Microbial fermentation process for improving the flavor of coffee fruit acids

Abstract

The application discloses a microbial fermentation process for improving coffee fruit acid flavor and belongs to the technical field of coffee processing. The coffee fermentation process is scientific and reasonable to improve the fruit acid flavor of roasted coffee by additionally adding screened strains under the condition of reserving original bacteria of coffee. The fermentation can improve the organic acid content of roasted coffee, wherein the content of citric acid is 8861.58 mu / g, the content of quinic acid is 9291.51 mu / g, the content of maleic acid is 204.32 mu / g, the content of fumaric acid is 190.23 mu / g, the content of succinic acid is 150.69 mu / g, and the content of nicotinic acid is 50.89 mu / g. The coffee fermentation process can improve the coffee bitterness and quality, and the total score of coffee reaches the level of high-quality coffee.

Description

Technical Field

[0001] This invention relates to a microbial fermentation process for enhancing the fruity acidity of coffee, belonging to the field of coffee processing technology. Background Technology

[0002] Coffee, originating in Ethiopia and the Congo region of Africa, is the world's most popular non-alcoholic beverage. Primarily grown in tropical regions between 20 degrees north and south latitude, it is a major economic crop in over 50 developing countries. There are three main varieties of coffee: Liberia (large bean), Robusta (medium bean), and Arabica (small bean). Arabica has the highest commercial value, accounting for over 75% of the coffee market. Due to its low latitude, high altitude, and large diurnal temperature range, Yunnan coffee is characterized by its rich aroma, full-bodied taste, fresh flavor, and mild yet potent character, making it popular with the market and consumers. However, the low premium rate of Catimor, the main variety grown in Yunnan, limits its high-value utilization. Research has found that the initial processing methods of fresh coffee cherries significantly impact the chemical composition and sensory characteristics of coffee, and are an important way to maintain and improve the flavor and quality of coffee beverages.

[0003] Currently, coffee primary processing methods include dry processing, wet processing, and honey processing. However, these methods are characterized by batch-specific and single-process characteristics, making it difficult to precisely control the quality of each batch of fresh coffee cherries. Li Liangwei et al. found that fermenting green coffee beans with *Rhizopus oligosporus* resulted in a 1.5-fold increase in proline and aspartic acid concentrations due to protein hydrolysis during fermentation, and a 2-fold increase in the total concentration of volatile phenolic derivatives due to extensive degradation of ferulic acid and caffeic acid. Furthermore, 36% of the detected total volatiles were produced during fermentation, indicating that *Rhizopus oligosporus* can induce changes in aroma precursors in green coffee. However, the above studies mainly evaluated the overall quality of fermented green coffee beans by molds, and there are no reports on improving specific flavor components of roasted coffee through mold fermentation of fresh coffee cherries. Research shows that organic acids are key intermediate products of carbohydrate decomposition and metabolism, playing a crucial role in taste characterization. They contribute to the acidity and fruitiness of coffee while suppressing bitterness. Current research on the impact of existing processing techniques on specific flavor components is limited; therefore, providing a processing technique that can significantly improve the organic acid content of coffee has significant application value. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a microbial fermentation process to improve the acid flavor of coffee fruit, aiming to solve the technical problem of the lack of a process in the prior art to improve the organic acid components in coffee fruit and thus optimize the taste and flavor of coffee.

[0005] The first technical solution provided by this invention is a Penicillium ovatum strain T1, which was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No. 41551.

[0006] The second technical solution provided by the present invention is a strain of Bacillus altitudinis T3, which was deposited at the China General Microbiological Culture Collection Center on October 24, 2024, with the accession number CGMCC No. 32324.

[0007] The third technical solution provided by the present invention is a compound microbial preparation, which contains Penicillium ovatum T1 as described in the first technical solution and Bacillus altitudinis T3 as described in the second technical solution.

[0008] In some embodiments, the concentration of Penicillium ovatum T1 in the compound microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0009] In some embodiments, the concentration of Bacillus saltitudinis T3 in the compound microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0010] The fourth technical solution provided by the present invention is a fermentation agent containing Penicillium ovatum T1 as described in the first technical solution and Bacillus saltitudinis T3 as described in the second technical solution.

