Microbial fermentation process for improving flavor of caffeolic acid

Abstract

The invention discloses a microbial fermentation process for improving the flavor of coffee tartaric acid, and belongs to the technical field of coffee processing. According to the coffee fermentation processing technology, screened strains are additionally added under the condition that original flora of coffee is reserved, the coffee fermentation processing technology is scientific and reasonable, the tartaric acid flavor of roasted coffee is improved, the organic acid content of the roasted coffee can be increased through enhanced fermentation, and the citric acid content is 8861.58 micrograms / g; the quinic acid content is 9291.51 [mu] g / g; the content of maleic acid is 204.32 mu g / g; the content of fumaric acid is 190.23 mu g / g, and the content of succinic acid is 150.69 mu g / g; the content of nicotinic acid is 50.89 mu g / g, the bitter taste of the coffee can be improved, the quality of the coffee is improved, and the total score of the coffee reaches the grade of high-quality coffee.

Description

Technical Field

[0001] The invention relates to a microbial fermentation process for improving the acid flavor of coffee, and belongs to the technical field of coffee processing. Background Art

[0002] Coffee, native to Ethiopia and Congo in Africa, is the most popular non-alcoholic beverage in the world. It is mainly grown in tropical areas between 20 degrees north and south latitude, and is the main cash crop in more than 50 developing countries. There are three main varieties of coffee: large-grain Liberia, medium-grain Robusta, and small-grain Arabica. Among them, Arabica has a higher commercial utilization value, accounting for more than 75% of the coffee market. Due to the climate characteristics of low latitude, high altitude, and large temperature difference between day and night, the coffee in Yunnan has the characteristics of rich aroma, mellow taste, fresh smell, and strong but not strong, which is deeply favored by the market and consumers. However, as the main planting variety in Yunnan Province, Catim has a low boutique rate, which limits its high-value utilization. Studies have found that the primary processing method of fresh coffee fruit has a great influence on the chemical composition and sensory composition of coffee, which is an important way to maintain and improve the flavor quality of coffee beverages.

[0003] At present, the primary processing methods of coffee include dry processing, wet processing, honey processing, etc. However, these primary processing methods have the characteristics of batch and single processing technology, and it is impossible to accurately control the quality of each batch of fresh coffee fruit. Li Liangwei et al. found that when raw coffee beans were fermented with oligosporus, protein hydrolysis during the fermentation process led to a 1.5-fold increase in the concentration of proline and aspartic acid, and the extensive degradation of ferulic acid and caffeic acid led to a 2-fold increase in the total concentration of volatile phenolic derivatives, and 36% of the total volatiles detected were produced during the fermentation process, indicating that oligosporus can induce changes in the aroma precursors of green coffee. However, the above studies mainly evaluated the comprehensive quality of coffee beans after fermentation by molds, and there have been no reports on improving the specific flavor quality components of roasted coffee by fermenting fresh coffee fruits with molds. Studies have shown that organic acids are key intermediates of carbohydrate decomposition and metabolism, and play a key role in taste characterization. They contribute to the sourness and fruitiness of coffee and inhibit bitterness. At present, there are few studies on the effects of existing processing technologies on specific flavor quality components. Therefore, providing a processing technology that can significantly improve the organic acid content of coffee has important application value. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a microbial fermentation process for improving the acid flavor of coffee berries, aiming to solve the technical problem that the prior art lacks the process of improving the organic acid components in coffee berries and further optimizing the taste and flavor of coffee.

[0005] The first technical solution provided by the present invention is a strain of Penicillium ovatum T1, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 24, 2024, and the strain collection number is: CGMCC No.41551.

[0006] The second technical solution provided by the present invention is a strain of Bacillus altitudinis T3, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 24, 2024, and the strain collection number is: CGMCC No.32324.

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

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

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

[0010] The fourth technical solution provided by the present invention is a starter culture, wherein the starter culture contains the Penicillium ovatum T1 described in the first technical solution and the Bacillus saltitudinis T3 described in the second technical solution.

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

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

[0013] The fifth technical solution provided by the present invention is the use of Penicillium ovatum T1 described in the first technical solution, Bacillus altitudinis T3 described in the second technical solution, the composite microbial preparation described in the third technical solution, or the starter described in the fourth technical solution in processing coffee.

