A strain of bay yeast, a strain of Hansenula spores in grape juice, a mixed bacteria anaerobic fermentation method using bay yeast and Hansenula spores in grape juice, and coffee beans

Abstract

The present invention provides a strain of Bayan yeast, a strain of Hansen yeast with spores in grape juice, a mixed bacteria anaerobic fermentation method using Bayan yeast and Hansen yeast with spores in grape juice, and coffee beans, and improves the processing technology of Catim coffee beans. The present invention selects mature and undamaged Catim coffee fresh fruits, washes, sorts, and peels to obtain fresh coffee beans. The mixed bacterial suspension is inoculated into fresh coffee beans at a ratio of 1-2%, and anaerobically fermented at 28±2°C for 24-36 hours; after fermentation, the coffee beans are dried at a specific temperature and humidity, and the surface water is dried at a specific temperature and humidity, and the moisture content is 10-12% after two pile treatments, and the beans are shelled and baked to a light or medium degree, and finally Catim coffee powder is obtained. This fermentation method significantly improves the flavor quality of Catim coffee beans, presenting a strong nutty, caramel, and citrus aroma, bright sourness, mellow sweetness, moderate bitterness, mellow and smooth, sweet and refreshing aftertaste, and a cup test score of up to 83.5 points, becoming an efficient and innovative coffee bean processing technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation and deep processing of food, and specifically relates to the screening, acquisition, microbial preservation and mixed preparation of a composite starter (mixed seed bacteria suspension) of a bay yeast strain and a grape juice spore Hansen yeast strain, which is then applied in the process of mixed bacteria anaerobic fermentation of Yunnan Catim coffee beans to obtain high-quality Catim coffee bean products. Background Art

[0002] Catimu coffee beans are a hybrid coffee bean that combines Arabica and Caturra (a variant of Bourbon), with 25% Robusta coffee ancestry. Catimu coffee beans are characterized by high yield, short plants, and dense planting; because they inherit the Robusta gene, Catimu coffee beans are better able to resist coffee berry disease, coffee leaf rust, and have stronger resistance to diseases and pests.

[0003] Catimu coffee beans carry Robusta genes and are considered to have a "devil's aftertaste". Some coffee tasters believe that Catimu coffee beans have a sour flavor, which affects the overall flavor balance; the diversity and concentration of aromatic compounds are insufficient, and Catimu coffee beans lack the complex flavor layers brought by sweetness and rich aromatic substances; after roasting, Catimu coffee beans have a relatively rough texture and lack a delicate, smooth, and mellow taste. However, the influence of coffee bean genes is smaller than the interaction between the planting environment and field management. The planting environment (altitude, soil, climate) and field management (fertilization, disease and pest control) play a key role in the overall quality of coffee beans.

[0004] The FAO Coffee Commodity Notes and the International Coffee Organization (ICO) both provide yield data and planting performance for different coffee varieties. The average yield of Catimum coffee beans is 2.5-3.5 tons per hectare, while the average yield of pure Arabica coffee is 1.5-2.0 tons per hectare. Catimum coffee beans generally begin to bear fruit after 2-3 years, while Arabica coffee takes 3-4 years to bear fruit. Therefore, the high yield and shorter growth cycle of Catimum coffee give it a significant advantage in commercial planting.

[0005] The field survey report of the Agricultural Technology Extension Center and the Coffee Growers Association records in detail the performance of Catimu coffee in the actual planting environment. Yunnan is the main production area of ​​Catimu coffee beans, which are generally planted at an altitude of 1,200 to 1,400 meters. After more than 20 years of cultivation, the Catimu coffee bean variety has adapted to the soil and water of Yunnan, and the grassy smell, mixed bitterness and astringency are not easy to taste. At present, the Catimu coffee beans in the Yunnan production area are of excellent quality, with a high body and a nutty and chocolate aroma. Yunnan Catimu coffee beans have been widely accepted by coffee bean hunters and coffee tasters, and Yunnan Catimu coffee beans are recognized as one of the boutique coffee bean varieties.

[0006] In the processing of Catim coffee beans, the two most commonly used processing techniques are dry processing (natural drying) and wet processing (washing). Dry processing, also known as natural processing or sun drying, is to dry the whole coffee fruit in the sun, and the coffee beans are fermented in the skin. Wet processing is to soak the harvested fruits in water, remove the bad beans floating on the water surface, then remove the skin and pulp, ferment and wash the coffee beans. There is also honey processing, which is a method between dry processing and wet processing; in honey processing, some or all of the pulp is retained around the coffee beans and then dried.

[0007] Kopi luwak is a representative product among the existing fermented coffees. After the coffee fruit is eaten by the civet, the civet's digestive system. In the civet's digestive system, the coffee beans come into contact with digestive juices and enzymes, and can only digest the surface of the coffee fruit. The hard coffee beans inside the fruit cannot be digested, and the undigested coffee beans are excreted with feces. The enzymes in the civet's gastric juice and saliva have a chemical effect on the coffee beans, and the digestive juice affects the chemical structure of the coffee beans, especially the decomposition of proteins. Therefore, the saliva and gastric juice of the civet are mixed with the coffee during the digestion process, and its protein is decomposed by the enzymes in the civet's intestines. Protein is one of the main sources of bitterness in coffee. After decomposition, it will reduce bitterness and increase sweetness and aroma. Therefore, Kopi luwak tastes sweet and has a special aroma. However, the Kopi luwak produced by combining biological digestion and fermentation has a small output and high cost, so it is expensive, the consumer group is not wide, the market share is small, and the popularity is low. Summary of the invention

[0008] The technical problems to be solved by the present invention include: the existing natural fermentation of coffee leads to uncontrollable coffee flavor and unstable coffee quality; the natural fermentation cycle is long, the fermentation degree is uncontrollable, over-fermentation or under-fermentation; the dominant bacteria species in the fermentation stage are uncontrollable, and molds and Fusarium are easy to contaminate coffee, produce mucormycetes and fumonisins, which affect the quality of coffee and endanger human health.

[0009] In order to solve the problems and shortcomings in the above-mentioned prior art, the present invention discloses a mixed anaerobic fermentation method using Saccharomyces bayanus and Hansenula spores in grape juice. According to the preparation method, the above-mentioned technical shortcomings can be effectively avoided; the obtained coffee beans are of excellent quality and have been professionally cupped with a tasting score of 83.5 points.

[0010] The first object of the present invention is to Saccharomyces bayanus ) strains and Hansenula sporogenes ( Hanseniaspora grapes ) strains were screened and obtained, and the strains were preserved. The acquisition methods and identification results of these two strains are attached.

[0011] The second purpose of the present invention is to prepare a mixed seed bacterial suspension using the two strains (Saccharomyces bayanus and Hansenula spores) obtained by the present invention.

[0012] The third object of the present invention is to provide a mixed anaerobic fermentation method using Saccharomyces bayanus and Hansenula vitis spores, by applying a composite fermentation agent (mixed seed bacteria suspension) to carry out mixed anaerobic fermentation in the preparation process of Yunnan Catim coffee beans, and combining the honey treatment method to process coffee, so as to avoid the disadvantages of the existing traditional processing methods and improve the flavor of Catim coffee beans.

[0013] The fourth purpose of the present invention is to obtain a high-quality Catim coffee through the fermentation method, which has the fructose sweetness naturally retained in the honey processing process, which is natural, long and delicate; the fruity aroma of the coffee itself is retained, and after the introduction of excellent yeast for fermentation, the flavor of tropical fruits is added, with the fresh aroma of citrus and rich nuts and caramel aroma, and the taste is fuller; in addition, the coffee has a bright acidity, a mellow sweetness, a moderate bitterness, a perfect balance, a smooth and delicate taste, a sweet and refreshing aftertaste, and a long and lasting aftertaste, forming a pleasant aftertaste; the tasting score is 83.5 points.

[0014] Specifically, the first purpose of the present invention is to screen and obtain endophytes or dominant bacteria, and to explore endophytes or dominant bacteria in coffee by mining coffee microbial resources. Therefore, two technical solutions are formed, as follows:

[0015] (1) A Bay's yeast Saccharomyces bayanus ) and strain preservation information.

[0016] A Bay's yeast Saccharomyces bayanus ), numbered CI-03, and deposited in the General Microbiological Center of China Microbiological Culture Collection Administration. CI-03 Saccharomyces cerevisiae was named Saccharomyces bayanus The deposit number is CGMCC No: 30565, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] (2) A kind of Hansen yeast with spores in grape juice ( Hanseniaspora grapes ) and strain preservation information.

[0018] A kind of Hansenula sporogenes of grape juice ( Hanseniaspora grapes ), numbered LI-08, named Hanseniaspora grapes , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 30563, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0019] A Bay's yeast Saccharomyces bayanus The method for screening and obtaining the strains of ) is as follows:

[0020] a. Select red grape samples: Select mature Binchuan red grapes from Dali Prefecture, Yunnan Province, China, collect them into sterilized sample bags, quickly transfer them to low-temperature boxes for storage, and transport them to the laboratory at low temperature;

[0021] b. Binchuan red grape sample separation: Weigh an appropriate amount of Binchuan red grape sample, place it in 90 mL of sterile physiological saline, and mix it by shaking to disperse the microbial cells in the Binchuan red grape sample;

[0022] c. Binchuan red grape sample gradient dilution: Use sterile saline to gradient dilute the Binchuan red grape mixed liquid;

[0023] d. Spreading and culture: Spread 200 μL of the dilution on a yeast extract peptone glucose agar plate and culture the yeast at 30°C;

[0024] e. Observe the colony morphology: Figure 1 As shown, the dominant strains were selected by observing the morphology, size, color, gloss, texture, surface and edge characteristics of the colonies;

[0025] f. Isolation and purification to obtain single colonies: The strain was further purified by the cross-streaking method to obtain single colonies;

[0026] g. Cryopreservation of dominant strains (isolates): Isolates with consistent morphology are cryopreserved for subsequent identification.

[0027] A kind of Hansenula vitis spores yeast ( Hanseniaspora grapes The strain screening and acquisition methods of ) are as follows:

[0028] A. Selection of coffee cherry samples: Mature coffee cherries (Coffea arabica var. Typica) were harvested at an altitude of 1,300 meters in Pu'er, Yunnan Province, China. After washing, peeling and degumming, they were collected into sterilized sample bags and quickly transferred to low-temperature boxes for storage. They were then transported to the laboratory at low temperature.

[0029] B. Isolation of Arabica coffee beans: Weigh 10 g of Arabica coffee beans; place in 90 mL of sterile saline and shake to disperse the microbial cells in the coffee bean sample;

[0030] C. Gradient dilution of Arabica coffee beans sample: gradient dilution of Arabica coffee beans mixed liquid with sterile saline;

[0031] D. Spreading and culture: Take 200 μL of the dilution and spread it on a yeast extract powder peptone glucose agar plate and culture the yeast at 30 ℃;

[0032] E. Observe the colony morphology: Figure 2 As shown, the dominant strains were selected by observing the morphology, size, color, gloss, texture, surface and edge characteristics of the colonies;

[0033] F. Isolation and purification to obtain single colonies: The strain was further purified by the cross-streaking method to obtain single colonies;

[0034] G. Cryopreservation of dominant strains (isolates): Isolates with consistent morphology are cryopreserved for subsequent identification.

[0035] Further, a Bayer's yeast ( Saccharomyces bayanus ) and a strain of Hansenula vitis spores ( Hanseniaspora grapes ) strain identification method is to accurately identify the species of microorganisms through a series of molecular biological technical steps, including DNA extraction, PCR amplification, sequencing and comparative analysis. This method can not only efficiently identify microorganisms, but also provide a reliable scientific basis for the classification, research and application of microorganisms. In the microbial fermentation process of Yunnan Catimu coffee beans, using this method to screen and identify dominant strains can significantly improve the fermentation efficiency and sensory quality of Yunnan Catimu coffee beans.

[0036] Binchuan red grape sample strain (a Bayer yeast ( Saccharomyces bayanus The main goal of the process is to isolate and extract endogenous microbial DNA, especially yeast, from coffee bean samples; the focus of the process is to avoid cross-contamination and false positive results and ensure DNA purity. Saccharomyces bayanus The specific steps of the strain identification method are as follows:

[0037] Phase 1: DNA Extraction

[0038] DNA extraction is to extract DNA from microbial samples using the TSINGKE Plant DNA Extraction Kit (Universal). The universal extraction method provided by the kit can adapt to a variety of sample types. Through appropriate pre-treatment (such as gradient dilution and removal of interfering substances), high-quality DNA can be effectively extracted to provide a reliable template for subsequent PCR amplification and sequencing.

[0039] (1) Add 200 μL Buffer ATL and 20 μL Proteinase K to the grinding tube, add half a spoon of 3 mm zirconium beads or one 5 mm steel bead (depending on the status of the bacterial plate, bacteria can be added with zirconium beads, fungal hyphae and anaerobic bacteria need to be added with steel beads), take a single colony from the bacterial plate into the grinding tube, and grind it on an automatic grinder at 60 Hz for 2 min;

[0040] (2) Centrifuge briefly, add 200 μL Buffer AL, and mix thoroughly;

[0041] (3) Incubate in a 70°C water bath for 15 min, oscillate and mix twice. Centrifuge at 12,000 rpm for 3 min after lysis is complete. Transfer 400 μL of supernatant to a new deep-well plate. Add 300 μL of Bufer BD and 20 μL of magnetic beads to the deep-well plate and place it in station 2 of the extractor.

[0042] (4) Take a new extraction plate and dispense 300 μL of Buffer BW1 into each well and place it at station 3 of the extraction instrument. Dispense 75% ethanol into two plates with 500 μL wells and place them at stations 4 and 5 respectively. Dispense 100 μL / 80 μL / 60 μL of elution into each well of the elution plate (dispense the corresponding volume according to the sample status) and place it at station 6.

[0043] Note: Buffer BD and Buffer BW1 need to be diluted with a specified amount of anhydrous ethanol before use;

[0044] (5) Select the corresponding program, check the instrument status and extraction plate information, and then run it;

[0045] (6) After the run is completed, remove the elution plate for DNA concentration and electrophoresis gel testing and store it at 4°C.

[0046] The second stage: PCR amplification. Use the universal primers for bacterial species identification to amplify, and analyze the amplified products by agarose gel electrophoresis to see whether the PCR product bands are consistent with the target size, whether they are single, and whether there are dragging bands. The universal primers for bacterial species identification are shown in Table 1 below:

[0047] Table 1

[0048]

[0049] The ITS site PCR amplification system and amplification procedure are shown in Table 2 below:

[0050] Table 2

[0051]

[0052] Phase 3: PCR product sequencing: After the PCR product is qualified, the target band is cut and purified, and the recovered product is used for Sanger sequencing.

[0053] Phase 4: Comparison and analysis of sequencing results

[0054] S1. The Sanger sequencing results were spliced ​​using the software ContigExpress, and the inaccurate parts at both ends were removed;

[0055] S2. Perform blastn (latest version v2.13) on the spliced ​​sequences in batches to compare with the nucleic acid database. The nucleic acid database uses the latest version of the nt library. By performing blastn comparison with the nt library, the AccessionNumber of the homologous sequence and the species identification and annotation can be obtained.

[0056] Phase 5: Sequencing results are shown in Table 3 below:

[0057] Table 3

[0058]

[0059] Specifically, Figure 1 As shown, morphological observation of the isolates cultured on the plate showed that Saccharomyces cerevisiae CI-03 exhibited typical yeast characteristics. The CI-03 colonies formed milky white round particles with a smooth surface. The Gram staining results of CI-03 were all positive.

[0060] Finally, based on the molecular biology and morphological results, CI-03 was identified as Saccharomyces bayanus ( Saccharomyces bayan ), and the above yeast was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and named CⅠ-03 Saccharomyces bayanus The deposit number is CGMCC No: 30565, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0061] Arabica coffee beans of the Typica variety were sampled from a strain of Hansenula viticola ( Hanseniaspora grapesThe main goal of the method is to isolate and extract endogenous microbial DNA, especially yeast, from coffee bean samples; the specific steps of a method for identifying the strain of Hanseniaspora uvarum are as follows:

[0062] Phase 1: DNA extraction. DNA extraction is to extract DNA from microbial samples using the TSINGKE Plant DNA Extraction Kit (Universal). The universal extraction method provided by the kit can adapt to a variety of sample types. Through appropriate pre-treatment (such as gradient dilution and removal of interfering substances), high-quality DNA can be effectively extracted to provide a reliable template for subsequent PCR amplification and sequencing.

[0063] S (1). Add 200 μL Buffer ATL and 20 μL proteinase K to the grinding tube, add half a spoon of 3 mm zirconium beads or one 5 mm steel bead (depending on the status of the bacterial plate, bacteria can be added with zirconium beads, fungal hyphae and anaerobic bacteria need to add steel beads), take a single colony from the bacterial plate into the grinding tube, and grind it on an automatic grinder at 60 Hz for 2 min;

[0064] (2) Centrifuge briefly, add 200 μL Buffer AL, and mix thoroughly;

[0065] (3) Incubate in a 70°C water bath for 15 min, oscillate and mix twice. Centrifuge at 12,000 rpm for 3 min after lysis is complete. Transfer 400 μL of supernatant to a new deep-well plate. Add 300 μL of Bufer BD and 20 μL of magnetic beads to the deep-well plate and place it in station 2 of the extractor.

[0066] 4). Take a new extraction plate and dispense 300 μL of Buffer BW1 into each well and place it at station 3 of the extraction instrument. Dispense 75% ethanol into two plates with 500 μL wells and place them at stations 4 and 5 respectively. Dispense 100 μL / 80 μL / 60 μL (dispense the corresponding volume according to the sample status) into each well of the elution plate and place the elution solution at station 6.

[0067] Note: Buffer BD and Buffer BW1 need to be diluted with a specified amount of anhydrous ethanol before use;

[0068] (5) Select the corresponding program, check the instrument status and extraction plate information, and then run it;

[0069] (6) After the run is completed, remove the elution plate for DNA concentration and electrophoresis gel testing and store it at 4°C.

[0070] The second stage: PCR amplification. PCR amplification. Use the universal primers for bacterial species identification shown in Table 1 for amplification, and analyze the amplified products by agarose gel electrophoresis to determine whether the PCR product bands are consistent with the target size, whether they are single, and whether there are dragging bands.

[0071] The ITS site PCR amplification system and amplification program are shown in Table 2:

[0072] Phase 3: PCR product sequencing: After the PCR product is qualified, the target band is cut and purified, and the recovered product is used for Sanger sequencing.

[0073] Phase 4: Comparison and analysis of sequencing results.

[0074] S1. The Sanger sequencing results were spliced ​​using the software ContigExpress, and the inaccurate parts at both ends were removed.

[0075] S2. Perform blastn (latest version v2.13) on the spliced ​​sequences in batches to compare with the nucleic acid database. The nucleic acid database uses the latest version of the nt library. By performing blastn comparison with the nt library, the AccessionNumber of the homologous sequence and the species identification and annotation can be obtained.