[0011] In some embodiments, the total bacterial concentration of Penicillium ovatum T1 and Bacillus altitudinis T3 in the fermentation agent is not less than 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL or 1×10 7 ~1×109 CFU / g.

[0012] In some embodiments, the ratio of Penicillium ovatum T1 to Bacillus altitudinis T3 is 1:1 to 1:2.

[0013] The fifth technical solution provided by this invention is the application of Penicillium ovatum T1 as described in the first technical solution, Bacillus altitudinis T3 as described in the second technical solution, the compound microbial preparation as described in the third technical solution, or the fermentation agent as described in the fourth technical solution in coffee processing.

[0014] In some embodiments, the application utilizes Penicillium ovatum T1 as described in the first technical solution, Bacillus altitudinis T3 as described in the second technical solution, the compound microbial preparation as described in the third technical solution, or the fermenting agent as described in the fourth technical solution to ferment coffee berries.

[0015] The sixth technical solution provided by the present invention is a fermented coffee processing method, wherein the method involves inoculating coffee cherries with Penicillium ovatum T1 as described in the first technical solution, Bacillus altitudinis T3 as described in the second technical solution, a compound microbial preparation as described in the third technical solution, or a fermenting agent as described in the fourth technical solution for fermentation, followed by drying and roasting.

[0016] In some embodiments, the method includes the following steps: (1) Inoculation: 500g to 600g of peeled coffee cherries are added to sterile water at a material-to-water ratio of 1:10, and then the fermentation agent is inoculated to make the total bacterial concentration in the entire system 1×10⁻⁶. 7 ~1×10 9 CFU / mL, with a mixed bacterial ratio of 1:1 to 1:2;

[0017] (2) Fermentation: Ferment at 28-30℃ for 12-36 hours;

[0018] (3) Washing: After fermentation, wash to remove all the pectin layer;

[0019] (4) Drying: Allow the washed coffee cherries to air dry naturally for 3 to 5 days, so that the moisture content of the coffee beans reaches 10 to 12%.

[0020] (5) Hulling and silverskin removal: Hull and silverskin are removed from the dried coffee beans, and defective beans are removed to make green coffee beans.

[0021] (6) Roasting: Roast the green coffee beans at 170°C to 180°C, with an air damper of 4 to 5, a heat of 1500 to 2000, for 7 to 9 minutes, resulting in a roasting degree of 55 to 65.

[0022] The seventh technical solution provided by the present invention is a method for improving the fruit acid flavor of coffee, wherein the method involves fermenting coffee cherries using Penicillium ovatum T1 as described in the first technical solution, Bacillus altitudinis T3 as described in the second technical solution, a third compound microbial preparation, or a fermenting agent as described in the fourth technical solution.

[0023] In some embodiments, the fruit acid flavoring substances include citric acid, quinic acid, maleic acid, fumaric acid, succinic acid, and niacin.

[0024] The technical effects of this invention are as follows:

[0025] This invention, while preserving the original coffee microbiota, adds selected strains of bacteria. This scientifically and rationally improves the fruit acid flavor of roasted coffee through fermentation, enhancing the organic acid content of roasted coffee. Specifically, the citric acid content is 8861.58 μg / g; quinic acid content is 9291.51 μg / g; maleic acid content is 204.32 μg / g; fumaric acid content is 190.23 μg / g; succinic acid content is 150.69 μg / g; and niacin content is 50.89 μg / g. Furthermore, it improves the bitterness of coffee, enhancing its overall quality and bringing it up to the level of specialty coffee.

[0026] Preservation of biological materials

[0027] A strain of Penicillium ovatum T1, classified as Penicillium ovatum, was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41551.

[0028] A strain of Bacillus altitudinis T3, classified as Bacillus altitudinis, was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32324. Attached Figure Description

[0029] Figure 1 A flavor evaluation chart for fermented coffee.

[0030] Figure 2 A chart showing the ten indicators and total score for coffee evaluation.