[0014] In certain embodiments, the application is to ferment coffee cherries using the Penicillium ovatum T1 described in the first technical solution, the Bacillus altitudinis T3 described in the second technical solution, the composite microbial preparation described in the third technical solution, or the starter described in the fourth technical solution.

[0015] The sixth technical solution provided by the present invention is a fermented coffee processing method, wherein the Penicillium ovatum T1 described in the first technical solution, the Bacillus altitudinis T3 described in the second technical solution, the composite microbial preparation described in the third technical solution, or the starter described in the fourth technical solution is inoculated into coffee cherries for fermentation, and then dried and baked.

[0016] In some embodiments, the method comprises the following steps: (1) inoculation: 500 g to 600 g of peeled coffee berries are added to sterile water at a material-water ratio of 1:10, and then the fermentation agent is added to make the total bacterial concentration in the whole system 1×10 7 ~1×10 9 CFU / mL, mixed bacteria ratio is 1:1 to 1:2;

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

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

[0019] (4) Drying: Dry the washed coffee cherries naturally for 3 to 5 days until the moisture content of the coffee beans reaches 10 to 12%;

[0020] (5) Shelling and silver skin removal: Shell and silver skin the dried coffee beans, remove defective beans, and make green coffee beans;

[0021] (6) Roasting: Roast the green coffee beans at 170°C to 180°C, with the air damper at 4 to 5, the heat at 1500 to 2000, and the roasting time at 7 to 9 minutes, and the roasting degree at 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 comprises fermenting coffee cherries using the Penicillium ovatum T1 described in the first technical solution, the Bacillus altitudinis T3 described in the second technical solution, the third composite microbial preparation or the starter described in the fourth technical solution.

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

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

[0025] The invention adds a screened strain while retaining the original bacterial flora of coffee. The coffee fermentation processing technology can scientifically and rationally improve the fruity acid flavor of roasted coffee. Strengthening fermentation can increase the organic acid content of roasted coffee, wherein the citric acid content is 8861.58 μg / g; the quinic acid content is 9291.51 μg / g; the maleic acid content is 204.32 μg / g; the fumaric acid content is 190.23 μg / g; the succinic acid content is 150.69 μg / g; and the nicotinic acid content is 50.89 μg / g. The bitterness of coffee can be improved, the quality of coffee can be improved, and the total score of coffee can reach the level of boutique coffee.

[0026] Biomaterial Deposit

[0027] A strain of Penicillium ovatum T1, classified as Penicillium ovatum, was deposited in the General Microbiology Center of China Culture Collection Administration on October 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is: CGMCC No.41551.

[0028] A strain of Bacillus altitudinis T3, classified as Bacillusaltitudinis, was deposited in the General Microbiology Center of China Culture Collection Administration on October 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is: CGMCC No.32324. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a taste evaluation chart of fermented coffee.

[0030] Figure 2 This is the evaluation chart of the ten coffee indicators and the total score.

[0031] Figure 3 This is a graph showing changes in organic acid content in fermented coffee. DETAILED DESCRIPTION

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

[0033] Test method:

[0034] 1. Taste evaluation: The evaluation team consists of 8 sensory members of the research group (4 females and 4 males) aged 20-30 years old. They evaluate the sourness, sweetness, bitterness, astringency, aftertaste and mellowness of coffee (as shown in Table 1), which are divided into three levels: low (0-5 points), medium (6-10 points) and high (11-15 points). Positive acidity represents good acidity, such as soft, bright and lively fruit acidity. Negative acidity represents bad acidity, which is not just dull and sharp, but some are similar to fermented acidity of overripe fruit or corrupt fruit, which is disgusting; dead acidity lacking connotation, chemical acidity; positive aftertaste represents aftertaste with distinct layers, sweetness and long-lasting, and will be given high scores; negative aftertaste represents astringency, roughness, bitterness and stickiness, or even miscellaneous taste, and the score is very low; mellowness is a tactile sensation of the mouth to the amount of effective substances dissolved in the liquid, not the taste. The feeling of water, tea, silkiness, oiliness, viscosity, and quality all constitute the feedback of this tactile sensation. It is generally believed that the higher the mellowness, the higher the score, and the lower the mellowness, the lower the score.

