A method for preparing flavored coffee by mixed culture fermentation combined with enfleurage and coffee beans

Abstract

The application discloses a method for preparing flavored coffee by mixed fermentation and scenting and coffee beans, belongs to the technical field of coffee bean scenting process technology, and selects natural fresh flowers, changes the coffee bean matrix gap through mixed fermentation of Saccharomyces cerevisiae and Bacillus pasteurii, and combines low-temperature scenting, and naturally integrates fresh flower aroma, realizes top aroma compensation and aroma clarity construction through mass transfer adsorption of volatile flavoring substances in the scenting stage, reduces the influence of top aroma attenuation after baking on the flavor consistency of finished products, so that stable expression of characteristic flavor and more suitable industrialization replication are realized.

Description

Technical Field

[0001] This invention relates to the field of coffee bean preparation technology, and in particular to a method and coffee beans for preparing flavored coffee by mixed fermentation combined with scenting, especially a method and coffee beans for preparing coffee beans by mixed fermentation of Saccharomyces cerevisiae and Acetobacter pasteurianus combined with low-temperature scenting. Background Technology

[0002] Controlled fermentation of commercial coffee beans with exogenous microorganisms and flavor compounds is an emerging processing method. By selecting suitable fermenting agents and optimizing the treatment of the native microorganisms present in coffee, the flavor-related components in green coffee beans that were previously fermented can be re-fermented and transformed into targeted modifications and improvements. Ultimately, this diversifies the coffee flavor, enhances the original flavor of the commercial beans, and expands the coffee consumer market. Patent CN115868566A discloses a method for directional flavor fermentation of coffee and its preparation. This invention uses gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) to determine the volatile components of roasted coffee beans, obtain sensory-related chemical components, select materials containing the aforementioned chemical components as flavor substrates, and co-ferment green coffee beans with the flavor substrates. Using green coffee beans as raw materials, coffee beverages with specific flavors (such as rose, osmanthus, jasmine, and tangerine peel flavors) are produced. However, this preparation process still has shortcomings in terms of engineering implementation and product consistency. Its flavor source largely depends on the natural composition and batch stability of exogenous flavor substrates. Substrates such as dried flowers and fruit peels are significantly affected by the place of origin, harvest period, drying and storage conditions. Fluctuations in the content of target aroma and its precursors can easily lead to deviations in the flavor intensity and profile of the finished product. At the same time, exogenous substrates enter the fermentation system in the form of powder / solids, which introduces additional non-bean-derived solid variables. This may produce unpredictable side reactions and off-flavor risks during fermentation and subsequent roasting, and increase the uncertainty of flavor substance retention in post-processing. In addition, characteristic aromas are generally volatile and heat-sensitive. Relying solely on "co-fermentation for aroma" in the green bean stage still carries the risk of top aroma attenuation and aroma clarity reduction after roasting, degassing and storage. This results in the "directional flavor" being greatly affected by the roasting curve and shelf life in the final cupping, which is not conducive to the formation of a low-cost and replicable process control system at the production end. Therefore, a special process is needed to overcome the above influences.

[0003] The scenting process is a mature technique for adding exogenous flavors, now commonly used to blend tea flavors with floral aromas. It utilizes the principle of tea leaves absorbing fragrance and fresh flowers releasing fragrance, a traditional technique for making scented tea by mixing tea leaves and fresh flowers, letting them stand, separating and drying them. Essentially, it treats fresh flowers as a small bioreactor that continuously releases volatiles and moisture. Under the conditions of openness at night, respiration releasing heat and humid heat migration, these aroma molecules diffuse and adsorb into the tea leaves and are "fixed" in fragrance. For example, Chinese patent CN202511703718.1 discloses a tea tree flower tea and its production method. This method includes a scenting process, where dried tea tree flowers with a moisture content of 35%–40% are scented. The scenting method involves mixing the tea tree flowers and dried flowers in a specific ratio, placing them in a breathable bag, transferring them to a scenting chamber, letting them stand for 24–30 hours, drying them, repeating the scenting process 2–3 times, followed by freeze-drying, and then warming them to room temperature to obtain the tea tree flower tea. This method produces a compound tea tree flower tea with low caffeine content, rich aroma layers, and complete nutrient retention. Furthermore, Chinese patent CN115024371A discloses a natural floral aroma coffee bean scenting process. This process uses natural fresh flowers (including jasmine, double-petaled red rose, pomelo blossom, bitter orange blossom, osmanthus, locust blossom, wintersweet, willow-leaf wintersweet, ginger flower, or magnolia) to scent coffee beans, naturally infusing them with floral aromas. This allows the coffee flavor and floral aromas to blend seamlessly, enhancing the coffee's taste and flavor. The scented coffee has increased natural floral sweetness, solves the problem of chemical additives, and adds dried tangerine peel for a more refreshing taste. It also incorporates traditional Chinese medicine for liver and stomach health. However, in the above preparation process, during the re-drying stage of coffee bean preparation, the newly absorbed highly volatile floral aromas may be prematurely evaporated, accelerating the aging of the coffee's own aroma and causing oxidation and flavor dulling. Summary of the Invention

[0004] To address the aforementioned technical deficiencies, this application provides a method for preparing flavored coffee using mixed-culture fermentation combined with scenting, as well as coffee beans. The specific technical solution is as follows:

[0005] The first objective of this application is to screen for dominant strains for mixed fermentation, including Saccharomyces cerevisiae and Acetobacter pasteurianus.