[0031] Figure 3 This is a graph showing the changes in organic acid content in fermented coffee. Detailed Implementation

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0033] Test method:

[0034] 1. Taste Evaluation: The evaluation panel consists of 8 sensory members (4 women and 4 men) aged 20-30 from the research group. They evaluate the acidity, sweetness, bitterness, astringency, aftertaste, and body of the coffee (as shown in Table 1). The evaluation is divided into three levels: low (0-5 points), medium (6-10 points), and high (11-15 points). Positive acidity represents good acidity, such as smooth, bright, and lively fruit acidity. Negative acidity represents bad acidity, which is not only dull and sharp, but also similar to the fermented acidity of overripe or rotten fruit, which is unpleasant. Dead acidity lacking in character is chemical acidity. Positive aftertaste represents a distinct aftertaste, sweet and refreshing, long-lasting and will be given a high score. Negative aftertaste represents astringency, roughness, bitterness, stickiness, or even off-flavors, and will be given a low score. Body is a tactile sensation in the mouth about the amount of effective substances dissolved in the liquid, not a taste. The tactile feedback is composed of various sensations such as wateriness, tea-like texture, silkiness, oiliness, viscosity, and quality. Generally, it is believed that a higher body thickness corresponds to a higher score, and a lower body thickness corresponds to a lower score.

[0035] Table 1 Flavor Evaluation Form

[0036]

[0037] 2. Sensory Evaluation: The sensory evaluation of the coffee samples was conducted according to the Specialty Coffee Association of America (SCAA) standard cupping method. The evaluation panel consisted of 8 sensory members (4 women and 4 men) aged 20-30 from the research group. They cupped and scored the coffee according to the SCAA standard cupping method and procedure, evaluating ten indicators: aroma (dry and wet), flavor, aftertaste, acidity, body, consistency, balance, cleanliness, sweetness, and overall evaluation. Each coffee sample was evaluated in five copies according to the SCAA standard, with scores ranging from 6 to 9.75 points in increments of 0.25 points. (Reference: Miao Xingyu, Wang Qinghua, Chai Chunrong, et al. Effects of Saccharomyces cerevisiae and Lactobacillus plantarum enhanced fermentation on volatile flavors of coffee [J / OL]. Food Industry Technology, 1-15 [2024-09-27]).

[0038] 3. Organic Acid Detection: 99.9% citric acid was purchased from SUPELCO Solution Within; 98.00% niacin, 98.00% succinic acid, and 98.00% fumaric acid were purchased from Shanghai Yuanye; 99.00% maleic acid and 98.00% quinic acid were purchased from Huijia Biotechnology. Amino acids in the coffee liquid were determined using liquid chromatography-triple quadrupole mass spectrometry. Standard and Sample Preparation: Accurately weigh 5 mg each of the six organic acid components (citric acid, quinic acid, fumaric acid, maleic acid, succinic acid, and niacin) into a 2 mL centrifuge tube and dissolve in an appropriate amount of ultrapure water. Then, transfer the solution from the centrifuge tube to a 5 mL volumetric flask and dilute to volume with ultrapure water. Finally, a mixed standard stock solution with a concentration of 500 μg / mL was obtained and stored in a refrigerator protected from light until use. The coffee was brewed into a liquid according to the sensory evaluation method. 1 mL of the coffee liquid was filtered through a 0.22 μm water membrane and transferred to a brown vial for injection analysis.

[0039] Detection conditions: UHPLC conditions: A Hypercrb C18 column (100 mm × 2.1 mm, 3 μm) was used to separate organic acid components in tea leaves. The column temperature was 45℃, and the injection volume was 1 μL. The mobile phase consisted of 0.1% formic acid, 1 mmol ammonium formate in water (phase A), and acetonitrile (phase B). The flow rate was 0.3 mL / min, and the specific elution gradients were as follows: 0–1 min, 5–15% B; 1–3.5 min, 15–25% B; 3.5–4.5 min, 25–35% B; 4.5–8 min, 35–40% B. Mass spectrometry conditions: An electrospray ionization source was used, and multiple reaction monitoring (MRM) was employed in negative ion mode for mass spectrometry scanning. The MRM detection ion pairs for each compound were optimized and selected, and the ion source parameters were also optimized. The optimized parameters are as follows: drying gas flow rate 15 L / min, purge gas flow rate 3 L / min, interface voltage 4.5 kV, CID gas pressure 230 kPa, ion transfer tube temperature 250 ℃, heating plate temperature 400 ℃, and detection voltage 2.08 kV.

