analysis of yeast-fermented green coffee beans and unfermented green coffee beans; C: Correlation analysis of metabolites among yeast-fermented green coffee beans.

[0032] Figure 12 These are the difference analysis diagrams of flavor components; A: Z-score distribution diagram of the difference in flavor components between yeast-fermented green beans and unfermented green beans; B: Correlation heatmap between flavor components of yeast-fermented green beans and unfermented green beans.

[0033] Biological Preservation Instructions The NCBI accession numbers for the following yeasts are: NR_111984.1; Brett's yeast: NR_125501.1; Bayer conjugate yeast: NR_442885.1; Candida astrologica: NR_111808.1; Pichia pastoris: NR_119085.1; Schizosoma: NR_044978.1; and Kluyveromyces clarkii: NR_119622.1. Detailed Implementation

[0034] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0035] Kombucha, also known as black tea, is a non-ethanol or low-ethanol beverage made by fermenting sugared tea water through a symbiotic culture of bacteria and yeast. Yeast, as a crucial component of the symbiotic flora in the kombucha fermentation system, metabolizes glycolysis to produce ethanol, carbon dioxide, and various secondary metabolites. By isolating and blending kombucha yeast, the fermentation process of Yunnan Arabica coffee has been improved. Coffee fermented using this process exhibits significantly enhanced sensory qualities, showing marked improvements in aroma complexity, acidity brightness, body, and flavor profile in the SCA cupping system.

[0036] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0037] Example 1 1) A dominant yeast strain was isolated from kombucha; Kombucha was prepared using tea samples collected from Jingmai Mountain, Huimin Township, Lancang Lahu Autonomous County, Pu'er City, Yunnan Province, and processed at the Pu'er Tea Deep Processing Center of Yunnan Agricultural University. The tea leaves (10g per liter of water) were steeped in boiling water for 5 minutes, filtered, and the sugar was dissolved while still hot. After naturally cooling to room temperature (25℃), 10% by volume of SCOBY (Symbiotic Culture of Bacteria and Yeast) and kombucha stock solution were added. The container was covered with gauze and placed in a dark, well-ventilated area at a controlled temperature of 25℃ for 10 days of fermentation.

[0038] Kombucha samples were collected and homogenized in sterile physiological saline (0.85% NaCl) using a homogenizer. The homogenized samples were serially diluted 10-fold. 100 µL of the diluted solution was evenly spread on solid YPD medium using a sterile glass spreader and incubated at 28°C for 72 hours to allow for sufficient colony growth.

[0039] 2) Morphological identification; On a sterile operating table, single colonies of different morphologies, colors, and sizes were selected and transferred to new YPD agar plates for further purification until colonies of a single morphology were obtained. Preliminary identification was performed by morphological observation, followed by extraction of total DNA from the strains. Two rounds of PCR amplification were conducted using universal primers, and sequencing was performed. The amplified products were analyzed for fragment size by 2% agarose gel electrophoresis. PCR products meeting the requirements were sent to Sangon Biotech (Shanghai) Co., Ltd. for high-throughput sequencing. Sequence alignment analysis was performed using bioinformatics tools such as BLAST, and the results were analyzed in conjunction with the NCBI database to determine the species composition and functional properties of the bacterial community.

[0040] Yeast strains cultured on five different culture media exhibited typical colony morphological characteristics, with milky white, turbid, or smooth and moist surfaces, neat and evenly distributed edges, showing good colony isolation, clear colony morphology, and relatively uniform size. The isolated and purified yeast strains were identified as mainly divided into seven species, namely... 酿酒酵母属 酿酒酵母 , 布鲁塞尔酒香酵母 , 拜耳接合酵母 , 假丝酵母属 星状 , 发酵毕赤酵母 , 粟酒裂殖酵母 , 尖端克勒克酵母 .

[0041] All the strains mentioned above have been published on NCBI, and the brewer's yeast ( 酿酒酵母属 酿酒酵母 The accession number of the described *Brethia brevicornu* is NR_111984.1; 酒香酵母属 布鲁塞尔The accession number of the Bayer conjugating yeast is: NR_125501.1; 接合酵母属 拜耳 The accession number for the *Candida astrosa* is NR_442885.1; the *Candida astrosa* ( 星状假丝酵母 The accession number of the fermented Pichia pastoris is: NR_111808.1; 发酵毕赤酵母 The accession number of the described fission yeast is NR_119085.1; 粟酒裂殖酵母 The accession number of the described Kluyveromyces clarithii is NR_044978.1; 尖端克勒克酵母 The accession number for this is NR_119622.1.