[0006] The brewer's yeast Saccharomyces cerevisiae, with the number F3, was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39125. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The Acetobacter pasteurianus strain 06YS was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37404, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] A second objective of this invention is to provide a method for preparing flavored coffee through mixed-culture fermentation combined with scenting, the method comprising the following steps:

[0009] S1, Coffee Bean Selection: Select undamaged Catim coffee green beans that have been washed and have a moisture content of 10-12%. Remove any damaged or worm-eaten beans.

[0010] S2, Pasteurization: The screened green coffee beans are mixed with pure water at a 1:1 mass ratio and then sterilized at 75°C for 30 minutes to initially change the pore structure of the green coffee beans.

[0011] S3, Inoculation and Fermentation: A compound microbial agent is introduced into the sterilized coffee beans. The compound microbial agent includes Saccharomyces cerevisiae F3 and Acetobacter pasteurellium 06YS as described above. The ratio of F3 to 06YS in the compound microbial agent is (1-3):(3-1).

[0012] S4, mixed fermentation: After inoculation, stir evenly and ferment at 28℃ for 24 hours;

[0013] S5, Initial Drying: The fermented green coffee beans are dried in a 45℃ drying oven for 24 hours to dry the surface of the green coffee beans and maintain the moisture content of the green beans at 8-10%.

[0014] S6, Fresh Flower Scenting: Select fresh flowers picked on the same day and still in bud. Layer the fresh flowers and the coffee beans after the first drying, and scent them at a low temperature for 30-60 hours. Turn the pile over to dissipate heat according to the temperature changes.

[0015] S7, Secondary Drying: After scenting, remove residual flowers and dry the green coffee beans at 45 ℃ for 12 h to restore the moisture content to 11%;

[0016] S8, Product Roasting: Standardized roasting of processed green coffee beans to create a harmonious coffee flavor profile while preserving floral aromas.

[0017] Furthermore, the mixed culture ratio of *Saccharomyces cerevisiae* and *Acetobacter pasteurellium* is F3:06YS=1:1, and the bacterial concentration is 1.0×10⁻⁶. 8 CFU / mL, inoculation amount is 1%.

[0018] Furthermore, in step S6, the mass ratio of fresh flowers to coffee beans after the first drying is 1:2.

[0019] Furthermore, the low-temperature curing temperature in S6 is 15°C.

[0020] Furthermore, the molding time in S6 is 48 hours.

[0021] Furthermore, the flowers include any one of jasmine, dark red rose, or osmanthus.

[0022] A third objective of this invention is to provide a coffee bean prepared by a method for producing flavored coffee using a combination of mixed microbial fermentation and scenting.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) During the fermentation stage, the complementary metabolism of multiple microbial strains is used to enhance the release and transformation of endogenous precursors in coffee;

[0025] (2) By sterilization and microbial metabolism, the matrix structure of coffee beans is changed on the basis of green coffee beans, thereby improving the controllability of the base aroma and mouthfeel structure and increasing the dependence on the aroma carried by exogenous substrates.

[0026] (3) During the scenting stage, the top aroma compensation and aroma clarity are achieved through the mass transfer and adsorption of volatile flavor substances, avoiding the off-flavors and retention uncertainty caused by the direct entry of solids into the system, and reducing the impact of the top aroma decay after baking on the flavor consistency of the finished product, thereby achieving stable expression of characteristic flavors and more suitable industrial replication. Attached Figure Description

[0027] Figure 1 This is a flowchart of a method for preparing flavored coffee using mixed microbial fermentation combined with scenting, as described in this invention.

[0028] Figure 2 This is the growth curve of yeast;

[0029] Figure 3 This is the growth curve of lactic acid bacteria;

[0030] Figure 4 This is a plate diagram of an interstrain antagonism experiment.

[0031] Figure 5 This is a preliminary sensory evaluation report; Figure 6 The effects of different mixed bacterial ratios and inoculum amounts on the cupping scores of coffee samples were investigated, where: a) the effect of different yeast to acetic acid bacteria ratios on the cupping scores of coffee samples; and b) the effect of different mixed bacterial inoculum amounts on the cupping scores of coffee.

[0032] Figure 7This is a standard curve graph of caffeine content in roasted coffee beans; the equation of the standard curve is: y = 25764x + 34577, R 2 =0.9992; where y represents the content of the substance, calculated in μg / mL, x represents the peak area of ​​the sample solution, and R 2 This represents the coefficient of determination for linear regression;

[0033] Figure 8 Electron micrograph of the surface morphology of coffee bean outer skin;

[0034] Figure 9 To measure the cross-sectional structure of the endosperm inside a coffee bean;

[0035] Figure 10 The effects of high temperatures on jasmine flowers;

[0036] Figure 11 Images of samples after 24 hours of curing at 15℃ and 35℃.

[0037] Figure 12 The total score and sensory score of floral fragrance were calculated for samples treated at different scenting temperatures. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0039] Saccharomyces cerevisiae, strain F3, was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39125, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0040] Acetobacter pasteurianus, strain 06YS, was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37404, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] Example 1: Screening of strains

[0042] 1. Screening of yeast strains

[0043] A type of brewing yeast (Saccharomyces cerevisiae) F3 was deposited on January 16, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39125 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Screening of brewer's yeast strains:

[0045] a. Selection of coffee pectin fermentation broth samples: Select mature fresh Catimor coffee pectin from Baoshan City, Yunnan Province, China. After collecting the samples into sterile sample bags, quickly transfer them to a low-temperature box for preservation and transport to the laboratory at low temperature.