[0040] Raw materials used in the examples:

[0041] 1. PDA medium: Weigh 200g of peeled potatoes, chop them, add 1000mL of distilled water and boil for 20min. After filtering through eight layers of gauze, add 20g of anhydrous glucose and 20g of agar, and add distilled water to 1000mL.

[0042] 2. LB medium: 5g peptone, 3g beef extract, 5g sodium chloride, 20g agar, 1000mL distilled water

[0043] 3. PDA Nutritional Broth: Peel 200g of potatoes, chop them, add 1000mL of distilled water and boil for 20 minutes. Filter through eight layers of gauze, add 20g of anhydrous glucose, and add distilled water to 1000mL.

[0044] 4. LB Nutritional Broth: 5g peptone, 3g beef extract, 5g sodium chloride, 1000mL distilled water.

[0045] 5. YEPD medium: 10g yeast extract, 20g peptone, 20g glucose, 2g agar, 1000mL distilled water

[0046] 6. MRS medium: Casein peptone 10g, yeast extract 5g, sodium acetate 5g, Tween 80 1g, magnesium sulfate heptahydrate 0.2g, beef extract 10g, glucose 20g, dipotassium hydrogen phosphate 2g, manganese sulfate heptahydrate 0.05g, calcium carbonate 20g, agar 20g, diamine citrate 2g, distilled water 1000mL

[0047] Example 1: Isolation and Identification of Strains

[0048] 1. Separation of Penicillium ovatum and Bacillus altitudinis

[0049] The prepared culture medium, an Erlenmeyer flask containing 100ml of water and glass beads, a 1ml pipette tip, seven test tubes containing 9ml of ultrapure water, and a spreader were autoclaved.

[0050] Place the prepared plates, spreaders, inoculation needles, pipettes, and pipette tips into a laminar flow hood and sterilize them under ultraviolet light.

[0051] When the sterile water cools to about 30°C, place the sample in the laminar flow hood for sampling. Take out the soil sample from the coffee plantation and pour it into an Erlenmeyer flask (try not to let the sample touch the mouth of the Erlenmeyer flask), and then stopper it.

[0052] Use a pipette to transfer the sample mixture into a test tube to prepare a 10-fold dilution (10... -1 ). Use a pipette to draw 1 mL of 10. -1 A 1:1 dilution was added to a test tube containing 9 mL of sterile water to obtain a 1:1 dilution. -2 Dilute the solution by 10 times, and so on, diluting to 10 times each time. -7 Take 1 mL of each dilution and add it dropwise to PDA, YEPD, MRS, and LB culture plates. Spread the solution evenly using a glass rod, first with a "+" sign and then with a "()" sign. Set up 3 replicates for each concentration.

[0053] Label the coated plates with numbers (date, serial dilution concentration, culture medium type, etc.), seal the labeled plates, and then incubate them upside down in an incubator at 28°C.

[0054] Observe the growth of colonies on the plates every day, isolate and purify the grown colonies, and select different colonies for subculture.

[0055] 2. Identification of Penicillium ovatum and Bacillus altitudinis

[0056] Genomic DNA extraction: Take a 2 ml centrifuge tube, add 200 μL of pretreatment solution and a few grinding beads, then add an appropriate amount of bacterial sample, and grind thoroughly in a grinder. Add 20 μL of Proteinase K and 200 μL of lysis buffer, mix thoroughly by inverting, and incubate at 70°C for 10 min. Add 200 μL of anhydrous ethanol, mix thoroughly by inverting, and centrifuge briefly to remove droplets from the inner wall of the tube cap. Pass through an adsorption column, wash once with washing buffer, and wash twice with rinsing buffer. Incubate the adsorption column at room temperature for 3–5 minutes to completely dry any residual rinsing buffer in the adsorption material. Transfer the adsorption to a new centrifuge tube, add 50–100 μL of ddH2O dropwise to the center of the adsorption membrane, incubate at room temperature for 3–5 min, centrifuge at 12000 rpm for 2 min, and collect the solution in the centrifuge tube (reference). 18S Amplification: PCR amplification was performed using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as primers. PCR system (30 μL): 1 μL template, 1 μL each of forward and reverse primers, 15 μL mixed solution, and ddH2O to make up to 30 μL. PCR conditions: 96℃ pre-denaturation for 5 min; 96℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min, storage at 4℃. 16S amplification: PCR amplification was performed using bacterial primers 16S27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), V4-515F (5'-GTGCCAGCAGCCGCGGTAA-3'), and V4-806R (5'-GGACTACCAGGGTATCTAA-3'). The PCR system and conditions were the same as for 18S amplification. PCR product detection: 3 μL of PCR product was analyzed using a 1.0% agarose gel electrophoresis to observe the banding characteristics. The purified PCR product was then analyzed using sequencing. Sequencing results were compared with NCBI-BLAST.