[0042] Table 1 Results of yeast strain screening As can be seen from the data in Table 2, 酿酒酵母 Saccharomyces cerevisiae exhibits a significant advantage in breaking down sugars and generating alcohols, and is the main metabolic driver in the fermentation process; 酒香酵母属 布鲁塞尔 Brett's yeast and 拜耳接合酵母 Bayer conjugated yeast plays an important role in the formation of complex alcohol and acid aromas in yeast ferments; 星状假丝酵母 Candida astrosa and 发酵毕赤酵母 Fermenting Pichia pastoris helps to improve the flavor profile of the fermentation system, enhancing its sweetness and richness. 粟酒裂殖酵母 fission yeast and 尖端克勒克酵母 Kluyveromyces kurlii participates in the transformation of chemical components, adding richness to the fermentation flavor. Different fermentation agent blend ratios have a significant impact on sensory scores and flavor characteristics. The HC-b group achieved the best results in both SCA cupping and sensory scores, exhibiting rich fruit, wine, and caramel aromas. Therefore, the HC-b group was used in subsequent process optimization analysis.

[0043] Table 2 Effects of different proportions of compound fermentation agents Note: Different letters indicate significant differences. P <0.05) Example 2 A method for making coffee, the method comprising: 1) Selection of raw materials: premium grade fresh coffee cherries, purchased from Xinzai Coffee Estate in Xinzai Village, Lujiang Town, Longyang District, Baoshan City, Yunnan Province. The variety is Arabica-Catim, at an altitude of 1650m. The fermentation agent obtained by compounding the strains screened in Example 1 is added to the coffee for fermentation to obtain coffee with better sensory quality and functional quality. 2) Optimization of fermentation conditions: Sensory quality was assessed using the SCA cupping method, and fermentation parameters (including initial pH, inoculum size, temperature, and time) were improved using single-factor experiments and response surface methodology to determine the proportion of starter culture and the proportion of each yeast strain. Single-factor experiments were designed with four different factors: inoculum dosage, initial fermentation pH, fermentation temperature, and fermentation time. The median values ​​for the remaining factors were used. Fresh specialty coffee cherries were skinned and then fermented with an inoculum mixture of various microbial strains. After fermentation, fermented green beans were obtained and medium-roasted at 200℃ to produce roasted coffee beans. The beans were ground and evaluated using the SCA cupping score of the brewed coffee to determine the optimal single-factor levels. Based on the single-factor experiments, the effects of inoculum dosage (A), initial fermentation pH (B), fermentation temperature (C), and fermentation time (D) on the SCA cupping score were investigated using the overall SCA cupping score (Y) as the response value. A four-factor, three-level Box-Behnken experimental design was employed to determine the optimal factor levels for response surface optimization. Using Design-Expert 13, a multiple regression analysis was performed to obtain the regression model equation for the sensory response score (Y): Y = 80.05 + 0.0833A + 2.52B - 0.3542C + 3.00D + 0.3125AB + 0.2500AC + 0.1875AD + 0.2500BC - 1.25BD - 0.3125CD - 0.4312A 2 +0.3500B 2 -1.77C 2 +1.94D 2 The experimental data were analyzed using ANOVA, and the results are shown in Table 3. The model was highly significant (P < 0.01), and the lack-of-fit term was not significant (P > 0.05), indicating a good fit between the experimental results and the regression model. Coefficient of determination R0 2 The adjusted R-value is 0.9314. 2 The p-value is 0.8629, indicating a high model fit. The p-value shows that the linear terms B and D, and the quadratic term C... 2 D 2 The interaction term BD significantly affected the results (P < 0.01), indicating that the factors interacted with each other in the fermentation of coffee by kombucha yeast. Comparing the F-values, the order of influence of each factor on sensory scores was: D (fermentation time) > B (fermenting agent dosage) > C (yeast temperature) > A (initial fermentation pH). The optimal fermentation conditions for yeast-fermented coffee, determined by a quadratic multiple regression model, were: initial fermentation pH 7.000, fermenting agent dosage 24.899%, fermentation temperature 35.177℃, and fermentation time 86.517 h.