[0076] Phase 5: The sequencing results are shown in Table 4.

[0077] Table 4

[0078]

[0079] Specifically, the morphological observation of the isolates cultured on the plate showed that Hansenula vitis spores LI-08 showed typical yeast characteristics. LI-08 colonies formed white, sticky particles with a shiny surface and rounded edges (see Figure 2 ). Gram staining results of LI-08 were all positive.

[0080] Finally, the molecular biology and morphological results were combined to identify LI-08 as Hansenula vitis spores ( Hanseniaspora grapes ), and the above-mentioned Hansenula spores in grape juice were preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and LI-08 was named Hansenula spores in grape juice Hanseniaspora grapes The deposit number is CGMCC No: 30563, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0081] Furthermore, the second object of the present invention is to prepare a mixed seed bacterial suspension using the two strains (Saccharomyces bayanus combined with Hansenula spores) obtained in the first object of the present invention.

[0082] Phase 1: Preparation of mixed seed suspension

[0083] S1. Frozen strains: Purified and identified Saccharomyces bayanus (CⅠ-03) and Hansenula spores (LI-08) strains were frozen in glycerol tubes at -80℃;

[0084] S2. Medium preparation: Prepare YPD liquid medium, which is composed of peptone, glucose, and yeast extract powder and is suitable for the growth of yeast; this medium provides rich nutrients and supports the rapid proliferation of Saccharomyces bayanus and Hansenula sporeifera;

[0085] Phase II: Inoculation and activation of strains in mixed seed suspension

[0086] S3. Thawing of strains: Take out two frozen strains (Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporangiophora vinifera (LI-08 strain) from a -80℃ freezer and thaw them quickly at room temperature; in this step, the thawing process of Saccharomyces cerevisiae and Hansenula sporangiophora vinifera is slightly different to ensure that each strain is inoculated in the best state. The thawing time of Saccharomyces cerevisiae is 60 minutes, and the thawing time of Hansenula sporangiophora vinifera is 40-60 minutes; and the inoculation amount needs to be adjusted according to the growth rate and activity of each strain to ensure the best growth. The inoculation amount of Saccharomyces cerevisiae is 50-100 microliters, and the inoculation amount of Hansenula sporangiophora vinifera is 50-100 microliters; the freezing and thawing process will affect the activity of each strain, and the subsequent two consecutive generations of cultivation will help to restore the growth vitality of the strain to ensure that it is in the best state for subsequent fermentation;

[0087] S4. strain inoculation: thawed Saccharomyces bayanus and Hansenula vitis spores were inoculated into YPD liquid culture medium, respectively. YPD culture medium usually contains peptone, glucose, and yeast extract powder to provide nutrition for the growth of yeast;

[0088] S5. Set the strain culture conditions: Place the inoculated culture medium in a constant temperature shaking incubator with a culture temperature of 28±2°C and a shaking speed of 180±20 rpm for 24-36 hours. In this step, although the culture time is set to 24-36 hours, it will be slightly adjusted each time based on the observed growth status and culture effect to ensure that the mixed strain achieves optimal activity.

[0089] S6. Monitoring the growth of strains: Observe the growth of each strain (Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores of grape juice (LI-08 strain) to ensure that the strains are in the logarithmic growth phase;

[0090] The third stage: Cultivate and activate the strain, improve the activity of the bacterial suspension, and ensure that the strain grows well. This continuous subculture stage requires strict control of the subculture time and inoculation volume to ensure that each generation of strains is subcultured during the logarithmic growth phase to obtain the best growth state.

[0091] S7. Primary culture: Incubate at 28±2℃ in a constant temperature shaking incubator at 180±20 rpm for 24-36 hours; monitor and observe the growth of each strain in real time; during the primary culture, regularly observe the turbidity and growth of the culture solution, and the increase in turbidity of the culture solution indicates the growth of yeast; within 24-36 hours of culture, the culture solution reaches the logarithmic growth phase, at which time the yeast grows fastest and has the highest activity;

[0092] S8. First subculture: Take an appropriate amount of bacterial liquid from the primary culture medium and inoculate it into a new YPD liquid culture medium at a ratio of 1:100. Ensure that the operation is carried out in a sterile environment to avoid contamination. At this time, the inoculated culture medium needs to be placed in a constant temperature shaking incubator, set the temperature to 28±2℃, the speed to 180±20 rpm, and continue to culture for 24-36 hours. Repeat the same method, monitor and observe the turbidity and growth of the culture medium in real time to ensure that the yeast reaches the logarithmic growth phase.

[0093] S9. Second subculture: Take an appropriate amount of bacterial liquid from the first generation culture medium and inoculate it into a new YPD liquid culture medium. The common ratio is 1:100. Ensure that the operation is carried out in a sterile environment to avoid contamination. At this time, the inoculated culture medium needs to be placed in a constant temperature shaking incubator, set the temperature to 28±2℃, the speed to 180±20 rpm, and continue to culture for 24-36 hours. Repeat the same method, monitor and observe the turbidity and growth of the culture medium in real time to ensure that the yeast reaches the logarithmic growth phase.

[0094] The fourth stage: centrifugation. The purpose of centrifugation is to separate the microorganisms in the culture medium from the liquid culture medium so as to collect the precipitated bacterial sludge (bacterial cells) for subsequent bacterial suspension preparation and fermentation process;

[0095] S10. Preparation before centrifugation: Pour the culture fluids after two generations of culture into centrifuge tubes respectively, ensure that the mass in each centrifuge tube is the same, and place the centrifuge tubes symmetrically in the rotor of the centrifuge to ensure balanced operation of the centrifuge and avoid mechanical damage or reduction of centrifugal efficiency due to imbalance;

[0096] S11. Set the centrifugation conditions and start: the centrifugation speed is 4000-6000 rpm, the centrifugation time is 10-15 minutes, and the centrifugation temperature is 4°C; after completing the settings, start the centrifuge, run the centrifuge according to the set conditions, and start centrifugation;

[0097] S12. Centrifugal operation process: The microorganisms in the suspension are separated by centrifugal force; the centrifugal force generated by high-speed rotation causes the microbial cells with higher density to move to the bottom of the centrifuge tube to form a precipitate;

[0098] S13. Remove the supernatant and collect the bacterial sludge: Gently pour off the supernatant and retain the bacterial sludge precipitated at the bottom of the centrifuge tube;

[0099] S14. Prepare mixed seed bacterial suspension: dilute the collected bacterial mud with sterile saline and mix them well, and adjust the concentration of single bacterial suspension to 10 8 CFU / mL, that is, the concentration of the Bayer Saccharomyces suspension is 10 8 CFU / mL, the concentration of Hansenula spore suspension in grape juice is 10 8 CFU / mL; the bacterial suspension of Bayan Saccharomyces cerevisiae (CⅠ-03) and Hansenula viticola (LI-08) were mixed in a volume ratio (v) of 1:1; the final concentration of the mixed seed bacterial suspension was 0.5×10 8 CFU / mL (i.e., each milliliter of bacterial suspension contains 10 8 colony forming units) for subsequent fermentation.

[0100] Furthermore, the third object of the present invention is to provide a mixed anaerobic fermentation method using Saccharomyces bayanus and Hansenula vitis spores, by applying a composite fermentation agent (mixed seed bacteria suspension) for anaerobic fermentation in the preparation process of Yunnan Catim coffee beans, and combining the honey treatment method to process coffee, so as to avoid the drawbacks of existing traditional processing methods and improve the flavor of Catim coffee beans.

[0101] Specifically, a coffee bean preparation method using mixed anaerobic fermentation of Saccharomyces bayanus and Hansenula spores of grape juice is realized by three stages: preparation before fermentation (pretreatment), fermentation and subsequent treatment:

[0102] (1) Pre-processing stage: selecting fresh Catim coffee berries, washing, sorting, and peeling.

[0103] (2) Fermentation stage: inoculation and anaerobic fermentation.

[0104] (3) Subsequent processing stage: drying of surface water, double fermentation, shelling, roasting and grinding of coffee beans after drying.

[0105] Specifically, the coffee beans and preparation method using mixed anaerobic fermentation of Saccharomyces bayanus and Hansenula spores in grape juice described in the present invention are achieved by the following technical solutions:

[0106] (1) Pre-processing stage: selecting fresh Catim coffee berries, washing, sorting, and peeling.

[0107] S1. Select Catimu coffee berries: Select ripe Catimu coffee berries, making sure they are full and have uniform color.

[0108] Remove immature fruits, shrunken fruits, fruit with diseases and insect pests, and other foreign objects; select fresh Catim fruit that is free of frost, microbial infection, and mechanical damage;

[0109] S2. Fresh fruit washing: Wash the picked Catim fruit with clean water to remove the dirt and impurities on the surface;

[0110] S3. Fresh fruit sorting: remove floating fruits and foreign objects to ensure that the remaining Catim fruit is healthy and undamaged;

[0111] S4. Peeling the fresh fruit: After cleaning, peel the Catim coffee fruit to obtain fresh coffee beans;

[0112] (2) Fermentation stage: using the prepared mixed bacterial suspension, inoculating (or inoculating), and anaerobic fermentation.

[0113] S5. Inoculation: Add the prepared mixed seed suspension to the fresh coffee beans and mix evenly at an inoculation rate of 1-2% (based on the weight of the fresh coffee beans);

[0114] S6. Anaerobic fermentation: Place the mixed coffee beans into a food-grade thickened plastic bag (15 filaments, filled with nitrogen); perform anaerobic fermentation at 28±2°C, mix once every 6-8 hours; fermentation time is 24-36 hours;

[0115] (3) Subsequent processing stage: drying of surface water, double fermentation, shelling, roasting and grinding of coffee beans after drying.

[0116] S7. Dry the surface water: Dry in an environment with a temperature of 24-32 ℃ and a humidity of 30-40%; spread the fermented coffee beans and place them evenly on the drying rack to dry the surface moisture for about 2-4 days; the thickness of each layer of coffee beans should be moderate, and each coffee bean has enough space for ventilation and drying, avoiding overlap and accumulation; the spreading thickness should be between 2-3 cm; turn the coffee beans at least 2-3 times a day to ensure uniform drying of the coffee beans;

[0117] S8. First fermentation: Pile the coffee beans to a certain height (30-40cm) in an environment with a temperature of 18-22 ℃ and a humidity of 50-60% for 24 hours; during the stacking process, ensure that the coffee pile is well ventilated to prevent local overheating and mold growth; spread the coffee beans to a thickness of about 2-3cm in an environment with a temperature of 28-32 ℃ and a humidity of 30-40% for 24 hours;

[0118] S9. After the first fermentation, turn the pile and turn the outer coffee beans to the inside for the second fermentation. The conditions and methods for the second fermentation are the same as S8, that is, in an environment with a temperature of 18-22 ℃ and a humidity of 50-60%, pile the coffee beans to a certain height (30-40cm) for 24 hours; during the stacking process, ensure that the coffee pile is well ventilated to prevent local overheating and mold growth; in an environment with a temperature of 28-32 ℃ and a humidity of 30-40%, spread the coffee beans to a thickness of about 2-3cm for 24 hours;

[0119] S10. Shelling after drying: Dry the coffee beans to a moisture content of 10-12%; when the coffee beans reach the required moisture content, shell them to obtain fermented coffee beans; ensure good ventilation in the spreading area to accelerate the evaporation of moisture and the drying process of the coffee beans; ensure that the entire process is carried out in an environment with good sanitary conditions to prevent the coffee beans from being contaminated by the outside world; regularly monitor and check the dryness of the coffee beans in real time, and adjust the fermentation and drying time and conditions when necessary;

[0120] S11. Roasting and grinding: roasting the shelled coffee beans to a degree ranging from light roasting to medium roasting; the roasted coffee beans are cooled and then ground to obtain the final Catim coffee powder product.

[0121] Furthermore, the fourth object of the present invention is to obtain a high-quality Catim coffee bean product (product) by an anaerobic fermentation method using mixed bacteria of Saccharomyces cerevisiae and Hansenula vitis spores; the Catim coffee prepared by the present invention has the fructose sweetness naturally retained in the honey processing process, which is natural, long and delicate; the fruity aroma of the coffee itself is retained, and after the introduction of excellent yeast for fermentation, the flavor of tropical fruits is added, with the fresh aroma of citrus and rich nuts and caramel aroma, and the taste is fuller; in addition, the coffee has a bright acidity, a mellow sweetness, a moderate bitterness, a perfect balance, a smooth and delicate taste, a sweet and refreshing aftertaste, and a long and lasting aftertaste, forming a pleasant aftertaste; the tasting score is 83.5 points.

[0122] In addition, according to the first object of the present invention, the biological characteristics of the Saccharomyces cerevisiae (CI-03) strain are as follows:

[0123] First, the single colony of Bayer's Yeast (CⅠ-03) is small, white to light yellow, round, smooth and shiny, and easy to pick up; the cell morphology of Bayer's Yeast (CⅠ-03) is oval to elliptical, about 5-10 microns long and about 3-6 microns wide, and it mainly reproduces by budding.

[0124] Secondly, Bay's yeast (CI-03) includes specific physiological and biochemical properties and polysaccharide components. Specifically, Bay's yeast (CI-03) was subjected to a variety of tests, and the test results are as follows: gelatin liquefaction test, H 2 O 2 The results of enzyme activity and pH culture tests were positive, while the results of hydrogen sulfide test and motility test were negative. Specifically, Saccharomyces cerevisiae (CⅠ-03) can ferment a variety of sugars and produce ethanol and carbon dioxide. The specific fermentation characteristics are: fermentation of glucose, fermentation of fructose, fermentation of sucrose, fermentation of maltose, fermentation of xylose, fermentation of mannose, fermentation of raffinose, fermentation of trehalose, fermentation of galactose, and fermentation of melibiose are positive, while fermentation of inulin and fermentation of lactose are negative.

[0125] Third, the main polysaccharides contained in Bay's yeast (CⅠ-03) and their corresponding functions are as follows: Glucans include α-glucan and β-glucan, which are important components of the cell wall and have the function of maintaining the mechanical strength of the cell wall; Mannan is one of the main polysaccharides in the cell wall, combining 5-50% of protein to form a mannan-protein complex, which constitutes the outer structure of the yeast cell wall; Galactan plays a structural support and stabilization role in the cell wall; Other storage polysaccharides such as glycogen are stored in the cytoplasm as energy reserves. In addition, although Bay's yeast (CⅠ-03) cannot ferment inulin, inulin exists in the cell during storage and metabolism.

[0126] It is concluded that Bayer's yeast (CⅠ-03) has a strong ability to decompose sugars, can efficiently produce ethanol, carbon dioxide and a variety of aromatic compounds (such as esters, alcohols and ketones), and shows efficient fermentation activity and aroma production during the fermentation process. Its acid resistance and alcohol resistance enable it to maintain activity at lower pH values ​​and higher alcohol concentrations, and it is an excellent strain commonly used in food and industrial fermentation.

[0127] According to the first object of the present invention, the biological characteristics of the Hansenula sporogenes (LI-08) strain are as follows:

[0128] First, the single colony of Hansenula sporangiophora (LI-08) is medium in size, light yellow to milky white, round, with a moist and smooth surface and flat edges; the cell morphology of Hansenula sporangiophora (LI-08) is oval to elliptical, about 4-8 microns in length and about 2-5 microns in width, and it mainly reproduces by budding.

[0129] Secondly, Hansenula sporangiophora (LI-08) has specific physiological and biochemical characteristics and polysaccharide components. Specifically, Hansenula sporangiophora (LI-08) was subjected to a variety of tests, and the test results are as follows: gelatin liquefaction test, H 2 O 2The results of enzyme activity and pH culture tests were positive, while the results of hydrogen sulfide test and motility test were negative. Specifically, Hansenia sporei in grape juice (LI-08) can utilize a variety of sugars during the fermentation process, and the specific fermentation characteristics are as follows: fermentation of glucose, fermentation of fructose, fermentation of sucrose, fermentation of maltose, fermentation of xylose, fermentation of mannose, fermentation of raffinose, fermentation of trehalose, fermentation of galactose, and fermentation of melibiose are positive, while fermentation of inulin and fermentation of lactose are negative.

[0130] Third, the main polysaccharides contained in Hansen yeast (LI-08) in grape juice and their corresponding functions are as follows: Glucans include α-glucan and β-glucan. β-glucan exists in the cell wall and has immune regulation and antioxidant functions; α-glucan also exists in the cell wall, but in a lower proportion. Mannan, as an important component of the cell wall, plays a key role in maintaining cell structure and function. Galactan plays a structural support and stabilization role in the cell wall, enhancing the toughness and elasticity of the cell wall. Glycogen can be stored in the cytoplasm as the main storage carbon source and energy source of yeast cells. It is broken down to provide energy during metabolism. Other storage polysaccharides include some special storage polysaccharides, such as trehalose, which play an important role in coping with environmental stress and protecting cell function.

[0131] It is concluded that the colony characteristics, cell morphology and physiological and biochemical test results of Hansenula spores in grape juice (LI-08) show that it is a yeast strain with strong adaptability and active metabolism. The main polysaccharide components contained in Hansenula spores in grape juice (LI-08) play an important role in maintaining cell structure and function, providing energy and coping with environmental stress, making Hansenula spores in grape juice (LI-08) have significant application value in the fermentation process.

[0132] In addition, according to the technical solution of the present invention, the mechanism of using the mixed seed suspension of Saccharomyces bayanus (CI-03) strain and Hansenula spores of grape juice (CIII-23) strain is as follows:

[0133] From the perspective of the metabolic pathway of mixed fermentation,

[0134] (1) Glycolysis and alcohol fermentation pathway (main contribution of Saccharomyces cerevisiae): Under anaerobic conditions, Saccharomyces cerevisiae (CⅠ-03) converts glucose into ethanol and carbon dioxide through glycolysis and alcohol fermentation pathways.

[0135] Glycolysis: Bay Saccharomyces cerevisiae converts glucose into pyruvate through glycolysis to generate ATP and NADH, which is a key process for energy generation. Adenosine triphosphate (ATP) is the main energy currency of the cell and provides energy for various biological activities of yeast cells; nicotinamide adenine dinucleotide (NADH) is reoxidized to NAD⁺ during the fermentation process, and the released energy is used for other metabolic activities.

[0136] (Glucose → Pyruvate) C 6 H 12 O 6 →2C 3 H 4 O 3 +2ATP+2NADH;

[0137] Alcohol fermentation: Under anaerobic conditions, pyruvate is converted into acetaldehyde and carbon dioxide, and then acetaldehyde is reduced to ethanol. This process regenerates NAD⁺, allowing glycolysis to continue. Ethanol is one of the main metabolites, which is produced during the fermentation process and accumulates in the fermentation environment. The carbon dioxide produced will escape the fermentation liquid and partially dissolve in the fermentation liquid, affecting the gas composition of the fermentation environment.