[0057] Example 2: Preparation of coffee using Penicillium ovatum and Bacillus altitudinis

[0058] A fermented coffee processing method includes the following steps:

[0059] (1) Strains culture: The two strains (Penicillium ovatum and Bacillus altitudinis) were streaked on PDA medium and LB medium and cultured at 30℃ for 2 days. The two activated strains were then cultured in PDA nutrient broth and LB nutrient broth at 30℃ and 160r / min for 1 day. (2) Preparation of bacterial suspension: The cultured strains were centrifuged at 8000rpm for 10min and placed in 10mL of sterile water to make the bacterial suspension concentration 1×10⁻⁶. 7 (3) Coffee peeling: Thaw the frozen coffee cherries 24 hours in advance, manually remove the coffee peels, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except coffee cherries. The original strains of coffee cherries should be preserved and the addition of other strains should be avoided. The sterilization parameters are 121℃ for 20min. (5) Inoculation: The inoculation step is carried out in a clean bench. Put 500g of coffee cherries into 500mL of sterile water, and then add the pre-prepared bacterial suspension to make the concentration of the whole system 1×10 7 CFU / mL / mL, the mixed bacteria ratio is 1:1 to 1:2. (6) Fermentation: Ferment the inoculated coffee at 30℃ for 36 hours in a constant temperature incubator. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layer. (8) Drying: Let the washed coffee cherries air dry naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Hulling and silverskin removal: Hull and silverskin of the dried coffee beans, remove defective beans, and make green coffee beans. (10) Roast the green coffee beans under the following conditions: put them in the pot at 170℃, air damper 4, heat 2000, roast for 9 minutes and 30 seconds, and roast degree value 59.

[0060] Example 3: Preparation of coffee using Bacillus altitudinis

[0061] A fermented coffee processing method includes the following steps:

[0062] (1) Strawberry culture: Bacillus altitudinis was streaked onto LB medium and incubated at 30℃ for 2 days. The activated bacteria were then cultured in LB nutrient broth at 30℃ and 160 rpm for 1 day. (2) Preparation of bacterial suspension: The cultured bacteria were centrifuged at 8000 rpm for 10 min and placed in 10 mL of sterile water to make the bacterial suspension concentration 1×10⁻⁶. 7(3) Coffee peeling: Thaw the frozen coffee cherries 24 hours in advance, manually remove the coffee peels, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except coffee cherries. The original strains of coffee cherries should be preserved and the addition of other strains should be avoided. The sterilization parameters are 121℃ for 20min. (5) Inoculation: The inoculation step is carried out in a clean bench. Put 500g of coffee cherries into 500mL of sterile water, and then add the pre-prepared bacterial suspension to make the concentration of the whole system 1×10 7 CFU / mL. (6) Fermentation: Ferment the inoculated coffee at 30℃ for 36 hours in a constant temperature incubator. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layer. (8) Drying: Allow the washed coffee cherries to air dry naturally for 3 days to bring the moisture content of the coffee beans to 10%. (9) Hulling and Silverskin Removal: Remove the hulls and silverskin from the dried coffee beans, and remove defective beans to make green coffee beans. (10) Roast the green coffee beans under the following conditions: 170℃ for roasting, air damper 4, heat 2000, roast for 9 minutes, and roast degree 59.