[0046] b. Separation of Catimor coffee fresh fruit pectin sample: Weigh an appropriate amount of Catimor coffee fresh fruit pectin fermentation sample, place it in 90 mL of sterile physiological saline, and mix by shaking to disperse the microbial cells in the Catimor coffee fresh fruit pectin fermentation sample.

[0047] c. Gradual dilution of Catimor coffee fresh fruit pectin fermentation sample: The Catimor coffee fresh fruit pectin fermentation mixture was serially diluted with sterile physiological saline;

[0048] d. Isolation and culture: 200 μL of the diluted solution was inoculated into coffee bean substrate medium and cultured at 30℃ for 24 h. 100 μL of the diluted solution was used to isolate the strain through a high-throughput micro-level droplet culture omics system and cultured at 30℃ for 24 h. The strain with the best growth was screened at an OD value of 600 nm. The isolated droplets were inoculated into yeast extract peptone glucose medium (YPD) and cultured at 32℃.

[0049] e. Observe colony morphology: Select dominant strains by observing the morphology, size, color, luster, texture, surface and edge characteristics of the colonies;

[0050] f. Isolation, purification, and single colony acquisition: The strain was further purified by streak plating to obtain single colonies;

[0051] g. Cryopreservation of dominant strains (isolated strains): Isolates with consistent morphology were cryopreserved for subsequent identification.

[0052] 2. Screening of acetic acid bacteria

[0053] Acetobacter pasteurianus 06YS was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37404, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0054] a. Selection of coffee pectin fermentation broth samples: Select mature fresh Catimor coffee pectin from Baoshan City, Yunnan Province, China. After collecting the samples into sterile sample bags, quickly transfer them to a low-temperature box for preservation and transport to the laboratory at low temperature.

[0055] b. Separation of Catimor coffee fresh fruit pectin sample: Weigh an appropriate amount of Catimor coffee fresh fruit pectin fermentation sample, place it in 90 mL of sterile physiological saline, and mix by shaking to disperse the microbial cells in the Catimor coffee fresh fruit pectin fermentation sample.

[0056] c. Gradual dilution of Catimor coffee fresh fruit pectin fermentation sample: The Catimor coffee fresh fruit pectin fermentation mixture was serially diluted with sterile physiological saline;

[0057] d. Spreading and culture: 200 μL of the diluted solution was inoculated into coffee bean substrate medium and cultured at 30℃ for 24 h. 100 μL of the diluted solution was used to isolate the bacterial strain through a high-throughput micro-level droplet culture omics system and cultured at 30℃ for 24 h. The strain with the best growth was screened at an OD value of 600 nm. The isolated droplets were inoculated onto 9-bacterial MRS agar plates and cultured at 36 ℃.

[0058] e. Observe colony morphology: Select dominant strains by observing the morphology, size, color, luster, texture, surface and edge characteristics of the colonies;

[0059] f. Isolation, purification, and single colony acquisition: The strain was further purified by streak plating to obtain single colonies;

[0060] g. Cryopreservation of dominant strains (isolated strains): Isolates with consistent morphology were cryopreserved for subsequent identification.

[0061] 3. Experiment on the fermentation performance of the strain

[0062] Following the above screening process, a total of 15 non-replicative microbial strains with good growth status were obtained, which can be used for fermentation products. Of these, 8 strains were identified as yeasts and 7 as lactic acid bacteria.

[0063] The concentration of the activated bacterial culture was adjusted to 1.0 × 10⁻⁶ using a microplate reader spectrophotometer. 8 CFU / mL was inoculated into a liquid culture medium with green coffee beans as the substrate at a 2% inoculum. The mixture was then incubated at a constant temperature of 28 ℃ for 48 h. The absorbance of the fermentation broth was measured at a wavelength of 600 nm every 4 h to plot the growth curve of the superior yeast strain. Figure 2 ) and lactic acid bacteria growth curve ( Figure 3 ),Depend on Figure 2 It can be seen that the selected yeast strains exhibited a significantly accelerated growth rate within 4-8 hours, entered a stationary phase around 20-24 hours, and maintained an absorbance of approximately 1.2. This indicates that the yeast strains are well-adapted to the coffee bean culture medium and can quickly reach the late logarithmic growth phase required for fermentation, making them suitable for industrial production. Among them, F3 (orange), E1 (orange-yellow), and H12 (rose-red) showed particularly good adaptability.

[0064] Depend on Figure 3 It can be seen that the selected lactic acid bacteria initially grew slowly and steadily, but the growth rate accelerated over time, entering the logarithmic growth phase around 24 hours, but did not enter the stationary phase within 48 hours. This indicates that the lactic acid bacteria have generally poor adaptability to coffee bean culture medium and require a relatively long time to reach the late logarithmic growth phase required for fermentation. However, 06YS (red), 44YS (green), and 61YS (brown) showed better adaptability.

[0065] 4. Strain antagonism experiment

[0066] Based on the adaptability of the strains in the coffee green bean culture medium, dominant strains were selected for antagonistic experiments. Therefore, yeast strains E1, F3, and H12 (Kluyveromyces lactis, Saccharomyces cerevisiae, and Saccharomyces cerevisiae) and dominant strains 06YS, 44YS, and 61YS (Acetobacter pastoris, Acetobacter pastodes, and Lactococcus lactis) were selected for the antagonistic experiments. The results are as follows: Figure 4 As shown, the colony edges of the test strains are interconnected, and the surrounding area of ​​the test strains grows normally without the appearance of transparent inhibition zones. The mycelial growth at the interface between the two colonies is uniform, with no obvious sparse or absent areas. The colony tips of the two strains can extend freely and come into contact, and may even overlap, without any hyphal dissolution or growth stagnation. This indicates that all the above strains can undergo mixed fermentation and have the basis for co-growth.