[0138] (pyruvate → acetaldehyde + carbon dioxide) C 3 H 4 O 3 →CH 3 CHO+CO 2 ;

[0139] Ethanol is one of the main fermentation products of Saccharomyces cerevisiae. It has antibacterial effects and can inhibit the growth of certain harmful microorganisms, thereby helping to maintain the stability of the fermentation environment.

[0140] (acetaldehyde → ethanol) CH 3 CHO+NADH→C 2 H 5 OH+NAD + .

[0141] (2) Pathway for the production of organic acids and aromatic compounds (main contribution of Hansenulax sporeans): Hansenulax sporeans (CⅢ-23) produces organic acids and aromatic compounds through a variety of metabolic pathways.

[0142] Lactic acid production: The lactic acid produced by Hansenula sporogenes in grape juice helps to lower the pH value of the fermentation environment, inhibit the growth of adverse microorganisms, and stabilize the fermentation environment.

[0143] (Pyruvate → Lactate) C 3 H 4 O 3 +NADH→C 3 H 6 O 3 +NAD + ;

[0144] Acetic acid production: Acetic acid has antibacterial effects at low concentrations, which can inhibit the growth of harmful microorganisms and maintain the health of the fermentation environment.

[0145] (Ethanol → Acetic acid) C2 H 5 OH+O 2 →CH 3 COOH+H 2 O;

[0146] Ethyl acetate production: Ethyl acetate is an important ester aromatic compound that gives fermented coffee its unique fruity and floral aroma.

[0147] (acetic acid + ethanol → ethyl acetate) CH 3 COOH+C 2 H 5 OH→CH 2 C0OC 2 H 5 +H 2 O;

[0148] Production of phenolic compounds (from phenylalanine): This phenolic compound is produced by Hansenula sporogenes in grape juice and has a unique aroma, enriching the flavor level of coffee.

[0149] (Phenylalanine → 4-ethylphenol) C 9 H 11 NO 2 →C 8 H 1O O+CO 2 +NH 3

[0150] Through the above metabolic pathways and chemical reactions, Saccharomyces bayanus and Hansenula sporei work together to produce rich metabolites, significantly improving the flavor and quality of coffee beans. These metabolic activities not only optimize the fermentation process, but also ensure the quality and flavor consistency and stability of the final product.

[0151] In addition, according to the technical solution of the present invention, the mechanism of mixed bacteria anaerobic fermentation using Saccharomyces bayanus (CI-03) strain and Hansenula spores of grape juice (LI-08) strain is as follows:

[0152] Specifically, the acquisition principles of Saccharomyces bayanus (CⅠ-03) and Hansenula sporei (LI-08) strains mainly include sample collection, microbial separation and purification, identification and preservation. Although the acquisition process of these two yeasts is different in specific samples and identification methods, their core principles are similar, and both use microbial separation and purification, morphological observation, physiological and biochemical identification, and molecular biological identification steps to obtain high-purity target strains for subsequent fermentation research and application.

[0153] Specifically, the advantages of Saccharomyces cerevisiae (CⅠ-03) are its strong ability to decompose sugars and produce aroma. On the one hand, Saccharomyces cerevisiae can efficiently decompose sugars in coffee beans and produce a variety of metabolites, including ethanol and acid compounds, which contribute to the fruity and winey flavors of coffee. On the other hand, Saccharomyces cerevisiae can produce a large number of aromatic compounds, such as esters, alcohols and ketones, during the fermentation process. These compounds can give coffee a unique floral and fruity aroma and sweetness, improving the overall flavor of coffee.

[0154] Specifically, the advantages of Hansenula sporangiophora (LI-08) are its acid and alcohol resistance and its ability to produce complex flavor precursors. On the one hand, Hansenula sporangiophora has good acid and alcohol resistance and can continue to be active during the fermentation process, especially in the late fermentation environment with high acidity and alcohol concentrations. It can still metabolize normally. On the other hand, Hansenula sporangiophora can degrade macromolecules such as sugars and lipids. During baking, it generates a variety of volatile flavor substances such as furans, aldehydes and pyrazines through the Maillard reaction, caramelization reaction, lipid oxidation and the interaction of intermediates. These ingredients can enhance the caramel flavor, floral aroma and nutty aroma of coffee and increase the richness of the taste.

[0155] Specifically, the mechanism of compounding Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores in grape juice (LI-08) is synergistic effect, co-fermentation, enriching the flavor of coffee beans, and improving the quality of Catim coffee beans. First, Saccharomyces cerevisiae and Hansenula spores in grape juice can produce a synergistic effect when they are co-fermented. Saccharomyces cerevisiae quickly decomposes nutrients and produces primary fermentation products, some of which can be used as substrates for Hansenula spores in grape juice, thereby improving the utilization rate of nutrients, enhancing metabolic activity, shortening the fermentation cycle, and improving fermentation efficiency. Second, yeast metabolism produces antibacterial substances or the two yeast strains compete for nutrients, inhibiting the growth of harmful microorganisms; in addition, yeast metabolites (such as alcohol) can change the pH value and redox state of the fermentation environment. These changes will also inhibit the growth of certain toxin-producing fungi, further stabilize the fermentation environment, and ensure the normal fermentation process. Third, different types of yeast have different metabolic characteristics and flavor contribution capabilities. Fermentation with a single yeast can often only produce limited flavors and aromas, while the mixed use of Saccharomyces cerevisiae and Hansenulax spores can bring diverse and complex flavors. This is because different yeasts can produce different types and concentrations of aromatic compounds. The combined use can better regulate the balance of acidity and sweetness, ultimately making the flavor characteristics of Catim coffee more balanced and harmonious.

[0156] In addition, according to the technical solution of the present invention, Saccharomyces bayanus and Hansenula spores of grape juice interact with each other during the fermentation process, degrade macromolecular substances in coffee beans, synthesize flavor precursors, improve the flavor of coffee, and increase the rate of fine products.

[0157] Specifically, Saccharomyces cerevisiae and Hansenula spores co-ferment to produce different metabolites, such as amino acids and small molecule carbohydrate nutrients, which provide necessary nutritional support for fermentation. In addition, some of these metabolites are growth factors for the other strain of bacteria, forming a synergistic effect. For example, the extracellular enzymes secreted by Saccharomyces cerevisiae can help Hansenula spores break down complex carbohydrates more effectively. Under limited resource conditions, there is also a complex competition and cooperation relationship between microorganisms. Yeast inhibits the growth of harmful microorganisms by producing antibiotic antibacterial substances, and promotes the fermentation process together with other beneficial microorganisms.

[0158] In addition, according to the technical solution of the present invention, the composite bacterial suspension of Saccharomyces bayanus (CI-03) strain and Hansenula spores of grape juice (LI-08) strain plays an important role in the fermentation process of Catim coffee. Through the cell wall breaking effect of microorganisms and the secretion of functional enzymes, the aroma and taste substances in the coffee beans are released, thereby improving the flavor characteristics of the coffee beans; specifically as follows:

[0159] From the microbial wall-breaking effect, Bayer's yeast (CⅠ-03) mainly secretes cellulase and hemicellulase, which can effectively degrade the cellulose in the cell wall and promote the release of cell contents; while Hansenula sporogenes (LI-08) mainly secretes pectinase and other polysaccharide enzymes, which can degrade pectin and other polysaccharide components in the mucus layer, provide substrates for subsequent chemical reactions, and facilitate the action of various enzymes in the coffee bean matrix.

[0160] From the secretion of functional enzymes and their dynamic changes, in the early and middle stages of fermentation, the activity of polygalacturonase gradually increases, promoting the degradation of pectin and optimizing the fermentation process; polygalacturonase can degrade pectin, reduce the sticky substances in coffee beans, and release more aromatic compound precursors. At the same time, in the middle and late stages of fermentation, the activity of mannanase gradually increases, promoting the decomposition of macromolecular substances and improving the flavor characteristics of coffee; mannanase can degrade mannan, reduce the bitterness of coffee, and improve sweetness and taste.

[0161] From the release and changes of flavor substances during the fermentation process, the degradation of cell walls and the action of enzymes cause the polysaccharides and proteins in coffee beans to be broken down into monosaccharides and amino acids. These components undergo Maillard reaction and Strecker degradation reaction during roasting to form new flavor substances. Under the action of yeast, the degraded cell wall components in coffee beans release a variety of aromatic compounds, such as alcohols, esters and phenols, which give coffee its unique aroma.

[0162] In terms of improving the flavor characteristics of Catim coffee beans, the synergistic effect of polygalacturonase and cellulase releases more aromatic compounds, increasing the flavor complexity of coffee; while the action of mannanase and other polysaccharidase decomposes bitter components, improving the sweetness and taste of coffee. In addition, the alcohol, ester and phenolic volatile compounds produced during the fermentation process significantly enhance the aroma of coffee, giving it a richer and more unique flavor.

[0163] In addition, according to the technical solution of the present invention, the source of the Saccharomyces cerevisiae (CI-03) strain is Binchuan red grapes; and the source of the Hansenula sporogenes (LI-08) strain is Arabica coffee beans of the Typica variety. By using the naturally existing microorganisms in coffee beans or microorganisms in the environment for fermentation, not only can the cost of adding external strains be saved, but also a unique flavor characteristic can be formed.

[0164] First, we use the natural endogenous microbial resources in coffee beans to form a diverse microbial community, optimize the fermentation process, and improve the flavor and quality of coffee. Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores (LI-08) significantly improve the flavor characteristics of coffee beans by breaking the coffee beans' cell wall and secreting functional enzymes during the fermentation process.

[0165] Secondly, microbial community succession promotes the interaction between microorganisms. Different microorganisms form a dynamic balance through competition and symbiosis, thus optimizing the fermentation process. The metabolites produced by the early microorganisms provide substrates for the growth of subsequent microorganisms, which gradually accumulate and transform to form complex flavor substances. Specifically, the process of microbial community succession is as follows:

[0166] S1. Initial stage: In the early stage of fermentation, aerobic microorganisms in the environment (such as lactic acid bacteria and acetic acid bacteria) begin to multiply. These microorganisms metabolize simple sugars and other easily degradable organic substances in coffee beans to produce lactic acid, ethanol and carbon dioxide.

[0167] S2. Intermediate stage: As fermentation progresses, the oxygen in the environment gradually decreases, and anaerobic microorganisms (such as lactic acid bacteria and acetic acid bacteria) begin to dominate. These microorganisms further degrade complex carbohydrates and proteins, producing more organic acids and volatile compounds.

[0168] S3. Late stage: In the late stage of fermentation, some acid-resistant and ethanol-resistant microorganisms (such as two specific yeasts and bacteria) take the lead and complete the final metabolic process, further enhancing the flavor and aroma of coffee.

[0169] Third, during the fermentation process, two different yeasts and other microorganisms metabolize to produce alcohols, esters, ketones and organic acids, while polyphenols, sugars and amino acids are transformed under the metabolism of microorganisms. After roasting, a series of complex chemical reactions occur to generate a large number of volatile flavor substances, further enriching the flavor level of coffee. Specifically, the process of flavor change:

[0170] S1. Initial stage: mainly lactic acid and ethanol are formed, increasing acidity and a slight aroma;

[0171] S2. Mid-term: produces rich esters and alcohols, increasing fruity and floral aromas;

[0172] S3. Late stage: complex ketones and organic acids are formed, which increase the layering and lasting aroma of coffee.

[0173] Fourthly, it has an advantage in the early and middle stages of fermentation. Saccharomyces cerevisiae (CⅠ-03) secretes cellulase and hemicellulase to break the coffee bean wall and release the flavor precursors in the coffee beans. It also produces alcohols and esters through metabolism, giving the coffee a fruity and winey aroma.

[0174] Hansen yeast (LI-08) plays an important role in the middle and late stages of fermentation. It further decomposes pectin and macromolecular sugars in the mucus layer by secreting pectinase and other polysaccharidases, which is beneficial for the synthesis of complex ketones and organic acids, increasing the layering and lasting aroma of coffee. Specifically, the dynamic changes of enzyme activity:

[0175] S1. Early stage: Enzyme activity gradually increases, and it begins to decompose complex macromolecules and release flavor precursors;

[0176] S2. Mid-term: Enzyme activity reaches its peak, breaking down cell wall components to the greatest extent, which is beneficial for microorganisms and other enzyme systems to function;

[0177] S3. Late stage: Enzyme activity gradually decreases, but still maintains a certain level to complete the degradation and transformation of remaining substances.

[0178] Furthermore, the anaerobic fermentation method using mixed bacteria of Saccharomyces bayanus and Hansenula spores in grape juice described in the present invention has the following beneficial effects:

[0179] The present invention utilizes microbial fermentation to improve the flavor of coffee, uses fresh Catimu coffee fruit as raw material, accesses composite microorganisms to first perform anaerobic fermentation, and makes full use of the wall-breaking effect and metabolic characteristics of different yeasts, which is conducive to the release of aromatic and flavorful substances in Catimu coffee. After the fermentation is completed, it is combined with a honey treatment method. The carbohydrates and other material components rich in the inner peel of Catimu coffee, in the process of reducing the moisture content during drying, the sweetness and sourness slowly penetrate into the Catimu coffee beans, and the resulting coffee flavor is balanced after baking, and the taste is soft and more mellow. At the same time, the method of the present invention is simple to operate, easy to control and adjust in the processing process, and is suitable for industrial production. And it effectively solves the problems of long natural fermentation cycle of Catimu coffee, difficult to control, easy to pollute, uncontrollable coffee flavor, and unstable quality of Catimu coffee products at this stage.

[0180] Furthermore, the biological characteristics of the aforementioned Saccharomyces cerevisiae (CⅠ-03) strain and the Hansenula sporangial yeast (LI-08) strain indicate that the two strains have many similarities in terms of sugar fermentation ability, fermentation products, environmental adaptability, colony and cell morphology, and tolerance, which enables the two strains to work synergistically in mixed fermentation, jointly optimize the fermentation process of coffee beans, and improve fermentation efficiency and coffee bean product quality.

[0181] Specifically, in terms of environmental adaptability, both Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores in grape juice (LI-08) have good environmental adaptability and stress resistance. Both yeasts can maintain activity between pH 4.5 and pH 5.4, and both can grow and reproduce in the temperature range of 20-30°C, with the optimal fermentation temperature being 28±2°C. Specifically, in terms of colony and cell morphology, the colonies formed by Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores in grape juice (LI-08) on the culture medium have a moist and smooth surface, and the cell morphology is oval to elliptical, and they reproduce by budding.

[0182] Furthermore, the complementarity of fermented sugars was shown in the combined fermentation of the two strains. Specifically, both Hansenia sporeans (LI-08) and Saccharomyces cerevisiae (CⅠ-03) can ferment a variety of sugars, and neither can ferment lactose and inulin. Specifically, the main metabolites of Saccharomyces cerevisiae (CⅠ-03) are ethanol and carbon dioxide, and it can also produce a variety of aromatic compounds (such as esters, alcohols and ketones) to enhance the aroma and flavor of fermented coffee beans. Hansenia sporeans (LI-08) can produce complex aroma components (such as phenols, aldehydes and organic acids) during the fermentation process. These compounds can enhance the aroma level and taste richness of fermented coffee beans.

[0183] Furthermore, from the perspective of optimizing the mixed fermentation process, the ability to regulate the fermentation environment and improve the fermentation dynamics is presented. Specifically, the ethanol produced by Saccharomyces cerevisiae (CⅠ-03) and the organic acids (such as lactic acid and acetic acid) produced by Hansenia spores in grape juice (LI-08) can adjust the pH value of the fermentation environment, inhibit the growth of miscellaneous bacteria, and provide a relatively stable and safe fermentation environment; the metabolites produced by the two yeasts can affect the redox state of the fermentation environment and contribute to the generation and stability of specific aroma components. Specifically, the rapid sugar decomposition and alcohol fermentation ability of Saccharomyces cerevisiae (CⅠ-03) and the complex metabolic pathway of Hansenia spores in grape juice (LI-08) improve the efficiency of the entire fermentation process; the mixed fermentation of these two yeasts can start quickly at the beginning of fermentation, form a dominant bacterial community, and maintain stable metabolic activity throughout the fermentation process.

[0184] Furthermore, in terms of improving the quality of coffee bean products, the combined action of Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporei (LI-08) can produce a variety of aromatic compounds, including esters, alcohols, ketones, phenols and organic acids, which together give the fermentation products a unique and complex aroma and flavor. Due to the synergistic effect of the two yeasts, the fermentation products have a richer taste and a stronger sense of layers.

[0185] Furthermore, from the perspective of the food fermentation industry, mixed fermentation of Saccharomyces cerevisiae (CI-03) and Hansenula sporangiophora (LI-08) can achieve fine regulation of the fermentation process by precisely controlling the ratio and fermentation conditions of the two yeasts, thereby producing high-quality and consistent fermentation products, thereby reducing the differences in coffee bean products between different batches and ensuring the quality stability of coffee bean products. BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Figure 1 This is a colony morphology diagram of the Bayan Saccharomyces cerevisiae (CI-03) described in the present invention;

[0187] Figure 2 This is a colony morphology diagram of the grape juice spore-bearing Hansen yeast (LI-08) of the present invention;

[0188] Figure 3 The present invention discloses a process flow of coffee beans and a preparation method thereof by anaerobic fermentation of mixed bacteria of Saccharomyces bayanus and Hansenula spores in grape juice;

[0189] Figure 4 This is a physical picture of the fresh Yunnan Baoshan Catim coffee beans of the present invention;

[0190] Figure 5 This is a physical picture of the fresh fruit peeling step of the anaerobic fermentation method using mixed bacteria of Saccharomyces bayanus and Hansenula spores in grape juice according to the present invention;

[0191] Figure 6 This is a physical picture of the surface water drying stage of the anaerobic fermentation method using mixed bacteria of Saccharomyces cerevisiae and Hansenula spores in grape juice as described in the present invention. DETAILED DESCRIPTION

[0192] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0193] The first object of the present invention is to Saccharomyces bayanus ) strains and Hansenula sporogenes ( Hanseniaspora grapes ) strains have been screened, obtained and preserved, and the acquisition methods and identification results of these two strains are attached.

[0194] The second object of the present invention is to prepare a mixed seed bacterial suspension using the two strains (Saccharomyces bayanus combined with Hansenula spores) obtained in the first object of the present invention. The third object of the present invention is to provide a mixed bacterial anaerobic fermentation method using Saccharomyces bayanus and Hansenula spores, by applying a composite fermentation agent (mixed seed bacterial suspension) to Yunnan Catim coffee beans for anaerobic fermentation, and combining the honey treatment method to process coffee, so as to avoid the drawbacks of the existing traditional processing methods and improve the flavor of Catim coffee beans. The fourth purpose of the present invention is to obtain a high-quality Catim coffee through the fermentation method, which has the fructose sweetness naturally retained in the honey processing process, which is natural, long and delicate; the fruity aroma of the coffee itself is retained, and after the introduction of excellent yeast for fermentation, the flavor of tropical fruits is added, with the fresh aroma of citrus and rich nuts and caramel aroma, and the taste is fuller; in addition, the coffee has a bright acidity, a mellow sweetness, a moderate bitterness, a perfect balance, a smooth and delicate taste, a sweet and refreshing aftertaste, and a long and lasting aftertaste, forming a pleasant aftertaste, and the tasting score is 83.5 points.