[0035] Table 1 Taste evaluation table

[0036]

[0037] 2. Sensory evaluation: The sensory evaluation of coffee samples was conducted in accordance with the standard cupping method of the Specialty Coffee Association of America (SCAA). The evaluation team consisted of 8 sensory members of the research group aged 20-30 (4 females and 4 males). They scored coffee aroma (dry and wet aroma), flavor, aftertaste, acidity, body, consistency, balance, cleanliness, sweetness, and overall evaluation according to the SCAA standard cupping method and process. Each coffee sample was evaluated in quintuplicate according to the SCAA standard and scored in the range of 6-9.75 points in increments of 0.25 points. (Reference: Miao Xingyu, Wang Qinghua, Chai Chunrong, et al. Effect of enhanced fermentation of brewer's yeast and Lactobacillus plantarum on volatile flavor of coffee [J / OL]. Food Industry Science and Technology, 1-15 [2024-09-27].)

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

[0039] Detection conditions: UHPLC conditions: A Hypercrb C18 column (100mm×2.1mm, 3μm) was used to separate the organic acid components in tea leaves, with a column temperature of 45°C and an injection volume of 1μL. The mobile phase consisted of 0.1% formic acid, 1mmol ammonium formate water (phase A) and acetonitrile (phase B), with a flow rate of 0.3mL / min. The specific elution gradient was as follows: 0–1min, 5–15% B; 1–3.5min, 15–25% B; 3.5–4.5min, 25–35% B; 4.5–8min, 35–40% B. Mass spectrometry conditions: The ion source was an electrospray ion source, and the multi-reaction monitoring (MRM) scanning mode in the negative ion mode was used for mass spectrometry scanning. First, the MRM detection ion pairs of each compound were optimized and selected, and the ion source parameters were optimized at the same time. The optimized specific 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 °C, heating plate temperature 400 °C, and detection voltage 2.08 kV.

[0040] The raw materials used in the examples are:

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

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

[0043] 3. PDA nutrient broth: Peel and weigh 200 g of potatoes, chop them, add 1000 mL of distilled water and boil for 20 min. After filtering through eight layers of gauze, add 20 g of anhydrous glucose and add distilled water to 1000 mL.

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

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

[0046] 6. MRS medium: casein peptone 10g, yeast powder 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, a conical flask containing 100 ml of water and glass beads, a 1 ml pipette tip, 7 test tubes containing 9 ml of ultrapure water, and a coating rod were sterilized by high-pressure steam.

[0050] Place the poured plates, applicators, inoculation needles, pipettes, and pipette tips into the clean bench and start ultraviolet sterilization.

[0051] When the sterile water cools to about 30°C, place the sample on the clean bench for sampling, take out the soil sample from the coffee planting area and pour it into a conical bottle (try not to let the sample get on the mouth of the conical bottle), and plug it.

[0052] Use a pipette to draw the sample mixture into a test tube to make a 10-fold dilution (10 -1 ). Use a pipette to draw 1mL10 -1 Add the dilution to a test tube containing 9 mL of sterile water to obtain 10 -2 dilution, and so on, dilute to 10 -7 . Take 1 mL of each dilution concentration and drop it on four culture medium plates: PDA, YEPD, MRS, and LB. Use a glass rod to spread it evenly in a “+” and then “()” manner. Set up 3 replicates for each concentration.

[0053] The coated plates were numbered and labeled (date, gradient dilution concentration, culture medium type, etc.), the labeled plates were sealed, and then placed in an incubator at 28°C for inversion culture.

[0054] Observe the growth of the colonies in the plate every day, separate 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 2ml centrifuge tube, add 200μL pretreatment solution and a small amount of grinding beads, then add an appropriate amount of bacterial sample, and grind it in a grinder until it is fully ground. Add 20μL Proteinase K, add 200μL lysis solution, mix thoroughly by inversion, and place at 70℃ for 10min. Add 200μL anhydrous ethanol, mix thoroughly by inversion, and centrifuge briefly to remove droplets on the inner wall of the tube cap. Pass through the adsorption column, wash once with washing solution, and wash twice with rinsing solution. Place the adsorption column at room temperature for 3 to 5 minutes to thoroughly dry the residual rinsing solution in the adsorption material. Transfer the adsorption to a new centrifuge tube, add 50 to 100μL ddH2O to the middle of the adsorption membrane in mid-air, place at room temperature for 3 to 5min, centrifuge at 12000rpm for 2min, and collect the solution in a centrifuge tube (literature). 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 upstream and downstream primers, 15 μL mixed solution, and 30 μL ddH2O. PCR conditions: 96°C pre-denaturation for 5 min; 96°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 10 min, and storage at 4°C. 16S amplification: PCR amplification was performed using bacterial 16S27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), V4-515F (5'-GTGCCAGCAGCCGCGGTAA-3'), and V4-806R (5'-GGACTACCAGGGTATCTAA-3') as primers. The PCR system and conditions were the same as those for 18S amplification. PCR product detection: 3 μL PCR product was tested on 1.0% agarose gel to observe the band characteristics. The purified PCR product was tested on the machine. The sequencing results were compared by NCBI-BLAST.