[0067] 5. Sensory experiment of cupping

[0068] Based on the above screening, the selected dominant strains E1, F3, H12 and strains 06YS, 44YS, and 61YS were arranged at a bacterial concentration of 1x10⁻⁶. 8 CFU / ml, mixed with bacteria at a 1:1 ratio, and 1% bacterial solution (actually added at 1ml / 100g green coffee beans) was inoculated into sterilized green coffee beans. Fermentation began at 28℃ and continued for 24 hours. After fermentation, the beans were then processed into the scenting stage. After scenting, the green coffee beans were roasted, and a preliminary sensory evaluation was conducted according to Table 1. The evaluation was scored according to the evaluation table in Table 1, and the preliminary sensory evaluation results are as follows: Figure 5 As shown, the F3-06YS mixed-culture fermentation group achieved significantly higher sensory evaluations in all five evaluation criteria compared to the other mixed-culture fermentation groups. Furthermore, the F3-43YS and F3-44YS fermentation groups also achieved high sensory evaluations in all five evaluation criteria. Therefore, strains F3 and 06YS were subsequently selected as fermentation strains to ferment fresh coffee beans, followed by scenting to prepare coffee beans with floral aromas.

[0069] Table 1. Preliminary sensory evaluation of coffee after fermentation and scenting with different fermenting agents.

[0070]

[0071] Example 2: A compound microbial agent for coffee fermentation

[0072] The compound microbial agent is prepared by mixing a suspension of Saccharomyces cerevisiae and Acetobacter pasteurellium in a 1:1 ratio, with a suspension concentration of 1.0 × 10⁻⁶. 8 The CFU / mL can be further processed into a freeze-dried microbial agent for the fermentation of green coffee beans, with an inoculation amount of 1% for the compound microbial agent.

[0073] The Saccharomyces cerevisiae strain mentioned above is the F3 strain of this invention, which was deposited on January 15, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39125. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0074] The *Acetobacter pasteurianus* strain described herein is the 06YS strain, which was deposited on January 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37404, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0075] (1) Determination of the bacterial solution ratio in compound bacterial agents

[0076] To further determine the effect of mixed-strain fermentation on coffee flavor, the experiment explored the effects of mixed-strain fermentation ratios (Saccharomyces cerevisiae: Acetobacter pasteurization) of 2:1, 3:2, 1:1, 1:2, and 2:3 on coffee cupping scores, with a bean-to-water ratio of 1:1, an inoculum size of 1%, a fermentation temperature of 28℃, and a fermentation time of 24h.

[0077] According to the scoring results ( Figure 6a) Different ratios of yeast to acetic acid bacteria significantly affected the cupping performance of coffee samples. When the ratio of *Saccharomyces cerevisiae* to *Acetobacter pastoris* was 1:1, the sample achieved the highest sensory score of 82 points. The sensory description revealed aromas of fragrant wood, gardenia, lemon, and honeysuckle, with a clear overall aroma that laid a good foundation for subsequent flavor integration. Simultaneously, the acidity was relatively mild, without any obvious pungent sourness or abnormal fermentation odor, indicating that the two strains could form a suitable synergistic metabolic relationship at this ratio. When the proportion of yeast was relatively high, the alcohols and some volatile flavor compounds produced by yeast metabolism could impart certain floral and fruity aromas to the sample. However, an insufficient proportion of acetic acid bacteria might limit the further conversion of alcohols into organic acids and esters, affecting the overall aroma complexity and flavor harmony of the sample. As the proportion of acetic acid bacteria increases, the accumulation of acidic substances in the system accelerates. When the ratio of *Saccharomyces cerevisiae* to *Acetobacter pasteurization* was 2:3, the average sensory score in cupping was the lowest, at 80.08 points. Negative flavors such as excessive acidity and an unpleasant fermented taste appeared in the sensory descriptions, indicating that a high proportion of acetic acid bacteria may lead to excessive accumulation of acidic metabolites, making the coffee acidity sharp and masking the original floral aroma, sweetness, and body. The reason for this phenomenon may be that yeast mainly utilizes fermentable sugars in coffee beans to produce ethanol, higher alcohols, and some aroma precursors during fermentation, while acetic acid bacteria can further utilize ethanol in the system to generate organic acids and promote the formation of some ester flavor compounds. When the ratio of the two strains is 1:1, alcohol production, organic acid accumulation, and esterification reactions may be in a relatively balanced state, which is conducive to the formation of a more harmonious floral and fruity aroma and a mellow acidity. When the proportion of acetic acid bacteria is too high, it easily leads to excessive accumulation of organic acids, producing excessive acidity and an unclean fermented aroma, reducing the overall sensory quality of the coffee. Therefore, the present invention ultimately selected 1:1 as the appropriate mixed culture ratio for mixed culture fermentation.

[0078] (2) The effect of mixed inoculum quantity on coffee cupping score

[0079] To further determine the effect of mixed culture fermentation on coffee flavor, the experiment investigated the effects of mixed culture inoculum amounts of 0.5%, 1%, 1.5%, 2%, and 2.5% on coffee cupping scores, with a bean-to-water ratio of 1:1, a mixed culture ratio of 1:1, a fermentation temperature of 28℃, and a fermentation time of 24 hours.