[0195] Furthermore, the coffee bean variety used in all embodiments of the present invention is Catimor, which comes from Baoshan.

[0196] Specifically, the fermentation strain used in the present invention is Saccharomyces cerevisiae ( Saccharomyces bayanus ) strains, as described above, Saccharomyces bayanus ( Saccharomyces bayanus ) strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and CⅠ-03 was named Saccharomyces bayanus Saccharomyces bayanus The deposit number is CGMCC No: 30565, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0197] The fermentation strain used in the present invention is Hansenula vitis spores ( Hanseniaspora grapes ) strains, as described above, Hansenula vitis spores ( Hanseniaspora grapes ) strain deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, LI-08 was identified as Hansenula vitis spores ( Hanseniaspora grapes ), the deposit number is CGMCC No: 30563, the deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0198] As mentioned above, the Bay's yeast used in the present invention ( Saccharomyces bayanus ) strains and Hansenula sporogenes ( Hanseniaspora grapes ) The methods of obtaining strains are divided into strain screening, strain isolation, purification and preservation, and strain identification.

[0199] First, the Bayer yeast used in the present invention Saccharomyces bayanus )The process of obtaining the strain is as follows:

[0200] (1) Bayer's yeast Saccharomyces bayanus ) Screening: Bayer Saccharomyces strain samples were collected from mature Binchuan red grapes in Dali Prefecture, Yunnan Province, China. After being collected into sterilized sample bags, they were quickly transferred to low-temperature boxes for preservation and transported to the laboratory at low temperature.

[0201] (2) Isolation, purification and preservation of Saccharomyces cerevisiae strains: Endogenous microorganisms were isolated from the Binchuan red grape suspension. 10 g of coffee bean sample was weighed and placed in 90 mL of sterile saline for oscillation and mixing. The coffee bean mixture was diluted with sterile saline in a gradient manner. 200 μL of the dilution was spread on a yeast extract powder peptone glucose agar plate and the yeast was cultured at 30°C. The dominant yeast was selected according to the colony morphology and further streaked to obtain single colonies. The isolates with consistent morphology were frozen and stored for identification.

[0202] (3) Identification of Saccharomyces cerevisiae strains:

[0203] DNA extraction: TSINGKE plant DNA extraction kit (universal type) was used.

[0204] PCR amplification: Use the universal primers for bacterial species identification shown in Table 1 for amplification, and analyze the amplified products by agarose gel electrophoresis to determine whether the PCR product bands are consistent with the target size, whether they are single, and whether there is any dragging.

[0205] The PCR product determination results were compared and analyzed, and the Sanger sequencing results were spliced ​​using the software ContigExpress, and the inaccurate parts at both ends were removed. Furthermore, the spliced ​​sequences were blastn (latest version v2.13) in batches to compare with the nucleic acid database. Among them, the latest version of the nt library was selected for the nucleic acid database. By blastn comparison with the nt library, the Accession Number of the homologous sequence and the species identification and annotation can be obtained.

[0206] Next, the Hansen yeast ( Hanseniaspora grapes )The process of obtaining the strain is as follows:

[0207] (1) Hansenula sporogenes in grape juice ( Hanseniaspora grapes ) Screening: Grape juice spore-bearing Hansen yeast strain samples were collected from ripe coffee cherries (Coffea arabica. var. Typica, Arabica small-grain coffee Typica variety) at an altitude of 1,300 meters in Pu'er City, Yunnan Province, China. After washing, peeling and degumming, the cherries were collected into sterilized sample bags and quickly transferred to low-temperature boxes for storage, and then transported to the laboratory for low-temperature storage.

[0208] (2) Isolation, purification and preservation of Hansenula spore-bearing strains in grape juice: Endogenous microorganisms were isolated from a suspension of Arabica coffee beans of the Typica variety. 10 g of coffee bean sample was weighed and placed in 90 mL of sterile saline for oscillation and mixing. The coffee bean mixture was diluted with sterile saline in a gradient manner, and 200 μL of the dilution was spread on a yeast extract powder peptone glucose agar plate, and the yeast was cultured at 30 °C. The dominant yeast was selected according to the colony morphology and further streaked to obtain single colonies. The isolates with consistent morphology were frozen and stored for identification.

[0209] (3) Identification of Hansenula sporogenes in grape juice:

[0210] DNA extraction: TSINGKE plant DNA extraction kit (universal type) was used.

[0211] PCR amplification: amplification was performed using the universal primers for bacterial species identification shown in Table 1. The amplified products were analyzed by agarose gel electrophoresis to determine whether the PCR product bands were consistent with the target size, whether they were single, and whether there was any dragging.

[0212] The PCR product test results were compared and analyzed, and the Sanger sequencing results were spliced ​​using the software ContigExpress, and the inaccurate parts at both ends were removed. Furthermore, the spliced ​​sequences were blastn (latest version v2.13) in batches to compare with the nucleic acid database. Among them, the nucleic acid database selected the latest version of the nt library, and by blastn comparison with the nt library, the Accession Number of the homologous sequence and the species identification and annotation can be obtained.

[0213] Example 1 A mixed anaerobic fermentation method using Saccharomyces bayanus and Hansenula spores of grape juice and coffee beans according to the present invention

[0214] In a specific embodiment, the present invention provides a mixed anaerobic fermentation method using Saccharomyces bayanus and Hansenula spores of grape juice, comprising the following steps:

[0215] S1. Step 1: Preparation of mixed seed suspension (Bay yeast: Hansenula vitis spores)

[0216] Bayer's yeast (CⅠ-03) and Hansenula viticola (LI-08) frozen in -80 ℃ glycerol tubes were inoculated into YPD liquid medium and cultured at 28±2 ℃ in a constant temperature shaking incubator at 180±20 rpm for 24-36 h to activate two generations;

[0217] After the culture was completed, the supernatant liquid culture medium was removed after centrifugation, and the bacterial sludge was kept for later use. The bacterial sludge was diluted with sterile saline and the concentration of the single bacterial suspension was adjusted to 10 8 CFU / mL, that is, the concentration of the Bayer Saccharomyces suspension is 10 8 CFU / mL, the concentration of Hansenula spore suspension in grape juice is 10 8 CFU / mL; and the bacterial suspensions of the two strains were mixed at a ratio of CI-03:LI-08 of 1:1 (v:v). At this time, the concentration of the combined seed suspension was 0.5×10 8 CFU / mL, reserve;

[0218] S2. Raw material selection: Pick and remove immature fruits, shriveled fruits, fruit with diseases and insect pests, and foreign objects, and select fresh coffee fruits that are frost-free, free of microbial infection, and with less mechanical damage for later use.

[0219] S3. Pre-fermentation treatment steps (cleaning, sorting, peeling): The picked coffee berries are washed with clean water, and floating berries and foreign objects are removed. After the remaining coffee berries are cleaned, they are taken out and spread out to drain the water, and then the coffee berries are peeled to obtain fresh coffee beans.

[0220] S4. Inoculation and fermentation: Add the mixed seed suspension prepared in step 1 to the coffee beans treated in step 3, mix well and place in a food-grade thickened plastic bag (15 filaments, filled with nitrogen) for anaerobic fermentation, mixing once every 6-8 hours. The inoculation amount of the suspension is 1-2% (based on the weight percentage of fresh coffee beans), the fermentation temperature is 28±2 ℃, and the fermentation time is 36 hours;

[0221] S5. Drying the surface water: After the fermentation in step 4 is completed, the surface water of the fresh coffee beans is dried on a drying rack in an open factory in an environment with a temperature of 24-32 ℃ and a humidity of 30-40%. It takes about 2-4 days.

[0222] S6. Piling: After the surface water is dried, the seeds are piled up, that is, piled up to a certain height of about 30-40 cm in an environment with a temperature of 18-22 ℃ and a humidity of 50-60%; spread out to dry in an environment with a temperature of 28-32 ℃ and a humidity of 30-40%, with a thickness of about 2-3 cm, and piled up 1-2 times.

[0223] S7. Shelling after drying: When the moisture content of the coffee beans is 10-12%, shelling is performed to obtain fermented coffee beans.

[0224] S8. Roasting and grinding: The final coffee beans are roasted and ground to obtain Catim coffee powder.

[0225] Using the mixed seed bacterial suspension includes preparing the mixed bacterial suspension separately, and the specific steps of preparing the mixed bacterial suspension are as follows:

[0226] S51. Frozen strains: Purified and identified Saccharomyces bayanus (CⅠ-03) and Hansenula viticola (LI-08) strains were frozen in glycerol tubes at -80℃;

[0227] S52. Medium preparation: Prepare YPD liquid medium, which is composed of peptone, glucose, and yeast extract powder and is suitable for the growth of yeast. This medium provides rich nutrients and supports the rapid proliferation of Saccharomyces bayanus and Hansenula spores. The formula of YPD medium is 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast extract powder, which are dissolved in 1 liter of distilled water and sterilized by high-pressure steam (121 ℃, 20 min) for later use.

[0228] S53. Thawing of strains: Take out two frozen strains (Saccharomyces cerevisiae (CⅠ-03) and Hansenula viticola (LI-08) strains) from a -80°C freezer and thaw rapidly at room temperature; the thawing time of Saccharomyces cerevisiae is 60 minutes, and the thawing time of Hansenula viticola is 40-60 minutes; the inoculation volume of Saccharomyces cerevisiae is 50-100 μL, and the inoculation volume of Hansenula viticola is 50-100 μL;

[0229] S54. Inoculation of strains: thawing Saccharomyces cerevisiae and Hansenula viticola were inoculated into YPD liquid culture medium, respectively. YPD culture medium usually contains peptone, glucose, and yeast extract powder to provide nutrition for the growth of yeast;

[0230] S55. Set the mixed strain culture conditions: place the inoculated culture medium in a constant temperature shaking incubator, the mixed strain culture temperature is 28±2℃, the shaking speed is 180±20 rpm, and the mixed strain culture time is 24-36 hours; in this step, although the culture time is set to 24-36 hours, it will be slightly adjusted each time according to the observed growth status and culture effect to ensure that the mixed strain achieves the best activity; the fermentation strain is a 1:1 volume ratio of Bayer Saccharomyces cerevisiae suspension and a grape juice spore Hansen yeast suspension. At this time, the final concentration of the mixed seed suspension is 0.5×10 8 CFU / mL;

[0231] S56. Monitoring the growth of strains: Observe the growth of each strain (Saccharomyces cerevisiae (CⅠ-03) and Hansenula spores of grape juice (LI-08 strain) to ensure that the strains are in the logarithmic growth phase;

[0232] S57. Primary culture: Incubate at 28±2°C in a constant temperature shaking incubator at 180±20 rpm for 24-36 hours;

[0233] Monitor and observe the growth of each strain in real time to ensure that the strain is in the logarithmic growth phase;

[0234] S58. First subculture: Take an appropriate amount of bacterial liquid from the primary culture medium and inoculate it into a new YPD liquid culture medium. The common ratio is 1:100. Ensure that the operation is carried out in a sterile environment to avoid contamination. At this time, the inoculated culture medium needs to be placed in a constant temperature shaking incubator, set the temperature to 28±2℃, the speed to 180±20 rpm, and continue to culture for 24-36 hours. Repeat the same method, monitor and observe the turbidity and growth of the culture medium in real time to ensure that the yeast reaches the logarithmic growth phase.

[0235] S59. Second subculture: Take an appropriate amount of bacterial liquid from the first generation culture medium and inoculate it into a new YPD liquid culture medium. The common ratio is 1:100. Ensure that the operation is carried out in a sterile environment to avoid contamination. At this time, the inoculated culture medium needs to be placed in a constant temperature shaking incubator, set the temperature to 28±2℃, the speed to 180±20 rpm, and continue to culture for 24-36 hours. Repeat the same method, monitor and observe the turbidity and growth of the culture medium in real time to ensure that the yeast reaches the logarithmic growth phase.

[0236] S510. Preparation before centrifugation: Pour the culture fluids after two generations of culture into centrifuge tubes respectively, ensure that the mass in each centrifuge tube is the same, and place the centrifuge tubes symmetrically in the rotor of the centrifuge to ensure balanced operation of the centrifuge to avoid mechanical damage or reduction of centrifugal efficiency due to imbalance;

[0237] S511. Set the centrifugation conditions and start: the centrifugation speed is 4000-6000 rpm, the centrifugation time is 10-15 minutes, and the centrifugation temperature is 4°C; after completing the settings, start the centrifuge, run the centrifuge according to the set conditions, and start centrifugation;

[0238] S512. Centrifugal operation process: The microorganisms in the suspension are separated by centrifugal force; the centrifugal force generated by high-speed rotation causes the microbial cells with higher density to move to the bottom of the centrifuge tube to form a precipitate;

[0239] S513. Remove the supernatant and collect the bacterial sludge: Gently pour off the supernatant and retain the bacterial sludge precipitated at the bottom of the centrifuge tube;

[0240] S514. Preparation of mixed seed bacterial suspension: Dilute the collected bacterial sludge with sterile saline and mix well, and adjust the concentration of the single bacterial suspension to 10 8 CFU / mL; the bacterial suspension of Bayan Saccharomyces cerevisiae (CⅠ-03) and Hansenula viticola (LI-08) were mixed in a volume ratio (v) of 1:1; the final concentration of the mixed seed bacterial suspension was 0.5*10 8 CFU / mL (i.e., each milliliter of bacterial suspension contains 10 8 colony forming units) for subsequent fermentation.

[0241] Example 2 (Control Example and Measurement Results) Effect of a novel method for fermenting fresh coffee fruit according to the present invention, using a single bacterial fermentation or not inoculating exogenous strains on the Yunnan Catim coffee bean product

[0242] In multiple groups of specific embodiments and control examples, based on the novel processing method for fermenting fresh coffee fruits described in the present invention, in order to compare the effects of single-bacteria fermentation or not inoculating exogenous strains on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0243] In multiple groups of control examples, the mixed fermentation of the present invention is a mixed anaerobic fermentation method, and the first group of Catim coffee beans is fermented with Saccharomyces bayanus (CⅠ-03) and Hansenula sporogenes (LI-08).

[0244] Specifically, the specific example of anaerobic fermentation with a single bacterium (CI-03 Saccharomyces cerevisiae) is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that anaerobic fermentation with a single bacterium (CI-03 Saccharomyces cerevisiae) is used, and the remaining steps are the same as those in Example 1, which is used as a control example.

[0245] Specifically, the specific example of anaerobic fermentation with a single bacterium (LI-08 Hansenula sporangiophora) is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that anaerobic fermentation with a single bacterium (LI-08 Hansenula sporangiophora) is used, and the remaining steps are the same as those in Example 1, which is used as a control example.

[0246] Specifically, the specific embodiment without inoculation treatment is as follows: the fermentation method of fresh coffee beans in this comparative example is different from that in Example 1 in that no inoculation treatment is performed in this example, and the native strain of coffee beans is used as the fermentation microbial community, and there is no artificial inoculation strain. The remaining steps are consistent with Example 1, which is used as a control example.

[0247] In four groups of specific examples and control examples, multi-dimensional measurements (water content measurement, pH content measurement, tannin content measurement and sensory evaluation) were performed, as follows:

[0248] The moisture content of the coffee beans of three different control examples and Example 1 of the present invention that were anaerobic fermented for 36 h was measured. The measuring method is as follows: using HS153 halogen moisture meter, accurately weighing 2.00 g of coffee sample, putting it into a constant weight aluminum box, heating and drying at 105 ° C until the weight does not change within 90 s, recording the moisture content displayed by the instrument, and repeating three times for each sample. The moisture content measurement results of different examples are shown in Table 5 below.

[0249] Table 5

[0250]

[0251] Specifically, the four groups of experimental results are analyzed as shown in Table 5:

[0252] (1) Relationship between different coffee bean processing methods and moisture content

[0253] The first group (mixed anaerobic fermentation) had a moisture content of 41.30%. This indicates that during the mixed anaerobic fermentation treatment, the pectin in the high-density matrix and the metabolic activities and product accumulation of yeast created a highly osmotic environment in the fermentation system, which led to a decrease in cell tension and loss of intracellular water, significantly reducing the moisture content of the coffee beans. This treatment method can effectively shorten the drying process of Catim coffee beans.

[0254] The second group (anaerobic fermentation with single-bacteria CⅠ-03 Bayer's Yeast) had a moisture content of 49.43%: This indicates that the coffee beans treated with single-bacteria fermentation (CⅠ-03 Bayer's Yeast) had the highest moisture content, indicating that this treatment method maintained a relatively high moisture content during the fermentation process.

[0255] The third group (anaerobic fermentation of grape juice with single bacteria LI-08 Hansen's yeast) had a moisture content of 42.06%. This showed that the moisture content of Catim coffee beans after single bacteria fermentation (LI-08 Hansen's yeast) was lower, but still higher than that of mixed bacteria anaerobic fermentation, indicating that its effect in controlling moisture was not as good as that of mixed bacteria anaerobic fermentation.

[0256] The fourth group (without inoculation of exogenous strains) had a moisture content of 45.90%. The moisture content of Catim coffee beans that had not been inoculated was in the middle, indicating that the control effect of moisture content by fermentation under natural conditions was not as good as that by fermentation with specific strains.

[0257] (2) Effect of moisture content on the flavor control of Catim coffee beans under different coffee bean processing methods

[0258] The results of the four experiments shown in Table 5 show that mixed anaerobic fermentation can effectively reduce the moisture content, indicating that the fermentation process is more complete and produces more aromatic compounds and flavor substances; the moisture content is the highest in the anaerobic fermentation of single-bacteria CI-03 Bayer's yeast, which is easy to lead to insufficient fermentation and produce less flavor substances; the moisture content is low in the anaerobic fermentation of single-bacteria LI-08 Hansen yeast, indicating that the fermentation is more complete, but not as good as mixed anaerobic fermentation; when no bacterial treatment is done, the moisture content is in the middle, indicating that the natural fermentation effect is limited and the flavor control is not as good as the fermentation treatment of specific bacterial species.

[0259] Furthermore, as shown in Table 5, the final conclusions of the four groups of experiments are as follows: by comparing the moisture content under different treatment methods, the best treatment method has been found, which provides a scientific basis and guidance for the fermentation of Catim coffee beans.

[0260] Specifically, different processing methods directly affect the moisture content of coffee beans, which in turn affects their fermentation effect and final quality.