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

[0058] A fermented coffee processing process comprises the following steps:

[0059] (1) Strain culture: Two strains (Penicillium ovatum and Bacillus altitudinis) were streaked with PDA medium and LB medium and cultured at 30°C for 2 days. The two activated strains were cultured in PDA nutrient broth and LB nutrient broth at 30°C and 160 r / min for 1 day. (2) Preparation of bacterial suspension: The cultured strains were centrifuged at 8000 rpm for 10 min and placed in 10 mL of sterile water to make the bacterial suspension concentration 1×10 7 CFU / mL, set aside. (3) Coffee peeling: Thaw the frozen coffee berries for 24 hours in advance, remove the coffee berries manually, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except the coffee berries. The original bacteria of the coffee berries should be retained and the addition of other bacteria should be avoided. The sterilization parameters are sterilization at 121°C for 20min. (5) Inoculation: The inoculation step is carried out in an ultra-clean workbench. Put 500g of coffee berries into 500mL of sterile water, and then add the bacterial suspension prepared in advance to make the concentration of the entire system 1×10 7 CFU / mL / mL, the mixed bacteria ratio is 1:1 to 1:2. (6) Fermentation: Ferment the inoculated coffee in a constant temperature incubator at 30℃ for 36h. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layers. (8) Drying: Dry the washed coffee cherries naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Shelling and silver skin removal: Shell and silver skin the dried coffee beans, remove the defective beans, and make green coffee beans. (10) Roast the green coffee beans. The roasting conditions are 170℃, air damper 4, firepower 2000, roasting 9min30s, and roasting degree value 59.

[0060] Example 3 Preparation of coffee using Bacillus altitudinis

[0061] A fermented coffee processing process comprises the following steps:

[0062] (1) Strain culture: Bacillus altitudinis was streaked with LB medium and cultured at 30°C for 2 days. The activated bacteria were cultured in LB nutrient broth at 30°C and 160 rpm for 1 day. (2) Preparation of bacterial suspension: The cultured strains were centrifuged at 8000 rpm for 10 min and placed in 10 mL of sterile water to a bacterial suspension concentration of 1×10 7CFU / mL, set aside. (3) Coffee peeling: Thaw the frozen coffee berries for 24 hours in advance, remove the coffee berries manually, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except the coffee berries. The original bacteria of the coffee berries should be retained and the addition of other bacteria should be avoided. The sterilization parameters are sterilization at 121°C for 20min. (5) Inoculation: The inoculation step is carried out in an ultra-clean workbench. Put 500g of coffee berries into 500mL of sterile water, and then add the bacterial suspension prepared in advance to make the concentration of the entire system 1×10 7 CFU / mL. (6) Fermentation: Ferment the inoculated coffee in a constant temperature incubator at 30°C for 36 hours. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layers. (8) Drying: Dry the washed coffee cherries naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Shelling and silver skin removal: Shell and silver skin the dried coffee beans, remove the defective beans, and make green coffee beans. (10) Roast the green coffee beans. The roasting conditions are 170°C, air damper 4, fire power 2000, roasting for 9 minutes, and roasting degree value 59.