[0080] According to the scoring results ( Figure 6(b) The optimal cupping sensory score of 82 points was achieved with a mixed inoculum concentration of 1%, described as having aromas of wood, gardenia, lemon, and honeysuckle. Conversely, the lowest average cupping sensory score of 80.75 points was achieved with a concentration of 2.5%, characterized by excessive fermentation, woodiness, and low body. This discrepancy is presumably due to the lower inoculum concentration resulting in fewer viable microorganisms in the initial fermentation stage. This requires a longer time for the microorganisms to adapt and multiply, potentially leading to insufficient conversion of available substrates within the coffee beans and limited release of flavor precursors, thus affecting the formation of floral, fruity, and fermented aromas. As the inoculum concentration increases, the metabolic rate of microorganisms in the mixed inoculum system accelerates, promoting the conversion of sugars, organic acids, and some macromolecules in the coffee beans and increasing the release of related flavor precursors. However, when the inoculum concentration was further increased to 2.5%, the higher inoculum concentration caused a rapid increase in the number of microorganisms in the fermentation system, accelerating the consumption of nutrients such as fermentable sugars and promoting the rapid accumulation of organic acids, alcohols, and other metabolites. Under the condition that the fermentation temperature and time remained constant, an excessively high inoculum concentration may cause the fermentation degree to exceed the suitable range required for coffee flavor formation, leading to the accumulation of acids and some undesirable fermentation products. Simultaneously, it masks the original aroma, sweetness, and floral notes of coffee, resulting in negative flavors such as over-fermentation and woody notes. In summary, an excessively low inoculum concentration may cause insufficient metabolism by the strains, while an excessively high inoculum concentration can easily lead to an excessively rapid fermentation rate and excessive accumulation of metabolites, which is detrimental to the harmonious expression of the overall coffee flavor. Among these, a mixed inoculum concentration of 1% resulted in samples that performed better in terms of floral aroma, acidity, fermentation degree, and overall cupping quality. Therefore, this invention ultimately selected 1% as the suitable inoculum concentration for mixed fermentation.

[0081] Example 3: Coffee processed using different methods

[0082] (I) To explore the effects of different processing methods on coffee bean quality, this experiment used fresh coffee cherries as raw material and set up a two-factor cross-experiment involving fermentation method and scenting process. The specific experiment is as follows:

[0083] Step 1, uniform pre-processing of coffee bean raw materials: Select fresh coffee cherries with consistent ripeness and uniform size, degumm them uniformly, rinse them with clean water, divide them into 8 equal portions, drain the surface moisture and set aside to ensure that there are no differences between the initial raw materials.

[0084] Step 2, Group fermentation treatment

[0085] Unfermented group (groups 1 and 2): Coffee beans were not inoculated with microorganisms and were soaked in room temperature water for the same duration as a fermentation blank control;

[0086] Acetic acid bacteria fermentation group (groups 3 and 4): Prepare standardized acetic acid bacteria fermentation liquid, spray it evenly onto coffee beans, and let it ferment at a constant temperature of 28℃ for 48 hours.

[0087] Yeast fermentation groups (groups 5 and 6): Prepare yeast fermentation broth and ferment at a constant temperature for 48 h under the same conditions;

[0088] Mixed fermentation groups (groups 7 and 8): Acetic acid bacteria and yeast were mixed in a fixed ratio to prepare a compound bacterial solution, and fermented at the same temperature and humidity for 48 hours.

[0089] After all groups have finished fermenting, wash off any residual bacterial liquid on the surface with clean water and dry with low-temperature hot air until the moisture content is uniform.

[0090] Step 3, Scented / Unscented Group Processing: Divide each type of fermented coffee beans into two equal portions for scenting and unscenting respectively. The unscented group is directly processed into the roasting process after drying; the scented group has the fermented and dried coffee beans placed in sealed ceramic jars and scented at a low temperature of 15℃ for 30-60 hours. The pile is turned over to dissipate heat according to the temperature change. The pile is turned over once every time the temperature is detected to be higher than 25℃, so as to keep the scenting temperature below 25℃.

[0091] Step 4, uniform roasting treatment (secondary drying): After scenting, remove residual flowers and dry the green coffee beans at 45℃ for 12 hours to restore the moisture content to 11%. After roasting, cool to room temperature, seal and store away from light, and test the caffeine content. The standard curve of caffeine content in roasted coffee beans is shown below. Figure 7 Based on the standard curve, the caffeine content in coffee beans is shown in Table 2 below:

[0092] Table 2. Caffeine content in roasted coffee beans under different processing methods

[0093] 1 Unfermented + Unscented 7.20±0.34 2 Unfermented + scenting 7.32±0.64 3 Acetic acid bacteria fermentation + no aging 6.53±0.68 4 Acetic acid bacteria fermentation + scenting 6.61±0.33 5 Yeast fermentation + no aging 6.48±0.11 6 Yeast fermentation + scenting 6.57±0.49 7 Mixed-culture fermentation + airless fermentation 5.81±0.78 8 Mixed-culture fermentation + scenting 6.02±0.42

[0094] Table 2 shows that the caffeine content in roasted coffee beans changed significantly after different fermentation and scenting treatments. The caffeine content in the unfermented + unscented group was 7.20 mg / g, while the caffeine content in the unfermented + scented group was 7.32 mg / g, indicating that the caffeine content increased slightly after scenting alone. The caffeine content in roasted coffee beans treated with different fermentation methods varied. Under unscented conditions, the caffeine content in the acetic acid bacteria fermentation group and the yeast fermentation group decreased from 7.20 mg / g to 6.53 mg / g and 6.48 mg / g, respectively, representing decreases of approximately 9.31% and 10.00%. The mixed fermentation group had the lowest caffeine content, decreasing from 7.20 mg / g to 5.81 mg / g, a decrease of approximately 19.31%. Under the scenting conditions, the caffeine content of the acetic acid bacteria fermentation + scenting, yeast fermentation + scenting, and mixed fermentation + scenting treatment groups were 6.61, 6.57, and 6.02 mg / g, respectively, all lower than the unfermented + scenting treatment group. The mixed fermentation + scenting group showed a reduction of approximately 17.76% compared to the unfermented + scenting group. This demonstrates that fermentation treatment can reduce the caffeine content of roasted coffee beans, with mixed fermentation showing a more significant reduction effect.