[0063] Example 4: Preparation of coffee using Penicillium ovatum

[0064] A fermented coffee processing method includes the following steps:

[0065] (1) Strawberry culture: Penicillium ovatum was streaked onto PDA medium and incubated at 30℃ for 2 days. The activated bacteria were then cultured in PDA nutrient broth at 30℃ and 160 rpm for 1 day. (2) Preparation of bacterial suspension: The cultured bacteria were centrifuged at 8000 rpm for 10 min and placed in 10 mL of sterile water to make the bacterial suspension concentration 1×10⁻⁶. 7 (3) Coffee peeling: Thaw the frozen coffee cherries 24 hours in advance, manually remove the coffee peels, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except coffee cherries. The original strains of coffee cherries should be preserved and the addition of other strains should be avoided. The sterilization parameters are 121℃ for 20min. (5) Inoculation: The inoculation step is carried out in a clean bench. Put 500g of coffee cherries into 500mL of sterile water, and then add the pre-prepared bacterial suspension to make the concentration of the whole system 1×10 7CFU / mL. (6) Fermentation: Ferment the inoculated coffee at 30℃ for 36h in a constant temperature incubator. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layer. (8) Drying: Let the washed coffee cherries air dry naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Hulling and silverskin removal: Hull and silverskin of the dried coffee beans, remove defective beans, and make green coffee beans. (10) Roast the green coffee beans under the following conditions: put them in the roaster at 170℃, air damper at 4, heat at 2000, roast for 9min17s, and roast degree value of 59.

[0066] Comparative example: Natural fermentation

[0067] Remove steps (1) and (2) in Example 1. (3) Peeling the coffee: Thaw the frozen fresh coffee cherries 24 hours in advance and manually remove the coffee cherries skin. The weight of the coffee after peeling is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water. Sterilize all materials except coffee cherries. The original strains of coffee cherries should be preserved and the addition of other strains should be avoided. The sterilization parameters are 121℃ for 20 minutes. Remove (5). No inoculation is required. It is carried out directly in the ultra-clean workbench. Put 500g of coffee cherries into 500mL of sterile water. (6) Fermentation: Ferment the inoculated coffee in a constant temperature incubator at 30℃ for 36 hours. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layer. (8) Drying: Let the washed coffee cherries air dry naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Remove the husk and silverskin: Remove the husk and silverskin from the dried coffee beans and remove defective beans to make green coffee beans. (10) Roast the green coffee beans under the following conditions: put them in the pot at 170°C, air damper at 4, heat at 2000, roast for 9 minutes and 13 seconds, and roast degree value is 59.

[0068] Test case

[0069] The flavors of the fermented coffees from Examples 1-3 and the comparative examples were evaluated. Figure 1As shown, based on six flavor indicators, coffee undergoing intensified fermentation exhibits enhanced acidity, sweetness, aftertaste, and body. The acidity score of naturally fermented coffee (comparative example) was 2.57, increasing to 4.86 for T1, 5.63 for T3, and 6.38 for T1-T3 after intensified fermentation. Similarly, the sweetness score of naturally fermented coffee (comparative example) was 1.67, increasing to 2.8 for T1, 2.33 for T3, and 2.6 for T1-T3 after intensified fermentation. The aftertaste score of naturally fermented coffee (comparative example) was 5.43, increasing to 6.57 for T1 and 5.75 for T3 after intensified fermentation. The T1-T3 score increased to 6.88 points; the body of the naturally fermented product was 4.14 points, which decreased to 3.71 points after enhanced fermentation, T3 increased to 5.25 points, and T1-T3 increased to 7.25 points; bitterness and astringency both decreased. The bitterness of the naturally fermented product (comparative example) was 10.43 points, which decreased to 8.29 points, 9.50 points, and 7.38 points after enhanced fermentation; the astringency of the naturally fermented product (comparative example) was 4.83 points, which decreased to 2.5 points, 2.17 points, and 2.60 points after enhanced fermentation.

[0070] Sensory evaluation (SCAA) was conducted on the coffee samples from Examples 1-3 and the comparative fermented coffee samples. Figure 2 As shown, based on the evaluation of ten indicators and the total score, the coffee improved after the enhanced fermentation in terms of dry aroma, wet aroma, flavor, aftertaste, acidity, body, consistency, cleanliness, sweetness, balance, overall evaluation, and total score. Furthermore, the T1-T3 groups reached the level of specialty coffee, with a total score of 82.07.