[0063] Example 4 Preparation of coffee using Penicillium ovatum

[0064] A fermented coffee processing process comprises the following steps:

[0065] (1) Strain culture: Penicillium ovatum was streaked with PDA medium and cultured at 30°C for 2 days. The activated bacteria were cultured in PDA nutrient broth at 30°C and 160 rpm for 1 day. (2) Preparation of bacterial suspension: The cultured strains were centrifuged at 8000 rpm for 10 min and placed in 10 mL of sterile water to a bacterial suspension concentration of 1×10 7 CFU / mL, set aside. (3) Coffee peeling: Thaw the frozen coffee berries for 24 hours in advance, remove the coffee berries manually, and the weight of the peeled coffee is 500g. (4) Sterilization: Sterilize the fermentation tank and tap water, and sterilize all materials except the coffee berries. The original bacteria of the coffee berries should be retained and the addition of other bacteria should be avoided. The sterilization parameters are sterilization at 121°C for 20min. (5) Inoculation: The inoculation step is carried out in an ultra-clean workbench. Put 500g of coffee berries into 500mL of sterile water, and then add the bacterial suspension prepared in advance to make the concentration of the entire system 1×10 7CFU / mL. (6) Fermentation: Ferment the inoculated coffee in a constant temperature incubator at 30°C for 36 hours. (7) Washing: Wash the fermented coffee with tap water to remove all the pectin layers. (8) Drying: Dry the washed coffee cherries naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Shelling and silver skin removal: Shell and silver skin the dried coffee beans, remove the defective beans, and make green coffee beans. (10) Roast the green coffee beans. The roasting conditions are: put them into the pot at 170°C, the air damper is 4, the fire is 2000, roasting is 9min17s, and the roasting degree value is 59.

[0066] Comparative example: natural fermentation

[0067] Steps (1) and (2) in Example 1 are removed. (3) Peeling of coffee: thaw the frozen fresh coffee berries for 24 hours in advance, remove the coffee berries manually, and the weight of the peeled coffee is 500 g. (4) Sterilization: sterilize the fermentation tank and tap water, and sterilize all materials except the coffee berries. The original strains of the coffee berries should be retained and the addition of other strains should be avoided. The sterilization parameter is sterilization at 121° C. for 20 min. Step (5) is removed. Inoculation is not required. The process is directly carried out in a clean bench. 500 g of coffee berries are placed in 500 mL of sterile water. (6) Fermentation: ferment the inoculated coffee in a constant temperature incubator at 30° C. for 36 hours. (7) Washing: wash the fermented coffee with tap water to remove all pectin layers. (8) Drying: dry the washed coffee berries naturally for 3 days to make the moisture content of the coffee beans reach 10%. (9) Shelling and silver skin removal: Shell and silver skin the dried coffee beans, pick out defective beans, and make green coffee beans. (10) Roast the green coffee beans. The roasting conditions are: put them into the pot at 170℃, the air damper is 4, the fire is 2000, roasting for 9min13s, and the roasting degree value is 59.

[0068] Test Case

[0069] The taste of Examples 1 to 3 and the comparative fermented coffee was evaluated. Figure 1As shown in the figure, from the perspective of the six taste indicators, the acidity, sweetness, aftertaste and mellowness of coffee are all enhanced after enhanced fermentation. The acidity of natural fermentation (comparative example) is 2.57 points, and after enhanced fermentation, T1 increases to 4.86 points, T3 increases to 5.63 points, and T1-T3 increases to 6.38 points; the sweetness of natural fermentation (comparative example) is 1.67 points, and after enhanced fermentation, T1 increases to 2.8 points, T3 increases to 2.33 points, and T1-T3 increases to 2.6 points; the aftertaste of natural fermentation (comparative example) is 5.43 points, and after enhanced fermentation, T1 increases to 6.57 points, T3 increases to 5.75 points, and T1-T3 increases to 5.82 points. points, T1-T3 increased to 6.88 points; the mellowness of natural fermentation was 4.14 points, after enhanced fermentation, T1 decreased to 3.71 points, T3 increased to 5.25 points, and T1-T3 increased to 7.25 points; the bitterness and astringency both decreased, the bitterness of natural fermentation (comparative example) was 10.43, after enhanced fermentation, T1 decreased to 8.29 points, T3 decreased to 9.50 points, and T1-T3 decreased to 7.38 points; the astringency of natural fermentation (comparative example) was 4.83 points, after enhanced fermentation, T1 decreased to 2.5 points, T3 decreased to 2.17 points, and T1-T3 decreased to 2.60 points.

[0070] Sensory evaluation (SCAA) was performed on Examples 1 to 3 and the comparative fermented coffee. Figure 2 As shown, based on the ten indicators and the total score, the dry aroma, wet aroma, flavor, aftertaste, acidity, body, consistency, cleanliness, sweetness, balance, overall evaluation and total score of the coffee are all improved after enhanced fermentation, and the T1-T3 group has reached the level of boutique coffee, with a total score of 82.07.