[0261] The mixed bacteria anaerobic fermentation shown in Table 5 had the lowest moisture content (41.30%), the best fermentation effect, and the coffee beans were fully dried; the single bacteria CI-03 Bayer's anaerobic fermentation had the highest moisture content (49.43%), the fermentation effect was poor, and the coffee beans were relatively wet; the single bacteria LI-08 Hansen's spores in grape juice anaerobic fermentation had a lower moisture content (42.06%), and the fermentation effect was better; when no bacterial treatment was performed, the moisture content was in the middle (45.90%), and the fermentation effect was average.

[0262] Specifically, mixed bacteria anaerobic fermentation shows obvious advantages in controlling moisture content and improving the quality of coffee beans. It can effectively reduce moisture content, ensure a sufficient fermentation process, and help shorten the subsequent drying time to produce rich aromatic compounds and flavor substances.

[0263] (2) pH determination

[0264] The pH of the coffee beans anaerobic fermented for 36 h in three different control examples and Example 1 of the present invention was measured by the following method: 5.00 g of coffee sample was weighed, 25 mL of distilled water was added, and the mixture was shaken for 20 min, and the pH was directly measured using a pH meter. The pH measurement results of different examples are shown in Table 6 below.

[0265] Table 6

[0266]

[0267] Specifically, the four groups of experimental results shown in Table 6 are analyzed (see Table 6):

[0268] (1) Relationship between different coffee bean processing methods and pH value

[0269] The first group (mixed anaerobic fermentation) has a pH value of 4.66: This indicates that the carbohydrate nutrients in the mixed anaerobic fermentation system are used as substrates for microbial growth, producing ethanol, acetic acid and lactic acid, which leads to a decrease in pH value. The metabolic activity of the microorganisms will also be more vigorous, thereby improving fermentation efficiency and reducing production costs. This treatment method can effectively control the acidity during the fermentation process and is conducive to improving flavor.

[0270] The second group (anaerobic fermentation of single bacteria CⅠ-03 Bay yeast) had a pH value of 5.45: This indicates that the pH value after single bacteria fermentation (CⅠ-03 Bay yeast) treatment was the highest, indicating that the amount of acidic substances accumulated during the fermentation process under this treatment method was average, and the acidity was relatively moderate.

[0271] The third group (anaerobic fermentation of grape juice with single bacteria LI-08 Hansen yeast with spores) had a pH value of 4.87: This indicates that the pH value after single bacteria fermentation (LI-08 Hansen yeast with spores) is moderate, indicating that it has a certain effect in controlling acidity, but is not as good as mixed bacteria anaerobic fermentation.

[0272] The fourth group (without exogenous strains) had a pH value of 3.95: This indicated that the pH value without bacterial treatment was the lowest, indicating that natural fermentation produced more acidic substances, which would make the sour taste overly prominent and affect the flavor balance of the coffee beans.

[0273] (2) Effect of pH value on the flavor control of Catim coffee beans under different coffee bean processing methods

[0274] The results of the four groups of experiments showed that mixed bacteria anaerobic fermentation can effectively control acidity and moderate pH value, giving coffee a unique sourness and bright acidity. In such a fermentation system, it is helpful to develop a more complex and delicate aroma, which is beneficial to the enhancement and balance of flavor; the acidity of anaerobic fermentation with single bacteria CⅠ-03 Bayer's yeast is the lowest and the pH value is the highest, which easily leads to a bland flavor and lack of layering; the acidity of anaerobic fermentation with single bacteria LI-08 Hansen yeast is moderate and the pH value is low, which can enhance the flavor, but the effect is not as good as mixed bacteria anaerobic fermentation; the acidity without inoculation treatment is the highest and the pH value is the lowest, which easily leads to over-fermentation and the production of undesirable flavor substances.

[0275] Furthermore, the final conclusion of the four groups of experiments was: by comparing the pH values ​​under different treatment methods, the best treatment method was found, which provided a scientific basis and guidance for the fermentation of Catim coffee beans.

[0276] Specifically, different treatment methods directly affect the pH value of coffee beans, and then affect their acidity and flavor performance. The pH value in mixed bacteria anaerobic fermentation is moderate (4.66), the acidity is well controlled, and the flavor enhancement effect is the best; the pH value in single bacteria CⅠ-03 Bayer's anaerobic fermentation is the highest (5.45), the acidity is the lowest, the flavor is relatively bland, and the layering is poor; the pH value in single bacteria LI-08 grape juice spore Hansen yeast anaerobic fermentation is low (4.87), the acidity is moderate, and the flavor is good; the pH value in the treatment without inoculation is the lowest (3.95), the acidity is the highest, and undesirable flavor substances are produced.

[0277] Specifically, mixed bacteria anaerobic fermentation shows obvious advantages in controlling acidity and improving the flavor of coffee beans. It can effectively control the pH value, ensure the fermentation process is sufficient, and produce rich aromatic compounds and flavor substances.

[0278] (3) Tannin content determination and analysis

[0279] The tannin content of the coffee beans subjected to anaerobic fermentation for 36 h in three different control examples and Example 1 of the present invention was measured by the following method:

[0280] S1. Preparation of standard curve: Accurately pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of tannic acid standard solution (200 mg / L) into 10 mL colorimetric tubes containing 6 mL of water, add 0.5 mL of forlin phenol, add 1 mL of 7.5% sodium carbonate solution, and make up to the scale with distilled water. Shake well, let stand for 30 min for color development, and use the reagent blank as the blank control. Measure the absorbance of the standard solution at a wavelength of 760 nm, and draw a standard curve with tannic acid concentration as the horizontal axis and absorbance value as the vertical axis.

[0281] S2. Determination of coffee sample: accurately measure 0.2 mL of coffee powder extract, place it in a 10 mL colorimetric tube containing 6 mL of water, add 0.5 mL of forlin phenol, add 1 mL of 7.5% sodium carbonate solution, distilled water to the scale, and shake well. After standing for 30 minutes to develop color, use the reagent blank as the blank control, measure the absorbance of the sample solution at a wavelength of 760 nm, and calculate the concentration of tannic acid in the test solution according to the standard curve.

[0282] Specifically, the tannin content results of each embodiment calculated by the tannic acid standard curve are shown in Table 7 below:

[0283] Table 7

[0284] .

[0285] Specifically, four groups of experimental results are analyzed (see Table 7):

[0286] (1) Relationship between different coffee bean processing methods and tannin content

[0287] The first group (mixed bacteria anaerobic fermentation) has a tannin content of 14.15 mg / g: This indicates that during the mixed bacteria anaerobic fermentation process, the action of microorganisms and their enzyme systems releases bioactive substances and increases antioxidant activity, which is beneficial to consumers' health, delays deterioration during storage, and improves the flavor and texture of coffee beans.

[0288] The second group (anaerobic fermentation of single bacteria CⅠ-03 Bayer's yeast) had a tannin content of 12.26 mg / g: This indicates that the tannin content after single bacteria fermentation (CⅠ-03 Bayer's yeast) treatment is lower, indicating that this treatment method is less effective in tannin synthesis and release.

[0289] The third group (anaerobic fermentation of grape juice with single bacteria LI-08 Hansen yeast with spores) had a tannin content of 12.62 mg / g: This indicates that the tannin content after single bacteria fermentation (LI-08 Hansen yeast with spores) is lower, but higher than that of single bacteria CⅠ-03 Bay yeast anaerobic fermentation, indicating that it has a certain effect in promoting tannin production.

[0290] The fourth group (without inoculation of exogenous strains) had a tannin content of 13.73 mg / g: This indicated that the tannin content of the untreated group was between that of mixed anaerobic fermentation and that of single anaerobic fermentation, indicating that natural fermentation could also promote the formation of tannins to a certain extent, but the effect was not as good as that of mixed anaerobic fermentation.

[0291] (2) Effect of tannin content on the flavor control of Catim coffee beans under different coffee bean processing methods

[0292] The results of the four groups of experiments showed that the tannin content in the mixed bacteria anaerobic fermentation was the highest, which could bring its unique bitterness to coffee, enhance the layering of coffee, and effectively improve the flavor and texture of coffee beans; the tannin content in the anaerobic fermentation of single bacteria CⅠ-03 Bayer's yeast was the lowest, and the flavor and texture were relatively bland; the tannin content in the anaerobic fermentation of single bacteria LI-08 Hansen's yeast with sporangium in grape juice was low, and the flavor and texture improvement effect was average; the tannin content in the uninoculated treatment was in the middle, and the flavor and texture improvement effect was limited.

[0293] Furthermore, the final conclusion of the four groups of experiments was: by comparing the tannin content under different treatment methods, the best treatment method was found, which provided a scientific basis and guidance for the fermentation of Catim coffee beans.

[0294] Specifically, different treatment methods directly affect the tannin content of coffee beans, and thus affect their flavor and texture performance. The tannin content in mixed bacteria anaerobic fermentation is the highest (14.15 mg / g), and the flavor and texture improvement effect is the best; the tannin content in single bacteria CⅠ-03 Bayer's anaerobic fermentation is the lowest (12.26 mg / g), and the flavor and texture are relatively bland; the tannin content in single bacteria LI-08 grape juice spore Hansen yeast anaerobic fermentation is low (12.62 mg / g), and the flavor and texture improvement effect is average; the tannin content in the treatment without inoculation is in the middle (13.73 mg / g), and the flavor and texture improvement effect is limited.

[0295] Specifically, mixed bacteria anaerobic fermentation shows obvious advantages in improving the tannin content and flavor texture of coffee beans, and can effectively promote the formation of tannins and improve the flavor and texture of coffee beans.

[0296] (4) Sensory evaluation

[0297] After anaerobic fermentation for 36 h in three different control examples and Example 1 of the present invention, coffee beans that were dried, piled and air-dried to a moisture content of 10-12% were roasted and then subjected to sensory evaluation. The specific evaluation process is as follows:

[0298] S1. Grinding: The roasted coffee beans are passed through a 20-mesh sieve, and the coffee powder passing rate should be 70-75%.

[0299] S2. Brewing: Pour 90-94 ℃ hot water into 11 g of coffee powder, break the dregs and pick them up after 4 minutes, and then taste them. The cupping samples are randomly coded, numbered, and presented.

[0300] The coffee samples were scored by five baristas with Q-graders in the sniffing, sipping and aftertaste procedures. The flavor characteristics were evaluated from ten comprehensive aspects: Fragrance / Aroma, Clean cup, Sweetness, Acidity, Body, Flavor, Aftertaste, Balance, Uniformity and Overall (see Table 8).

[0301] Table 8

[0302]

[0303] According to the SCA scoring requirements, coffee with a score of 80 or above is considered a specialty coffee. The use of mixed strain anaerobic fermentation and honey processing has a relatively significant improvement in the coffee flavor. The review team scored the coffee beans according to the SCA international cupping standards, and the coffee received a high score of 83.5 (see Table 8).

[0304] Further, the scoring results are shown in the table. Example 1 obtained a total score of 83.5 points, single bacteria (CI-03 Bay's yeast) anaerobic fermentation obtained a total score of 81.0 points, single bacteria (LI-08 grape juice spore Hansenula) anaerobic fermentation obtained a total score of 81.75 points, and no inoculation treatment obtained a total score of 79.0 points (see Table 8).

[0305] Furthermore, the panelists unanimously described the coffee beans fermented with mixed yeasts of Saccharomyces cerevisiae CⅠ-03 and Hansenula vitis spores CⅢ-23 as having strong nutty, caramel and citrus aromas, bright acidity, mellow sweetness, moderate bitterness, mellow and smooth taste, sweet and refreshing aftertaste, and long-lasting flavor. The intensity and flavor of the coffee fermented with no inoculation were weaker.

[0306] Specifically, coffee fermented by Saccharomyces cerevisiae CI-03 has a nutty, light cocoa aroma, good sweetness, but a relatively thin juice texture and low fruitiness. Specifically, coffee fermented by Hansenia sporeans LI-08 has a nutty, caramel aroma, a light lemon aroma, a slightly sour taste, and a sweet aftertaste, but a poor mellowness, with a bitter aftertaste and mixed flavors.

[0307] In addition, the coffee flavor of the non-inoculated group is lighter, with flavors of nuts and caramel, slightly stimulating acidity, slightly strong sourness, light aftertaste, and a short aftertaste.

[0308] Example 3 (Control Example and Measurement Results) Effect of Mixed Fermentation of Different Strains on Yunnan Catim Coffee Beans by Using a New Processing Method for Fermenting Fresh Coffee Fruits of the Present Invention

[0309] In multiple groups of specific embodiments and control examples, based on the novel processing method for fermenting fresh coffee fruits described in the present invention, in order to compare the effects of mixed fermentation of different strains on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0310] In multiple groups of control examples, the mixed fermentation of the present invention is a mixed anaerobic fermentation method, and the sixth group of Catim coffee beans uses Saccharomyces cerevisiae (CI-03) and Hansenia sporei (LI-08) for mixed fermentation. Specifically, the specific example of mixed fermentation of Saccharomyces cerevisiae (CI-03) and Saccharomyces cerevisiae is as follows: The fermentation method of fresh coffee beans in this comparative example is different from that in Example 1 in that mixed strains (Saccharomyces cerevisiae + Saccharomyces cerevisiae) are used for anaerobic fermentation, and the remaining steps are consistent with Example 1, as a control example. The cupping score of the coffee bean product prepared according to this example is 79.5 points.

[0311] The specific example of mixed fermentation of Saccharomyces bayanus (CI-03) and Pichia kluyveri is as follows: The fermentation method of fresh coffee beans in this comparative example is different from that in Example 1 in that mixed strains (Saccharomyces bayanus + Pichia kluyveri) are used for anaerobic fermentation, and the remaining steps are consistent with those in Example 1, which is used as a control example. The cupping score of the coffee bean product prepared according to this example is 78.0 points.

[0312] The specific example of mixed fermentation of Hansenula sporangiophora (LI-08) and Saccharomyces cerevisiae is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that mixed strains (Hansenula sporangiophora + Saccharomyces cerevisiae) are used for anaerobic fermentation, and the remaining steps are the same as those in Example 1, which is used as a control example. The cupping score of the coffee bean product prepared according to this example is 81.0 points.

[0313] The specific example of mixed fermentation of Hansenula sporangiophora (LI-08) and Pichia kluyveri is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that mixed strains (Hansenula sporangiophora + Pichia kluyveri) are used for anaerobic fermentation, and the remaining steps are the same as those in Example 1, which is used as a control example. The cupping score of the coffee bean product prepared according to this example is 78.25 points.

[0314] The specific example of mixed fermentation of Saccharomyces cerevisiae and Pichia kluyveri is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that mixed strains (Saccharomyces cerevisiae + Pichia kluyveri) are used for anaerobic fermentation, and the remaining steps are the same as those in Example 1, which is used as a control example. The cupping score of the coffee bean product prepared according to this example is 79.75 points.

[0315] In six groups of specific embodiments and control examples, the experimental data obtained are shown in Table 9 below:

[0316] Table 9

[0317]

[0318] Note: The data comes from specific experimental examples and control examples. The scores are based on the SCA international cupping standards, which are professional and credible and can objectively reflect the quality of coffee beans.

[0319] Specifically, six groups of experimental results are analyzed (see Table 9):

[0320] (1) Relationship between strain combination and cupping score

[0321] The first group (Bayan Saccharomyces cerevisiae + CI-03 + Saccharomyces cerevisiae) scored 79.5 points in the cupping test: This strain combination can produce better flavor and aroma during the fermentation process, but the overall score is slightly lower than the highest score.

[0322] The second group (Saccharomyces bayanus + CI-03 + Pichia kluyveri), the cupping score was 78.0 points: the fermentation effect of this combination was average, and the cupping score was low, which was due to the poor synergy between the strains.

[0323] The third group (Hansenula sporeensis LI-08 + Saccharomyces cerevisiae) scored 81.0 points in the cupping test: The flavor and aroma produced by this strain combination during the fermentation process are more prominent and scored higher.

[0324] The fourth group (Hansenula LI-08 + Pichia kluyveri), the cupping score was 78.25 points: The fermentation effect of this strain combination was average, and the cupping score was slightly higher than the second group, but still low.

[0325] The fifth group (Saccharomyces cerevisiae + Pichia kluyveri), cupping score of 79.75 points: The combination of these two yeasts produced a relatively medium flavor and aroma, with a score close to the first group.

[0326] Group 6 (Bay Saccharomyces CⅠ-03 + Hansen Saccharomyces LI-08), cupping score: 83.5 points: This combination has the best fermentation effect. The synergistic effect between the strains significantly enhances the flavor and aroma of the coffee beans, and the sensory score is the highest.

[0327] (2) Effect of strain combination on flavor control of Catim coffee beans

[0328] Specifically, the mixed fermentation under the combination of Saccharomyces cerevisiae + CI-03 + Saccharomyces cerevisiae resulted in coffee beans with good flavor and aroma, but not as good as the best combination; the mixed fermentation under the combination of Saccharomyces cerevisiae CI-03 + Pichia kluyveri resulted in average fermentation effect and poor flavor and aroma of the coffee beans.

[0329] Specifically, the mixed fermentation of grape juice with the combination of Hansenula spores LI-08 + Saccharomyces cerevisiae resulted in a coffee bean product with more prominent flavor and aroma and a higher score; the mixed fermentation of grape juice with the combination of Hansenula spores LI-08 + Pichia kluyveri resulted in a general fermentation effect of the coffee bean product, with a score slightly higher than that of Saccharomyces bayanus CI-03 + Pichia kluyveri.

[0330] Specifically, the mixed fermentation under the combination of Saccharomyces cerevisiae + Pichia kluyveri resulted in a coffee bean product with a relatively medium flavor and aroma, with a score close to that of Saccharomyces bayanus CI-03 + Saccharomyces cerevisiae.

[0331] Specifically, the mixed fermentation of Saccharomyces cerevisiae CI-03 and Hansenulax sporeans LI-08 produced coffee beans with the best flavor and aroma, the highest cupping score, and significant synergy between the strains.

[0332] Furthermore, the final conclusion of the six groups of experiments is: by comparing the cupping scores under different strain combinations, the best combination has been found, which provides a scientific basis and guidance for the fermentation of Catim coffee beans.

[0333] Specifically, different strain combinations directly affect the fermentation effect of coffee beans and the final cupping score. The cupping score of the combination of Saccharomyces cerevisiae CI-03 and Hansenula spores LI-08 was the highest, and the synergistic effect between the strains significantly improved the flavor and aroma of the coffee beans; the combination of Hansenula spores LI-08 and Saccharomyces cerevisiae had a higher score and a better fermentation effect; the cupping scores of other combinations were relatively low, and the fermentation effect was average.

[0334] Specifically, the best strain combination is Saccharomyces cerevisiae CI-03 + Hansenula spores LI-08, that is, the mixed bacteria anaerobic fermentation method provided by the present invention. The strain combination of Saccharomyces cerevisiae CI-03 + Hansenula spores LI-08 shows obvious advantages in improving the flavor and aroma of coffee beans, and has the highest sensory score.