[0044] Table 3. Analysis of variance of the response surface regression model Note: Indicates a significant difference ( P <0.05); This indicates that the difference is highly significant ( P <0.01); Amount of starter culture added (A), initial pH of fermentation (B), fermentation temperature (C), fermentation time (D) Figure 9 The results showed that the interaction between initial fermentation pH and temperature significantly affected the score, reaching its maximum when the pH was 6.0-7.0 and the temperature was around 35℃. The interaction between inoculum size and fermentation time indicated that the optimal flavor profile was achieved with an inoculum size of 20%-25% and a fermentation time of 72-90 hours. The synergistic effect of temperature and time showed that longer fermentation times within the medium-high temperature range significantly improved the score, but excessively high or low temperatures inhibited fermentation. A quadratic multiple regression model yielded the optimal fermentation conditions for kombucha yeast-fermented coffee as follows: initial fermentation pH 7.000, starter culture addition of 24.899%, fermentation temperature 35.177℃, and fermentation time 86.517 hours. Considering practical operation, the conditions were modified to an initial fermentation pH of 7, a starter culture addition of 24.9%, a fermentation temperature of 35.2℃, and a fermentation time of 86.5 hours. Under these conditions, three verification experiments were conducted, and the resulting score was 86.5±0.057, which was in good agreement with the model's prediction. This indicates that the model's analysis and prediction of the fermentation process conditions of Kombucha yeast in coffee are accurate and reliable.

[0045] Considering the actual operation process, the process conditions were modified to: initial fermentation pH 7, starter culture addition of 24.9% of the coffee cherry weight, fermentation temperature 35.2℃, and fermentation time 86.5 h. The proportions of different starter cultures significantly affected sensory scores and flavor characteristics. Group B achieved the best results in both SCA cupping and sensory evaluations, exhibiting rich fruity, wine-like, and caramel aromas. The final determined mass ratio of the starter culture was 1:2:1:2:1:2:1:1:

[0046] Table 4 Single-factor experimental design Note: The percentages of added amounts in the table are all based on the weight fraction of fresh coffee cherries. Table 5 Response Surface Factor Level Design Note: All percentages added in the table are mass fractions. Table 6 Sensory Evaluation Criteria for Fermented Coffee Table 7 Effects of different proportions of compound fermentation agents Note: Different letters in the same column indicate significant differences (P < 0.05). 3) Preparing coffee: The prepared starter culture (Breton Brønsted yeast: Bayer zygosacchari: Candida astrosa: Pichia pastoris: Schizosomal yeast: Kluyveromyces kurine) was prepared in a mass ratio of 1:2:1:2:1:2:1:1. Coffee was fermented using modified fermentation parameters (initial pH 7, 24.9% starter culture, 35.2℃, 86.5h fermentation time) to produce coffee (YC group). Its SCA cupping score, physicochemical properties, volatile components, and flavor components were measured and compared with unfermented coffee (NFC group) and coffee produced through natural fermentation (relying on environmental microorganisms (wild yeast, lactic acid bacteria, acetic acid bacteria, and other microorganisms) attached to the fresh fruit skin (NC group). Figure 5 As shown in Figure A, the scores of the fermented group (YC group) were significantly higher than those of the unfermented group (NFC group) and the naturally fermented group (NC group), reaching a maximum of over 80%, indicating that the yeast-fermented coffee group had a significant advantage in overall flavor performance. Figure 5 The radar chart in section B further evaluated the sensory characteristics of each sample. The fermented group (YC group) performed exceptionally well in aroma, aftertaste, and body, scoring close to 8 points or higher. Its balance of sweetness and acidity was also superior to the other two groups, with lower bitterness, demonstrating a balanced flavor profile. In contrast, the unfermented group (NFC group) and the naturally fermented group (NC group) exhibited relatively bland sensory characteristics, with lower scores in aroma and aftertaste, and higher bitterness, resulting in an overall flavor profile significantly lower than the fermented group (YC group). The pH value of the fermented group decreased to 4.56, and the total acid content increased to 26.43 meq / L, indicating a significantly enhanced acidification effect during fermentation. The fermentation group (YC group) showed increased flavonoid, polyphenol, and chlorogenic acid content to 13.68 mg / L, 7.38 mg / L, and 7.38 mg / L, respectively, exhibiting strong antioxidant properties. The amino acid content increased significantly to 27.36 mg / g, indicating that its fermentation products contained more flavor compounds and nutrients. The caffeine content decreased significantly to 10.62 mg / g, which is related to the metabolic transformation of caffeine during fermentation. This demonstrates that the fermentation group (YC group) fermented with yeast starter exhibits a richer flavor and higher nutritional value overall.