[0071] The organic acids in the coffee of Examples 1 to 3 were detected. Figure 3As shown in the figure, after enhanced fermentation, the citric acid, quinic acid, maleic acid, fumaric acid, succinic acid and nicotinic acid in coffee are all enhanced. The citric acid content of natural fermentation is 4364.51μg / g, which increases to 7684.73μg / g in T1, 6362.84μg / g in T3 and 8861.58μg / g in T1-T3 after enhanced fermentation. The quinic acid content of natural fermentation (comparative example) is 4527.39μg / g, which increases to 769 in T1 after enhanced fermentation. 9.62μg / g, T3 increased to 7059.89μg / g, T1-T3 increased to 9291.51μg / g; the maleic acid content of natural fermentation (comparative example) was 81.97μg / g, after enhanced fermentation, T1 increased to 190.17μg / g, T3 increased to 148.68μg / g, T1-T3 increased to 204.32μg / g; the fumaric acid content of natural fermentation (comparative example) was 84.74μg / g, after enhanced fermentation, T1 increased to 121. 92μg / g, T3 increased to 139.52μg / g, T1-T3 increased to 190.23μg / g; the succinic acid content of natural fermentation (comparative example) was 62.24μg / g, after enhanced fermentation, T1 increased to 139.65μg / g, T3 increased to 96.33μg / g, T1-T3 increased to 150.69μg / g; the nicotinic acid content of natural fermentation (comparative example) was 34.02μg / g, after enhanced fermentation, T1 increased to 62.17μg / g , T3 increased to 54.33μg / g, T1-T3 increased to 50.89μg / g; organic acid is the key intermediate product of carbohydrate metabolism, and plays a key role in taste characterization. It contributes to the sourness and fruitiness of coffee, while inhibiting bitterness. Relevant studies have shown that one of the characteristics that citric acid can impart is sourness, fruitiness and berry flavor. Both in terms of sensory perception and material content, it can be seen that after the enhanced fermentation of coffee by these two strains of bacteria, the fruity flavor of coffee can be enhanced, thereby improving the quality of coffee.

[0072] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Penicillium ovatum T1, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 24, 2024, and the culture collection number is: CGMCC No.41551.

2. A strain of Bacillus altitudinis T3, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 24, 2024, and the culture collection number is: CGMCC No.32324.

3. A composite microbial preparation, characterized in that: The composite microbial preparation contains the Penicillium ovatum T1 according to claim 1 and the Bacillus subtilis T3 according to claim 2.

4. The composite microbial preparation according to claim 3, characterized in that: In the composite microbial preparation, the concentration of Penicillium ovatum T1 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g; the concentration of the highland Bacillus T3 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

5. A leavening agent, characterized in that The starter contains the Penicillium ovale T1 according to claim 1 and the Bacillus subtilis T3 according to claim 2.

6. The leavening agent according to claim 5, characterized in that In the fermentation agent, the total bacterial concentration of the Penicillium ovale T1 and the Bacillus subtilis T3 is not less than 1×10 7 ~1×10 9 CFU / mL or 1×10 7 ~1×10 9 CFU / g; the addition ratio of the Penicillium ovatum T1 and the Bacillus subtilis T3 is 1:1 to 1:

2.

7. Use of the Penicillium ovale T1 according to claim 1, the Bacillus subtilis T3 according to claim 2, the composite microbial preparation according to claim 3 or 4, or the starter according to claim 5 or 6 in processing coffee, characterized in that: The application is to ferment coffee cherries using the Penicillium ovale T1 described in claim 1, the Bacillus subtilis T3 described in claim 2, the composite microbial preparation described in claim 3 or 4, or the starter described in claim 5 or 6.

8. A method for processing fermented coffee, characterized in that: The method comprises the steps of inoculating the Penicillium ovatum T1 according to claim 1, the Bacillus subtilis T3 according to claim 2, the composite microbial preparation according to claim 3 or 4, or the starter according to claim 5 or 6 into coffee cherries for fermentation, followed by drying and baking.

9. A method for increasing the fruit acid flavor of coffee, characterized in that: The method comprises fermenting coffee cherries using the Penicillium ovatum T1 according to claim 1, the Bacillus subtilis T3 according to claim 2, the composite microbial preparation according to claim 3 or 4, or the starter according to claim 5 or 6.

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

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