[0335] Example 4 (Control Example and Measurement Results) Effects of Different Mixed Seed Bacteria Suspension Inoculation Amounts on Yunnan Catim Coffee Bean Products in a New Processing Method for Fermentation of Fresh Coffee Fruits According to the Present Invention

[0336] In multiple groups of specific embodiments and control examples, based on the novel processing method for fresh coffee fruit fermentation described in the present invention, in order to compare the effects of different mixed seed fungus suspension inoculation amounts on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0337] In multiple groups of control examples, the mixed fermentation of the present invention is a mixed anaerobic fermentation method, and the second group of Catim coffee beans uses Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporogenes (LI-08) for mixed fermentation, and the inoculation amount of the mixed seed bacteria suspension is 1-2%.

[0338] The specific embodiment of the mixed seed bacteria suspension inoculation amount <1% is as follows: The coffee bean fermentation method of this comparative example (first group) is different from that of Example 1 in that the mixed seed bacteria suspension inoculation amount is <1%, and the remaining steps are consistent with Example 1, which is used as a control example.

[0339] A specific embodiment in which the inoculation amount of the mixed seed bacteria suspension is greater than 2% is as follows: The fermentation method of green coffee beans in this comparative example (the third group) is different from that in Example 1 in that the inoculation amount of the mixed seed bacteria suspension is greater than 2%, and the remaining steps are consistent with those in Example 1, which is used as a control example.

[0340] In three groups of specific embodiments and control examples, the experimental data obtained are shown in Table 10 below:

[0341] Table 10

[0342]

[0343] (Note: The data comes from specific experimental examples and control examples. The scores are based on the SCA international cupping standards, which are professional and credible and can objectively reflect the quality of coffee beans.)

[0344] Specifically, three groups of experimental results are analyzed (see Table 10):

[0345] (1) Relationship between inoculation volume and cupping score

[0346] The first group (inoculum size <1%) had a cupping score of 80.75 points: This indicates that when the inoculum size is low, the exogenous bacteria do not form an advantage in the fermentation system, the fermentation effect is not significant, the fermentation cycle is extended, the risk of contamination by foreign bacteria is increased, the effect of the bacteria is limited, and the metabolites that can be produced per unit time are reduced, resulting in a low cupping score.

[0347] The second group (inoculum size 1-2%) scored 83.5 points in the cupping test: This indicates that the moderate inoculum size enables the strain to quickly take a dominant position in the early stage of fermentation, forming a competitive advantage, using the nutrients in the system for metabolic activities more quickly, accelerating the fermentation process, maintaining an efficient metabolic state, being able to fully function, achieving the best fermentation effect, and producing relatively rich aromatic compounds and flavor substances, resulting in the highest cupping score.

[0348] The third group (inoculum size > 2%) had a cupping score of 82.0 points: This indicates that the inoculum size is too high and the microorganisms multiply rapidly in the early stages of fermentation, consuming a large amount of oxygen, resulting in insufficient dissolved oxygen. It also causes a large amount of metabolic waste to be produced during the fermentation process, leading to over-fermentation. This will subsequently inhibit the growth and metabolism of the microorganisms, affecting the final flavor and quality, and resulting in a lower cupping score.

[0349] (2) Effect of inoculation amount on the flavor control of Catim coffee beans

[0350] The results of the three groups of experiments showed that when the inoculation amount was too low (<1%), the bacteria were insufficient and the flavor of the Catim coffee beans was poor; when the inoculation amount was moderate (1-2%), the bacteria were sufficient and the Catim coffee beans had the best flavor and the highest sensory score; when the inoculation amount was too high (>2%), excessive fermentation occurred, affecting the flavor consistency and quality of the Catim coffee beans.

[0351] Furthermore, the final conclusion of the three groups of experiments is: by comparing the fermentation effects under different inoculation doses, the optimal inoculation dose has been found, which provides a scientific basis and guidance for the mixed bacteria fermentation of Catim coffee beans.

[0352] Specifically, changes in inoculation amount directly affect the fermentation effect of the strain and the flavor performance of Catim coffee beans. When the inoculation amount is <1%, the strain is not effective enough and the cupping score is low; when the inoculation amount is 1-2%, the strain is fully effective, the fermentation effect is the best, and the cupping score is the highest; when the inoculation amount is >2%, excessive or uneven fermentation affects the flavor consistency and the cupping score is reduced.

[0353] Specifically, the novel processing method for fermenting fresh coffee fruit of the present invention has an optimal inoculation amount of 1-2%. Within this range, the strain can fully exert its function, produce rich aromatic compounds and flavor substances, and make the quality of Catim coffee beans reach the best state. In the actual production of Catim coffee beans, the fermentation effect of different batches of coffee beans under the same inoculation amount can ensure the consistency and stability of production.

[0354] Example 5 (Control Example and Measurement Results) Effects of Different Anaerobic Fermentation Times on Yunnan Catim Coffee Bean Products in a New Processing Method for Fermenting Fresh Coffee Fruits According to the Present Invention

[0355] In multiple groups of specific embodiments and control examples, based on the novel processing method for fermenting fresh coffee fruits of the present invention, in order to compare the effects of different anaerobic fermentation times on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0356] In multiple groups of control examples, the mixed fermentation of the present invention is a mixed anaerobic fermentation method, and the second group of Catim coffee beans uses Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporogenes (LI-08) for mixed fermentation, and the anaerobic fermentation time is anaerobic 36 hours.

[0357] The specific embodiment in which the anaerobic fermentation time is 24 h is as follows: The fermentation method of green coffee beans in this comparative example (first group) is different from that in Example 1 in that the anaerobic fermentation time is 24 h, and the remaining steps are consistent with those in Example 1, which is used as a control example.

[0358] The specific embodiment in which the anaerobic fermentation time is 48 h is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that the anaerobic fermentation time is 48 h, and the remaining steps are consistent with those in Example 1, which is used as a control example.

[0359] In three groups of specific embodiments and control examples, the experimental data obtained are shown in Table 11 below:

[0360] Table 11

[0361]

[0362] (Note: The data comes from specific experimental examples and control examples. The scores are based on the SCA international cupping standards, which are professional and credible and can objectively reflect the quality of coffee beans.)

[0363] Specifically, three groups of experimental results are analyzed (see Table 11):

[0364] (1) Relationship between fermentation time and cupping score

[0365] The first group (anaerobic fermentation for 24 hours) had a cupping score of 80.25 points: This indicates that the fermentation time was too short, the microorganisms were unable to fully utilize the substrate for metabolism, the fermentation process was incomplete, and fewer aromatic compounds and flavor substances were produced, resulting in a lower cupping score.

[0366] The second group (anaerobic fermentation for 36 hours) had a cupping score of 83.5 points: This indicates that within the appropriate fermentation time, the microorganisms can make full use of the substrate for metabolism, produce abundant metabolites and flavor substances, and have the best fermentation effect. The aromatic compounds and flavor substances produced are relatively rich, which helps to ensure the stability and consistency of the coffee quality, so the cupping score is the highest.

[0367] The third group (anaerobic fermentation for 48 hours) had a cupping score of 81.0 points: This indicates that the fermentation time was too long, resulting in over-fermentation, producing undesirable flavor substances or causing the decomposition of flavor substances, affecting the final flavor and quality, and resulting in a lower cupping score. In addition, over-fermentation also causes the decomposition or loss of certain bioactive substances, or increases the risk of contamination by foreign bacteria, posing a threat to the safety and stability of coffee.

[0368] (2) Effect of fermentation time on the flavor control of Catim coffee beans

[0369] The results of the three groups of experiments showed that when the fermentation time was too short (24 hours), the bacteria did not fully exert their effect and the flavor was poor; when the fermentation time was appropriate (36 hours), the bacteria exerted their effect fully, the fermentation effect was best, and the cupping score was the highest; when the fermentation time was too long (48 hours), over-fermentation occurred, affecting the flavor consistency and quality.

[0370] Furthermore, the final conclusion of the three groups of experiments is: by comparing the fermentation effects under different fermentation times, the optimal fermentation time has been found, which provides a scientific basis and guidance for the mixed bacteria anaerobic fermentation of Catim coffee beans.

[0371] Specifically, when the fermentation time is 24 hours, the bacteria are not fully active and the cupping score is low; when the fermentation time is 36 hours, the bacteria are fully active, the fermentation effect is optimal, and the cupping score is the highest; when the fermentation time is 48 hours, excessive fermentation produces undesirable flavor substances, affecting flavor consistency, and the cupping score is reduced.

[0372] Specifically, the novel processing method for fermenting fresh coffee fruit of the present invention has an optimal fermentation time of 36 hours: within this time range, the bacteria can fully exert their effects, produce rich aromatic compounds and flavor substances, and make the quality of Catim coffee beans reach the best state. Therefore, mixed bacteria anaerobic fermentation for 36 hours shows the best effect in all dimensions, which is an efficient, economical and environmentally friendly processing method that can significantly improve the overall quality of Catim coffee beans.

[0373] Example 6 (Control Example and Measurement Results) Effects of Whether to Perform Piling and Different Piling Times on Yunnan Catim Coffee Bean Products in a New Processing Method for Fermenting Fresh Coffee Fruits Described in the Present Invention

[0374] In multiple groups of specific embodiments and control examples, based on the novel processing method for fermenting fresh coffee fruits described in the present invention, in order to compare the effects of whether or not to perform pile fermentation and different pile fermentation times on Catimu coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0375] In multiple groups of control examples, the mixed fermentation of the present invention is a mixed anaerobic fermentation method, and the second group of Catim coffee beans uses Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporogenes (LI-08) for mixed fermentation, and the number of fermentations is 1-2 times.

[0376] The specific example of not performing pile fermentation and directly drying is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that pile fermentation is not performed and the coffee beans are directly dried until the moisture content of the coffee beans is 10-12%. The remaining steps are consistent with those in Example 1, which is used as a control example.

[0377] The specific embodiment of the fermentation number of 3-4 times is as follows: The fermentation method of green coffee beans in this comparative example is different from that in Example 1 in that the fermentation number of 3-4 times is used, and the other steps are consistent with those in Example 1, which is used as a control example.

[0378] In three groups of specific embodiments and control examples, the experimental data obtained are shown in Table 12 below:

[0379] Table 12

[0380]

[0381] (Note: The data comes from specific experimental examples and control examples. The scores are based on the SCA international cupping standards, which are professional and credible and can objectively reflect the quality of coffee beans.)

[0382] Specifically, three groups of experimental results are analyzed (see Table 12):

[0383] (1) Relationship between number of fermentation times and cupping scores

[0384] The first group (no pile fermentation) has a cupping score of 81.25 points: pile fermentation can promote fermentation or other chemical reactions. Without pile fermentation at all, the fermentation effect of the coffee beans is weak, the acidity of the coffee beans is relatively high, and the sweetness and complexity are slightly inferior to the coffee beans with proper pile fermentation, resulting in a lower cupping score.

[0385] The second group (fermentation 1-2 times), the cupping score is 83.5 points: the moderate number of fermentation times allows the bacteria to fully play their role, which can promote the internal chemical reaction and component transformation of the coffee beans, increase the sweetness and layering of the coffee beans, and thus the cupping score is the highest.

[0386] The third group (3-4 fermentations) scored 81.75 points in the cupping test: Too many fermentations lead to over-fermentation, which may cause the coffee beans to have unpleasant sour or musty tastes, causing the coffee beans to deteriorate, affecting the final flavor and quality, resulting in a lower cupping score. In addition, too many fermentations increase the complexity and uncertainty of the processing process, making it difficult to control the final flavor.

[0387] (2) Effect of fermentation times on the flavor control of Catim coffee beans

[0388] If pile fermentation is not carried out, the bacteria strain is not fully effective and the flavor is weak; if pile fermentation is carried out 1-2 times, the bacteria strain is fully effective, the fermentation effect is best, and the cupping score is the highest; if pile fermentation is carried out 3-4 times, excessive fermentation or uneven fermentation will occur, affecting the flavor consistency and quality.

[0389] Furthermore, the final conclusion of the three groups of experiments is: by comparing the fermentation effects under different fermentation times, the optimal fermentation times have been found, which provides a scientific basis and guidance for the fermentation treatment of Catim coffee beans.

[0390] Specifically, if no fermentation is performed, the bacteria strain is not fully effective and the cupping score is low; if the fermentation is performed 1-2 times, the bacteria strain is fully effective, the fermentation effect is optimal, and the cupping score is highest; if the fermentation is performed 3-4 times, excessive fermentation or uneven fermentation affects the flavor consistency and the cupping score is reduced.

[0391] Specifically, in the novel method for fermenting fresh coffee fruit of the present invention, the optimal number of fermentation is 1-2 times. Within this range, the bacteria can fully play their role, produce rich aromatic compounds and flavor substances, and make the quality of Catim coffee beans reach the best state.

[0392] Example 7 (Control Example and Measurement Results) Comparison of the Effects of Mixed Bacteria Aerobic Fermentation and Mixed Bacteria Anaerobic Fermentation on Yunnan Catim Coffee Bean Products

[0393] In multiple groups of specific embodiments and control examples, in order to compare the effects of the mixed bacteria aerobic fermentation treatment method and the mixed bacteria anaerobic fermentation method of the present invention on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0394] Among the multiple groups of control examples, the mixed fermentation of the present invention is the second group of experiments, and the second group of Catim coffee beans are subjected to mixed anaerobic fermentation using Saccharomyces bayanus (CⅠ-03) and Hansenula sporogenes (LI-08).

[0395] The experimental method and steps of mixed bacteria aerobic fermentation treatment are as follows:

[0396] S1. Sample preparation: Select the same batch of Catim coffee beans, 15 kg per group;

[0397] S2. Mixed aerobic fermentation method: A group of coffee beans were aerobic fermented using Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporogenes (LI-08) at a temperature of 28±2°C for 36 hours, with stirring every 6-8 hours to mix well.

[0398] The experimental method and steps of mixed bacteria anaerobic fermentation are as follows:

[0399] S1. Sample preparation: Select the same batch of Catim coffee beans, 15 kg per group;

[0400] S2. Mixed bacteria anaerobic fermentation treatment method (the method of the present invention): another group of coffee beans is subjected to anaerobic fermentation using Saccharomyces cerevisiae (CⅠ-03) and Hansenula sporangiophora vitis vinifera (LI-08) at a fermentation temperature of 28±2°C for 36 hours, with stirring every 6-8 hours to mix well.

[0401] Furthermore, professional identification and scoring were performed for the three groups of experimental results. The cupping score was scored by professional coffee tasters according to the SCA standard, and the average value was taken for each treatment. The aroma characteristics were analyzed by gas chromatography-mass spectrometry (GC-MS) to analyze the main aromatic compounds in coffee beans. The acidity, sweetness, and taste scores were scored by coffee tasting and analyzed using an electronic tongue. The physical properties recorded the appearance, color, and uniformity of the Catim coffee beans in different experimental groups during the experiment. The specific experimental results are shown in Table 13:

[0402] Table 13

[0403]

[0404] (Note: The data comes from specific experimental examples and control examples. The SCA international cupping standard includes multiple indicators such as aroma, flavor, acidity, balance, sweetness, and aftertaste. The comprehensive score can objectively reflect the quality of coffee beans.)

[0405] Specifically, two groups of experimental results are analyzed (see Table 13):

[0406] Based on the cupping scores, the flavor score of mixed bacteria anaerobic fermentation was the highest (83.5 points), significantly better than mixed bacteria aerobic fermentation (79.75 points), indicating that anaerobic fermentation can produce richer and more complex flavors.

[0407] In terms of the main aroma compounds, the conditions of mixed bacteria anaerobic fermentation are highly controllable, and the concentration of aroma compounds produced is the highest. The content of acid and ester compounds is relatively high. For example, ethyl acetate and isoamyl acetate help to form the sensory characteristics of fruit flavor, furfuryl alcohol and 2-furfural furan compounds provide floral and caramel aroma, and acetic acid, as the main acid substance after anaerobic fermentation, contributes to fruity sourness. These volatile components give coffee a complex and balanced aroma. The mixed bacteria aerobic fermentation process is relatively fast and easily affected by the environment. The concentration of aroma compounds produced is relatively low, and the content of some flavor substances is not strong enough.

[0408] In terms of acidity, the acidity score of mixed bacteria anaerobic fermentation is the highest (8.2). Yeast undergoes anaerobic respiration, and sugars are converted into ethanol and carbon dioxide. In this process, the sugar degradation rate is appropriate, and the production of ethanol neutralizes some acidic metabolites, making the coffee taste softer, the acidity is soft and bright, and the fruity acidity is natural and harmonious, which enhances the flavor level of the coffee. The acidity score of mixed bacteria aerobic fermentation is lower (7.6). During aerobic fermentation, there is sufficient oxygen, and the active aerobic respiration of microorganisms produces a large amount of acidic metabolites. The acidity of the coffee is too high and is unacceptable, resulting in a lower score.

[0409] In terms of sweetness, the mixed bacteria anaerobic fermentation has the highest sweetness score (8.0). The sugar decomposition rate in this process is most suitable, and the anaerobic environment increases the activity of certain enzymes, which decomposes the polysaccharides in the coffee beans into small molecule sugars, retaining and enhancing the sweetness of the coffee and making the flavor more balanced. The mixed bacteria aerobic fermentation has a lower sweetness score (7.7). During the aerobic fermentation process, the sugar decomposition rate is too fast and converted into ethanol and other flavor substances. Therefore, the final sweetness of the coffee is lower and the sweetness is poor.

[0410] In terms of the taste of coffee beans, mixed bacteria anaerobic fermentation scored the highest (9.3), with a mellow and full taste and a strong sense of layers, and was more popular with consumers; mixed bacteria aerobic fermentation scored a lower (8.0), with a more average taste.

[0411] In terms of the appearance, color and uniformity of the coffee bean products, the mixed bacteria anaerobic fermentation had the highest uniformity score (9.2) and the coffee beans had the best appearance consistency; the mixed bacteria aerobic fermentation had a lower uniformity score (8.5) and average appearance consistency.

[0412] Furthermore, the coffee bean characterization during the two experiments reflected the shortcomings of the barrel treatment method and the advantages of mixed fermentation, as shown in Table 14 below:

[0413] Table 14

[0414]

[0415] Experimental conclusions (see Table 14):

[0416] The mixed bacteria aerobic fermentation treatment method has obvious shortcomings. From the final quality of coffee beans, the coffee bean products obtained by the mixed bacteria aerobic fermentation treatment method are difficult to control the flavor, and need frequent stirring to ensure oxygen supply, poor flavor consistency, and low concentration of aroma compounds; the flavor diversity is low, the types and concentrations of aroma compounds are small, and the flavor levels are not rich. From the process flow of coffee beans, the coffee bean products obtained by the mixed bacteria aerobic fermentation treatment method have a long processing time, require frequent stirring, the fermentation process is unstable, and the time is difficult to control; the process manufacturing cost is high, and continuous oxygen supply and stirring equipment are required, the energy consumption is high, and the equipment maintenance cost is high; the preparation environment is difficult to control, continuous oxygen supply is required, and it is highly dependent on the external environment and is significantly affected by environmental changes.