[0047] Table 8. Results of Physicochemical Index Analysis Note: Different letters in the same column indicate significant differences (P < 0.05); YC: yeast-fermented coffee, NFC: unfermented coffee, NC: naturally fermented coffee. Figure 6 and Figure 7 The results show that yeast-fermented roasted beans (YC group) are significantly superior to unfermented roasted beans (NFC group) and naturally fermented roasted beans (NC group) in most physicochemical indicators. The pH value of the YC group decreased to 4.56, and the total acid content increased significantly to 26.43 meq / L. This indicates a significantly enhanced acidification effect during fermentation. Yeast produces a large amount of organic acids through glycolysis and other pathways, leading to a sharp drop in the system pH. This not only inhibits the growth of unwanted microorganisms and ensures the safety of fermentation, but more importantly, it is the direct material basis for shaping the unique acidic flavor of coffee (such as bright fruity acidity) and creates favorable conditions for the subsequent transformation of flavor compounds. The contents of flavonoids, polyphenols, and chlorogenic acid in the YC group increased to 13.68 mg / L, 7.38 mg / L, and 7.38 mg / L, respectively, demonstrating strong antioxidant properties. During natural fermentation, the enzyme system is disordered, and phenolic substances are easily degraded or oxidized by other microorganisms. However, β-glucosidase secreted by yeast hydrolyzes bound phenols, releasing free flavonoids and polyphenols, improving bioavailability, and promoting the release of large phenolic molecules or the conversion of specific chlorogenic acid isomers. The amino acid content increased significantly to 27.36 mg / g. This high amino acid level provides ample precursors for the formation of key aroma compounds such as pyrazines (nutty aroma) and furans (caramel aroma) during roasting, directly explaining the prominent aroma and enrichment of flavor amino acids in the YC group cupping, supporting the harmonious sweet and sour sensory characteristics of the YC group. The caffeine content decreased significantly to 10.62 mg / g, catering to the demand of some consumers for decaffeinated coffee, and may also have altered the interaction between caffeine and other flavor compounds, positively impacting the overall smoothness and balance of the flavor. There were no significant differences in TDS among the three groups, indicating that the changes in these components and the specific relationships between groups were relatively small, but the YC group showed a more balanced overall performance.

[0048] This result is consistent with emerging research in existing technologies regarding the regulation of coffee quality by microbial fermentation. Compared to unfermented (NFC group) and naturally fermented (NC group), YC beans showed a 20.3% reduction in caffeine content, highlighting the efficiency of yeast metabolism in the degradation of methylxanthine (Wang Y, et al. Anaerobic germination of green coffee beans. Curr Res Food Sci. 2023.). The increased free amino acid content (27.36 mg / g) provided a rich substrate for the Maillard reaction, explaining the enhanced nutty / caramelized flavor in YC, consistent with the role of amino acid-derived styraldehyde in roasted coffee (Ariefandie NF, Fan Z. Coffee bean processing: Emerging methods. Food Chem. 2023.). The increase in free phenolic compounds (flavonoids increased by 26.5% compared to the NC group) may be due to the hydrolysis of bound forms by yeast β-glucosidase activity, thereby improving bioavailability and antioxidant capacity (Carillo P, et al. Enhancing sustainability by improving plant salt tolerance through algal biostimulants. Biology. 2020.). The dominant role of Saccharomyces cerevisiae in driving ester synthesis (such as ethyl acetate) and acidification (final pH 4.56) confirms its role in enhancing fruity / floral aroma complexity, a point previously mentioned in kombucha fermentation (Villarreal-Soto, SA, et al. Understanding kombucha tea fermentation: A review. J Food Sci. 2018.). 酒香酵母属 The decrease in abundance may reduce undesirable volatile phenols and improve flavor balance; this phenomenon has also been observed in controlled wine fermentation (Cascone G, et al. Formulation of chestnut flour-enriched snacks. Foods. 2024.). It is worth noting that... 酿酒酵母属 and 威克汉姆酵母属The synergistic effects between them enhance ester diversity (such as ethyl isovalerate), which echoes reports of their co-metabolism in fermented beverages (Suffys S, et al. Characterization of aroma active compound production during kombuchafermentation. Foods. 2023. Zhao Y, et al. Evaluation of non-Saccharomycesyeasts for aroma compositions. J Fungi. 2022.).