[0095] The trends in caffeine content across different processing methods showed that the scented samples all had slightly higher caffeine content than their unscented counterparts. Specifically, the unscented group increased from 7.20 mg / g to 7.32 mg / g, the acetic acid bacteria fermented group from 6.53 mg / g to 6.61 mg / g, the yeast fermented group from 6.48 mg / g to 6.57 mg / g, and the mixed-culture fermented group from 5.81 mg / g to 6.02 mg / g. This phenomenon may be related to moisture migration, moisture absorption by the coffee bean tissue, and changes in pore structure during the scenting process, making some of the caffeine in an embedded or bound state easier to extract and detect. On the other hand, fermentation shows the opposite trend, which is presumably because microorganisms secrete related enzyme systems during growth and metabolism, and utilize and transform the internal substrates of coffee beans, causing some caffeine to migrate, degrade or be further transformed. Compared with single-strain fermentation, there may be complementary effects between yeast and acetic acid bacteria in mixed fermentation system in terms of substrate utilization and metabolite transformation, thereby accelerating the release, decomposition and transformation of caffeine and its related complexes.

[0096] Caffeine is a major contributor to the bitterness of coffee; therefore, reducing caffeine content can, to some extent, weaken the bitterness in the coffee liquid, making the taste of the sample more mellow. In the context of this patented process, pasteurization and fermentation alter the pore structure and internal matrix of green coffee beans, providing a foundation for the diffusion, adsorption, and retention of volatile aroma compounds during the subsequent scenting process. The scenting stage, in turn, replenishes the top aroma through the mass transfer and adsorption of floral aroma compounds, enhancing the clarity of the floral fragrance. The mixed-culture fermentation + scenting treatment group, while maintaining a lower caffeine content, facilitates the formation of a mellower bitterness, a clearer floral aroma, and a more harmonious overall flavor, indicating a synergistic effect between mixed-culture fermentation and floral scenting. By combining mixed-culture fermentation with scenting, the floral aroma can be better integrated into the coffee beans.

[0097] Table 3. Cupping scores for scented and unscented coffee beans.

[0098]

[0099] An analysis of cupping scores for roasted beans using scenting and fermentation processes (Table 3) shows that coffee treated with scenting had significantly higher scores than those without. Specifically, the cupping score for untreated coffee without scenting or fermentation was 79.5; for coffee fermented with acetic acid bacteria without scenting, the score was 79.75; for coffee fermented with yeast without scenting, the score was 81; and for coffee fermented with mixed cultures without scenting, the score was 82.00. However, the addition of scenting processes increased the cupping scores for all the above-mentioned samples. The cupping score for untreated coffee with scenting was 82.5; the cupping score for coffee fermented with acetic acid bacteria was 82.5; the cupping score for coffee fermented with yeast was 83.75; and the cupping score for coffee fermented with mixed cultures after scenting increased to 85.00. Furthermore, changes in the fermentation process significantly affect coffee cupping scores. Based on the data analysis above, single-strain fermentation results in lower cupping scores than mixed-strain fermentation samples. Acetic acid bacteria fermentation alone leads to lower scores, presumably because its growth and metabolism result in a large accumulation of acetic acid flavor compounds and flavor precursors, potentially leading to an unpleasant sourness and lower cupping scores. In contrast, mixed-strain fermentation produces esters from the acids generated by acetic acid bacteria and alcohols from yeast fermentation. These esters further decompose during roasting, enriching the overall flavor of the coffee and reducing the overly sour taste often associated with acetic acid bacteria fermentation alone. Co-fermentation with yeast enhances the body and sweetness. Therefore, single lactic acid fermentation results in lower cupping scores, while co-fermentation improves the overall cupping score. Regardless of whether green coffee beans are fermented or not, the cupping scores of coffee beans combined with the scenting process show an upward trend. The fermentation stage utilizes the complementary metabolism of multiple microorganisms to enhance the release and transformation of endogenous precursors in coffee, improving the controllability of the base aroma and mouthfeel structure. The scenting stage achieves top aroma compensation and aroma clarity through the mass transfer and adsorption of volatile flavor substances. The interaction between the two results in a cupping score that is high or even close to that of specialty coffee.

[0100] Example 4: The effect of different processing techniques on the surface structure and subsequent cross-sectional structure of coffee beans.

[0101] Electron microscopy analysis of coffee beans was performed. Sample preparation: The required green bean samples were pre-frozen at -4℃ for 24 hours, and then freeze-dried at -40℃ for 7 days in a vacuum freeze dryer. Rapid freezing was used to lock in the interstitial water flow in the samples.

[0102] Sample photography: Take the processed sample, whole raw beans / slices are pasted in a dry vacuum environment, directly pasted on conductive adhesive, sprayed with gold and then tested. The testing equipment is a German ZEISS-GeminiSEM 500.