[0417] The mixed bacteria anaerobic fermentation treatment method of the present invention presents unique advantages. From the final quality of coffee beans, the coffee bean product obtained by the mixed bacteria anaerobic fermentation treatment method has precise flavor control. By controlling the anaerobic environment, a variety of aroma compounds are produced with high concentrations, and the complexity and layering of flavor are significantly improved; at the same time, the coffee bean product obtained by the mixed bacteria anaerobic fermentation treatment method has high flavor diversity, many types of aroma compounds, high concentrations, rich flavor layers, and a stronger and more unique coffee flavor. From the process flow of coffee beans, the coffee bean product obtained by the mixed bacteria anaerobic fermentation treatment method has a stable fermentation process and easy-to-control fermentation time; and the processing cost is low, no oxygen supply is required, energy consumption is low, equipment maintenance is simple, and cost advantages are obvious; the anaerobic environment is stable, less dependent on the external environment, and less affected by environmental changes.

[0418] Overall, the mixed bacteria anaerobic fermentation treatment method shows obvious advantages in flavor control, processing time, cost, environmental factors and flavor diversity. It is a more efficient, economical and environmentally friendly treatment method that can significantly improve the overall quality of Catim coffee beans.

[0419] Example 8 (Control Example and Measurement Results) Comparison of the Effects of Wine Barrel Fermentation and Mixed Bacteria Anaerobic Fermentation on Yunnan Catim Coffee Bean Products

[0420] In multiple groups of specific embodiments and control examples, in order to compare the effects of the whiskey barrel treatment method, the red wine barrel treatment method and the mixed culture method of the present invention on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0421] Among the multiple groups of control examples, the mixed fermentation of the present invention is the third group of experiments, and the third group of Catim coffee beans uses Saccharomyces bayanus (CⅠ-03) and Hansenula sporogenes (LI-08) to carry out mixed anaerobic fermentation.

[0422] Furthermore, the barrel fermentation treatment method is a treatment method in which coffee beans are aged in barrels so that they absorb the residual flavor in the barrels. Many estates and bean seed bases in Honduras, a famous coffee bean producing area, use the barrel treatment method to treat coffee beans after harvest. This embodiment requires different barrel treatment methods for Catim coffee beans as a control example.

[0423] Specifically, the whiskey barrel treatment method and the red wine barrel treatment method are as follows for Catim coffee beans:

[0424] S1. Sample preparation: Select the same batch of Catimu coffee and divide them into two groups, each group has 15 kg; and manually select the ripe Catimu coffee berries, wash them with water to remove branches, residual leaves, soil, over-ripe and under-ripe beans, remove the peel and pulp, and obtain fresh coffee beans;

[0425] S2. Fermentation in different wine barrels: Put the treated Catim coffee beans with a mucilage layer into oak barrels (whiskey barrels and red wine barrels) that have been used to brew wine and seal them;

[0426] S3. Barrel fermentation: Regularly tumble different oak barrels (whisky barrels and red wine barrels) to allow the coffee beans to evenly absorb the aroma of the wine; whiskey barrel treatment is to place a group of coffee beans in a whiskey barrel for aging for 30 days; red wine barrel treatment is to place another group of coffee beans in a red wine barrel for aging for 30 days to ensure the degree of fermentation and complete absorption of the aroma of the wine;

[0427] S4. Drying: After the barrel fermentation is completed, the Catim coffee beans are taken out for drying, such as taking them to a greenhouse for drying or using other suitable drying methods.

[0428] Furthermore, professional identification and scoring were performed for the three groups of experimental results. The cupping score was scored by professional coffee tasters according to the SCA standard, and the average value was taken for each treatment. The aroma characteristics were analyzed by gas chromatography-mass spectrometry (GC-MS) to analyze the main aromatic compounds in coffee beans. The acidity, sweetness, and taste scores were scored by coffee tasting and analyzed using an electronic tongue. The physical properties recorded the appearance, color, and uniformity of the Catim coffee beans in different experimental groups during the experiment.

[0429] The specific experimental results are shown in Table 15 below:

[0430] Table 15

[0431]

[0432] (Note: The data comes from specific experimental examples and control examples. The SCA international cupping standard includes multiple indicators such as aroma, flavor, acidity, balance, sweetness, and aftertaste. The comprehensive score can objectively reflect the quality of coffee beans.)

[0433] Specifically, three groups of experimental results are analyzed (see Table 15):

[0434] From the cupping score, after being treated with whiskey barrels, the coffee beans had a flavor score of 79.75 points. The vanilla aroma and complex fermentation in the barrels increased the flavor level. After being treated with red wine barrels, the coffee beans had a higher score of 80.0 points. The tannins and lactic acid in the red wine barrels gave the coffee a richer flavor level and higher acidity. After mixed fermentation, the coffee beans had the highest score of 83.5 points. Mixed fermentation produced abundant esters, alcohols and furan compounds, making the coffee beans rich in flavor and rich in levels.

[0435] From the perspective of the main aroma compounds, the coffee beans are processed in whiskey barrels, which allow a very small amount of air to pass through the barrel wall, creating a low-temperature and low-oxygen fermentation environment. The unique fermentation method gives the finished product a whiskey-like aroma and oak fragrance, making the coffee more layered and long-lasting. In addition, the high concentrations of ethyl acetate and vanillin give the coffee the sweetness of vanilla, macarons and cream; after being processed in red wine barrels, the concentrations of lactic acid and ethyl acetate in the coffee bean product are high, bringing a complex and high-quality sour taste and a unique slightly astringent feeling. The aroma of the coffee itself is combined with the flavor substances to form a unique mixed flavor of fruit and wine. In addition, the barrel contains a certain amount of tannin, which penetrates into the coffee beans, giving the coffee a rich wine aroma and layered texture. The aroma compounds produced by mixed bacteria anaerobic fermentation have the highest concentration, and the content of acids and ester compounds is relatively high. For example, ethyl acetate and isoamyl acetate help to form the sensory characteristics of fruit flavor, furfuryl alcohol and 2-furfural furan compounds provide floral and caramel aromas, and acetic acid, as the main acid substance after anaerobic fermentation, contributes to fruity sourness. These volatile components give coffee a complex and balanced aroma.

[0436] In terms of acidity and sweetness, after being treated with whiskey barrels, the coffee acidity is no longer so sharp, but softer, more balanced and easier to accept (7.5); coffee treated with whiskey barrels can clearly feel the sweetness of vanilla and cream when tasting (7.5), and has a subtle wine aroma; the fruit acid and wine acid contained in the red wine barrel treatment can slightly increase the acidity of the coffee and present a more obvious and pleasant fruit acidity (8.0); this method presents more of a mixed flavor of fruit and wine, and the sweetness is slightly inferior to the whiskey barrel treatment method (7.2); after mixed bacteria anaerobic fermentation, the coffee The acidity and sweetness of the coffee bean products are optimal (8.2 and 8.0). In terms of acidity, the yeast undergoes anaerobic respiration, and sugars are converted into ethanol and carbon dioxide. In this process, the sugar degradation rate is appropriate, and the production of ethanol neutralizes some acidic metabolites, making the coffee taste softer, the acidity soft and bright, and the fruity taste natural and harmonious, which enhances the flavor level of the coffee. In terms of sweetness, the sugar decomposition rate in this process is optimal, and the anaerobic environment increases the activity of certain enzymes, which decomposes the polysaccharides in the coffee beans into small molecule sugars, retaining and enhancing the sweetness of the coffee and making the flavor more balanced.

[0437] In terms of the taste of the coffee beans, after being treated with whiskey barrels, the coffee beans have a fuller, richer, mellower and smoother taste (8.7), with strong vanilla and fruity aromas. After being treated with red wine barrels, the coffee beans have a mellower taste, are more refreshing and bright (9.0), have a balanced sweet and sour taste, and have a unique red wine flavor and astringent tannins. After mixed fermentation, the coffee beans have the most mellow taste (9.3), with a balanced sweet and sour taste and a strong sense of layering.

[0438] In terms of the appearance color and uniformity of the coffee beans, after being processed in whiskey barrels, the appearance of the coffee beans was light brown, with average uniformity (8.5); after being processed in red wine barrels, the appearance of the coffee beans was light brown, with a higher uniformity (9.0); after mixed fermentation, the appearance of the coffee beans was brown-yellow, with the highest uniformity (9.2).

[0439] Furthermore, the coffee bean characterization during the three experimental groups reflected the shortcomings of the barrel treatment method and the advantages of mixed fermentation, as shown in Table 16 below:

[0440] Table 16

[0441] .

[0442] Experimental conclusions (see Table 16):

[0443] In terms of the flavor and aroma of the coffee beans, the mixed fermentation treatment significantly improved the complex flavor and aroma of Catim coffee beans, with the highest score; the whiskey barrel treatment and red wine barrel treatment also significantly improved the flavor, but the mixed fermentation had a stronger flavor complexity and layering.

[0444] In terms of the taste and appearance of coffee beans, mixed fermentation performs best, with a mellow taste and rich layers; red wine barrel treatment is second, with a unique red wine flavor and astringent tannins; and whiskey barrel treatment brings a full taste and rich aroma.

[0445] From the overall evaluation of coffee bean products, compared with whiskey barrels and red wine barrels, mixed fermentation has obvious advantages in flavor complexity, taste roundness and aroma level. In addition, for food industry production, barrel processing requires a long aging time and high cost, while the mixed fermentation process is relatively controllable and efficient, suitable for large-scale production of high-quality coffee products.

[0446] Example 9 (Control Example and Measurement Results) Comparison of the Effects of Water-Washing Treatment, Semi-Dry Treatment and Mixed Bacteria Anaerobic Fermentation on Yunnan Catim Coffee Bean Products

[0447] In multiple groups of specific embodiments and control examples, in order to compare the effects of the water-washed treatment method, the semi-washed treatment method and the mixed culture method of the present invention on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0448] Among the multiple groups of control examples, the mixed fermentation of the present invention is the third group of experiments, and the third group of Catim coffee beans uses Saccharomyces bayanus (CⅠ-03) and Hansenula sporogenes (LI-08) to carry out mixed anaerobic fermentation.

[0449] The water-washing treatment method is a method of removing the pectin layer of coffee beans through wet fermentation, and then washing with water and drying. The water-washing treatment method can effectively control the quality and consistency of coffee beans, reduce the appearance of defective beans, and make the coffee present a brighter, clean, and higher acidity taste. Ethiopia and Colombia, famous coffee bean producing areas, mostly use the water-washing treatment method, so that the produced coffee has a fresh taste and bright acidity. This example requires the water-washing treatment method for Catim coffee beans as a control example.

[0450] Specifically, the treatment method of Catim coffee beans by the washed processing method is as follows:

[0451] S1. Picking: Select the same batch of Catim coffee and pick the ripe fruits, 15 kg per group;

[0452] S2. Mechanical degumming: Using a machine to remove the skin and part of the pulp of Catim coffee berries;

[0453] S3. Fermentation: Put the fresh Catimu coffee beans with pectin into the fermentation tank, add appropriate amount of water, and let it ferment naturally; during the fermentation process, microorganisms will decompose the pectin in the Catimu coffee beans;

[0454] S4. Washing: After fermentation is completed, rinse the Catim coffee beans with plenty of water to completely remove residual pectin and impurities;

[0455] S5. Drying: Place the washed Catim coffee beans in the sun or use drying equipment to reduce their moisture content to 10-12%.

[0456] The semi-dry method, also known as the honey treatment method, is a method of direct drying after partially removing the pulp. The flavor characteristics of coffee beans processed by the semi-dry method are between the washed method and the sun-dried method. They have a lower sour taste than washed beans, a higher cleanliness than sun-dried beans, and retain a certain sweetness. This example requires the conventional semi-dry treatment of Catim coffee beans as a control example.

[0457] Specifically, the semi-dry method for processing Catim coffee beans is as follows:

[0458] S1. Screening: Select the same batch of Catimu coffee, 15 kg per group; put the picked coffee berries into the pool, the overripe / overripe coffee berries will float on the water surface, and the ripe Catimu coffee beans will settle to the bottom of the pool, and the ripe Catimu coffee will be screened out;

[0459] S2. Remove the peel and pulp: Use a machine to remove the peel and pulp, and the fresh coffee beans still retain their mucus layer;

[0460] S3. Drying: Take the smooth-surfaced Catim coffee beans with a pectin layer outdoors to dry until the moisture content drops to about 10%-12%.

[0461] Furthermore, professional identification and scoring were performed for the three groups of experimental results. The cupping score was scored by professional coffee tasters according to the SCA standard, and the average value was taken for each treatment. The aroma characteristics were analyzed by gas chromatography-mass spectrometry (GC-MS) to analyze the main aromatic compounds in coffee beans. The acidity, sweetness, and taste scores were scored by coffee tasting and analyzed using an electronic tongue. The physical properties recorded the appearance, color, and uniformity of the Catim coffee beans in different experimental groups during the experiment.

[0462] The specific experimental results are shown in Table 17 below:

[0463] Table 17

[0464]

[0465] (Note: The data comes from specific experimental examples and control examples. The SCA international cupping standard includes multiple indicators such as aroma, flavor, acidity, balance, sweetness, and aftertaste. The comprehensive score can objectively reflect the quality of coffee beans.)

[0466] Specifically, three groups of experimental results are analyzed (see Table 17):

[0467] From the cupping score, the coffee beans produced by the washed processing method have a clearer, brighter acidity and a clean flavor, and the original flavor of the coffee beans is prominent (76 points), but its flavor is relatively simple, the body is light, and it lacks complexity; the coffee beans produced by the semi-dry processing method have a slightly higher flavor score (78.25 points). This processing method allows the coffee beans to absorb more sugar during the drying process, making the finished coffee product sweeter and improving the flavor, but the cleanliness is relatively low; the coffee beans produced by the mixed fermentation method have the highest flavor score (83.5 points), which is significantly better than the other two processing methods, with a strong flavor and rich layers.

[0468] From the perspective of the main aroma compounds, in the coffee beans that have been washed, butyl butyrate and ethyl acetate have fruity aroma, and 2-ethylfuran and 2-furanmethanol have caramel aroma; in the coffee beans that have been semi-dry-cleaned, 2-pentanone and 2-acetylfuran make the coffee sweet and fruity, and hexanal has fruity characteristics and a richer aroma; the aroma compounds produced by mixed bacteria anaerobic fermentation are at the highest concentration, with a high content of acids and ester compounds, such as ethyl acetate and isoamyl acetate, which help to form the sensory characteristics of fruity flavor, furfuryl alcohol and 2-furfural furan compounds provide floral and caramel aroma, and acetic acid, as the main acid substance after anaerobic fermentation, contributes to fruity acidity. These volatile components give coffee a complex and balanced aroma.

[0469] In terms of acidity, sweetness, and taste, the washed method is also a natural fermentation process. Microbial metabolism during soaking leads to a decrease in the content of fenugreek, glucose, and fructose. The anoxic conditions of the washed method promote alcoholic or lactic acid fermentation of sugars, producing different metabolites and organic acids. The acidity score of washed coffee beans is 8.0, with obvious sourness and bright acidity. The lowest sweetness score is 6.5, which is insufficient sweetness. The taste score is 7.5, which is relatively general. The coffee bean products obtained by the semi-dry method have the sweetness of honey or caramel because more sugar is attached or retained in the coffee beans during the drying process. The sweetness score is 7.5, the acidity score is 7.8, the sourness is slightly weak, and the taste score is 8.0, which is more mellow and mellow. The mixed bacteria anaerobic fermentation combined with honey processing has a soft and balanced acidity, bright and pleasant (8.2). Anaerobic fermentation controls the fermentation process, so that the sugar in the coffee beans is better retained and converted, thereby producing more sweet substances. The honey processing method further increases the sweetness of the coffee during the fermentation process by retaining some pectin. Therefore, the coffee obtained by the mixed anaerobic fermentation combined with the honey processing method has a higher sweetness, and the sweetness is natural, full and long-lasting (8.0). Anaerobic fermentation gives the coffee a fuller body and wine aroma, while the honey processing method adds fruity and fermented flavors to the coffee. The combination of these two processing methods makes the coffee have both depth and breadth in taste, and can show multiple levels of flavor (9.3).

[0470] In terms of appearance color and uniformity, the coffee bean product obtained by the washed processing method has a blue-green appearance, while the coffee bean product obtained by the semi-dry method has a yellow-green appearance, and the coffee bean product obtained by the mixed fermentation has a light green appearance; the coffee bean product obtained by the washed processing method has a uniformity score of 8.5, and the coffee bean product obtained by the semi-dry method has a uniformity score of 8.8, which are relatively consistent, while the coffee bean product obtained by the mixed fermentation has the highest uniformity score of 9.2, with the best appearance consistency.

[0471] Furthermore, the characterization of coffee beans during the three groups of experiments reflected the shortcomings of the washed and semi-washed processing methods and the advantages of mixed fermentation, as shown in Table 18 below:

[0472] Table 18

[0473]

[0474] Experimental conclusion:

[0475] According to Table 18, in terms of flavor control, mixed fermentation performs best in flavor control, and can achieve flavor consistency and precise control by controlling the dominant strains and fermentation conditions; the washed processing method and the semi-dry method are more difficult to control in flavor and are easily affected by the environment and processing conditions. In terms of flavor diversity, the mixed fermentation flavor is the most diverse and complex, giving the coffee rich flavor and layers; while the washed processing method has a relatively simple flavor, and the semi-dry method has a more diverse flavor but poorer stability.

[0476] In terms of process environment requirements, mixed fermentation has the least impact on the environment and the production process is environmentally friendly; while the water-washing method and semi-dry method have a greater impact on the environment, requiring a large amount of water resources and long drying time. In terms of processing time, mixed fermentation has the shortest processing time and the highest production efficiency; the water-washing method and semi-dry method have a longer processing time and lower production efficiency. In terms of the cost of preparing coffee beans, mixed fermentation has a lower cost and lower production cost; while the water-washing method and semi-dry method have a higher cost and high production cost.

[0477] From the perspective of the overall process, mixed fermentation has shown obvious advantages in all dimensions. It is a more efficient, economical and environmentally friendly processing method that can significantly improve the overall quality of Catim coffee beans.

[0478] Example 10 (Control Example and Measurement Results) Comparison of the Effects of Sun-drying, Four Honey Treatments and Mixed Fermentation on Yunnan Catim Coffee Bean Products

[0479] In multiple groups of specific embodiments and control examples, in order to compare the effects of the sun-drying method, the four honey treatment methods and the mixed culture method of the present invention on Catim coffee beans, the following experiments were conducted and the corresponding data were recorded.

[0480] Among the multiple groups of control examples, the mixed fermentation of the present invention is the sixth group of experiments, and the sixth group of Catim coffee beans are subjected to mixed anaerobic fermentation using Saccharomyces bayanus (CⅠ-03) and Hansenula sporogenes (LI-08).