[0049] Although Buitrago (Buitrago DFL, et al. Modulating coffee fermentation quality using microbial inoculus. Sustainability. 2025.) and others have reported similar improvements in SCA scores achieved using coffee by-product inoculants, the kombucha complex microbial community of this application offers unique advantages: (1) systematic reshaping of flavor chemistry through multi-strain enzyme activity (such as β-glucosidase); (2) microbial succession ( Saccharomyces and Wickerhamomyces (2) Dynamically optimize metabolite profile; (3) This process also enhances health-related compounds (chlorogenic acid, flavonoids). This fermentation strategy provides a sustainable solution for enhancing sensory appeal and health components by targeting and reshaping the chemical composition of coffee. It clarifies the key role of microbial community structure and its metabolic function in the regulation of coffee flavor quality. Further research needs to promote industrial scale-up verification and analyze the specific contribution of single strains to flavor formation.

[0050] Figures 10 - 11 The pie chart of metabolite classification shows that the main metabolites in green coffee beans are heterocyclic compounds (52%), primarily including alkaloids such as caffeine and trigonelline, as well as pyrazine precursors, which constitute the chemical basis for coffee bitterness and roasted flavor. Following these are esters (12%), ketones (7%), and alcohols (5%), etc. Figure 10 ). Figure 11 The PCA scatter plot of A shows that the metabolic profiles of the YR (yeast-fermented raw beans) group and the NFR (unfermented raw beans) group are generally similar, indicating that yeast fermentation did not significantly alter primary metabolism, and core metabolic pathways (such as the TCA cycle and alkaloid synthesis) remained in homeostasis, consistent with the microecological disturbance characteristics of fermentation. However, Figure 11The volcano plot of B shows that there are significantly different metabolites between the YR group and the NFR group. Among them, several metabolites are significantly upregulated or downregulated in the YR group. Metabolites with large VIP values ​​(such as phenethyl acetate: yeast β-glucosidase hydrolyzes bound aromatic aglycones to release free esters, and caffeine: yeast organic acid environment promotes the demethylation of methylxanthine) make important contributions to flavor changes. Figure 11 The network diagram in section C illustrates the correlations between various metabolites, revealing that yeast fermentation exerts a complex regulatory effect on the metabolic network of green coffee beans. The co-expression of caffeine and trigonelline is weakened, reducing the synergistic effect of bitterness. Therefore, yeast fermentation does not completely overturn the chemical composition of green beans, but rather enhances flavor potential through targeted regulation of key nodes (such as β-glucosidase activation and methyltransferase inhibition).