[0071] The organic acids in the coffee from Examples 1-3 were detected, such as... Figure 3As shown, after intensified fermentation, the levels of citric acid, quinic acid, maleic acid, fumaric acid, succinic acid, and nicotinic acid in coffee all increased. The citric acid content in naturally fermented coffee was 4364.51 μg / g, while after intensified fermentation, T1 increased to 7684.73 μg / g, T3 increased to 6362.84 μg / g, and T1-T3 increased to 8861.58 μg / g. The quinic acid content in naturally fermented coffee (comparative example) was 4527.39 μg / g, while after intensified fermentation, T1 increased to 769 μg / g. The maleic acid content in the naturally fermented (comparative) sample was 81.97 μg / g, which increased to 190.17 μg / g in T1, 148.68 μg / g in T3, and 204.32 μg / g in T1-T3 after enhanced fermentation. The fumaric acid content in the naturally fermented (comparative) sample was 84.74 μg / g, which increased to 121 μg / g in T1 after enhanced fermentation. The succinic acid content in the naturally fermented (comparative) sample was 62.24 μg / g, which increased to 139.52 μg / g at T1 and 190.23 μg / g at T3 after enhanced fermentation. The nicotinic acid content in the naturally fermented (comparative) sample was 34.02 μg / g, which increased to 62.17 μg / g at T1 after enhanced fermentation. T3 increased to 54.33 μg / g, and T1-T3 increased to 50.89 μg / g. Organic acids are key intermediate products of carbohydrate decomposition and metabolism, playing a crucial role in taste characterization. They contribute to the acidity and fruitiness of coffee while suppressing bitterness. Furthermore, relevant studies have shown that one of the characteristics that citric acid can impart is acidity, fruitiness, and berry flavor. Both sensory and material content indicate that coffee can enhance its fruity acidity and improve its quality after being fermented by these two strains of bacteria.

[0072] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Penicillium ovoidis ( Penicillium ovatum T1, characterized in that, It was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41551.

2. A strain of Highland Bacillus ( Bacillus altitudinis T3, characterized in that, It was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32324.

3. A compound microbial preparation, characterized in that, The compound microbial preparation contains Penicillium ovale T1 as described in claim 1 and Bacillus hygroscopicus T3 as described in claim 2.

4. The compound microbial preparation according to claim 3, characterized in that, In the compound microbial preparation, the concentration of Penicillium ovale T1 is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g; the concentration of the *Bacillus hygroscopicus* T3 is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

5. A fermenting agent, characterized in that, The fermentation agent contains Penicillium ovale T1 as described in claim 1 and Bacillus hygroscopicus T3 as described in claim 2.

6. The fermenting agent according to claim 5, characterized in that, In the fermentation spawn, the total bacterial concentration of Penicillium ovale T1 and Bacillus hygroscopicus T3 is 1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g; the ratio of Penicillium ovale T1 to Bacillus hygroscopicus T3 is 1:1 to 1:

2.

7. The application of Penicillium ovale T1 as described in claim 1, Bacillus hygroscopicus T3 as described in claim 2, the compound microbial preparation as described in claim 3 or 4, or the fermenting agent as described in claim 5 or 6 in coffee processing, characterized in that, The application involves fermenting coffee berries using Penicillium ovale T1 as described in claim 1, Bacillus hygroscopicus T3 as described in claim 2, the compound microbial preparation as described in claim 3 or 4, or the fermenting agent as described in claim 5 or 6.

8. A method for processing fermented coffee, characterized in that, The method involves inoculating coffee cherries with the Penicillium ovale T1 as described in claim 1, Bacillus hygroscopicus T3 as described in claim 2, the compound microbial preparation as described in claim 3 or 4, or the fermenting agent as described in claim 5 or 6, followed by drying and roasting.

9. A method for enhancing the fruity acid flavor compounds in coffee, characterized in that, The method involves fermenting coffee berries using Penicillium ovale T1 as described in claim 1, Bacillus hygroscopicus T3 as described in claim 2, the compound microbial preparation as described in claim 3 or 4, or the fermenting agent as described in claim 5 or 6.

10. The method according to claim 9, characterized in that, The fruit acid flavoring substances include citric acid, quinic acid, maleic acid, fumaric acid, succinic acid, and niacin.

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