[0481] The sun-drying method uses sunlight to directly dry the coffee cherries, reducing their moisture content to a storable level.

[0482] The traditional sun drying method is to directly lay the picked coffee fruits on the drying bed, dry them naturally in the sun, and turn them over regularly during the period; the trellis sun drying method is to dry the coffee fruits on a specially built trellis, so that the ventilation and light conditions can be better controlled and the influence of rain and dust can be reduced; the covering sun drying method is to use shade cloth or other coverings to adjust the intensity and time of sunlight exposure during the drying process to achieve a more ideal drying effect. The Yirgacheffe and Sidamo regions of Ethiopia, famous coffee bean producing areas, pick the ripe fruits of coffee beans and spread them flat on the elevated net bed for drying; Yemen, a famous coffee bean producing area, is famous for its unique sun drying process, which is to let the coffee fruits grow naturally on the tree and dry before picking them up, and then directly place them in the front yard of the coffee drying field to be dried in the sun. When the coffee beans are dried, the outer pulp and peel are removed and the coffee beans are taken out. This embodiment requires the conventional sun drying method for Catim coffee beans as a control example.

[0483] Specifically, the sun-drying method for treating Catim coffee beans is as follows:

[0484] S1. Picking: Select and pick the same batch of Catim coffee with better maturity, 15 kg per group;

[0485] S2. Screening: Remove unripe, damaged or defective berries and keep only good quality Catim coffee berries;

[0486] S3. Sun drying: Spread the screened Catim coffee berries evenly on the drying bed and dry them naturally in a sunny and well-ventilated environment. During the drying process, turn the berries frequently to ensure even drying and prevent mold or over-fermentation. This process usually lasts for several weeks (20 days), depending on weather conditions and the moisture content of the berries.

[0487] S4. Removing the shell: When the moisture content of Catim coffee beans is reduced to a certain level (specifically 10-12%), a shelling machine is used to remove the dried peel, pulp and inner peel to obtain coffee beans.

[0488] S5. Screening and grading: Screen and grade Catim coffee beans to remove defective beans and impurities, and classify them according to the size, shape and quality standards of the coffee beans.

[0489] Honey treatment is a method for processing green coffee beans, which is between the sun-drying method and the washing method. In the honey treatment process, after the coffee berries are picked and screened, the peel and pulp are removed, but part or all of the pectin is retained (this layer of pectin is called "honey" because it is sticky and has a high sugar content), and then the coffee beans with pectin are dried in the sun to reduce their water content to a certain extent. This method is used in coffee plantations in Costa Rica, Panama and Guatemala. Specifically, the honey treatment method is based on the amount of pectin retained and the length of the sun exposure time. In this embodiment, three different honey treatment methods are conducted, namely yellow honey, red honey and black honey. Specifically, the yellow honey treatment method has about 25% of the inner peel, the red honey treatment method has about 50% of the inner peel, and the black honey treatment method has almost no pectin removed, and the pectin layer is thicker by about 80-100%. Therefore, it takes longer and the conditions are more stringent to go from "yellow honey" to "black honey". Generally, the drying time of coffee depends on the local climate, temperature and humidity conditions. This embodiment requires different honey treatment methods for Catim coffee beans as a control example.

[0490] Specifically, the yellow honey treatment method for Catim coffee beans is as follows:

[0491] S1. Flotation sorting: Select the same batch of Catimu coffee beans, 15 kg per group; put the coffee berries into the water tank and select the ripe Catimu coffee beans (ripe berries will sink, while unripe berries, branches and leaves will float on the water);

[0492] S2. Remove the peel and pulp: Use a pulp sifter to remove the outer peel and pulp of the Catim coffee bean fruit;

[0493] S3. Drying twice: Drying the peeled coffee cherries for 2-3 days without turning them over, after which the moisture content of the peeled coffee cherries is about 30-35% and the color turns yellow; then drying the peeled coffee cherries for 1-3 days, repeatedly turning the peeled coffee cherries over during the second drying process, after which the moisture content of the peeled coffee cherries is 16-20%, to obtain dried coffee cherries;

[0494] S4. Storage: Store the dried coffee cherries in a dry and cool room for 2-4 days to produce honey flower aroma and fruit aroma, forming fragrant coffee cherries;

[0495] S5. Third drying: Dry the dried coffee cherries for the third time. It is best to spread the coffee cherries on a drying rack for the third drying until the moisture content is 10-12%, thereby obtaining finished yellow honey small-grain coffee beans.

[0496] Specifically, the red honey method for processing Catim coffee beans is as follows:

[0497] S1. Flotation sorting: Select Catimu coffee beans of the same batch, 15 kg per group; put the coffee berries into a water tank and select the ripe Catimu coffee beans (ripe berries will sink, while unripe berries, branches and leaves will float on the water);

[0498] S2. Remove the peel and pulp: Use a machine to remove the outer peel and part of the pulp of the Catim coffee fruit;

[0499] S3. Drying twice: Dry the peeled coffee cherries for the first time (in direct sunlight) for 1.5-2.5 hours without turning them over during the first drying process; then collect the peeled coffee cherries into piles and place them for 5-16 hours; finally dry them for the second time for 3-4 days, repeatedly turn the peeled coffee cherries over during the second drying process, about 4-5 times a day, so that the moisture content of the dried coffee cherries is about 20-26%;

[0500] S4. Storage: Store the dried coffee cherries in a dry and cool room for 2-4 days to produce fruity and sweet floral aromas, forming fragrant coffee cherries;

[0501] S5. Third drying: The dried coffee cherries are dried for the third time. It is best to spread the coffee cherries on the drying rack for the third drying, and turn them over about 4-5 times a day until the moisture content is 10-12%, and the finished red honey small-grain coffee beans are obtained.

[0502] Specifically, the black honey method for processing Catim coffee beans is as follows:

[0503] S1. Flotation sorting: Select Catimu coffee beans of the same batch, 15 kg per group; put the coffee berries into a water tank and select the ripe Catimu coffee beans (ripe berries will sink, while unripe berries, branches and leaves will float on the water);

[0504] S2. Remove the peel and pulp: Use a machine to remove the outer peel and part of the pulp of the Catim coffee fruit;

[0505] S3. Drying twice: Dry the peeled coffee cherries for 24 hours without turning them over during the first drying process; then collect the peeled coffee cherries into piles and place them for 8-16 hours; finally, dry them for 3-4 days for the second time, and turn the peeled coffee cherries over repeatedly during the second drying process, about 6-8 times a day, so that the moisture content of the dried coffee cherries is about 25-35%;

[0506] S4. Third drying: The dried coffee cherries are dried for the third time. It is best to spread the coffee cherries on the drying rack for the third drying, and turn them over about 4-5 times a day until the moisture content is 10-12%, and the finished black honey small-grain coffee beans are obtained.

[0507] Furthermore, professional identification and scoring were performed for the three groups of experimental results. The flavor score was scored by professional coffee tasters according to the SCA standard, and the average value was taken for each treatment. The aroma score was analyzed by gas chromatography-mass spectrometry (GC-MS) to analyze the main aromatic compounds in coffee beans. The acidity, sweetness, and taste scores were scored by coffee tasting and quantitatively analyzed using an electronic tongue meter. The physical properties were recorded by recording the appearance color, hardness, and uniformity of the Catim coffee beans in different experimental groups during the experiment.

[0508] The specific experimental results are shown in Table 19 below:

[0509] Table 19

[0510]

[0511] Specifically, the five groups of experimental results are analyzed (as shown in Table 19):

[0512] From the flavor score, the sun-drying method uses natural sunlight to dry the coffee beans, and the resulting coffee beans have a strong fruity and fermented aroma with a rich flavor; however, the flavor changes of the coffee beans obtained by the sun-drying method are difficult to control and are greatly affected by the weather and environment, resulting in poor flavor consistency; the long drying time causes the coffee beans to become damp and moldy, affecting the overall flavor quality of the Catim coffee beans. The coffee beans obtained by the yellow honey treatment method retain about 20% of the mucus layer and then dry. The resulting coffee beans are golden in appearance, oily, sweet, and have a strong honey flower aroma; the flavor control of the resulting coffee beans is still difficult, and the drying conditions have a great influence on the final flavor. The coffee beans obtained by the red honey treatment retain about 50% of the mucus layer and then dry, with candy aroma, chocolate aroma, sweet flower aroma, moderate acidity, good mellowness, smoothness, and long aftertaste; however, the temperature and humidity requirements during the drying process are higher, the processing difficulty increases, and the flavor consistency still needs to be controlled. The coffee beans obtained by the black honey treatment method retain about 80-100% of the mucus layer and then dry. During this period, a higher concentration of aromatic compounds is produced and penetrates into the coffee beans, making the flavor layer richer. The resulting coffee tastes full and smooth, with lively acidity and high mellowness; black honey treatment is the most difficult and requires a very high drying environment. A little carelessness will lead to flavor degradation and a long processing time. The mixed bacteria anaerobic fermentation method of the present invention controls the fermentation conditions and strain combination, and the resulting coffee bean product produces a variety of aromatic compounds, such as pyrazines, furans and esters, with precise flavor control, good balance of acidity and sweetness, and a mellow and full taste, which has an overall advantage.

[0513] From the main aroma compounds, the mixed bacteria anaerobic fermentation method produces the most aroma compounds and the highest concentration. For example, ethyl acetate and isoamyl acetate help to form the sensory characteristics of fruit flavor. Furfuryl alcohol and 2-furfural furan compounds provide floral and caramel aromas. Acetic acid, as the main acid substance after anaerobic fermentation, contributes to fruity sourness. These volatile components give coffee a complex and balanced aroma. The coffee processed by yellow honey, red honey and black honey methods has prominent honey aroma, sweet floral aroma and fruity aroma. Ketone compounds are mainly creamy and fruity. Damascus is one of the compounds that contributes mainly to the flavor of roasted coffee beans, which will produce floral and honey aromas. Furfural provides caramel aroma and sweet floral aroma, ester compounds contribute fruity aroma, such as furfuryl acetate; acid compounds are mainly sour, such as acetic acid; the sun-drying method has the least variety and concentration of aroma compounds. Linalool gives coffee fruit aroma, and ketone compounds are mainly creamy and fruity.

[0514] In terms of sweetness, the sweetness of sun-cured coffee is combined with fruit acid (6.5). The sweetness of yellow honey processed coffee is more obvious than that of sun-cured coffee, but it is still moderate compared to red honey and black honey (7.6). The red honey processed coffee is sweeter and can show a rich fruity sweet aroma (7.8). The black honey processed coffee has the highest sweetness, with a full and rich sweetness (7.9). Anaerobic fermentation controls the fermentation process so that the sugar in the coffee beans is better retained and converted, thereby producing more sweet substances. The honey processing method further increases the sweetness of the coffee during the fermentation process by retaining some pectin. Therefore, the coffee obtained by the mixed bacteria anaerobic fermentation combined with the honey processing method has a higher sweetness, and the sweetness is naturally full and long-lasting (8.0).

[0515] In terms of taste, coffee processed by the sun is usually richer, with a certain winey fermentation flavor and sweetness, while retaining more fruit flavor (6.5). Coffee processed by the yellow honey method has a relatively balanced taste, with both bright fruit acidity and moderate sweetness, and an overall refreshing taste (7.4). Coffee processed by the red honey method has a mellower taste, higher sweetness, and a strong fruity sweet aroma (7.8). Coffee processed by the black honey method has a full and diverse sweetness, with a variety of flavors such as fruit, cream, and caramel (8.0). Anaerobic fermentation gives coffee a fuller body and wine aroma, while the honey method adds fruity and fermented flavors to coffee. The combination of these two processing methods gives the coffee both depth and breadth in taste, and can show multiple levels of flavor (9.3).

[0516] Furthermore, the coffee bean characterization during the six experimental groups reflected the shortcomings of the sun-drying method and the four honey-drying methods and the advantages of mixed fermentation, as shown in Table 20 below:

[0517] Table 20

[0518]

[0519] Experimental conclusion (according to Table 20):

[0520] In terms of flavor control of coffee beans, the mixed bacteria anaerobic fermentation method achieves the best flavor control through the combination of bacteria and fermentation conditions; the sun-drying method and yellow honey processing method are second, with richer and more complex flavors. The flavor control of black honey processing and red honey processing methods is more difficult and is greatly affected by the environment.

[0521] In terms of the flavor diversity of coffee bean products, the flavor of the mixed bacteria anaerobic fermentation method described in the present invention is the most diverse and complex, with rich aromatic compounds and layered texture; the flavor of the black honey processing method is second, with the richest and most complex flavor, while the flavor of the red honey processing method is richer with enhanced layered texture, and the flavor of the yellow honey processing method is relatively fresh and sweet; the flavor of the sun-drying processing method is relatively simple.

[0522] In terms of process environment requirements, the mixed bacteria anaerobic fermentation method of the present invention has flexible environmental control and minimal impact on the environment; the sun-drying method is greatly affected by weather and environment and has high environmental requirements; the three honey processing methods are relatively complicated and time-consuming, and the drying conditions need to be controlled. The drying time is very important. If it is too short, the sweetness will be poor; if it is too long, it will have a musty smell. In terms of processing time, the mixed bacteria anaerobic fermentation method has a shorter processing time, and the fermentation process only takes 36-48 hours; while the sun-drying method and the traditional honey processing method have longer processing times, especially the black honey processing method, which has the longest drying time. In terms of coffee bean processing costs, the sun-drying method has a lower cost, but requires a lot of drying space and manpower; the mixed bacteria anaerobic fermentation method of the present invention has a moderate cost, but requires fermentation equipment and technology; the yellow honey processing method, the red honey processing method and the black honey processing method have higher costs, and the processing difficulty and drying time increase.

[0523] From the perspective of the overall process, the mixed bacteria anaerobic fermentation method described in the present invention shows obvious advantages in flavor control, processing time, cost, environmental factors and flavor diversity. It is a more efficient, economical and environmentally friendly processing method, which can significantly improve the overall quality of Catim coffee beans.

Claims

1. A composite fermentation agent for fermenting coffee beans, the composite fermentation agent being Saccharomyces cerevisiae ( Saccharomyces bayanus )CI-03 and Hansenula sporogenes ( Hanseniaspora uvarum ) LI-08 mixed seed suspension; The Bayan yeast CI-03 was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 9, 2024, with the deposit number CGMCC No: 30565, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The grape juice spore-bearing Hansen yeast LI-08 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on May 9, 2024, with a deposit number of CGMCC No: 30563, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method of using Saccharomyces cerevisiae ( Saccharomyces bayanus )CI-03 and Hansenula sporogenes ( Hanseniaspora uvarum ) LI-08 mixed bacteria fermentation method for preparing coffee powder, characterized in that: The following steps are involved: S1. Select fresh Catim coffee berries; S2. Wash fresh fruits; S3. Fresh fruit sorting; S4. Preparing fresh coffee beans; S5. Inoculation: A mixed seed suspension was prepared using Saccharomyces cerevisiae CI-03 and Hansenula vitis spores LI-08, and the mixed seed suspension was evenly added to the fresh coffee beans at an inoculation rate of 1-2% by weight, so that the mixed seed suspension was evenly distributed; S6. Anaerobic fermentation: Anaerobic fermentation is carried out in an environment of 24-32°C for 10-50 hours; S7. Dry the surface water: dry the surface water in an environment with a temperature of 24-32 ℃ and a humidity of 30-40%; S8. First fermentation: Pile the coffee beans to a height of 30-40 cm in an environment with a temperature of 18-22 °C and a humidity of 50-60% for 20-24 hours; S9. Second pile fermentation: After turning the pile, the second pile fermentation is carried out, and the conditions for the pile fermentation are the same as those in S8; S10. Shelling after drying: drying the coffee beans until the moisture content is 10-12%, and then shelling to obtain fermented coffee beans; S11. roasting and grinding to obtain the final Catim coffee powder product; The Bayan yeast CI-03 was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 9, 2024, with the deposit number CGMCC No: 30565, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The grape juice spore-bearing Hansen yeast LI-08 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on May 9, 2024, with a deposit number of CGMCC No: 30563, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

3. The method for preparing coffee powder according to claim 2, characterized in that: The mixed seed bacterial suspension is mixed with a suspension of Saccharomyces cerevisiae CI-03 and a suspension of Hansenula spores in grape juice LI-08. The ratio of the suspension of Saccharomyces cerevisiae CI-03 to the suspension of Hansenula spores in grape juice LI-08 is 1:1 by volume. The concentration of the suspension of Saccharomyces cerevisiae CI-03 is 10 8 CFU / mL, the concentration of the grape juice Hansen yeast LI-08 bacterial suspension is 10 8 CFU / mL.

4. The method for preparing coffee powder according to claim 2, characterized in that: The anaerobic fermentation in step S6 is to place the mixed coffee beans into a food-grade thickened plastic bag filled with nitrogen; perform anaerobic fermentation in an environment of 28±2°C, and mix once every 6-8 hours; the total duration of anaerobic fermentation is 24-36 hours.

5. The method for preparing coffee powder according to claim 2, characterized in that: The variety of the coffee beans is Yunnan Catim coffee beans.

6. The method for preparing coffee powder according to claim 2, characterized in that: The step S1 of selecting fresh Catimu coffee berries is to select ripe fresh Catimu coffee berries to ensure that the berries are full and have uniform color; remove immature berries, shrunken berries, berries with diseases and insect pests, and other foreign matter; and select fresh Catimu berries that are free of frost, microbial infection, and less mechanical damage for later use.

7. The method for preparing coffee powder according to claim 2, characterized in that: The step S2 of fresh fruit cleaning is to clean the picked Catimun fresh fruit with clean water to remove dirt and impurities on the surface.

8. The method for preparing coffee powder according to claim 2, characterized in that: The step S4 of preparing fresh coffee beans is to peel fresh Catim coffee berries to obtain fresh coffee beans.

9. The method for preparing coffee powder according to claim 2, characterized in that: The step S10 of drying and shelling is to dry the coffee beans until the moisture content is 10-12%; when the coffee beans reach the required moisture content, shelling is performed to obtain fermented coffee beans; and good ventilation is ensured in the spreading area to accelerate the evaporation of moisture and the drying process of the coffee beans; Ensure that the entire process is carried out in a hygienic environment to prevent the coffee beans from being contaminated by the outside world.

10. The method for preparing coffee powder according to claim 2, characterized in that: The step S11 of roasting and grinding is to roast the shelled coffee beans, with the degree of roasting ranging from light roasting to medium roasting; the roasted coffee beans are cooled and then ground to obtain the final Catim coffee powder product.

11. A Bay's yeast as claimed in any one of claims 2 to 10 ( Saccharomyces bayanus )CI-03 and Hansenula sporogenes ( Hanseniaspora uvarum ) Catim coffee powder is prepared by the method of preparing coffee powder by LI-08 mixed bacteria fermentation.

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