[0051] Figure 12 This demonstrates the differential regulation of flavor components in green coffee beans by yeast fermentation, and the Z-score distribution was observed. Figure 12 A) It can be seen that there are significant differences in the abundance distribution of flavor metabolites between the YR (yeast-fermented green beans) group and the NFR (unfermented green beans) group. In particular, the Z-scores of phenolic compounds and pyrazine compounds in the YR group are significantly higher than those in the NFR group, indicating the expansion of the Maillard reaction precursor library. These two types of substances generate guaiacol (smoky aroma) and alkylpyrazine (nutty aroma) respectively during roasting, which are directly related to the "aroma complexity" of the YC roasted beans cupping test. Meanwhile, alcohols and esters showed higher negative Z-scores in the NFR group, reflecting the yeast's ability to remove off-flavors: converting n-hexanol (grassy aroma) into ethyl hexanoate (fruity aroma), thus achieving flavor purification. Figure 12 B further illustrates the differences in the correlation patterns between flavor metabolites in the two groups. The significant positive correlation between phenolic and alcohol metabolites in the YR group may be related to the conversion of phenylalanine to phenylethanol (rose flavor) by aromatic amino acid decarboxylases during yeast metabolism, simultaneously releasing phenolic glycosides to generate free phenols, thus forming metabolic coupling. In contrast, the weaker correlation between ester and alcohol metabolites in the NFR group reflects the lower activity of the metabolic network under unfermented conditions. This phenomenon may be due to the direct regulation of key enzyme activity during yeast fermentation, such as the catalytic action of esterases and aromatic compound invertases, leading to dynamic changes in the synthesis and degradation efficiency of flavor substances in the metabolic pathway. Yeast fermentation may also indirectly influence the coordination of metabolic channels by altering the pH and redox conditions of the surrounding environment, thus further affecting the distribution of flavor metabolites and their interactions.

[0052] The composition of the microbial community changed significantly between day 0 and day 4 during the fermentation process using yeast preparations. Figure 8 This dynamic change is clearly presented. SaccharomycesThis genus maintained its dominant position throughout the fermentation process, with its relative abundance peaking at D4. Saccharomyces The metabolic activity of this genus increases the amount of alcohols and esters (ethyl acetate, isoamyl alcohol), thereby enhancing the complexity of the aroma. Brettanomyces The percentage of genus species showed a significant downward trend. Meanwhile... Wickerhamomyces Minor bacterial communities begin to show some niche occupancy at stage D4. The dynamic evolution of the bacterial community structure is closely related to the formation of flavor compounds. Saccharomyces As a dominant microbial species, it can generate key flavor substances such as alcohols and esters by metabolizing carbon sources, which plays a decisive role in enhancing the flavor of fermentation products. Brettanomyces A decrease in genus abundance may reduce the formation of volatile phenolic compounds, thus improving flavor harmony. Wickerhamomyces The increase in the number of genera is due to the enzymatic reaction promoting the accumulation of esters, which further enhances the aroma complexity of the fermentation products.

[0053] This study improved the fermentation process of Yunnan Arabica coffee by isolating and compounding kombucha yeast, and explored the relationship between microbial dynamics and flavor compound production. The optimal parameters established by response surface methodology (pH 7.0, inoculum size 24.9%, 35.2℃, 86.5 h) resulted in an SCA score of 86.5, an improvement of 18.5% compared to natural fermentation. The fermented beans exhibited harmonious acidity, high amino acid content (27.36 mg / g), and antioxidant flavonoids (13.68 mg / L), directly correlated with increased aroma complexity and reduced bitterness. Caffeine content decreased by 20.3%, meeting the market demand for low-decaffeinated specialty coffee. Volatile component analysis revealed yeast-specific upregulation of pyrazines and phenolic precursors (such as alkylpyrazines and guaiacol), establishing the nutty and smoky aroma base of the roasted beans. Simultaneously, volatile phenols such as n-hexanol (grassy flavor) and 4-ethylguaiacol were suppressed, optimizing flavor balance. Saccharomyces Dominant acidification (pH 4.56) and ester synthesis, Wickerhamomyces Increase ester diversity by enzymatically converting primary metabolites. Brettanomyces The reduced abundance significantly lowers the risk of off-flavors. Coffee fermented with kombucha yeast showed a significant improvement in sensory quality, with marked enhancements in aroma complexity, acidity brightness, body, and flavor profile in the SCA cupping system.