[0103] Changes in the surface structure of coffee beans, such as Figure 8As shown, untreated green coffee beans have a dense, continuous wavy fiber texture, an intact epidermis without large pores, and tightly stacked fiber layers, forming a complete barrier. However, after pasteurization and mixed fermentation, the surface of green coffee beans is enzymatically decomposed by microorganisms, resulting in numerous large, round pores. The integrity of the epidermis is broken, and a large amount of debris is produced on the surface. This indicates that the heat treatment process can damage the cell walls and some tissue structures of coffee beans, loosening the originally relatively compact internal matrix. After further mixed fermentation, the number of pores on the surface of coffee beans increases, and phenomena such as lamellae, pore enlargement, and crack extension appear in local areas. This suggests that yeast and acetic acid bacteria may further weaken the cell walls and intercellular binding structures of coffee beans through acid production, enzyme production, and substrate transformation during their growth and metabolism. This results in a more loose and porous microstructure. This structural change can increase the contact area between coffee beans and external volatile aroma substances, shorten the diffusion path of aroma molecules into the interior, and provide more binding sites for the adsorption, retention, and subsequent flavor fusion of volatile compounds such as jasmine and rose during the scenting process. Therefore, the electron microscopy results provide microscopic structural evidence that sterilization and mixed fermentation can open the pores of coffee beans, improve the matrix structure of coffee beans, significantly increase the permeability of the external barrier, and facilitate the entry of floral aroma substances into the interior of coffee beans during the subsequent scenting stage, thereby enhancing the expression of floral aroma and the overall flavor harmony.

[0104] Four treatment gradients (step-by-step superposition of processing steps) were applied to green coffee beans: ① Green beans (blank control group); ② Sterilized green beans; ③ Sterilized + fermented green beans; ④ Sterilized + fermented + cellar-aged green beans. The endosperm cross-sections of the four groups of coffee beans were observed to reflect the degree of damage to cell wall polysaccharides and fiber structure caused by processing. The results are as follows: Figure 9 As shown:

[0105] (1) The internal matrix of green beans (unsterilized, unfermented native coffee beans) is generally dense and flat, with only a few tiny shallow pores; the cell outline is complete and clear, the cell wall is thick and continuous, and there is almost no large-area lysis or porous structure.

[0106] (2) Sterilized raw beans are damaged by high temperature during sterilization, resulting in a large number of long cracks and cavities. The cell boundaries partially collapse, but large intact cell wall lamellae are still retained. The pores are large in size and few in number, resulting in only physical heat damage and no microbial degradation.

[0107] (3) The fermentation process continues. The microorganisms in the sterilized and fermented raw beans significantly degrade the cell wall polysaccharides, resulting in a large number of honeycomb-like fine porous structures. The cell walls are dissolved, broken, and thinned over a large area, forming an interconnected network of pores. In some areas, the cytoskeleton is completely disintegrated, with fine and dense pores. The fermenting microorganisms secrete extracellular enzymes (cellulase, pectinase) to decompose cell wall pectin and cellulose, generating a large number of pores. This structure is conducive to the dissolution and degradation of caffeine and flavor substances.

[0108] (4) Sterilization + fermentation + cellaring of raw beans. After cellaring, the honeycomb porous structure is further reshaped, the small pores are fused and the pore distribution is more uniform. The broken cell wall matrix undergoes a certain degree of adhesion and reconstruction. The porous network structure is retained, but the pore shape is more fragmented and loose. The temperature and humidity environment of cellaring continuously and slowly decomposes the residual polysaccharides. At the same time, soluble substances migrate and redistribute, and the looseness of the microstructure is further improved.

[0109] In summary, scanning electron microscopy (SEM) observation of the coffee bean endosperm cross-section reveals that the internal structure of untreated raw coffee beans is dense and intact. Sterilization alone only produces a few long, narrow fissures, and the overall cell wall structure remains largely intact. After microbial fermentation, cell wall polysaccharides are enzymatically hydrolyzed, the endosperm forms a dense, honeycomb-like porous network, and the cytoskeleton is significantly ruptured. Adding a fermentation process further enhances the looseness of the matrix, resulting in a finer and more uniform pore distribution. These microscopic structural results demonstrate that fermentation effectively disrupts the dense barrier of coffee beans, providing ample contact space for microbial degradation of caffeine.

[0110] Example 5: Exploratory Study on Curing Temperature

[0111] Unlike the scenting process for tea, where the temperature is typically controlled between 35 and 37°C, the condition of the flowers significantly impacts the final aroma quality during the scenting of green coffee beans and fresh jasmine. Higher temperatures can easily cause the jasmine flowers to develop a rotten aroma, resulting in a yellowing and discoloration. (The actual product may appear...) Figure 10 .

[0112] To determine the appropriate scenting temperature in this process, while maintaining consistent sterilization and fermentation conditions, three temperature conditions of 35℃, 25℃ and 15℃ were set for scenting jasmine flowers and green coffee beans. The scenting time was 48 h, and the total score of the samples and the performance of the floral aroma were used as evaluation indicators. The scoring criteria for the performance of the floral aroma are shown in Table 4.