[0054] These findings are consistent with emerging research on the role of microbial fermentation in regulating coffee quality. Compared to unfermented (NFC) and naturally fermented (NC) beans, YC beans showed a 20.3% reduction in caffeine content, highlighting the efficiency of yeast metabolism in the degradation of methylxanthine. The increased free amino acid content (27.36 mg / g) provided a rich substrate for the Maillard reaction, explaining the enhanced nutty / caramelized flavors in YC beans, consistent with the role of amino acid-derived stigmasteraldehyde in roasted coffee (Ariefandie NF, Fan Z. Coffee bean processing: Emerging methods. FoodChem. 2023.). The increase in free phenolic compounds (flavonoids increased by 26.5% compared to the NC group) may be due to the hydrolysis of bound forms by yeast β-glucosidase activity, thereby improving bioavailability and antioxidant capacity (Carillo P, et al. Enhancing sustainability by improving plant salt tolerance through algalbiostimulants. Biology. 2020.). The dominant role of Saccharomyces cerevisiae in driving ester synthesis (such as ethyl acetate) and acidification (final pH 4.56) confirms its role in enhancing fruity / floral complexity, a point also mentioned in kombucha fermentation (Villarreal-Soto, SA, et al. Understanding kombucha teafermentation: A review. J Food Sci. 2018.). Brettanomyces The decrease in abundance may reduce undesirable volatile phenols and improve flavor balance; this phenomenon has also been observed in controlled wine fermentation (Cascone G, et al. Formulation of chestnut flour-enriched snacks. Foods. 2024). It is worth noting that... Saccharomyces and Wickerhamomyces The synergistic effect between them enhances ester diversity (such as ethyl isovalerate), which echoes reports of their co-metabolism in fermented beverages.

[0055] Although Buitrago (Buitrago DFL, et al. Modulating coffee fermentation quality using microbial inoculus. Sustainability. 2025.) and others have reported similar improvements in SCA scores achieved using coffee by-product inoculants, the kombucha complex microbial community of this application offers unique advantages: (1) systematic reshaping of flavor chemistry through multi-strain enzyme activity (such as β-glucosidase); (2) microbial succession ( Saccharomyces and Wickerhamomyces (2) Dynamically optimize metabolite profile; (3) This process also enhances health-related compounds (chlorogenic acid, flavonoids). This fermentation strategy provides a sustainable solution for enhancing sensory appeal and health components by targeting and reshaping the chemical composition of coffee. It clarifies the key role of microbial community structure and its metabolic function in the regulation of coffee flavor quality. Further research needs to promote industrial scale-up verification and analyze the specific contribution of single strains to flavor formation.

[0056] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A leavening agent, characterized in that, The fermenting agent is compounded by Saccharomyces cerevisiae Saccharomyces cerevisiae , Brettanomyces bruxellensis Brettanomyces bruxellensis , Zygonema bayeri Zygosaccharomyces bailii , Candida stellata Candida stellata , Pichia fermentans Pichia fermentans , Schizosaccharomyces Schizosaccharomyces pombe and Kluyveromyces wickerhamii Kloeckera apiculata . 1:2:1:2:1:2:1 or 1:1:1:1:1:1:

1.

2. The fermenting agent according to claim 1, characterized in that, The preservation number of the Saccharomyces cerevisiae is NR_111984.1; The preservation number of the Brettanomyces bruxellensis is NR_125501.1; The preservation number of the Zygosaccharomyces bailii is NR_442885.1; The preservation number of the Candida stellata is NR_111808.1; The preservation number of the Pichia fermentans is NR_119085.1; The preservation number of the Schizosaccharomyces pombe is NR_044978.1; The preservation number of the Kloeckera apiculata is NR_119622.

1.

3. A method of preparing coffee, characterized in that, The preparation method comprises the following steps: 1) adding the fermenting agent of any one of claims 1-2 into the processed coffee fresh fruit for fermentation; obtaining fermented green coffee beans after the fermentation is completed; The fermentation condition is that the initial pH of the fermentation is 7, the adding amount of the fermenting agent is 24.9%, the temperature is 35.2℃, and the time is 86.5h. 2) roasting the fermented green coffee beans in the 1) to medium roasting to obtain coffee beans, and then grinding and brewing to obtain coffee.

4. The production method according to claim 3, characterized by, The mass ratio in the 1) is 1:2:1:2:1:2:

1.

5. The preparation method according to claim 3, characterized in that, The processing method of the coffee fresh fruit in the 2) is to remove the skin to obtain the processed coffee fresh fruit.

6. The preparation method according to claim 3, characterized in that, The temperature of the medium roasting in the 2) is 200℃.

7. Use of the fermenting agent of any one of claims 1-2 in the preparation of coffee.

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