[0113] Table 4. Fragrance intensity of samples at different scenting temperatures

[0114] The samples had no obvious floral fragrance or a weak floral fragrance, and were accompanied by unpleasant odors such as musty, rotten, or grassy smells. 1-3 The sample has a distinct floral aroma that is perceptible and is generally well-integrated with the coffee aroma, with no obvious unpleasant odors. 4-6 The sample has a clear and natural floral aroma with good characteristics of fresh flowers, and it blends well with the aroma of the coffee itself, resulting in a high degree of overall aroma harmony. 7-9

[0115] The result is Figure 10 It is evident that higher temperatures significantly affect the appearance of fresh jasmine flowers during the scenting process. Compared to lower temperatures, jasmine flowers treated at 35℃ are more prone to yellowing, browning, and wilting, accompanied by a certain putrid odor. This indicates that while this temperature is close to the traditional tea scenting temperature, it is not suitable for the system of scenting green coffee beans and fresh flowers together in this process. Further combining... Figure 11It can be seen that after 24 hours of scenting, the overall condition of the fresh flowers remained relatively good at 15℃, while the discoloration and deterioration of the flowers were more obvious at 35℃. This indicates that the increase in temperature will accelerate the decay of the flower tissue and have an adverse effect on the aroma quality of the subsequent coffee samples.

[0116] Coffee beans were scented with jasmine flowers to prepare coffee beans. Temperature was the controlled variable, and three temperature conditions were set. The sensory evaluation results are as follows: Figure 12 As shown, there were differences in the total score and floral aroma performance of the samples after different scenting temperatures. The total scores of all three temperature treatment groups were above 80 points, but the total score of the 15℃ treatment group was the highest, indicating that low-temperature scenting is more conducive to maintaining the overall flavor quality of the samples. In terms of floral aroma score, the 15℃ treatment group was significantly higher than the 25℃ and 35℃ treatment groups, while the 35℃ treatment group was the lowest. This indicates that although high temperature conditions may promote the release of floral aroma substances, they will also accelerate the decay, browning and unpleasant odors of the flowers, resulting in a decrease in the clarity of the floral aroma and interference with the aroma of the coffee itself. The 25℃ treatment group showed some improvement compared to the 35℃ group, but the floral aroma coordination and stability were still not as good as the 15℃ treatment group.

[0117] The reason for these results is that jasmine flowers experience increased respiration at higher temperatures, making their tissues more prone to softening and deterioration. This leads to a rapid release of aroma compounds and an increase in musty and stale odors, resulting in unclean sensory characteristics in the coffee sample. At 15℃, the decay and browning processes of the flowers are inhibited, and the release of aroma compounds is relatively slow and stable, which is conducive to the continuous migration, adsorption, and retention of volatile floral aroma compounds into the pores of green coffee beans. Combined with the improvement of the pore structure of coffee beans by the previous sterilization and fermentation treatments, 15℃ scenting can reduce the deterioration of the flowers while allowing floral aroma compounds to penetrate more fully into the coffee bean matrix, thereby improving the clarity of the floral aroma and the overall flavor harmony.

[0118] In summary, fresh flowers deteriorate significantly at 35℃, easily developing a putrid odor, making it unsuitable for this process; while a certain floral aroma can be formed at 25℃, the flavor stability is insufficient; the 15℃ treatment group performed better in terms of fresh flower condition, floral aroma expression, and overall cupping score. Therefore, this invention ultimately selected 15℃ as the suitable temperature for the scenting stage of floral-flavored coffee.

Claims

1. Saccharomyces cerevisiae F3 strain was deposited at the China General Microbiological Culture Collection Center on January 15, 2026, with accession number CGMCC No. 39125.

2. Acetobacter pasteurianu strain 06YS was deposited at the China General Microbiological Culture Collection Center on January 15, 2026, with accession number CGMCC No. 37404.

3. A fermentation method for mixed-culture fermentation combined with scenting flavored coffee, characterized in that, Includes the following steps: S1, selection of green coffee beans; S2, pasteurization; S3, Inoculation and Fermentation: Inoculating sterilized coffee beans with a compound microbial agent, wherein the compound microbial agent includes the Saccharomyces cerevisiae F3 strain as described in claim 1 and the Acetobacter pasteurianus O6YS strain as described in claim 2. S4, mixed fermentation: after inoculation, stir evenly and ferment; S5, Initial Drying: Drying the green coffee beans after fermentation; S6, Fresh Flower Scenting: Fresh flowers and initially dried coffee beans are layered alternately and scented at a low temperature for 30-60 hours; S7, Secondary Drying: After the scenting process, sift out any remaining flowers and dry the green coffee beans; S8, Product Roasting: Roasting green coffee beans after secondary drying to create a harmonious coffee flavor profile.

4. The method according to claim 3, characterized in that, The compound bacterial agent was prepared by mixing strain F3 and strain 06YS in a ratio of 1:

1.

5. The method according to claim 1, characterized in that, The inoculum amount of the compound bacterial agent in S3 is 1%, and the bacterial concentration is 1.0 × 10⁻⁶. 8 CFU / mL, fermentation temperature 28℃.

6. The method according to claim 1, characterized in that, In step S6, the mass ratio of fresh flowers to coffee beans after the first drying is 1:

2.

7. The method according to claim 1, characterized in that, The low-temperature curing temperature in S6 is 15℃, and the curing time is 48h.

8. The method according to claim 1, characterized in that, The flowers mentioned include any one of jasmine, dark red rose, or osmanthus.

9. A method for preparing flavored coffee beans using mixed-culture fermentation combined with scenting according to any one of claims 1-8.

10. The flavored coffee beans according to claim 9, characterized in that, The flavored coffee beans include: jasmine-scented coffee beans, dark red rose-scented coffee beans, and osmanthus-scented coffee beans.

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